TUGAS MATA KULIAH

ILMU PENYAKIT VIRAL

“AFRICAN SWINE FEVER (ASF)”

Oleh:

PALAGAN SENOPATI SEWOYO (1709511062)

MARTINA TIODORA SITOHANG (1709511063)
NI MADE ERNAWATI (1709511065)
SAMUEL EVAN (1709511066)
IDA BAGUS KRISNA PRADNYADANA (1709511098)
I PUTU DWI KOMALA PUTRA (17095110103)
AGUSTINHO MOREIRA BELO (17095110127)
ZELIA XIMENES (17095110133)
LABORATORIUM VIROLOGI
FAKULTAS KEDOKTERAN HEWAN
UNIVERSITAS UDAYANA
TAHUN
2019
 KATA PENGANTAR

Puji syukur kehadirat Tuhan Yang Maha Esa berkat limpahan rahmat dan
karunia-Nya, paper yang berjudul “AFRICAN SWINE FEVER (ASF)” dapat
diselesaikan dengan baik dan tepat waktu dalam tujuan pemenuhan tugas mata
kuliah ilmu penyakit viral. Tak lupa kami ucapkan terima kasih kepada para dosen
mata kuliah ilmu oenyakit viralyang telah membimbing dan mengajar kami:

1. Prof. drh. I NYOMAN MANTIK ASTAWA, Ph.D.

2. Prof. Dr. drh. GUSTI AYU YUNIATI KENCANA, MP.
3. Prof. Dr. drh. I Gusti Ngurah Kade Mahardika
4. Dr. drh. Ida Bagus Kade Suardana, M.Si.

Penulis sadar masih banyak kekurangan yang terdapat pada paper ini, jadi
sangat diperlukan saran dan kritik membangun dari pembaca. Besar harapan paper
ini dapat bermanfaat bagi mahasiswa Fakultas Kedokteran Hewan pada khususnya,
ataupun bidang-bidang saintek lain yang menekuni tentang parasitologi pada
umumnya. Akhir kata kami ucapkan terima kasih

Penulis

Denpasar, 2019

ii
 DAFTAR ISI

KATA PENGANTAR .................................................................................... ii

DAFTAR ISI ................................................................................................... iii
DAFTAR GAMBAR ...................................................................................... iv
DAFTAR TABEL ......................................................................................... v
BAB I PENDAHULUAN ............................................................................... 1
1.1 Latar Belakang ............................................................................... 3
1.2 Rumusan Masalah .......................................................................... 1
1.3 Tujuan ............................................................................................ 1

BAB II PEMBAHASAN ................................................................................ 2

2.1 Etiologi ........................................................................................... 2
2.2 Epidemiologi .................................................................................. 3
2.2.1 Spesies Terdampak ............................................................... 4
2.2.2 Cara penularan dan penyebaran ............................................ 4
2.3 Patogenesis .................................................................................... 5
2.4 Patologi .......................................................................................... 6
2.5 Gejala Klinis................................................................................... 9
2.6 Diagnosis ........................................................................................ 11
2.7 Diagnosis Banding ......................................................................... 11
2.8 Imunitas, Pencegahan, dan Kontrol ............................................... 13
BAB III PENUTUP ........................................................................................ 15
3.1 Kesimpulan ................................................................................... 15
3.2 Saran ............................................................................................... 15
DAFTAR PUSTAKA .................................................................................... 16

iii
 DAFTAR GAMBAR

Gambar 2.1 .................................................................................................... 2

Gambar 2.2 ..................................................................................................... 3

Gambar 2.3 ..................................................................................................... 5

Gambar 2.4 ..................................................................................................... 8

Gambar 2.5 ..................................................................................................... 8

Gambar 2.6 ..................................................................................................... 9

iv
 DAFTAR TABEL

Tabel 2.1 ......................................................................................................... 6

Tabel 2.2 .......................................................................................................... 12

v
 1

BAB I

PENDAHULUAN

1.1. Latar Belakang

African swine fever (ASF) merupakan penyakit viral pada babi yang
mengakibatkan angka mortalitas yang tinggi pada babi Hal ini menyebabkan
kehilangan ekonomi yang luarbiasa dan tidak bisa dicegah karena tidak adanya
vaksin yang efektif dan metode kontrol penyakit. Penyakit ini disebabkan oleh virus
yang pertama kali diidentifikasi di Kenya pada tahun 1920-an. Kemudian,
penyebaran terjadi begitu pesat sampai ke Eropa, dan sampai sekarang di Amerika
Selatan, dan karibia. Penyakit ini diberantas dari Eropa (kecuali Sardinia) pada
tahun 1990-an melalui kontrol drastis dan program pemberantasan. Pada paper ini
akan dibahas lebih lanjut mengenai penyakit viral ini.

1.2. Rumusan Masalah

1. Apa agen penyebab (etiologi) dari penyakit african swine fever ?
2. Bagaimana epidemiologi dari penyakit ini?
3. Bagaimana patogenesis dari penyakit ini?
4. Bagaimana gejala klinis dari penyakit ini?
5. Bagaimana diagnosis dari penyakit ini?
6. Apa diagnosis banding dari penyakit ini?
7. Bagaimana pengobatan dan pencegahan dari penyakit ini?
1.3. Tujuan Penulisan
1. Untuk mengetahui agen penyebab dari penyakit african swine fever (ASF)
2. Untuk mengetahui epidemiologi dari penyakit ini.
3. Untuk mengetahui patogenesis dari penyakit ini
4. Untuk mengetahui gejala klinis dari penyakit ini
5. Untuk mengetahui bagaimana cara mendiagnosis penyakit ini
6. Untuk mengetahui diagnosis banding dari penyakit ini
7. Untuk mengetahui bagaimana pengobatan dan pencegahannya.
 2

BAB II

PEMBAHASAN

2.1. Etiologi

Gambar 2.1. Pohon filogenik yang membandingkan protein dUTPase yang

dienkode oleh African swine fever virus (AFSV) dengan virus DNA lain.
African swine fever virus (ASFV) merupakan virus DNA beramplop yang
merupakan anggota dari genus Asfivirus dan famili Asfarviridae (Asfar = African
swine fever dan virus terkait). African swine fever virus merupakan satu-satunya
arbovirus DNA yang diketahui dan ditransmisikan melalui caplak dari genus
Ornithodoros. Strain virus dibedakan berdasarkan virulensi terhadap babi, yang
mana memiliki rentang dari yang penyakit sangat letal atau mematikan hingga
penyakit yang subklinis. Strain juga dapat dibedakan berdasarkan sekuens genetik,
dan geb enkode-virus p72 (yang juga dikenal sebagai p73) yang dapat digunakan
untuk genotiping virus.
 3

Gambar 2.2. (A) Pewarnaan negatif virion menunjukkan garis heksagonal dari
kapsid dengan amplop (B dan C) Pewarnaan negatif dari kapsid yang rusak yang
menunjukkan susunan kapsomer. (D) Bagian tipis dari virion menunjukkan
lapisan ganda yang mengelilingi inti. (Skala 100nm) (Sumber: J.L. Carraascosa)
Asfarvirus berdiameter kurang lebih 200 nm, dan memiliki nukleokapsid
yang dikelilingi oleh lapisan lipid internal dan sebuah kapsid ikosahedral kompleks.
Kapsid terdiri atas sebuah susunan heksagonal prisma dengan lubang sentral.
Genom terdiri atas molekul tunggal dari DNA untai ganda linier. Lebih dari 50
protein terdapat di dalam virion, termasuk beberapa enzim dan faktor yang
diperlukan di dalam proses transkripsi mRNA.

2.2. Epidemiologi

African swine fever (ASF) pertama kali ditemukan di Kenya pada tahun
1921, dan sejak itu telah menyebar secara cepat ke negara-negara afrika lain. ASF
pertama kali lolos dari afrika pada tahun 1957 dan mencapai Portugal melalui
kontaminasi produk pangan yang digunakan untuk babi. Setelah wabah ini yang
secara cepat dikontrol, ASFV masuk Portugal pada tahun 1960 dan pada waktu ini
menyebar ke seluruh semenanjung Iberian, dan persisten lebih dari 30 tahun. ASFV
menyebar secara sporadis ke negara lain di Eropa dan Amerika (Brazil, Republik
Dominika, Kuba, dan Haiti.) ASF telah diberantas pada negara-negara ini kecuali
kepulauan Italia Sardinia, yang telah terdampak sejak 1978.
 4

Pada tahun 2007, ASFV memasuki Georgia kemungkinan melalui kontaminasi

pangan yang digunakan untuk babi. Dari bagian ini, ASFV menyebar secara cepat
melalui negara dan memberi dampak negara tentangga seperti Armenia,
Azerbaijan, dan Federasi Russia. Pada daerah geografis ini, ASFV mempengaruhi
babi domestik dan liar dan menyebar dari utara ke barat. Pada tahun 2012, wabah
pertama di deklarasi di Ukraina, dan 2013 pada Belarus. Dari negara ini, ASF
berlanjut menyebar sebelum mencapai perbatasan Uni Eropa pada tahun 2014, yang
mana ditemukan bangkai babi liar pada negara Lithuania dan Polandia.

2.2.1 Spesies terdampak

African swine fever menginfeksi babi domestik dan anggota dari famili
Suidae, termasuk babi hutan (Potamochoerus aethiopicus, P. porcus, dan
Sus scrofa ferus). Semua usaha untuk menginfeksi hewan lain tidak
berhasil.

2.2.2 Cara penularan dan penyebaran

Rute transmisi Lokasi dan tanggal

Limbah daging babi mentah pada Lisbon, 1957
bandara Malta. 1978
Sardinia, 1978
Georgia, 2007
Pemindahan produk pangan babi Portugal, 1960
Spanyol, 1960
Italia, 1983
Belgia, 1985
Penularan secara alami oleh babi Federasi Russia, 2007
liar yang terinfeksi Lithuania, 2014
Polandia, 2014
Estonia, 2014
Caplak yang terinfeksi Portugal, 1999
 5

Penularan virus ini adalah melalui caplak Ornithodoros moubata (tick-

borne disease) sebagai vektor penyakit. Diansumsikan munculnya pertama
kali penyakit ini adalah akibat siklus alami caplak argasid dan babi hutan,
akan tetapi virus menyebar diluar lingkungan tradisional dan menginvasi
babi pada Eropa. Penyebaran virus ini juga karena akibat transportasi
produk-produk pangan babi. Produk yang terinfeksi ini kemungkinan dapat
diimpor secara ilegal untuk mendapatkan keuntungan.

2.3. Patogenesis

Gambar 2.3. Interaksi virus ASF dengan makrofag host.

Infeksi african swine fever pada babi domestik mengakibatkan leukopenia,
limfopenia, trombositopenia, dan apoptosis baik limfsoit maupun sel-sel fagositik
mononuklear. Kemampuan virus African swine fever untuk secara efisien
menyebabkan sitopatologi pada makrofag merupakan faktor penting dalam
virulensi virus. Pada makrofag yang terinfeksi, virus secara efektif menghambat
ekspresi sitokin pro-inflammatory seperti TNF (Tumor Necrosis Factor), interferon
(IFN)-1, dan interleukin (IL)- 8, akan tetapi menyebabkan ekspresi mengubah
faktor pertumbuhan (growth factor) – beta. Sebaliknya, peningkatan ekspresi TNF
juga dilaporkan pada kasus infeksi ASF secara in vitro dan in vivo. Strain dari virus
ASF dengan virulensi yang berbea memiliki kemampuan ekspresi memicu ataupun
menghambat sitokin pro-inflammatory atau IFN yang berbeda. Penghambatan
 6

peradangan dimediasi paling tidak oleh bagian gen viral A238L, yang mana
mengenkode sebuah protein yang mirip dengan faktor penghambat transkripsi
seluler, nuklear faktor kB (NFkB).

Jika infeksi terjadi melalui sistema pernafasan, virus bereplikasi pertama

kali pada tonsil faringeal dan limfe nodus mukosa nasal, sebelum menyebar secara
cepat ke dalam tubuh melalui viremia primer yang mana virion dihubungkan
dengan eritrosit maupun leukosit. Infeksi umum berlanjut, dengan titer virus yang
tinggi (sampai 109 dosis infeksius per ml darah atau per gram jaringan) dan semua
cairan sekresi dan eksresi mengandung jumlah virus infeksius yang banyak.

Babi yang lolos dari infeksi akut mungkin terlihat sehat atau terlihat kronis,
namun kedua grup ini tetap terjadi infeksi yang persisten. Infeksi yang persisten ini
bahkan tidak menunjukkan gejala klinis. Durasi infeksi persisten belum diketahui,
namun kadar virus yang rendah telah di deteksi pada jaringan sampai lebih dari
setahun setelah paparan.

2.4. Patologi

Tabel 2.1. Persebaran lesi yang diamati pada bentuk-bentuk ASF

ASF ASF Akut ASF Subakut ASF Kronik

Perakut
Demam Tinggi Tinggi Moderat Irreguler
ataupun
tidak ada
Trombositopenia - Tidak ada / Sementara Tidak ada
ringan
Kulit Eritema Eritema Eritema Area
nekrotik
Limfonodus - Gastrohepatik Gambaran umum Bengkak
dan renal seperti bekuan
dengan aspek darah
kelereng
 7

Limpa - Splenomegali Hiperemi parsial Terjadi

hiperemik splenomegali perbesaran
atau infark fokal dengan
warna
normal
Ginjal - Hemoragi Hemoragi -
Petekie pada petekie pada
korteks korteks, medulla,
dan pelvis,
edema perirenal
Paru-paru - Edema - Pleuritis
alveolar hebat dan
pneumonia
Kantung - Hemoragi Edema dinding -
empedu petekie
Jantung - Hemoragi Hemoragi pada Perikarditis
pada epikardium, fibrinosa
epikardium endokardium,
dan dan
endokardium hidroperikardium
Tonsil - - - Nekrotik
fokal
Alterasi - - Abortus Abortus
reproduksi
Sumber: Danrdiri, 1979, Gomez Villamandos et al., 1995a, Hervas et al., 1996,
Carrasco et al. 1997a, Sances Viszaino dan Aria, 2012.
 8

Gambar 2.4. (A) Hemoragi subkutan pada telinga (B) Splenomegali

Sumber: R. Harutyan, Pusat Diagnostik dan Epizootik Universitas Illinois
Pada kasus akut yang fatal pada babi domestik, lesi berat paling menonjol
pada sistem limfoid dan vaskular. Hemoragi terjadi secara luas, limfo nodus
visceral menyerupai gumpalan darah. Terdapat ptekie yang ditandai pada semua
permukaan serosa, limfo nodus, epikardium dan endokardium, kortek ginjal, vesica
urinaria, edema dan kongesti pada kolon dan paru-paru. Limpa seringkali
mengalami perbesaran dan rapuh. Penyakit yang kronik ditandai oleh ulkus kulit,
pneumonia, perikarditis, pleuritis, dan arthritis.

Gambar 2.5. (A) Splenomegali dengan warna keungu-unguan pada seluruh

rongga abdominal (hiperemik splenomegali) (B) Beberapa limfo nodus yang
menunjukkan derajat keparahan hemoragi. (C) Potongan limfo nodus dengan
tampilan kelereng (D) hemoragi petekia pada korteks renalis
 9

Gambar 2.6. ASF subakut. Edema yang intens pada dinding kantung empedu (A)
dan di sekeliling ginjal. (B) Limpa dengan hiperemik splenomegali parsial.
Hemoraagik dan edematus gastrohepatic (D) dan ginjal (E) limfonodus (F) ginjal
dengan hemoragik intens pada korteks, medulla, dan pelvis.
2.5. Gejala Klinis

Gambaran klinis ASF dapat berbeda-beda, bergantung pada virulensi isolat

virus, rute dan dosis infeksi dan karakteristik host. Tampilan klinis dari ASF pada
peternakan biasa dengan dosis kecil infeksi, tidak akan menyebabkan angka
motalitas yang tinggi ataupun gejala klinis, kecuali demam dan kematian akibat
perdarahan pada limfo nodus. Biasanya, beberapa hari kemudian, karena
peningkatan sirkulasi virus, ledakan infeksi dapat diamati dengan mortalitas yang
meningkat dan ciri-ciri lesi ataupun gejala klinis.

Perakut

Bentuk ini dipicu oleh strain ASFV yang sangat virulen yang ditandai
demam yang sangat tinggi (41-42 derajat celsius), kehilangan nafsu makan, tidak
 10

aktif, hipernoea, dan hiperemi kutaneus. Hewan biasanya mati secara mendadak
setelah 1-4 setelah menunjukkan gejala klinis dan tidak ada lesi terdapat pada organ.

Akut

Gejala akut atau hiperakut dari African swine fever pada babi yang rentan
ditandai dengan hemoragi yang hebat dengan mortalitas tinggi. Setelah masa
inkubasi 5 – 15 hari, babi mengalami demam (40.5-42 derajat C) yang persisten
selama sekitar 4 hari. Dimulai 1-2 hari setelah demam, terjadi tidak nafsu makan,
diare, inkoordinasi, dan lesu. Babi akan mati pada stadium ini tanpa gejala lain.
Pada beberapa babi terjadi dispnea, muntah-muntah, leleran (discharge) nasal dan
konjungtival, sianosis pada telinga dan moncong, dan hemoragi pada hidung dan
anus. Babi bunting biasanya terjadi abortus. Angka mortalitas seringkali 100%,
dengan babi domestik mati setelah 1-3 hari setelah demam.

Subakut

Bentuk penyakit ini disebabkan oleh isolat virus dengan virulensi moderat
/ sedang, dan memberikan dampak pada hewan yang menunjukkan gejala klinis
yang mirip dengan bentuk akut. Babi yang terdampak biasanya mati setelah 7 – 20
hari dan tingkat mortalitas memiliki rentang 30 sampai 70%. Babi yang berhasil
selamat biasanya akan pulih setelah 3 sampai 4 minggu. Babi yang telah pulih masih
dapat mengeksresikan virus sampai 6 minggu post infeksi. Kematian hewan dapat
terjadi dalam dua stadium berbeda: trombositopenia yang intens / fase leukopenia
atau pada fase pemulihan ketika hemoragi muncul akibat eritodiapedesis oleh
vasodilatasi, khususnya pada hewan muda. Babi yang mengalami ASF sub akut
akan mengalami demam moderat sampai tinggi, ascites, hidroperikardium dan
edema pada dinding kantung empedu. Limpa biasanya menunjukkan hiperemi
parsial splenomegali.

Kronis

Bentuk kronis dari ASF diakibatkan oleh infeksi virus dengan virulensi
rendah, yang telah diobservasi pada Spanyol dan Portugal. Bentuk ini ditandai
dengan lesi nekrotik pada kulit dan arthritis. Maka dari itu, bentuk ini belum pernah
 11

dideskripsikan pada negara-negara dengan ASFV jangka panjang (seperti Afrika

dan Sardinia). Telah dihipotesiskan bentuk kronis mungkin berasal dari evolusi
alami isolat ASFV pada studi vaksin yang dilakukan di semenjanjung Iberian pada
tahun 1960-an.

2.6. Diagnosis

Gejala klinis dari African swine fever mirip dengan beberapa penyakit,
termasuk bakterial septikemia seperti eriseplas, salmonellosis akut, akan tetapi
masalah utama dalam mendiagnosis adalah membedakan dengan swine fever klasik
(hog cholera). Setiap penyakit demam pada babi yang berhubungan dengan
hemoragi yang menyebar (hemoragik diatesis) dan angka kematian yang tinggi
harus meningkatkan kecurigaan terhadap African swine fever. Diagnosis infeksi
kronis bermasalah karena gejala klinis dan lesi pada babi yang terdampak berbeda-
beda dan sangat bervariasi. Konfirmasi atau peneguhan laboratorium sangat
penting, dari sampel darah, limfa, ginjal, limfo nodus viseral, dan tonsil harus
dikumpulkan untuk isolasi virus, deteksi antigen dengan PCR (Polymerase Chain
Reaction) untuk mendeteksi gen p72. Isolasi virus dilakukan di sumsum tulang babi
atau kultur leukosit darah perifer. Setelah isolasi awal, virus dapat diadaptasi untuk
tumbuh di berbagai lapisan sel, seperti sel Vero. Deteksi antigen didapatkan dengan
pewarnaan immunofluorescence pada jaringan.

2.7 Diagnosis Banding

Ketika terjadi stadium awal dari ASF, terutama ketika jumlah yang sedikit
hewan terdampak, diagnosis belum pasti. Keberadaan lesi non-spesifik dan
presentasi kematian yang kecil dapat dengan mudah dibingungkan oleh penyakit-
penyakit hemoragik babi. Karena itu, diagnosis banding berdasarkan lesi patologi
anatomi penyakit babi berikut: classical swine fever (CSF) / hog cholera, high-
patogenic procine reproductive and respiratory syndrome (HP-PRRS), eriseplas
babi, salmonelosis septikemia, dan procine dermatitis nephropathy syndrome
(PDNS). Karakteristik spesifik telah dirangkum pada tabel berikut.
 12

Tabel 2.2. Diagnosis Banding

ASF Akut ASF CSF HP- Erisepla Salmonel DNS

Subakut PRRS s babi osis
Septikem
ia
Kulit Eritema Eritema Eritema Sianosi Lesi Sianosis Makula
s diamond dan
Papula
Limfo Aspek Hemora Multiple Bengka Aspek Bengkak -
nodus “marbled” gi infark k “marble
pada d”
tepi
Limpa Hiperemik Hiperem Henorag Hemora Hiperem Splenom Glomeru
splenomeg i Parsial i petekie gi ik egali onephrit
ali dengan ptekie splenom is
fokal egali
infark
Ginjal Hemoragi Hemora Hemora - Hemora Hemorag -
petekie gi gi gi i ptekie
Petekie petekie petekie
Kantung Hemoragi Edema Hemora Bengka - - -
empedu petekie dinding gi ptekie k
dengan
hemora
gi
Tonsil - - Area - - - -
nekrotik
Intestin Hemoragi Petekie Button - - Enterokol -
petekie ulcers itis
nekrotik
 13

Trombo Tidak ada / Sementa Intens Atrofi - - -

sitopeni Ringan ra intersiti
a al
pneumo
nia
Lesi lain - - Gejala Arthirits Gejala -
Saraf dan saraf,
Kongeni endokar Kongesti
tal ditis mukosa
Malform vegetatif gastric,
asi nekrosis
fokal
pada liver
dan
bronchop
neumonia

2.8. Imunitas, Pencegahan dan kontrol

Baik komponen humoral dan seluler (termasuk limfosit spesifik CD8+)

berkontribusi dalam respon imun protektif terhadap virus African swine fever.
Respon antibodi terhadap virus ASF telah terbukti melindungi babi dari tantangan
mematikan. Namun, penawar antibodi untuk protein virion p30, p54, dan p72 tidak
cukup memberikan perlindungan yang diperantarai oleh antibodi.

Pencegahan dan pengendalian African swine fever dapat diperumit oleh

beberapa faktor, termasuk kurangnya vaksin yang efektif, penularan virus dalam
daging segar dan beberapa produk daging babi yang disembuhkan, adanya infeksi
persisten pada beberapa babi, kebingungan diagnostik dengan agen yang
menyebabkan sindrom penyakit serupa seperti hog cholera dan peranan caplak
lunak di berbagai belahan dunia. Keberadaan virus dalam caplak dan babi hutan di
 14

banyak negara Afrika sub-sahara menjadikan sulit, bukan tidak mungkin untuk
memutus siklus penyebaran virus.

Di belahan dunia lain, pada negara yang bebas African swine fever, mereka
memanajemen status bebas virus dengan melarang impor babi hidup dan produk
babi dari negara-negara terinfeksi, dan dengan memonitor penghancuran sisa-sisa
makanan dari kapal maupun pesawat yang terlibat dalam rute internasional. Vaksin
belum tersedia untuk mencegah infeksi ASF. Konsekuensinya, pencegahan
berdasarkan penghindaran dari penyakit itu sendiri. Rute-rute penyakit sebisanya
harus ditutup / di blok untuk menurunkan resiko.
 15

BAB III

PENUTUP

3.1 Kesimpulan

African swine fever virus (ASFV) merupakan virus DNA beramplop yang
merupakan anggota dari genus Asfivirus dan famili Asfarviridae. Penularannya
dapat melalui caplak dari genus Ornithodoros. Strain virus dibedakan berdasarkan
virulensi terhadap babi. Virus ini pertama kali di Kenya pada tahun 1921, setelah
itu menyebar secara cepat ke negara-negara lain. Gambaran patologis dari ASF
berbeda, bergantung dari bentuk ASF, yakni perakut, akut, subakut, dan kronis.
Diagnosis dari penyakit ini bisa dengan deteksi antigen menggunakan PCR untuk
mendeteksi gen. p72. Diagnosis banding dari penyakit ini adalah CSF, HP-PRRS,
Eriseplas babi, salmonellosis septikemia, dan PDDNS. Vaksin belum tersedia
sehingga diperlukan penghindaran terhadap ASF dengan penutupan / memblok
rute-rute penularan.

3.2 Saran
 16

DAFTAR PUSTAKA

MachLachlan, N.J & Edward J.D. (2010). Fenner’s Veterinary Virology. 4th Ed.
USA: Elsevier Inc.
United States Departement of Agriculture (2019). Disease Response Strategy
African Swine Fever. USA: U.S. Departement of Agriculture
Alcrudo D.B. et al. (2017). African Swine Fever: Detection and Diagnosis – A
Manual for Veterinarians. USA: Food and Agriculture Organization of the
United Nations.
Galindo I. & Covadonga A. (2017). African Swine Fever Virus: A Review. Viruses.
Vol. 9: 130
Vizcaino J.M.S et al. (2015). An Update on the Epidemiology and Pathology of
African Swine Fever. J. Comp. Path. Vol. 152:9-21
Blome S. et al. (2013). Pathogenesis of African swine fever in domestic pigs and
European wild boar. Virus Research. Vol. 122:130
Howey E.B. et al. (2013). Pathogenesis of highly virulent African swine fever virus
in domestic pigs exposed via intraoropharyngeal, intranasopharyngeal, and
intramuscular inoculation, and by direct contact with infected pigs. Virus
Research. Vol 328:339
J. Comp. Path. 2015, Vol. 152, 9e21 Available online at www.sciencedirect.com

ScienceDirect

www.elsevier.com/locate/jcpa
INFECTIOUS DISEASE: REVIEW ARTICLE

An Update on the Epidemiology and Pathology

of African Swine Fever
anchez-Vizca
ıno*, L. Mur*, J. C. Gomez-Villamandos†
J. M. S

and L. Carrasco†
* Centro VISAVET and Animal Health Department, Universidad Complutense de Madrid, Av. Puerta de Hierro SN 28040,
Madrid and † Department of Comparative Pathology, Universidad de C
ordoba, C
ordoba, Spain

Summary
African swine fever (ASF) is one of the most important infectious diseases of swine and has major negative con-
sequences for affected countries. ASF is present in many sub-Saharan countries, Sardinia and several countries
of eastern and central Europe, where its continuous spread has the swine industry on heightened alert. ASF is a
complex disease for which no vaccine or treatment is available, so its control is based on early detection and
rapid control of spread. For a robust and reliable early detection programme it is essential to be able to recog-
nize the clinical signs and pathological changes of ASF, keeping in mind that in most cases the first introduc-
tions don’t show high mortality nor characteristic clinical signs or lesions, but fever and some hemorrhagic
lymph nodes. Knowledge of the main characteristics of this infection, including its current distribution and
routes of transmission, is also essential for preventing and controlling ASF. This review addresses each of these
topics and aims to update knowledge of the disease in order to improve early detection of ASF in the field and
allow implementation of public health programmes.
Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: African swine fever; clinical signs; differential diagnosis; lesions

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
African Swine Fever Epidemiology and Main Routes of Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Clinical Presentation and Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Peracute African Swine Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Acute African Swine Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Subacute African Swine Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Chronic African Swine Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Differential Diagnosis between African Swine Fever and Other Haemorrhagic Diseases . . . . . . . . . . . . . . . . . . . . . . . 14
Classical Swine Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Porcine Reproductive and Respiratory Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Swine Erysipelas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Septicaemic Salmonellosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Porcine Dermatitis and Nephropathy Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Laboratory Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Early Detection, Prevention and Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Correspondence to: J. M. S
anchez-Vizca
ıno (e-mail: jmvizcaino@ucm.es).

0021-9975/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcpa.2014.09.003
10 anchez-Vizca
ıno et al.
J.M. S

Introduction Despite efforts made over past decades, there is no

vaccine available for preventing and controlling
African swine fever (ASF) is one of the most impor-
ASFV infection. Several strategies have been studied;
tant infectious diseases of swine and is present in
however, the lack of neutralizing antibodies, genetic
many African countries, some eastern and central Eu-
variability and presence of some gaps in knowledge
ropean countries and Sardinia. ASF must be notified
about ASF pathogenesis and immune modulation
to the World Organization of Animal Health (OIE)
make discovery-based approaches difficult.
and its presence leads to immediate restrictions on
the pig and pork trade.
ASF is caused by infection with a complex DNA vi- African Swine Fever Epidemiology and Main
rus, the ASF virus (ASFV), which is the only member Routes of Introduction
of the family Asfarviridae (Dixon et al., 2005). ASFV
ASF was first described in Kenya in 1921
is a large enveloped virus of approximately 200 nm in
(Montgomery, 1921) and since then it has spread
diameter that contains double-stranded DNA of
rapidly to other African countries. ASF first escaped
170e193 kilobase pairs (Dixon et al., 2013). ASFV
from Africa in 1957 to reach Portugal via contami-
is composed of more than 50 structural proteins and
nated waste containing infected pig products that
produces more than 150 proteins in infected macro-
were used to feed pigs. After this incursion, which
phages (Salas and Andr
es, 2013), many of which
was rapidly controlled, ASFV re-entered Portugal
are highly immunogenic. Hence, infection causes a
in 1960 and this time it spread to the whole Iberian
strong humoral immune response that persists for
Peninsula, where it persisted for more than 30 years
long periods of time. Nevertheless, the antibodies pro-
(Arias and S
anchez-Vizca
ıno, 2002). During this
duced are not able to neutralize ASF infection effec-
period (1960e1995), ASFV spread sporadically to
tively (Neilan et al., 2004) and serotyping is not
other countries in Europe and America (i.e. Brazil,
possible. Consequently, classification is based on gen-
Dominican Republic, Cuba and Haiti). ASF has
otyping procedures through the analysis of some
been eradicated from all of these countries except
genome regions, such as the C-terminal region of
from the Italian island of Sardinia, which has been
the gene encoding vp72 (Bastos et al., 2003). Based
affected since 1978 (S
anchez-Vizca
ıno and Arias,
on the differences observed in this region, circulating
2012; Costard et al., 2013a, b).
isolates of ASFV have been classified into 22 geno-
ASF has also continued to spread on the African
types (Boshoff et al., 2007).
continent and reached West African countries and
ASFV replicates in mononuclear phagocytic cells
some islands that were previously free of the disease.
of both domestic and wild swine. The virus infects
Some authors have stated that this spread started in
monocytes and macrophages (Malmquist and Hay,
1994 due to a combination of factors including: (1)
1960), but infection of T or B lymphocytes has never
increasing pig production on the continent, (2) pres-
been observed (Minguez et al., 1988). The virus also
ence of non-symptomatic pigs that could act as reser-
replicates in endothelial cells (Carrasco et al.,
voirs to spread the disease without being noticed and
1996a), hepatocytes, renal tubular epithelial cells
(3) globalization. These factors, together with the eco-
(G
omez-Villamandos et al., 1995a) and neutrophils.
nomic crisis of the present century, could be the origin
After initial replication in these primary sites, ASFV
of the spread of ASFV to Eastern Europe (Penrith and
spreads through the blood or the lymphatic system,
Vosloo, 2009; S
anchez-Vizca
ıno et al., 2013).
where it persists for long periods of time in the absence
In 2007, ASFV entered Georgia through the port of
of neutralizing antibodies and moves towards second-
Poti (Beltran-Alcrudo et al., 2008), potentially via
ary sites of replication.
contaminated food used to feed pigs. From this region,
ASFV also replicates in soft ticks of the genus
ASFV spread rapidly through the country and
Ornithodoros, which act as virus reservoirs. These ticks
affected neighbouring countries including Armenia,
are involved in the epidemiological cycle of ASF in
Azerbaijan and the Russian Federation. In this
eastern and southern Africa (Ornithodoros moubata)
geographical region, ASFV affects domestic and
and have also been observed during infection on the
wild boar and has spread to the north and west. In
Iberian Peninsula (Ornithodoros erraticus). Other Orni-
2012, the first outbreaks were declared in Ukraine,
thodoros spp. have been demonstrated to be susceptible
followed by Belarus in 2013 (S
anchez-Vizca
ıno
to ASF infection (European Food Safety Authority,
et al., 2013). From these countries, ASF continued
2010). ASFV strain Georgia 2007/1 can also replicate
to spread before reaching the European Union
in O. erraticus ticks (Diaz et al., 2012), but the potential
(EU) borders in 2014, when several dead wild boars
role of these ticks in virus transmission is still unknown
were found in Lithuania and Poland. Since then,
in other European regions.
several cases have been reported in Estonia and
 Epidemiology and Pathology of African Swine Fever 11

Latvia and repeatedly in Lithuania and Poland Clinical Presentation and Lesions
(OIE, 2014).
ASF can have different clinical presentations and
At this time, the critical situation of ASF in East
pathological lesions, depending on the virulence of
and Central Europe poses a serious risk to other EU
the virus isolate, the route and dose of infection and
countries, which could suffer the severe consequences
the host characteristics. The clinical presentation of
of trade restrictions, as has already happened with
ASF in a naive farm with a low dose of virus infection,
Russian Federation bans. However, it is important
will not cause high mortality nor characteristic clin-
to remember that the main source of ASFV is pres-
ical signs, except fever and deaths with some hemor-
ently Africa and from there, it could spread to other
rhagic lymph nodes. Usually, few days later, due to
disease-free countries/regions and could potentially
the increased viral circulation, more explosive infec-
spread and cause high-impact consequences, as have
tions could be observed with more mortality and
occurred previously in Europe.
characteristic clinical signs and lesions. Therefore,
The main routes for the spread of ASF involve the
every dead animal that present fever in a high risk
transport of infected animals and, more frequently,
area, should be tested for ASF.
the transport of infected products (Table 1). These in-
ASFV strains are usually classified as highly viru-
fected products could be imported illegally to make
lent, moderately virulent and low virulent (Pan and
profit or could involve catering waste containing in-
Hess, 1984). Highly virulent strains are usually respon-
fected pig products. Indeed, this was the means by
sible for the peracute (pigs dead 1e4 days post infec-
which ASF was introduced into many disease-free
tion [dpi]) and acute forms (animals dead 3e8 dpi)
countries in the 1970s (S
anchez-Vizca
ıno and Arias,
of the disease, while moderately virulent strains are
2012) and the cause of the last introduction in 2007
involved in the acute (pigs dead 11e15 dpi) and sub-
(Beltran-Alcrudo et al., 2008). Other sources of infec-
acute (animals die after 20 dpi) forms. In clinical
tion are related to contaminated fomites and trans-
terms, acute ASF develops over a 7-day period,
port, including livestock vehicles or infected
compared with 10e20 days for the subacute form of
material. Natural ranging of wild boar is one of the
the disease. Chronic ASF has been associated with
most serious concerns for the EU due to the proximity
infection by moderate-to-low virulence isolates
of affected territories, as demonstrated by the recent
(Mebus and Dardiri, 1979; McVicar, 1984), which
cases detected at EU borders and risk assessment an-
were only described in Spain, Portugal and the
alyses (De la Torre et al., in press). However, this route
Dominican Republic when ASF infection in these
is not likely to cause distant outbreaks in disease-free
areas was endemic. The main lesions observed in the
territories such as Asia without prior suspicion or
different forms of ASF are summarized in Table 2.
detection. Other factors, such as infected ticks or
airborne transmission, are also important, but only
at a local level and should be ruled out as potential Peracute African Swine Fever
sources for ASF infection in distant disease-free terri- This form is induced by highly virulent ASFV strains
tories. and is characterized by high fever (body temperature
Table 1
41e42
C), loss of appetite, inactivity, hyperpnoea
Main sources of ASF infection into free areas of Europe and cutaneous hyperaemia. Animals usually die sud-
denly 1e4 days after the onset of clinical signs and no
Transmission route Location and date of lesions are evident in organs.
occurrence

Raw pork waste at Lisbon, 1957 Acute African Swine Fever

airport/port Malta, 1978
Sardinia, 1978 This is the most usual form of the disease and is
Georgia, 2007 induced by highly or moderately virulent virus
Movement of pork or pig Portugal, 1960 strains. Animals with acute ASF display fever (body
product Spain, 1960 temperature 40e42
C) and a tendency to crowd
Italy, 1983 together; they also exhibit loss of appetite, inactivity,
Belgium, 1985 apathy and early leucopenia, induced by lymphope-
Natural ranging of Russian Federation, 2007 nia and changes in monocyte numbers (Colgrove
infected wild boar Lithuania, 2014 et al., 1969; Pan and Hess, 1984; G
omez-
Poland, 2014 Villamandos et al., 1995a; Carrasco et al., 1996b;
Estonia, 2014 S
anchez-Vizca
ıno and Arias, 2012). Severe
Infected ticks Portugal, 1999 pulmonary oedema, giving rise to respiratory
changes, is a characteristic finding in pigs infected
12 anchez-Vizca
ıno et al.
J.M. S

Table 2
Main lesions observed in the different forms of ASF
Peracute ASF Acute ASF
Fever High High
Thrombocytopenia Absent Absent or slight (late)
Skin Erythema Erythema
Lymph nodes e Gastrohepatic and renal with
marbled aspect
Spleen e Hyperaemic splenomegaly
Kidney e Petechial haemorrhages, mainly
in cortex
Lung e Severe alveolar oedema
Gall bladder e Petechial haemorrhages
Heart e Haemorrhages in epicardium and
endocardium
Tonsils e e
Reproductive alteration e e
with highly virulent strains of ASF. Affected animals
die in shock, usually 1 week after fever begins and
foam is generally observed around the mouth and
nose (Sierra et al., 1990; Carrasco et al., 1996a, 2002).
Affected pigs show erythema, which particularly
affects the skin of the ears, tail, distal extremities,
chest, abdomen and perianal area. Cyanosis may
also be observed 1e2 days prior to death on the skin
of the ears, abdomen and perianal areas. Small foci
of cutaneous necrosis (more characteristic of infec-
tions with strains of moderate virulence) and subcu-
taneous haematomas also occur. Other clinical signs
include mucoid nasal discharge, epistaxis, vomiting,
abdominal pain, constipation or diarrhoea, which is
initially mucoid, but may later become bloody (mel-
aena) (Moulton and Coggins, 1968; Mebus and
Dardiri, 1979; G
omez-Villamandos et al., 2013).
Abortion (caused by fever) may occur in pregnant
sows; in some cases, abortion may be the first sign of
an outbreak. Almost 90e100% of pigs with these
signs will die within 7 days.
Together with erythema and cyanosis of the skin, the
animals that develop acute ASF present with charac-
teristic hyperaemic splenomegaly in which the spleen
can be up to six times larger than normal. An affected
spleen may have rounded edges, be friable in consis-
tency and purpleeblack in colour and may occupy
the entire abdominal cavity from one side to the other
(Fig. 1A). Lymph nodes, mainly the gastrohepatic and
renal nodes, display haemorrhages in the medulla
(Fig. 1B), which is why sections of affected lymph nodes
sometimes have a marbled appearance (Fig. 1C). Kid-
neys usually show petechial haemorrhages in the cortex
(Fig. 1D) and renal pelvis. Other lesions that pigs usu-
ally show with acute forms of the disease are petechial
haemorrhages in the mucosa of the urinary bladder,
epicardium, endocardium and pleura (Mebus and
Subacute ASF Chronic ASF
Moderate Irregular or absent
Transient Absent
Erythema Necrotic areas
The majority of lymph nodes Swollen
resemble a blood clot
Partial hyperaemic splenomegaly Enlarged with normal
or focal infarct colour
Petechial haemorrhages in cortex, e
medulla and pelvis; perirenal oedema
e Pleuritis and pneumonia
Wall oedema e
Haemorrhages in epicardium and Fibrinous pericarditis
endocardium; hydropericardium
e Necrotic foci
Abortion Abortion
Dardiri, 1979; G
omez-Villamandos et al., 1995a;
Herv
as et al., 1996; Carrasco et al., 1997a, b; S

anchez-
Vizca
ıno and Arias, 2012).
Subacute African Swine Fever
This form of the disease is caused by moderately
virulent isolates and affected animals show similar clin-
ical signs to those animals with the acute form of the dis-
ease, although clinical signs tend to be less marked.
However, the vascular changes observed in subacute
forms of ASF, mainly haemorrhage and oedema, are
more intense than those reported in the acute form of
the disease (G
omez-Villamandos et al., 1995a, 2013).
Haemorrhages related to the development of intense,
although transient, thrombocytopenia are observed
in the early and mid stages of the disease (Villeda
et al., 1993a, b; G
omez-Villamandos et al., 1998).
Abortion is usually the first clinical sign of these
forms. Affected pigs usually die within 7e20 days and
the mortality rate can range from 30 to 70%.
Survivors usually recover within 3e4 weeks.
Recovered pigs can still excrete the virus up to 6
weeks after infection. Death of animals may occur at
two different stages: in the intense thrombocytopenia/
leucopenia phase or in the recovery phase when
haemorrhages appear due to erythrodiapedesis by
vasodilation, especially in young animals (G
omez-
Villamandos et al., 1998, 2013).
Pigs suffering from the subacute form of ASF display
a moderate to high fever, ascites, hydropericardium
and characteristic oedema of the wall of the gall-
bladder and bile duct (Fig. 2A), as well as in the sur-
rounding area of kidneys (perirenal oedema)
(Fig. 2B). The spleen usually shows initial partial hy-
peraemic splenomegaly (Fig. 2C) that progressively re-
verses, leaving some focal damage, which eventually
 Epidemiology and Pathology of African Swine Fever 13

Fig. 1. Acute ASF. (A) An enlarged, purpleeblack spleen crosses the entire abdominal cavity (hyperaemic splenomegaly). (B) Several
lymph nodes display degrees of haemorrhage, most intensely in the medulla. (C) Cut sections of lymph nodes with marbled appear-
ance. (D) Petechial haemorrhages in the renal cortex.

disappears, or focal infarction with different stages of
organization. Lymph nodes, mainly the gastrohepatic
(Fig. 2D) and renal nodes (Fig. 2E), as well as the sub-
mandibular, retropharyngeal, mediastinal, mesenteric
and inguinal nodes, are haemorrhagic, oedematous
and friable, which is why they often look like dark red
haematomas (S
anchez-Vizca
ıno and Arias, 2012;
G
omez-Villamandos et al., 2013). Renal
haemorrhages (Fig. 2F) are more intense (petechiae
and ecchymoses) and more extensive (cortex, medulla
and pelvis) than in acute forms, but are not linked to
the endothelial lesions reported in acute ASF
(G
omez-Villamandos et al., 1995a; Herv
as et al., 1996).
Chronic African Swine Fever
The chronic form of ASF is caused by infection by low
virulence strains, observed in Spain and Portugal, as
well as the Dominican Republic. This form is charac-
terized by necrotic lesions of the skin and by arthritis
(S
anchez-Botija, 1982). However, this form of the dis-
ease has never been described in those countries where
ASFV has been present for long periods of time (e.g.
Africa and Sardinia). Therefore, it has been hypothe-
sized that the chronic form may have originated
through the natural evolution of the ASFV isolates em-
ployed in the vaccination studies performed on the Ibe-
rian Peninsula in the 1960s. To confirm this hypothesis,
several molecular studies to elucidate the similarity be-
tween chronic forms and vaccine isolates are underway
(S
anchez-Vizca
ıno, unpublished observations).
The chronic form of the disease, which has no spe-
cific clinical signs, was mainly detected during sero-
logical screening to eradicate the disease in Spain, in
carrier animals, of which around 10% can spread
the virus for long periods of time, which likely plays
a key role in the persistence of the disease (Arias
and S
anchez-Vizca
ıno, 2002). These animals usually
have necrotic lesions of the skin and arthritis
(S
anchez-Botija, 1982), delayed growth, emaciation,
lameness, respiratory signs, abortion and low mortal-
ity (Arias et al., 1986). Unlike other forms of ASF,
chronic forms are characterized by the absence of
vascular lesions and by the presence of lesions in
which bacteria are involved, such as fibrinous pleuri-
tis and/or pericarditis, pleural adhesions, necrotic
pneumonia, fibrinous arthritis/periarthritis and
necrotic skin lesions, as well as necrotic areas on the
tonsils and the tongue (Moulton and Coggins, 1968;
14 anchez-Vizca
ıno et al.
J.M. S

Fig. 2. Subacute ASF. Intense oedema of the wall of the gall bladder (A) and surrounding the kidney (B). (C) Spleen with partial hyper-
aemic splenomegaly. Haemorrhagic and oedematous gastrohepatic (D) and renal (E) lymph nodes. (F) Kidneys with intense hae-
morrhage in the cortex, medulla and pelvis.

Arias et al., 1986). This form of the infection is not
currently in circulation.
Differential Diagnosis between African
Swine Fever and Other Haemorrhagic
Diseases
As discussed previously, during the early stages of
ASF, particularly when low numbers of animals are
affected, diagnosis is not straightforward. The pres-
ence of non-specific lesions and a small percentage
of deaths can easily be confused with other porcine
haemorrhagic diseases. Therefore, differential diag-
noses based on the gross lesions include the following
swine diseases: classical swine fever (CSF), high-
pathogenic porcine reproductive and respiratory syn-
drome (HP-PRRS), swine erysipelas, septicaemic
salmonellosis and porcine dermatitis nephropathy
syndrome (PDNS). The specific characteristics of
these differential diagnoses are summarized in Table
3 and descriptions of each disease, emphasizing the
similarities and differences with ASF, are presented
below.
Classical Swine Fever
CSF, also known as ‘hog cholera’, is a fatal disease
caused by a small enveloped RNA virus belonging
to the family Flaviviridae, genus Pestivirus (Van
Regenmortel et al., 2000). CSF takes various clinical
forms (i.e. peracute, acute, chronic and congenital)
depending on the virus strain involved, the environ-
ment, the time of infection and the individual host
response (Trautwein, 1988).
However in the acute and chronic forms of the dis-
ease, affected pigs show similar clinical signs:
 Table 3
Differential diagnosis of African swine fever on the basis of gross lesions

Epidemiology and Pathology of African Swine Fever

ASF (acute) ASF (subacute) CSF HP-PRRS Swine erysipelas Septicaemic salmonellosis DNS

Skin Erythema Erythema Erythema Cyanosis Diamond skin lesions Cyanosis Macules and papules
Lymph nodes Marbled aspect Haemorrhagic Marbled aspect Swelling or marbled Marbled aspect Swelling Marbled aspect
aspect
Spleen Hyperaemic Partial hyperaemic Multiple infarcts Scattered infarcts or Hyperaemic Splenomegaly e
splenomegaly splenomegaly or focal at the margin white spots on the surface splenomegaly
infarct
Kidney Petechial Petechial haemorrhages; Petechial Petechial Petechial Petechial Glomerulonephritis
haemorrhages perirenal oedema haemorrhages haemorrhages haemorrhages haemorrhages
Gall bladder Petechial Wall oedema Petechial e e e e
haemorrhages haemorrhages
Tonsils e e Necrotic areas Swelling or with e e e
haemorrhages
Intestine Petechial Petechial Button ulcers e e Necrotic enterocolitis e
haemorrhages haemorrhages
Thrombocytopenia Absence or Transient Intense (early) Absent Absent Absent Absent
slight (late)
Other lesions e e Nervous signs Thymic atrophy Arthritis and Nervous signs; congestion e
Congenital Interstitial pneumonia vegetative endocarditis of gastric mucosa;
malformation (chronic form) necrotic foci in the liver
(congenital form) and bronchopneumonia

African swine fever, ASF; classical swine fever, CSF; high-pathogenic porcine reproductive and respiratory syndrome, HP-PRRS; dermatitis and nephropathy syndrome, DNS; e, no change.

15
16
J.M. S

anorexia, lethargy, conjunctivitis, respiratory
signs and constipation followed by diarrhoea. These
clinical signs, together with the haemorrhages (pete-
chiae and ecchymoses) of the skin, kidneys, tonsils,
gallbladder and ileocaecal junction observed in the
acute form, could be confused easily with ASF.
Some of the most characteristic lesions of CSF
include the presence of multifocal infarction of the
margin of the spleen, which takes a triangular shape,
and the presence of necrotic foci on the tonsils
(Cheville and Mengeling, 1969; Trautwein, 1988;
Potier et al., 2006). Although these lesions are not
always present together, they represent, together with
the neurological signs (induced by non-purulent
meningoencephalitis), the best differentiation between
CSF and the acute and subacute forms of ASF.
Another characteristic lesion of the subacute and
chronic forms of CSF, which allows a differential diag-
nosis to be made with ASF, is the presence of ‘button
ulcers’ in the small (mainly ileum and ileocaecal
valves) and large intestines (caecum and colon)
(Cheville and Mengeling, 1969; Trautwein, 1988;
Van Oirschot, 1999; Potier et al., 2006).
Additionally, the onset of thrombocytopenia and
haemorrhages also differs between CSF and ASF.
Haemorrhages in CSF are associated with early
intense thrombocytopenia, which coincides with the
appearance of fever and the start of viraemia
(Cheville and Mengeling, 1969; Trautwein, 1988;
Bautista et al., 2002). However, in ASF, slight
thrombocytopenia in the late stage can be detected
in acute forms or transient thrombocytopenia may
be present in the early and mid stages of subacute
disease (Villeda et al., 1993a, b).
Porcine Reproductive and Respiratory Syndrome
PRRS is caused by two genotypes of the PRRS virus
(PRRSV), a member of the family Arteriviridae
(Nelsen et al., 1999). PRRS is characterized by respi-
ratory problems in growing and finishing pigs and by
reproductive failure in sows, with late-term abortion,
stillbirth and increased preweaning mortality.
Recently, however, highly pathogenic strains (HP-
PRRSV) have been isolated in China and south-
east Asia and in Eastern Europe (Garc
ıa-Nicol
as
et al., 2013; G
omez-Laguna et al., 2013). Infection
with HP-PRRSV strains is associated with severe
clinical signs, pulmonary lesions and aberrant host
immune responses (G
omez-Laguna et al., 2013).
Pigs infected with HP-PRRS strains display high
mortality accompanied by high fever (body tempera-
ture 40e42
C) severe lethargy, anorexia, cough, se-
vere dyspnoea, lameness and cyanosis of the ears,
limbs and perineum (Zhou et al., 2009; Han et al.,
anchez-Vizca
ıno et al.
2014). The main lesions are found in the lungs,
which display interstitial pneumonia (Han et al.,
2014), and in lymphoid organs, where severe thymic
atrophy and swelling and haemorrhage in the tonsils
and lymph nodes may be observed (located in the me-
dulla with a cut surface showing a marbled appear-
ance), together with scattered infarcts or white spots
on the surface of the spleen. Petechial haemorrhages
may be observed in the renal cortex (Wang et al.,
2011). Although haemorrhages in kidneys and lymph
nodes are present in HP-PRRSV, the absence of hy-
peraemic splenomegaly and the intensity of respira-
tory distress, due to interstitial pneumonia, are used
to make the differential diagnosis with ASF.
Swine Erysipelas
Swine erysipelas (SE) manifests commonly as acute or
subacute septicaemia and chronic proliferative lesions
caused by Erysipelothrix rhusiopathiae, a small gram-
positive bacillus (Sneath et al., 1986). Acute SE is
characterized by sudden onset, with occasional sud-
den death, in which animals have a high fever
(body temperature 40e42
C) for 5e7 days and also
display severe depression and leucopenia. The sub-
acute forms are less severe than the acute form and
abortion may occur in sows that come into contact
with acute or subacute SE during pregnancy (Wood
and Henderson, 2006).
The most characteristic lesion of SE is the presence
of rhomboid urticarial lesions (or ‘diamond skin’ le-
sions) in the acute non-fatal form from days 2e3 of
infection, which appear as small square or rhomboid
areas with light pink to dark purple colouration that
are usually raised and firm to the touch. In the acute
fatal form of SE lesions are similar to those of septicae-
mia, with extensive dark purple discolouration over
the ears, tail, abdomen, the posterior aspect of the
thighs, and the jowls. The lungs are congested and oe-
dematous and haemorrhages (petechial to ecchy-
motic) may be seen in the cortex of the kidneys, the
heart (particularly in the epicardium and the muscu-
lature of the atria) and the serosa of the stomach. The
spleen may be congested and markedly enlarged (hy-
peraemic splenomegaly) (Wood and Henderson,
2006), which is similar to the acute form of ASF.
The involvement of lymph nodes depends on the
area that they drain; usually the peripheral nodes
show marked congestion to haemorrhage of the
medulla and the cut surface reveals a marbled
appearance (Wood and Henderson, 2006).
Despite the similarities between acute SE and ASF
(i.e. clinical course, presence of haemorrhages, similar
spleen lesions), there are several characteristics that
differ between these diseases. Firstly, the presence of
 Epidemiology and Pathology of African Swine Fever
diamond skin lesions is typical in acute SE, but not in
ASF. Additionally, the peripheral lymph nodes are
usually the most affected in acute SE, while the gas-
trohepatic and renal nodes are mainly affected in
acute ASF.
Septicaemic Salmonellosis
Swine salmonellosis is caused by Salmonella cholerae-
suis and has been related to the development of sep-
ticaemia or enterocolitis (Baskerville and Dow,
1973). Pigs with septicaemic salmonellosis are inap-
petent, lethargic, febrile (body temperature
40.5e41.6
C), have a moist cough with slight expira-
tory dyspnoea, uncoordinated gait, tremor, paraly-
sis, convulsions and recumbency. These clinical
signs may result in death. At 3e4 dpi, animals suffer
from diarrhoea with watery yellow faeces. In most
outbreaks, which are frequently associated with
stressful situations, morbidity is variable but usually
<10%, although the mortality rate is high (Griffith
et al., 2006).
Pigs that die from septicaemic salmonellosis display
cyanosis of the ears, feet and tail and the ventral
abdomen. Cutaneous icterus can be severe, although
is not present consistently. Petechial haemorrhages
can be present in the renal cortex and on the epicar-
dium. There is splenomegaly, with crossing of the
abdominal cavity, but the spleen has normal colour.
The gastrohepatic and mesenteric lymph nodes are
swollen. Other lesions include hepatomegaly and
miliary foci of necrosis in the liver and congestion of
the gastric fundic mucosa. The lungs are diffusely con-
gested, often with interlobular oedema and sometimes
haemorrhage. Cranioventral bronchopneumonia is
not uncommon. In pigs that survive the first days, a
serous to necrotic enterocolitis develops (Griffith
et al., 2006).
The main differences between septicaemic salmo-
nellosis and the acute and subacute forms of ASF
are the absence of vascular lesions in the spleen and
lymph nodes, the existence of nervous signs and the
presence of necrotic enterocolitis and miliary foci of
necrosis in the liver.
Porcine Dermatitis and Nephropathy Syndrome
PDNS is a haemorrhagic syndrome associated with
infection with porcine circovirus type 2 (Rosell et al.,
2000; Segal
es et al., 2006). This syndrome affects
nursing, growing and adult pigs. Acutely affected
pigs usually die within a few days of onset of clinical
signs, but surviving pigs tend to recover and gain
weight 7e10 days after the syndrome begins (Segal
es
et al., 1998).
17
Affected pigs are anorexic and depressed, with
little or no pyrexia (Drolet et al., 1999). There
are redepurple macules and papules on the skin
caused by necrotizing vasculitis, mainly affecting the
hindlimbs and perineal area. These occasionally coa-
lesce to form large, irregular patches and plaques.
With time, these lesions become covered by dark
crusts and gradually fade (usually after 2e3 weeks),
sometimes leaving scars. In severe cases, the flank
and lateral thoracic wall may also be involved. On
rare occasions, multifocal lesions are observed
randomly over the entire body. Pigs that die from
acute PDNS have bilaterally enlarged kidneys with
small red dots in the renal cortex that correspond to
fibrinonecrotizing glomerulonephritis. Lymph nodes
may be enlarged and the medulla may be congested,
which is why the cut surface sometimes has a marbled
appearance (Segal
es et al., 2006).
Laboratory Diagnosis
Considering the similarities with other swine diseases,
and the potential appearance of unspecific clinical
signs, the laboratory diagnosis of ASF is essential in
order to make a correct diagnosis. There are several
techniques that can distinguish the 22 different
ASFV genotypes. A correct diagnosis of ASF should
include virological and serological detection to obtain
a complete picture of disease status (S
anchez-
Vizca
ıno and Mur, 2013). If ASF is suspected, collec-
tion of samples of serum and blood is required in addi-
tion to the main target organs, including spleen,
lymph nodes, kidney, lung and bone marrow. Oral
fluid samples can also be used to detect ASF anti-
bodies (Mur et al., 2013).
For virological detection, the most commonly used
techniques are virus isolation and haemadsorption
(HAD) tests, polymerase chain reaction (PCR) tech-
niques and direct immunofluorescence. Virus isolation
in macrophages and HAD (Malmquist and Hay,
1960) is the gold standard for ASF diagnosis and
required for the first detection of the disease in
disease-free territories or regions. However, as it is a
laborious and time-consuming process, it is normally
used only in reference laboratories. PCRs are currently
the mostly widely used tests thanks to the reliability of
the technology, even with badly preserved tissues.
There are conventional (Ag€ uero et al., 2003) and
real-time PCR (King et al., 2003) tests, both of which
have been validated by the OIE. Another virus detec-
tion test is direct immunofluorescence (DIF), which is
designed to detect ASF antigens in smears or sections of
infected tissue. For this purpose, antibodies against
ASFV conjugated with fluorescein are used for visual-
izing the presence of antigens.
18 anchez-Vizca
ıno et al.
J.M. S

Serology has been employed widely in eradication
and control programmes for ASF (Arias and
S
anchez-Vizca
ıno, 2002). As no vaccine is available,
the presence of ASF antibodies implies previous infec-
tion. Consequently, serology is appropriate for detect-
ing potential carrier animals in endemic situations
(Arias and S
anchez-Vizca
ıno, 2002). The enzyme
linked immunosorbent assay (ELISA) is the most
commonly used serological test as it can be widely em-
ployed for screening purposes. Several ELISA tests
are available. Presently, the OIE uses a validated
in-house soluble ASF antigen (S
anchez-Vizca
ıno
et al., 1982) and a commercial ELISA. Other ELISAs
based on recombinant antigens (Gallardo et al., 2009)
are also used. The immunoblotting (Pastor et al.,
1989) and immunoperoxidase (Pan et al., 1978) tests
are employed for confirmation purposes or to comple-
ment the detection of ASF antibody.
Early Detection, Prevention and Control
There is no vaccine available for preventing ASF
infection. Consequently, prevention is based on
avoiding introduction of the disease. Several routes
of entry and risk assessments have been described
for different EU countries (Mur et al., 2012a, b,
2014; Costard et al., 2013a; De la Torre et al., in
press). The main control measures are to monitor
the entry of pigs and pig products and to forbid the
use of food waste for feeding pigs. Food waste from
infected areas should be incinerated and never used
to feed pigs (S
anchez-Vizca
ıno and Arias, 2012).
Contact made between wild boar and domestic pigs
is also an important risk for ASF-free countries
located near infected areas. Separating domestic
free-ranging pig production from areas containing
wild boars is also important, as well as increasing bio-
security by fencing pig farms to control against this
route of transmission. A publication on assessing the
risk of introduction of ASF into the EU by wild
boar has described the importance of this transmission
route for ASFV (De la Torre et al., in press).
If risk assessments are performed for estimating the
risk of introduction of ASF, they should be used to in-
crease awareness and surveillance in the critical loca-
tions/times/pathways indicated by the analysis
(Mur et al., 2014). Another important control mea-
sure is access to a good early detection programme
that includes raising awareness among veterinarians
and farmers by providing them with updated infor-
mation on risks. Basic ASF concepts, especially
regarding transmission routes, how to avoid intro-
duction and clinical signs and lesions, should be ad-
dressed in leaflets, posters and by other information
campaigns.
Such information campaigns help counteract one of
the most important problems in the control of animal
diseases: delayed detection in the field. Knowledge of
the clinical presentation and pathological lesions
compatible with ASF is essential for effective control.
However, as it was mentioned before, the presentation
of the disease in the field does not always present the
typical clinical characteristics and lesions. Conse-
quently, every animal in a high risk area presenting
high fever and dead should be analysed for ASF to
make an early detection and avoid the spread of the
explosive infection. Otherwise, disease can spread (as
has already occurred with other animal diseases such
as CSF or foot-and-mouth disease in the EU) for weeks
or even months before suspected samples arrive at the
laboratory.
The control measures for ASF are based on rapid
field detection and diagnosis confirmation, slaughter-
ing animals in infected holdings and banning move-
ments of pigs and pig-derived products in affected
areas. Other factors, such as the involvement of wild
boar and/or ticks in disease appearance, should be an-
alysed as potential sources of infection and the mea-
sures required to control them should be applied
whenever necessary.
Acknowledgements
This study was funded partially by the European
Research Project ASFORCE (‘Targeted research
effort on African swine fever’, grant agreement
311931). L. Mur holds a scholarship (FPU program)
from the Spanish Ministry of Education. The authors
would like to dedicate this paper to all researchers
who have worked or collaborated with our team for
the last 30 years in studying ASF; particularly Prof.
A. Jover and Prof. M. A. Sierra, and the importance
contribution of Dr. I. Minguez and Dr. C. Patta who
have now both died, but their work on ASF research
and control will not be forgotten.
References
Ag€
uero M, Fernandez J, Romero L, Mascaraque CS,
Arias M et al. (2003) Highly sensitive PCR assay for
routine diagnosis of African swine fever virus in clinical
samples. Journal of Clinical Microbiology, 41, 4431e4434.
Arias ML, Escribano JM, Rueda A, S
anchez-Vizca
ıno JM
(1986) La peste porcina Africana. Medicine Veterinaire, 3,
333e350.
Arias M, S
anchez-Vizca
ıno JM (2002) African swine fever
eradication: the Spanish model. In: Trends in Emerging
Viral Infections of Swine, A Morilla, K Jin,
J Zimmerman, Eds., Iowa State University Press,
Ames, pp. 133e139.
 Epidemiology and Pathology of African Swine Fever
Baskerville A, Dow C (1973) Pathology of experimental
pneumonia in pigs produced by Salmonella choleraesuis.
Journal of Comparative Pathology, 83, 207e215.
Bastos AD, Penrith ML, Cruciere C, Edrich JL,
Hutchings G et al. (2003) Genotyping field strains of Af-
rican swine fever virus by partial p72 gene characterisa-
tion. Archives of Virology, 148, 693e706.
Bautista MJ, Ruiz-Villamor E, Salguero FJ, S
anchez-
Cord
on PJ, Carrasco L et al. (2002) Early platelet aggre-
gation as a cause of thrombocytopenia in classical swine
fever. Veterinary Pathology, 39, 84e91.
Beltran-Alcrudo D, Lubroth J, Depner K, De la Roque S
(2008) African swine fever in the Caucasus. FAO
EMPRES Watch,1e8, April 2008.
Boshoff CI, Bastos AD, Gerber LJ, Vosloo W (2007) Ge-
netic characterisation of African swine fever viruses
from outbreaks in southern Africa (1973e1999). Veteri-
nary Microbiology, 121, 45e55.
Carrasco L, Bautista MJ, G
omez-Villamandos JC, Mart
ın
de las Mulas J, Chac
on-M et al. (1997a) Development of
microscopic lesions in splenic cords of pigs infected with
African swine fever virus. Veterinary Research, 28, 93e99.
Carrasco L, Chac
on M, de Lara F, Mart
ın de las Mulas J,
G
omez-Villamandos JC et al. (1997b) Ultrastructural
changes related to lymph node haemorrhages in acute
African swine fever. Research in Veterinary Science, 62,
199e204.
Carrasco L, de Lara FC, G
omez-Villamandos JC,
Bautista MJ, Villeda CJ et al. (1996a) The pathogenic
role of pulmonary intravascular macrophages in acute Af-
rican swine fever. Research in Veterinary Science, 61, 193e198.
Carrasco L, de Lara FC, Mart
ın de las Mulas J, G
omez-
Villamandos JC, Perez J et al. (1996b) Apoptosis in
lymph nodes in acute African swine fever. Journal of
Comparative Pathology, 115, 415e428.
Carrasco L, N
~ez A, Salguero FJ, D
ıaz San Segundo F,
un
S
anchez-Cord
on PJ et al. (2002) African swine fever:
expression of interleukin-1a and tumour necrosis fac-
tor-a by pulmonary intravascular macrophages. Journal
of Comparative Pathology, 126, 194e201.
Cheville NF, Mengeling WL (1969) The pathogenesis of
chronic hog cholera (swine fever). Histologic, immuno-
fluorescent and electron microscopic studies. Laboratory
Investigation, 20, 261e274.
Colgrove GS, Haelterman ED, Coggins L (1969) Patho-
genesis of African swine fever in young pigs. American
Journal of Veterinary Research, 30, 1343e1359.
Costard S, Jones BA, Martinez-Lopez B, Mur L, de la
Torre A et al. (2013a) Introduction of African swine fe-
ver into the European Union through illegal importa-
tion of pork and pork products. PLoS One, 8, e61104.
Costard S, Mur L, Lubroth J, S
anchez-Vizca
ıno JM,
Pfeiffer DU (2013b) Epidemiology of African swine fe-
ver virus. Virus Research, 173, 191e197.
De la Torre A, Bosch J, Iglesias I, Mu~ noz MJ, Mur L et al.
(in press) Assessing the risk of African swine fever intro-
duction into the European Union by wild boar. Trans-
boundary and Emerging Disease, http://dx.doi.org/10.
1111/tbed.12129.
19
Diaz AV, Netherton CL, Dixon LK, Wilson AJ (2012) Afri-
can swine fever virus strain Georgia 2007/1 in Ornithodoros
erraticus ticks. Emerging Infectious Diseases, 18, 1026e1028.
Dixon LK, Chapman DAG, Netherton CL, Upton C
(2013) African swine fever virus replication and geno-
mics. Virus Research, 173, 3e14.
Dixon LK, Escribano JM, Martins C, Rock DL, Salas ML
et al. (2005) Asfarviridae. In: Eighth Report of the Interna-
tional Committee on Taxonomy of Viruses, CM Fauquet,
MA Mayo, J Maniloff, U Desselberger, LA Ball, Eds.,
Elsevier Academic Press, London, pp. 135e143.
Drolet R, Thibault S, D’Allaire S, Thomson JR, Done SH
(1999) Porcine dermatitis and nephropathy syndrome
(PDNS): an overview of the disease. Journal of Swine
Health and Production, 7, 283e285.
European Food Safety Authority. (2010) Scientific opinion
on the role of tick vectors in the epidemiology of
Crimean-Congo hemorrhagic fever and African swine
fever in Eurasia. EFSA Journal, 8, 1703.
Gallardo C, Reis AL, Kalema-Zikusoka G, Malta J,
Soler A et al. (2009) Recombinant antigen targets for
serodiagnosis of African swine fever. Clinical and Vaccine
Immunology, 16, 1012e1020.
Garc
ıa-Nicol
as O, Baumann A, Vielle NJ, G
omez-
Laguna J, Quereda JJ et al. (2013) Virulence and
genotype-associated infectivity of interferon-treated
macrophages by porcine reproductive and respiratory
syndrome viruses. Virus Research, 179, 204e211.
G

omez-Laguna J, Salguero FJ, Pallar
es FJ, Carrasco L
(2013) Immunopathogenesis of porcine reproductive
and respiratory syndrome in the respiratory tract. Veter-
inary Journal, 195, 148e153.
G

omez-Villamandos JC, Bautista MJ, Carrasco L,
Chac
on M, De Lara F et al. (1998) Thrombocytopenia
associated with apoptotic megakaryocytes in a viral hae-
morrhagic syndrome induced by a moderately virulent
strain of African swine fever virus. Journal of Comparative
Pathology, 118, 1e13.
G

omez-Villamandos JC, Bautista MJ, S
anchez-Cord
on PJ,
Carrasco L (2013) Pathology of African swine fever: the
role of monocyte-macrophage. Virus Research, 173,
140e149.
G

omez-Villamandos JC, Herv
as J, M
endez A, Carrasco L,
Mart
ın de las Mulas J et al. (1995a) Experimental Afri-
can swine fever: apoptosis of lymphocytes and virus
replication in other cells. Journal of General Virology, 76,
2399e2405.
Griffith RW, Schwartz KJ, Meyerholz DK (2006) Salmo-
nella. In: Disease of Swine, 9th Edit., BE Straw,
JJ Zimerman, S D’Allaire, DJ Taylor, Eds., Blackwell
Publishing, Ames, pp. 739e754.
Han D, Hu Y, Li L, Tian H, Chen Z et al. (2014) Highly
pathogenic porcine reproductive and respiratory syn-
drome virus infection results in acute lung injury of the
infected pigs. Veterinary Microbiology, 169, 135e146.
Herv
as J, G

omez-Villamandos JC, M
endez A, Carrasco L,
Sierra MA (1996) The lesional changes and pathogen-
esis in the kidney in African swine fever. Veterinary
Research Communications, 20, 285e299.
20 anchez-Vizca
ıno et al.
J.M. S

King DP, Reid SM, Hutchings GH, Grierson SS,
Wilkinson PJ et al. (2003) Development of a TaqManÒ
PCR assay with internal amplification control for the
detection of African swine fever virus. Journal of Virolog-
ical Methods, 107, 53e61.
Malmquist WA, Hay D (1960) Hemadsorption and cyto-
pathic effect produced by African swine fever virus in
swine bone marrow and buffy coat cultures. American
Journal of Veterinary Research, 21, 104e108.
McVicar JW (1984) Quantitative aspects of the transmis-
sion of African swine fever. American Journal of Veterinary
Research, 45, 1535e1541.
Mebus CA, Dardiri AH (1979) Additional characteristics
of disease caused by the African swine fever viruses iso-
lated from Brazil and the Dominican Republic. Proceed-
ings of the Annual Meeting of the US Animal Health
Association, 83, 227e239.
Minguez I, Rueda A, Dominguez J, S
anchez-Vizca
ıno JM
(1988) Double labeling immunohistological study of Af-
rican swine fever virus-infected spleen and lymph nodes.
Veterinary Pathology, 25, 193e198.
Montgomery ER (1921) On a form of swine fever occurring
in British East Africa (Kenya Colony). Journal of Compar-
ative Pathology and Therapeutics, 34, 159e191.
Moulton J, Coggins L (1968) Comparison of lesions in
acute and chronic African swine fever. Cornell Veteri-
narian, 58, 364e387.
Mur L, Gallardo C, Soler A, Zimmermann J, Pelayo V et al.
(2013) Potential use of oral fluid samples for serological
diagnosis of African swine fever. Veterinary Microbiology,
165, 135e139.
Mur L, Mart
ınez-L
opez B, Costard S, de la Torre A,
Jones BA et al. (2014) Modular framework to assess the
risk of African swine fever virus entry into the European
Union. BMC Veterinary Research, 10, 145.
Mur L, Martinez-Lopez B, Martinez M, Costard S,
Wieland B et al. (2012a) Quantitative risk assessment
for the introduction of African swine fever virus into
the European Union by legal import of live pigs. Trans-
boundary and Emerging Diseases, 59, 134e144.
Mur L, Mart
ınez-L
opez B, S
anchez-Vizca
ıno JM (2012b)
Risk of African swine fever introduction into the Euro-
pean Union through transport-associated routes: re-
turning trucks and waste from international ships and
planes. BMC Veterinary Research, 8, 149.
Neilan JG, Zsak L, Lu Z, Burrage TG, Kutish GF et al.
(2004) Neutralizing antibodies to African swine fever vi-
rus proteins p30, p54, and p72 are not sufficient for
antibody-mediated protection. Virology, 319, 337e342.
Nelsen CJ, Murtaugh MP, Faaberg KS (1999) Porcine
reproductive and respiratory syndrome virus compari-
son: divergent evolution on two continents. Journal of
Virology, 73, 270e280.
OIE (2014) WAHID Database. Disease Information. Available
at: http://web.oie.int/wahis/public.php?page¼disease_
immediate_summary (accessed 25 June 2014 2012).
Pan IC, Hess WR (1984) Virulence in African swine fever:
its measurement and implications. American Journal of
Veterinary Research, 45, 361e366.
Pan IC, Shimizu M, Hess W (1978) African swine fever:
microplaque assay by an immunoperoxidase method.
American Journal of Veterinary Research, 39, 491e497.
Pastor M, Laviada M, S
anchez-Vizca
ıno JM, Escribano J
(1989) Detection of African swine fever virus antibodies
by immunoblotting assay. Canadian Journal of Veterinary
Research, 53, 105e107.
Penrith M-L, Vosloo W (2009) Review of African swine fe-
ver: transmission, spread and control. Journal of the South
African Veterinary Association, 80, 58e62.
Potier MF, Mesplede A, Vannier P (2006) Classical swine
fever and other pestiviruses. In: Disease of Swine, 9th
Edit., BE Straw, JJ Zimerman, S D’Allaire,
DJ Taylor, Eds., Blackwell Publishing, Ames,
pp. 291e298.
Rosell C, Segal
es J, Ramos-Vara JA, Folch JM, Rodr
ıguez-
Arrioja GM et al. (2000) Identification of porcine circo-
virus in tissues of pigs with porcine dermatitis and ne-
phropathy syndrome. Veterinary Record, 146, 40e43.
Salas ML, Andr
es G (2013) African swine fever virus
morphogenesis. Virus Research, 173, 29e41.
S

anchez-Botija C (1982) African swine fever. New develop-
ments. Revue Scientifique et Technique, 4, 1065e1094.
S

anchez-Vizca
ıno JM, Arias M (2012) African swine fever.
In: Diseases of Swine, 10th Edit., JJ Zimerman,
L Karriker, A Ramirez, K Schwartz, G Stevenson,
Eds., John Wiley & Sons, Ames, pp. 396e404.
S

anchez-Vizca
ıno JM, Mur L (2013) African swine fever
diagnosis update. In: Vaccines and Diagnostics for Trans-
boundary Animal Diseases, JA Roth, JA Richt,
IA Morozov, Eds., Karger, Basel, pp. 159e165.
S

anchez-Vizca
ıno JM, Mur L, Mart
ınez-L
opez B (2013)
African swine fever (ASF): five years around Europe.
Veterinary Microbiology, 26, 45e50.
S

anchez-Vizca
ıno JM, Tabar
es E, Salvador E, S
anchez-
Bojita C (1982) Semipurified structural viral protein
for the detection of African swine fever antibodies by
the indirect ELISA technique. Current Topics in Veterinary
Medicine and Animal Science, 22, 214e222.
Segal
es J, Allan GM, Domingo M (2006) Porcine circovi-
rus diseases. In: Disease of Swine, 9th Edit., BE Straw,
JJ Zimerman, S D’Allaire, DJ Taylor, Eds., Blackwell
Publishing, Ames, pp. 299e307.
Segal
es J, Piella J, Marco E, Mateu-de-Antonio EM,
Espuna E et al. (1998) Porcine dermatitis and nephrop-
athy syndrome in Spain. Veterinary Record, 142,
483e486.
Sierra MA, Carrasco L, G
omez-Villamandos JC, Martin
de las Mulas J, Mendez A et al. (1990) Pulmonary intra-
vascular macrophages in lungs of pigs inoculated with
African swine fever virus of differing virulence. Journal
of Comparative Pathology, 102, 323e334.
Sneath PHA, Mair NS, Sharpe ME, Holt JG (1986)Bergey’s
Manual of Systematic Bacteriology. Williams & Wilkins,
Baltimore, pp. 1245e1249.
Trautwein G (1988) Classical swine fever and related viral
diseases. 3. Pathology and pathogenesis of the diseases.
In: Developments in Veterinary Virology, B Liess, Ed., Mar-
tinus Nijhoff Publishing, Boston, pp. 27e54.
 Epidemiology and Pathology of African Swine Fever
Van Oirschot JT (1999) Hog cholera. In: Diseases of Swine,
8th Edit., BE Straw, S D’Allaire, WL Mengeling,
DJ Taylor, Eds., Blackwell Science, Malden,
pp. 159e172.
Van Regenmortel MHV, Fauquet CM, Bishop DHL,
Carstens EB, Estes MK et al. (2000) Virus taxonomy,
classification and nomenclature of viruses. Seventh Report
of the International Committee on Taxonomy of Viruses, Aca-
demic Press, London, pp. 867e872.
Villeda CJ, Williams SM, Wilkinson PJ, Vi~ nuela E (1993a)
Haemostatic abnormalities in African swine fever. A
comparison of two virus strains of different virulence
(Dominican Republic 78 and Malta 78). Archives of
Virology, 130, 71e83.
Villeda CJ, Williams SM, Wilkinson PJ, Vi~ nuela E (1993b)
Consumption coagulopathy associated with shock in
acute African swine fever. Archives of Virology, 133,
467e475.
21
Wang G, Song T, Yu Y, Liu Y, Shi W et al. (2011) Immune
responses in piglets infected with highly pathogenic
porcine reproductive and respiratory syndrome virus.
Veterinary Immunology and Immunopathology, 142, 170e178.
Wood RL, Henderson LM (2006) Erysipelas. In: Diseases of
Swine, 8th Edit., BE Straw, S D’Allaire, WL Mengeling,
DJ Taylor, Eds., Blackwell Science, Malden, pp. 629e638.
Zhou L, Zhang J, Zeng J, Yin S, Li Y et al. (2009) The 30-
amino-acid deletion in the Nsp2 of highly pathogenic
porcine reproductive and respiratory syndrome virus
emerging in China is not related to its virulence. Journal
of Virology, 83, 5156e5167.
½ Received,
Accepted, September 23rd, 2014 
September 4th, 2014
Journal of General Virology (1998), 79, 1439–1443. Printed in Great Britain
.......................................................................................................................................................................................................... SHORT COMMUNICATION

The pathogenesis of African swine fever in the resistant

bushpig
C. A. L. Oura, P. P. Powell, E. Anderson† and R. M. E. Parkhouse
Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Surrey GU24 0NF, UK

eating warthog tissue or whole ticks containing virus

Bushpigs and warthogs are natural reservoir hosts (Thomson et al., 1980). The susceptibility of bushpigs to ASFV
of African swine fever virus (ASFV) in the wild, was first demonstrated by Montgomery (1921). Since then,
showing no clinical signs of disease when infected however, the one further study on bushpigs was limited to the
with the same highly virulent isolates of ASFV that isolation of ASFV from several animals (DeTray, 1963).
induce rapid, haemorrhagic death in domestic pigs. Bushpigs are thought to be natural reservoir hosts of ASFV.
In contrast to domestic pigs, infection of bushpigs They show no clinical signs of disease when infected with
with Malawi isolate results in low levels of virus virulent and haemorrhagic isolates of ASFV that kill domestic
replication and lymphocyte apoptosis within the pigs within 5–7 days of infection. The aim of this study was to
spleen, and a relatively low spread of virus to other investigate the role of the bushpig as a reservoir host of ASFV
lymphoid tissues. However, at 10 days post-in- and to compare the pathogenesis of ASF in bushpigs and
fection, a high degree of apoptosis was seen in B domestic pigs. Specifically, the levels of ASFV replication in
lymphocytes of the B cell follicles in bushpig lymph tissues of bushpigs and pigs were assessed by virus titrations ;
nodes. Virus infected cells were present amongst the pathology, cell tropism and organ distribution of ASFV
the apoptotic B cells of these follicles, suggesting were investigated by immunocytochemistry, and compared to
that indirect factors released from ASFV infected similar studies in the domestic pig which are reported in the
macrophages signal surrounding lymphocytes to accompanying paper (Oura et al., 1998 a). Such comparisons of
enter apoptosis. The susceptibility/resistance of disease immunopathology may lead to an understanding of
domestic pigs/bushpigs to ASFV may serve as a why bushpigs survive infections with ASFV whereas domestic
unique veterinary model for the recently emerging pigs die, and provide important clues for the design of future
haemorrhagic disease of man. control strategies, such as a vaccine.
All bushpig infection studies were carried out on an island
on Lake Kariba, Northern Zimbabwe, with two separate
African swine fever virus (ASFV) is a non-pathogenic DNA groups of bushpigs. The bushpigs were captured from an
virus of warthogs and bushpigs which persists in Africa via a ASFV-free area in Zimbabwe. Of the first group of four
natural cycle of transmission between the warthog (Phaco- bushpigs, around 6 months old, three were infected intra-
choerus aethiopicus), bushpig (Potamchoerus porcus) and the soft muscularly with 10% HAD (50 % haemadsorbing units per g
&!
tick (Ornithodorus moubata). Warthogs are born free from tissue or per ml blood) Malawi isolate (Haresnape et al., 1984)
infection and may be infected early in life following the bite of of ASFV and sacrificed 5, 10 and 15 days post-infection (p.i.),
an infected tick. Virus replicates in the warthog and produces and the fourth was an uninfected control. The second group of
a low degree of viraemia for a few weeks, which is sufficient to two bushpigs, also 6 months old, was infected intramuscularly
infect a proportion of ticks that feed on the viraemic young with 10% HAD Malawi isolate of ASFV and sacrificed at 5
&!
warthogs (Thomson, 1985). Domestic pigs in Africa acquire and 10 days p.i. Blood, liver, spleen, kidney, gastro-hepatic
infection from wildlife reservoirs of the virus primarily by the lymph node, mesenteric lymph node, parotid lymph node,
bite of an infected tick (Plowright, 1977). Also, under submandibular lymph node, thymus, lung and tonsil were
experimental conditions, domestic pigs have been infected by removed and rapidly frozen (liquid N ) in OCT compound
#
(Tissue Tec) or fixed in 4 % paraformaldehyde in PBS for 18 h,
Author for correspondence : P. P. Powell. then paraffin embedded. Sections (5 µm) were placed onto
Fax ­44 1483 232448. e-mail penny.powell!bbsrc.ac.uk Superfrost Plus slides (BDH). As a direct comparison, Large
White¬Landrace pigs weighing 30 kg were inoculated intra-
† Present address : Veterinary Research Laboratory, PO Box 8101, muscularly with the highly virulent, 100 % fatal and haem-
Causeway, Harare, Zimbabwe. adsorbent Malawi isolate of ASFV (10% HAD per pig). One
&!

0001-5319 # 1998 SGM BEDJ

C. A. L. Oura and others
Fig. 1. Comparison of virus titrations in bushpigs and domestic pigs
infected with the Malawi isolate of ASFV. Virus titrations (log10 50 %
haemadsorbing units per g tissue or per ml blood, HAD50) in blood,
spleen and tonsil removed from bushpigs and domestic pigs infected with
Malawi isolate of ASFV.
pig was used as the uninfected control, one pig was sacrificed
at 2 days p.i., and pigs were sacrificed in pairs at 3, 4, 5, 6 and
7 days p.i.
Virus levels in bushpig and domestic pig tissues and blood
were titrated by the haemadsorption assay described pre-
viously by Wilkinson et al. (1977). Titres of between 5 and 7
log HAD were measured in bushpig tissues (blood, spleen,
"! &!
liver, tonsil, lymph nodes, kidney and lung) at 5, 10 and 15
days p.i. When compared with tissue virus titres in Malawi
infected domestic pig tissues, the bushpig titres were around 2
log HAD lower (Fig. 1). These results indicate that
"! &!
considerably less virus replication occurred in ASFV infected
bushpig tissues compared to domestic pig tissues. Similar
levels of blood and tissue virus titrations (³1 log HAD )
"! &!
were measured in the two separate groups of infected bushpigs.
Cell tropism and organ distribution of ASFV Malawi isolate
were determined by immunocytochemistry on bushpig tissues
taken from two bushpigs at 5 and 10 days p.i. and one bushpig
at 15 days p.i. Standard immunohistochemical techniques were
carried out as described previously (Oura et al., 1998 b). The
levels of virus replication in tissues stained with anti-vp73
MAb 4H3 (Cobbold et al., 1996) were quantified by counting
the number of infected cells per mm#. The mean number of
infected cells and standard error from 30 fields were calculated.
As might have been predicted from the virus titrations, results
reveal that there was considerably less virus replication in
bushpig tissues compared to domestic pig tissues. There was
an initial low level of virus replication of (3±5³0±5 infected
cells}mm#) at 5 days p.i. in cells in the red pulp of the spleen
(Fig. 2 A). At this time-point there was no detectable replication
in the lymph nodes ; however, in the tonsil there was virus
replication (3±2³0±5 infected cells}mm#) in cells located within
the epithelium of tonsillar crypts (Fig. 2 C). This was in marked
contrast to the domestic pig in which, at 5 days p.i., there was
extensive replication of ASFV (Malawi isolate) in all lymphoid
BEEA
tissue. A high degree of virus replication (87³5 infected
cells}mm#) was seen in the red pulp of the spleen (Fig. 2 B) and
in cells located both outside and within the epithelium of
crypts in the tonsil (13³1±1 infected cells}mm#) (Fig. 2 D).
Interestingly, in the bushpig ASFV replication was seen in cells
of the tonsillar crypt at 5, 10 and 15 days p.i. These infected
cells are shed into the pharynx and, if they are coughed up,
may be a mode of transmission of the virus. Indeed, direct
transmission of ASFV (Malawi isolate) from bushpig to
domestic pig has been achieved under experimental conditions
(Anderson et al., 1998).
The organ distribution of the virus was significantly
different in bushpigs compared to domestic pigs. In bushpig
lymphoid tissues, virus replication was seen in the spleen but
there was very limited escape of the virus to other lymphoid
organs. This was in marked contrast to the domestic pig, where
initial high levels of virus replication in the spleen were rapidly
followed by escape of the virus and high levels of virus
replication in other lymphoid organs (Oura et al., 1998 a). The
higher degree of infection in the lymphoid tissue of domestic
pigs was associated with severe irreversible pathological
damage and apoptosis of lymphocytes which were not seen in
the bushpig lymphoid tissues. The number of ASFV infected
cells correlates with the degree of lymphocyte apoptosis,
indicating that ASFV infected macrophages, possibly through
the release of cytokines or other apoptotic mediators, may be
responsible for the extensive lymphocyte apoptosis. Therefore,
the reason why bushpigs survive infection with virulent
isolates of ASFV may be because the virus is not so extensively
disseminated throughout the mononuclear phagocytic system
as clearly occurs in the domestic pig. With fewer infected
macrophages in the peripheral lymphoid tissue there would be
less release of cytokines or vasoactive substances such as
reactive oxygen intermediates or nitric oxide, and thus less
destructive pathology and lymphocyte apoptosis.
There was a striking difference between bushpig lymph
nodes at 5 and 10 days p.i. The terminal deoxynucleotidyl
transferase (TdT)-mediated dUTP end labelling technique
(TUNEL) was used to label DNA strand breaks in apoptotic
cells in tissue sections (Oura et al., 1998 a). At 10 days p.i. many
B cells in many of the follicles had pyknotic nuclei and were
apoptotic (Fig. 3 A, B). In some B cell follicles 70–80 % of B
lymphocytes were apoptotic. Also, interestingly, virus rep-
lication was detected in cells in the B cell follicles (Fig. 3 C). The
most likely phenotypes of infected cells in the follicles are
tingible body macrophages or follicular dendritic cells. In order
to identify the phenotype of the apoptotic cells in the follicles,
lymph node cryosections from bushpigs, 10 days p.i. with
Malawi, were immunostained with anti-CD21 B cell specific
MAb C35 (Saalmuller, 1996). The vast majority of the cells in
the follicles were B cells (Fig. 3 D). This demonstrated that the
apoptotic cells in the follicles were B lymphocytes. The degree
of apoptosis of B lymphocytes in B cell follicles of bushpig
lymph nodes, at 10 days p.i., was considerably higher than the
 Pathogenesis of ASF in the bushpig

Fig. 2. Immunocytochemical localization of vp73 protein in bushpig and domestic pig tissues infected with ASFV (Malawi
isolate). Paraffin embedded sections, stained with anti-vp73 MAb 4H3, visualized with DAB (brown, arrow) and counterstained
with haematoxylin. (A) Bushpig spleen, 5 days p.i. with Malawi (¬80) ; (B) domestic pig spleen, 5 days p.i. with Malawi
(¬80) ; (C) bushpig tonsil, 5 days p.i. with Malawi (¬80) ; (D) domestic pig tonsil, 5 days p.i. with Malawi (¬160). W, White
pulp ; R, red pulp ; C, crypt.

degree of apoptosis seen in follicles of lymph nodes from
domestic pigs, 10 days p.i. with the moderately virulent Malta
isolate of ASFV (Oura et al., 1998 a). In order to stimulate an
exuberant immune response in lymph node germinal centres, a
pig was vaccinated twice with foot-and-mouth disease virus
vaccine (O1 serotype, Bayer) 3 weeks apart. Seven days after
the second vaccine the pig was sacrificed and the draining
lymph node was removed, paraffin embedded and stained with
TUNEL. There was considerably less apoptosis observed in
germinal centres of the vaccinated pig compared to the
bushpig at 10 days p.i. (data not shown). While not conclusive,
this indicates that the observed exuberant apoptosis is more
likely to result from a pathological component than a normal
immune activation.
Apoptosis of lymphocytes and ASFV infection of cells in
bushpig lymph nodes were mostly confined to the B cell

BEEB
C. A. L. Oura and others

Fig. 3. Apoptosis in bushpig lymph node infected with ASFV (Malawi isolate). Paraffin embedded lymph node sections from a
bushpig, 10 days p.i. with Malawi isolate (¬160). (A) Stained with haematoxylin and eosin ; (B) stained with TUNEL ;
(C) immunostained with anti-vp73 MAb 4H3 ; (D) lymph node cryosection from a bushpig, 10 days p.i. with Malawi isolate,
immunostained with anti-CD21 MAb C35. In immunohistochemical and TUNEL staining, signal was visualized with DAB
substrate (brown) and sections were counterstained with haematoxylin. F, B cell follicle.

follicles, with low levels of apoptosis and ASFV infection
occurring in T cell zones of the cortex and paracortex. This
observation is in marked contrast to the situation in domestic
pigs infected with Malawi isolate where the majority of ASFV
infected cells and apoptosis of lymphocytes were located in the
T cell areas (Oura et al., 1998 a ; Carrasco et al., 1996). There are
many possible causes of this B lymphocyte apoptosis which
include : (1) ASFV infected tingible body macrophages are
indirectly, perhaps through the release of cytokines or other
apoptotic mediators, inducing surrounding B cells to enter
apoptosis ; (2) the lymphocyte selection process is impaired
through damage to follicular dendritic cells so most of the B
cells in the germinal centres are selected for programmed cell
death, with few lymphocytes being positively selected for
continued survival ; or (3) the process of affinity maturation is
compromised so that only low affinity antibody-producing B
lymphocytes are produced. Because only high affinity
antibody-producing lymphocytes are selected for survival in

BEEC

the germinal centres, the majority of B lymphocytes enter
apoptosis.
ASFV has evolved in a natural cycle between the reservoir
hosts : the bushpig, the warthog and the soft tick. Sufficiently
high viraemias occur in the bushpig to allow infection of
feeding ticks, but ASFV replication in lymphoid tissues is kept
in check, and pathological damage and apoptosis in lymphoid
tissue are kept to a minimum so that the hosts survive infection
with no clinical signs of disease. Also, a controlled level of
apoptosis in uninfected lymphocytes may result in a diminished
immune response enabling ASFV infected cells to survive the
immune system for sufficient time to produce a productive
infection. In addition, bushpigs may also control the apoptosis
of B lymphocytes at the time of selection of high affinity
antibody producing B lymphocytes (10 days p.i.). This would
halt the production of high affinity anti-ASFV antibody and
facilitate long term survival of extracellular virus adsorbed to
the surface of red blood cells, enabling its transmission to
feeding ticks. This may also have important implications for
the persistence of the ASFV in the bushpig and suggests that
antibody mediated immunity may not be critically important
for bushpig survival. Characterization of bushpig and domestic
pig antibody responses are being carried out.
In contrast, the domestic pig is not a natural host of ASFV.
The factors that keep both the virus and apoptosis in check in
bushpigs are ‘ out of control ’ in domestic pigs. High levels of
virus replication in lymphoid tissue result in uncontrolled
apoptosis of lymphocytes, a severely impaired immune
response, and perhaps aberrant levels of cytokines contributing
to the haemorrhagic pathology. Therefore domestic pigs die
within 5–7 days of infection.
ASF with its differential pathology, haemorrhagic in the
susceptible domestic pig versus non-pathogenic in the resistant
bushpig, may be considered as a veterinary model for the
recently emerging haemorrhagic diseases of man.
We thank Dr Stuart Hargreaves, Director of Veterinary Services,
Harare, Zimbabwe for providing laboratory space in Zimbabwe. Thanks
to Mick Denyer for help with the animal work. The field work was funded
by the Overseas Development Administration (ODA) and the work was
also funded in part by MAFF Contract 1506.
Pathogenesis of ASF in the bushpig
References
Anderson, E. C., Hutchings, G., Mukarati, N. & Wilkinson, P. J. (1998).
African swine fever virus infection of the bushpig (Potamchoerus porcus)
and their significance in the epidemiology of the disease. Veterinary
Microbiology (in press).
Carrasco, L., Chacon, M., de Lara, F., Martin de las Mulas, J., Gomez-
Villamandos, J. C., Perez, J., Wilkinson, P. J. & Sierra, M. A. (1996).
Apoptosis in lymph nodes in acute African swine fever. Journal of
Comparative Pathology 115, 415–428.
Cobbold, C., Whittle, J. T. & Wileman, T. (1996). Involvement of the
endoplasmic reticulum in the assembly and envelopment of African swine
fever virus. Journal of Virology 70, 8382–8390.
DeTray, D. E. (1963). African swine fever. Advances in Veterinary Science
8, 299–333.
Haresnape, J. M., Wilkinson, P. J. & Mellor, P. S. (1984). Isolation of
African swine fever virus from ticks of the Ornithodorus moubata complex
(Ixodoidae : Argasidae) collected within the African swine fever enzootic
area of Malawi. Epidemiology and Infection 101, 173–185.
Montgomery, R. E. (1921). On a form of swine fever occurring in British
East Africa (Kenya colony). Journal of Comparative Pathology 34, 243–262.
Oura, C. A. L., Powell, P. P. & Parkhouse, R. M. E. (1998 a). African
swine fever : a disease characterized by apoptosis. Journal of General
Virology 79, 1427–1438.
Oura, C. A. L., Powell, P. P. & Parkhouse, R. M. E. (1998 b). Detection
of African swine fever virus in infected pig tissues by immuno-
cytochemistry and in situ hybridisation. Journal of Virological Methods (in
press).
Plowright, W. (1977). Vector transmission of African swine fever virus.
In Hog Cholera}Classical Swine Fever and African Swine Fever, EEC
Publication EUR 5904 EN, pp. 575–587. Edited by B. Liess. Luxembourg :
Office for Official Publications of the European Communities.
Saalmuller, A. (1996). Characterisation of swine leukocyte differ-
entiation antigens. Immunology Today 17, 352–354.
Thomson, G. R. (1985). The epidemiology of ASF : the role of free-
living hosts in Africa. Onderstepoort Journal of Veterinary Research 52, 201.
Thomson, G. R., Gainaru, M. D. & Van Dellen, A. F. (1980). Ex-
perimental infection of warthog (Phacochoerus aethiopicus) with ASFV.
Onderstepoort Journal of Veterinary Research 47, 19–22.
Wilkinson, P. J., Donaldson, A. I., Greig, A. & Bruce, W. (1977).
Transmission studies with African swine fever virus. Infection of pigs by
airborne virus. Journal of Comparative Pathology 87, 487–495.
Received 11 November 1997 ; Accepted 3 February 1998
BEED
 Virus Research 173 (2013) 122–130

Contents lists available at SciVerse ScienceDirect

Virus Research
journal homepage: www.elsevier.com/locate/virusres

Review

Pathogenesis of African swine fever in domestic pigs and European wild boar
Sandra Blome ∗ , Claudia Gabriel, Martin Beer
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifswald-Insel Riems, Germany

a r t i c l e i n f o a b s t r a c t

Article history: African swine fever (ASF) is among the most important viral diseases that can affect domestic and feral
Available online 6 November 2012 pigs. Both clinical signs and pathomorphological changes vary considerably depending on strain viru-
lence and host factors. Acute infections with highly virulent virus strains lead to a clinical course that
Keywords: resembles a viral haemorrhagic fever that is characterized by pronounced depletion of lymphoid tissues,
African swine fever apoptosis of lymphocyte subsets, and impairment of haemostasis and immune functions. It is generally
Pathogenesis
accepted that most lesions can be attributed to cytokine-mediated interactions triggered by infected
Domestic pigs
and activated monocytes and macrophages, rather than by virus-induced direct cell damage. Neverthe-
European wild boar
less, most pathogenetic mechanisms are far from being understood. This review summarizes the current
knowledge and discusses implications and research gaps.
© 2012 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
2. Clinical and pathological observations upon ASFV infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2.1. ASFV infection results in a wide range of clinical pictures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2.2. Gross pathological findings and virus detection mirror clinical courses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2.3. Blood count changes and phenotypic characteristics vary in the course of acute and chronic infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
3. Changes in primary target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
3.1. Susceptibility of monocyte/macrophages to ASFV infection increases upon maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
3.2. Secretory and phagocytic activation of monocyte/macrophages leads to proinflammatory and procoagulant responses . . . . . . . . . . . . . . . . 125
4. Viral distribution, secondary replication sites and pathomorphological correlates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.1. Viral replication leads to widespread lymphoid depletion through destruction and apoptosis of monocyte/macrophages and
uninfected lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.2. Haemorrhagic lesions are associated with release of cytokines by infected monocyte/macrophages rather than direct endothelial
cell damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
4.3. Impairment of platelets and coagulation changes contribute to evolution of a haemorrhagic syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4.4. Additional cytokine and acute phase responses link pathogenesis and immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5. Résumé and gap analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

1. Introduction family (Takamatsu et al., 2011). Due to the fact that soft ticks of
the genus Ornithodoros can be involved in the infection cycle, ASFV
Due to its relevance for animal health and pig industry, African is regarded as the only DNA virus that can be classified as an ARBO
swine fever (ASF) is one of the most important viral diseases of (arthropod borne) virus (Kleiboeker and Scoles, 2001).
domestic pigs. It was shown that it impairs pig production consid- In the vertebrate host, the virus replicates in cells of the
erably, especially in Africa (Penrith and Vosloo, 2009). The causative mononuclear phagocyte system (in older literature referred to
agent, African swine fever virus (ASFV), is a large and complex as reticuloendothelial system), predominantly monocytes and
DNA virus of the genus Asfivirus within the Asfarviridae virus macrophages although several other cell types can be infected,
especially in the later stages of the disease (Carrasco et al., 1996b,
1996d; Fernandez et al., 1992a; Mebus, 1987, 1988; Perez et al.,
∗ Corresponding author. Tel.: +49 38351 7 1144; fax: +49 38351 7 1275. 1994; Ramiro-Ibanez et al., 1995). It could be demonstrated in
E-mail addresses: sandra.blome@fli.bund.de (S. Blome), new-born piglets that primary viraemia occurs approximately 8 h
claudia.gabriel@fli.bund.de (C. Gabriel), martin.beer@fli.bund.de (M. Beer). post infection. Secondary viraemia is detected from 15 to 24 h post

0168-1702/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.virusres.2012.10.026
 S. Blome et al. / Virus Research 173 (2013) 122–130
infection and after 30 h, virus is found in almost all organs (Col-
grove et al., 1969, cited by the EFSA Scientific Report 2009). During
viraemia, haemadsorbing ASFV isolates are found associated with
erythrocytes (Quintero et al., 1986; Wardley and Wilkinson, 1977),
but also with lymphocytes and neutrophils (Plowright et al., 1994).
Infection correlates with a wide range of clinical syndromes from
almost inapparent disease to a haemorrhagic fever like illness with
high mortality (Penrith and Vosloo, 2009; Takamatsu et al., 2011).
Nowadays it is generally accepted that the massive destruc-
tion of macrophages plays a major role in pathogenesis, especially
in the impaired haemostasis due to the release of active sub-
stances including cytokines, complement factors and arachidonic
acid metabolites (Penrith et al., 2004b). The genesis of thrombo-
cytopenia that occurs particularly during the course of acute and
subacute ASFV infection is controversially discussed. While some
authors show evidence that infection and destruction of megakary-
ocytes may play a role (Rodriguez et al., 1996a), other researchers
did not find structural or ultrastructural correlates for an
impaired thrombocytopoiesis (Gomez-Villamandos et al., 1997a).
Pigs infected with ASFV generally suffer from severe lymphopenia
that could be attributed to apoptosis of lymphocytes. Production of
pro-inflammatory cytokines by infected macrophages is strongly
implicated in induction of apoptosis in lymphocyte populations
(Gomez-Villamandos et al., 1995a, 2003; Oura et al., 1998a).
The also described chronic forms of ASF might have an auto-
immune component, and lesions could result from the deposition of
immune-complexes in tissues such as kidneys, lungs and skin with
their subsequent binding to complement (EFSA Scientific Report
2009; Plowright et al., 1994).
Despite intensive research efforts, most of these pathogenetical
aspects are still far from being understood. This review compiles the
current knowledge of pathogenetical mechanisms and their clinical
and pathomorphological correlates, discusses their implications,
and identifies research gaps.
2. Clinical and pathological observations upon ASFV
infection
2.1. ASFV infection results in a wide range of clinical pictures
In both domestic pigs and wild boar, clinical signs of ASF vary
considerably from peracute deaths to inapparent courses and cor-
relate to a wide range of pathomorphological observations. Reports
on naturally resistant domestic pigs in Africa remain controver-
sially discussed and do not seem to have a genotypic correlate as
the offspring of less susceptible pigs does not inherit this advan-
tage (Penrith et al., 2004a). Upon infection with a virulent strain,
clinical signs develop after an incubation period of 2 to 7 (sel-
dom up to 14) days (Mebus, 1988). They may include high fever,
reddened skin at the acra, severe depression, anorexia, conjunctivi-
tis, vomiting, watery to bloody diarrhoea, accelerated respiratory
and pulse rate, abortion in pregnant sows, cyanosis, and incoor-
dination (EFSA Scientific Report, 2009). Acute lethal forms can
be accompanied by haemorrhagic lesions (petechiae, epistaxis)
(Gomez-Villamandos et al., 2003). Characteristics are thrombo-
cytopenia, petechial bleedings, and apparently increased vascular
permeability with extravasation of blood components. In less acute
disease courses, respiratory signs (coughing, sneezing, and dysp-
noea) and gastrointestinal lesions (mainly watery diarrhoea but
also obstipation) are frequently seen. Depending on strain viru-
lence, mortality ranges from 3 to 100%.
The ASFV strain currently affecting the Trans-Caucasian
countries, Russia, and Ukraine, is highly virulent and leads to
the above mentioned clinical picture with high fever, reddened
skin and cyanosis, especially upon agitation, severe depression
123
up to somnolence, anorexia, slight catarrhal conjunctivitis, vomi-
ting, watery diarrhoea, accelerated respiratory and pulse rate, and
ataxia/incoordination prior to death (Gabriel et al., 2011; Kolbasov
and Blome, unpublished data). In these studies, it could be demon-
strated that the clinical course in domestic pigs was irrespective of
the route of infection (oro-nasal, intramuscular or infection through
direct contact with infected pen-mates). In all experimental infec-
tions, mortality was 100% in less than 10 days. A similar picture
was observed upon oral or intramuscular infection of European
wild boar although skin colour and perfusion could not be assessed
(Blome et al., 2012; Gabriel et al., 2011). In wild boar, severe depres-
sion, anorexia, diarrhoea, and respiratory distress predominated.
Furthermore, central nervous symptoms including ataxia and con-
vulsions as well as epistaxis were observed in some cases prior to
death (Gabriel et al., 2011; Kolbasov and Blome, unpublished data).
2.2. Gross pathological findings and virus detection mirror
clinical courses
Like the major clinical signs, pathological findings vary consid-
erably depending on the course of disease. Frequently observed
lesions in domestic pigs include enlarged and haemorrhagic gas-
trohepatic and renal lymphnodes, splenomegaly with necrotic foci,
petechiae in kidneys, ecchymoses in serosae, alveolar haemor-
rhages and oedema of the lungs (Kleiboeker, 2002; Maurer and
Griesemer, 1958; Colgrove et al., 1969, reviewed by Rodriguez et al.,
1996a). Secondary infections may complicate the clinical and path-
omorphological picture. The same is seen in European wild boar of
all age classes upon highly virulent infection under experimental
conditions (Blome et al., 2012; Gabriel et al., 2011), and also in other
feral pigs (McVicar et al., 1981). Field data showed that all courses
of the disease including almost inapparent ones are possible (Perez
et al., 1998). Exemplary pathological findings in European wild boar
are depicted in Figs. 1 and 2.
Particularly during the clinical phase of infection, virus and viral
DNA can be detected by virus isolation and real-time PCR in all se-
and excretions of infected pigs, but the viral load varies consid-
erably (Gabriel et al., 2011). While high amounts of virus or viral
DNA can be detected in blood samples, all swab samples (rectal,
pharyngeal, nasal, and preputial/vaginal) show considerably lower
viral loads (Ekue et al., 1989; Gabriel et al., 2011). In detail, up to
3 × 106 copies can be detected per ␮l DNA from 200 ␮l of blood,
whereas only 4–8 × 103 copies per ␮l can be found in rectal swabs,
and 2–6 × 102 copies per ␮l in oropharyngeal swabs (Gabriel and
Blome, unpublished data). Exemplary data depicting the detection
of viral DNA in blood and swab samples from European wild boar
and domestic in-contact pigs are shown in Table 1. It is not sur-
prising that contagiosity is mainly linked to blood and that contact
alone is less efficient (Heuschele, 1967).
2.3. Blood count changes and phenotypic characteristics vary in
the course of acute and chronic infections
In general, infection with ASFV is accompanied by leukopenia
and thrombocytopenia, but extent and nature may vary with strain
virulence and host factors.
It has been reported by Wardley and Wilkinson (1977) that
infection with the highly virulent Kirawira isolate (KWH/12) (Greig
and Plowright, 1970) leads to a decrease in leucocyte counts while
the red blood count remains almost unchanged. In this study,
lymphocyte numbers decreased while an increased number of neu-
trophils, especially juvenile subsets, was observed. Virus could be
demonstrated in all major blood fractions, but over 90% were asso-
ciated with red blood cells. These findings are in agreement with
earlier observations by Detray and Scott (1957). In contrast, Gomez-
Villamandos et al. (1997a) did not observe significant changes
124 S. Blome et al. / Virus Research 173 (2013) 122–130

Table 1
Real-time PCR results from EDTA blood, oropharyngeal and faecal swab samples depicted as quantification cycle (cq) values. Wild boar were infected orally with 2 ml of
a spleen suspension containing 106 tissue culture infectious doses 50% of the highly virulent Caucasian ASFV strain per ml. Domestic pigs were co-housed two days after
inoculation of the wild boar. Wb: Wild boar; Dp: Domestic Pig; nd: not done. Grey shaded values indicate fever (±40 ◦ C) at that time point. −Negative result in qPCR; †Animal
was dead at this time point; *Serum instead of EDTA-blood sample.

Days post infection of wild boar

Animal Samples 0 1 2 3 4 5 6 7 9 13 17 20

Wb 1 Blood – nd – nd nd 23
Oropharyngeal swab – – – – nd – †
Faecal swab – – – – nd –
Wb 2 Blood – nd – nd nd 22 20 24
Oropharyngeal swab – – – – nd 37 37 37 †
Faecal swab – – – – nd – 38 -
Wb 3 Blood – nd – nd nd 28 22 23
Oropharyngeal swab – – – – nd – 38 34 †
Faecal swab – – – – nd 37 34 33
Wb 4 Blood – nd – nd nd 25 26 26
Oropharyngeal swab – – – 37 nd – 34 37 †
Faecal swab – – – – nd 30 29 33
Wb 5 Blood – nd 39 nd nd 25 23
Oropharyngeal swab – – – – nd 39 35 †
Faecal swab – – – – nd – 29
Wb 6 Blood – nd – nd nd 23 24
Oropharyngeal swab – – – 37 nd – 34 †
Faecal swab – – – – nd 35 32
Dp 1 Blood – nd nd nd nd nd 39 nd – 21 20 †
Dp 2 Blood – nd nd nd nd nd – nd – 23 nd †
Dp 3 Blood – nd nd nd nd nd – nd – – nd 29*

in total white blood cell counts, percentages of monocytes and
platelets counts upon infection with the highly virulent Malawi
1983 ASFV isolate (Haresnape et al., 1988), but lymphopenia and
neutrophilia was seen. This discrepancy may arise from slightly
different clinical courses. Moreover, interpretation of tendencies
by the authors varied to a certain extent. Even after infection with
the Kirawira isolate of ASFV, leukopenia barely reached patholog-
ical values (i.e. values <10 × 103 cells per ␮l) before the animals
succumbed to infection.
Other studies showed that acute infection with highly vir-
ulent viruses leads to a severe decrease of macrophages and
B-lymphocytes (reduction of 60% with respect to pre-infection
values) after a short period with increased B-lymphocyte counts
(probably due to polyclonal activation period of B-lymphocytes)
and an increase in macrophages at the beginning of infection
(Ramiro-Ibanez et al., 1997). In this study, severe leukopenia was
observed in global white blood cell counts 7 days post infection with
the highly virulent E70 isolate of ASFV. These findings coincided
with reduced relative numbers of lymphocytes and monocytes.
Affected lymphocyte subsets were, apart from the already men-
tioned B-lymphocytes, CD4+ T-helper cells. Furthermore, CD8+
cytotoxic T-cells increased by the end of the first week.
Investigation of phenotypic antigens and activation markers
revealed that at the peak of viral titres, an increase is seen in both

Fig. 1. Gross pathological lesions in a European wild boar piglet. The boar (approx-
imately six months of age) was infected oronasally with 3 × 106 tissue culture
infectious doses 50% (see table above) of the highly virulent Caucasian ASFV isolate.
This animal was euthanized in a moribund state at eight days post infection, show-
ing severe depression, high fever (41.8 ◦ C), tachypnoea, uncontrolled movements of
hind legs and inability to stand. (A) Acute diffuse ecchymosis and petechiae on the
mucosal surface of the stomach. (B) Kidney with numerous acute petechiae on the
renal cortex.
Fig. 2. Mandibular lymph nodes of a crossbred weaner domestic pig (approximately
30 kg). This animal acted as in-contact control to European wild boar piglets which
were orally inoculated with 2 × 106 tissue culture infectious doses 50% (see table
above) of the Caucasian ASFV isolate. The animal was brought into the same pen
three days after inoculation, and died 16 days after initial contact. Note high-grade,
acute, diffuse haemorrhages and enlargement.
 S. Blome et al. / Virus Research 173 (2013) 122–130
SLA II (MHC-II) and CD8+ expression. These findings are accom-
panied by down regulation of IL-2 (CD25) receptor expression
(Ramiro-Ibanez et al., 1997). As IL-2 is crucial for the modulation
of regulatory and effector lymphocyte functions, T-cell response
seems to be influenced. Similar changes in IL-2 receptor and SLA II
expression were reported by Canals et al. (1995) after investigation
of ASFV-infected cell cultures.
Studies using a moderately virulent ASFV strain from the
Dominican Republic (DR II, isolated in 1979) showed that virus
could be found in the isolated erythrocyte fraction as well as in
plasma and mononuclear leukocytes from peripheral blood after
oro-nasal infection (Knudsen and Genovesi, 1987). In this case,
virus infection had only minor effects on the number of circulat-
ing leukocytes as just slight increases in neutrophils and decreases
in lymphocyte counts were observed early upon infection. Respon-
siveness of mononuclear cells to mitogenic stimuli was preserved
throughout the trial. The latter disagrees with studies by other
authors where severe impairment of responsiveness to mitogenic
stimuli was observed (Gonzalez et al., 1990; Wardley, 1982).
Chronically infected animals were shown to exhibit marked
differences in blood count changes. During this disease course, B-
lymphocytes and macrophages showed an increase of 100% within
the first week after infection with the attenuated E75CV1 4 strain
with a peak that coincided with high viraemia, onset of fever, and
detectable antibodies (Ramiro-Ibanez et al., 1997). These values
were back to normal after the second week post infection. Both
CD8+ and CD4+ T cells showed an elevation during the second week
of infection. In this case, both SLA I (MHC-I) and SLA II showed
an elevated expression on peripheral mononuclear cells indica-
tive for stimulation of the immune system. These findings were
in agreement with in vitro studies and findings in spleen tissue sec-
tions (Gonzalez-Juarrero et al., 1992a, 1992b). At the third week
post infection, the expression was significantly decreased. This may
have implications for viral clearance through an impaired antigen
presentation. Here again, a decrease in IL-2 receptor expression
was observed, underlining the possible role in the pathogenesis of
lesions (Canals et al., 1995).
3. Changes in primary target cells
3.1. Susceptibility of monocyte/macrophages to ASFV infection
increases upon maturation
Without tick involvement, ASFV enters the body via the tonsils
or dorsal pharyngeal mucosa to the mandibular or retropharyngeal
lymph nodes, from where the virus spreads systemically through
viraemia (Greig, 1972; Plowright et al., 1994). Thereafter, the virus
is detectable in almost all tissues. Highest titres are observed in tis-
sues containing a large component of the mononuclear phagocyte
system (reticuloendothelial cellular elements) like the spleen and
lymphnodes (Heuschele, 1967).
The main target cells of ASFV, which are entered by endo-
cytosis, probably involving the mechanism of macropinocytosis
(Sanchez et al., 2012), belong to the monocyte/macrophage lineage
(Malmquist and Hay, 1960; Sanchez-Torres et al., 2003; Sierra et al.,
1990). This cell population is highly diverse and comprises different
phenotypes, activation and maturation stages. In addition, infection
of dendritic cells is observed which may interfere with humoral
immune responses (Gregg et al., 1995).
It was observed that different subsets of monocytes and
macrophages show a different susceptibility to ASFV, both in vivo
(Carrasco et al., 1992; Rodriguez et al., 1996a) and in vitro.
Bone marrow derived cells and fresh blood monocytes are less
susceptible to in vitro infection than alveolar macrophages. In
general, it could be shown that cells of higher maturation stages
125
expressing higher levels of macrophage specific markers and SLA
II antigens were most susceptible to ASFV infection. Especially
expression of CD163, an acute phase regulated receptor, and sur-
face antigen 4E9 (now known as porcine CD107a, or lysosomal
associated membrane protein 1) correlates with permissiveness to
ASFV infection (Sanchez-Torres et al., 2003). The fact that infection
could be inhibited by anti-CD163 or anti-4E9 monoclonal antibod-
ies demonstrates that both antigens play a role in the initial stages
of viral infection (Sanchez-Torres et al., 2003). This is an interesting
finding as it is known that CD163+ monocytes produce more TNF-␣,
express high levels of adhesion molecules and are better at pre-
senting antigens to T-cells when compared to CD163− monocytes
(Chamorro et al., 2005). This may have an impact on the influence
on endothelial cells and apoptosis induction capacity as well as
the timeframe of pathogenesis. The more mature scavenger type
macrophage seems to fulfil the needs for ASFV replication much
better than monocytes. One explanation could be the need for rapid
vesicle acidifications and lysosomal enzyme activity (Basta et al.,
1999). Nevertheless, further investigations are needed to confirm
and consolidate these results.
In peripheral blood of experimentally infected animals, mainly
monocytes and macrophages are positive for viral p30 antigen as
shown by flow cytometric analyses with a maximum percentage
ranging from 6 to 31% at days 7–9 post infection with an attenuated
strain and at day 5 after virulent ASFV infection (Ramiro-Ibanez
et al., 1995). The above mentioned influence of the maturation
state may explain the relatively low number of infected cells on
the peak of infection and thus viral replication. In addition to the
monocyte/macrophage subset, a small proportion of granulocytes
(7–21% of all infected cells) is also infected. Neither time point after
infection nor virus isolate led to significant differences regarding
affected cell subsets (Ramiro-Ibanez et al., 1995). Infection of neu-
trophils was confirmed by Carrasco et al. (1996d) who could show
that both mature and immature neutrophils can harbour virus.
Especially the latter are discussed as transport vehicles for virus
spreading (Gomez-Villamandos et al., 1997a).
3.2. Secretory and phagocytic activation of
monocyte/macrophages leads to proinflammatory and
procoagulant responses
Upon ASFV infection, several macrophage subpopulations
show signs of secretory and/or phagocytic activation. Moreover,
destruction of monocytes/macrophages leads to release of cel-
lular components. Activated monocytes/macrophages secrete a
wide range of mediators including proinflammatory cytokines
such as IL-1, IL-6, and TNF-␣ (Murtaugh et al., 1996). These
cytokines can trigger acute phase reactions, inflammation, acti-
vation of endothelial cells, and apoptosis. Among these factors,
TNF-␣ is particularly important (Gomez del Moral et al., 1999).
It can induce vascular changes (vasodilatation and increased per-
meability) and modulates the activation status of the vascular
endothelium (procoagulant/anticoagulant). Moreover, TNF-␣ is
involved in the control of apoptosis.
It could be demonstrated that an increase in the production of
TNF-␣, IL-1␣ and IL-1␤, and IL-6 coincides with onset of fever, vas-
cular damage, and changes in lymphoid structures (Salguero et al.,
2002). Detection of cytokines in tissues correlates with detection
of VP73 antigen in cells of the monocyte/macrophage lineage and
an increase in serum levels of TNF-␣ and IL-1␤.
Comparison of cytokine responses in macrophages infected
in vitro with low and highly virulent ASFV strains indicate that
attenuated strains show an altered response favouring cytokines
involved in cellular immunity, namely IFN-␣ and IL12p40 (Gil et al.,
2008). Thus, survival of animals may at least partly depend on the
nature of cytokine responses. An interesting finding in line with
126 S. Blome et al. / Virus Research 173 (2013) 122–130
both above mentioned considerations is that pulmonary intravas-
cular macrophages but not alveolar macrophages are infected
(Carrasco et al., 2002; Oura et al., 1998b). Infection of these cells
leads to intense activation and consequently expression of IL-1␣
and TNF-␣. A clear correlation was observed between this cytokine
response and interstitial oedema and fibrin microthrombi forma-
tion in septal capillaries (Carrasco et al., 2002). These findings were
supported by earlier studies of the same group that showed that
activation of pulmonary intravascular macrophages could play a
major role in the genesis of lung-associated lesions (Carrasco et al.,
1996a).
4. Viral distribution, secondary replication sites and
pathomorphological correlates
4.1. Viral replication leads to widespread lymphoid depletion
through destruction and apoptosis of monocyte/macrophages and
uninfected lymphocytes
Immunohistochemical studies upon infection with moder-
ately and highly virulent ASFV strains showed that infection
and depletion is initially found in monocytes/macrophages in
lymphoid organs, especially lymphnodes and spleen (Ramiro-
Ibanez et al., 1997; Rodriguez et al., 1996a). Later on, additional
cell types in various organs including megakaryocytes (Edwards
et al., 1985), tonsillar epithelial cells (Gomez-Villamandos et al.,
1997b; Rodriguez et al., 1996a), hepatocytes, kidney cells
and endothelial cells (Gomez-Villamandos et al., 1995a) get
infected. In vivo haemadsorption is observed in hepatic sinu-
soids, lymph sinuses, and red splenic pulp (Sierra et al.,
1991).
In the liver, Kupffer cell infection is accompanied by activa-
tion, erythrophagocytosis, in vivo haemadsorption, and lymphocyte
attachment (Gomez-Villamandos et al., 1995b). It was observed
that Kupffer cell and sinusoidal circulating cell counts increase
initially, and that the former show secretory activation in terms
of IL-1␣, TNF-␣, and IL-6 expression (Sanchez-Cordon et al.,
2008). Thus, infectable cells are concentrated. Fibrin networks and
microthrombi are found in sinusoidal lumina of peripheral lobular
areas. Beside this macrophage population, infection of endothelial
cells is observed from seven days post inoculation with highly viru-
lent ASFV such as the Malawi ’83 strain (Gomez-Villamandos et al.,
1995b).
The kidney is another organ with severe pathomorphologi-
cal changes including haemorrhages. In acute ASF, proliferative
glomerulonephritis including hyperplasia of collecting ducts
(Gomez-Villamandos et al., 1995c, 1995d) is seen while in sub-
acute forms immune-mediated, subendothelial and mesangial
deposits of immunoglobulins and complement prevail (Hervas
et al., 1996a). Haemorrhages seen in the kidney are attributed by
different authors to endothelial dysfunction, aggravated by virus
replication in endothelial cells in the finals stages of the disease
(Gomez-Villamandos et al., 1995c). In contrast, other groups dis-
cuss endothelial damage as direct cause for the observed petechiae
(Sierra et al., 1989). Upon acute infection with highly virulent
ASFV strains, the renal interstitium exhibits intense oedema and
macrophage infiltrates. ASFV replication in mesangial cells and
renal collecting duct epithelial cells could account for virus in
urine (without haematuria) (Gomez-Villamandos et al., 1995d).
In addition, virus replication in fibroblasts and smooth muscle
cells of small blood vessels is seen in later stages. In case of
subacute and chronic forms, immune mediated events including
immune-complex deposits are discussed as a cause of intersti-
tial haemorrhages associated with diapedesis (Hervas et al., 1996a,
1996b).
Besides general lymphoid depletion in the spleen, infected
macrophages attached to smooth muscle cells were observed in
the splenic cords of affected animals upon highly virulent infection.
These cells rapidly disappeared and were replaced by numerous
erythrocytes associated with fibrin deposits. Fibrin deposition and
platelet activation was probably due to the exposure of collagen
upon destruction of infected macrophages (Carrasco et al., 1997a).
In the tonsils of acutely infected animals, an increased number
of monocytes/macrophages was observed along with an increase
in the expression of proinflammatory cytokines (especially TNF-␣
and IL-1␣). This finding was accompanied by lymphocyte apoptosis
(Fernandez de Marco et al., 2007).
Comparison of moderately and highly virulent ASFV strains
showed no general differences in terms of cell tropism or organ
distribution, but significantly more severe tissue destruction with
increasing virulence (Oura et al., 1998a). The cellular depletion
described above was previously attributed to necrosis, but it could
be shown beyond doubt that the changes are attributable to apop-
totic mechanisms (Carrasco et al., 1996c; Gomez-Villamandos et al.,
1995a; Oura et al., 1998a; Ramiro-Ibanez et al., 1996, 1997). It
concerns both infected cells of the monocyte/macrophage lineage
as well as a large number of uninfected lymphocytes (Ramiro-
Ibanez et al., 1996). Apoptosis is also present in lymphoid tissues
associated with tonsils, bronchia, and the gastrointestinal system
(Ramiro-Ibanez et al., 1997) as well as in the liver and kidney tissues
(Gomez-Villamandos et al., 1995a). Apoptotic cells have been found
from 2 to 4 days post infection onwards in spleen and lymphnodes,
and from day 3 post infection in the thymus (Oura et al., 1998a;
Salguero et al., 2002). These cells were mainly lymphocytes, both
in B- and T-cell areas of lymphoid organs (Salguero et al., 2002).
Order and extent of depletion of these cell subsets varied among
studies using different ASFV strains (Carrasco et al., 1996c; Oura
et al., 1998a; Ramiro-Ibanez et al., 1997).
In line with the findings in the tonsils, the appearance of lym-
phocyte apoptosis in splenic and thymic structures as well as
lymphnodes coincided with expression of TNF-␣, IL-1␣ and IL-1␤
by activated monocytes/macrophages (Salguero et al., 2005). Thus,
proinflammatory cytokines seem to be responsible for apoptosis
following ASFV infection.
Based on these findings, research groups subsequently identi-
fied several ASFV genes involved in programmed cell death both in
an inhibitory or an inducing manner (Afonso et al., 1996; Brun et al.,
1996; Chacon et al., 1995; Galindo et al., 2008; Hernaez et al., 2004a,
2004b; Hurtado et al., 2004; Nogal et al., 2001; Portugal et al., 2009;
Revilla et al., 1997; Rodriguez et al., 2002; Zsak and Neilan, 2002).
In contrast to changes seen in the course of acute-lethal
infection, the chronic course is characterized by a rather severe
hyperplasia of lymphoid organs as evidenced by enlarged lymphn-
odes with hypercellularity (Ramiro-Ibanez et al., 1997). In general,
immune complexes may play a role in subacute ASFV infections
(Fernandez et al., 1992c).
In recovering pigs, Oura et al. (1998a) found that virus persisted
in lymphnodes and tonsils up to 48 days post infection. Virus was
located in cells surrounded by apoptotic lymphocytes (up to 32 dpi).
This confirms again the role of apoptosis of uninfected lymphocytes
induced by cytokines released by infected macrophages.
4.2. Haemorrhagic lesions are associated with release of
cytokines by infected monocyte/macrophages rather than direct
endothelial cell damage
Infection and destruction of monocytes/macrophages in lym-
phoid organs coincides with the appearance of first haemorrhagic
lesions in these organs starting from day 3 post infection
with a virulent ASFV strain (Salguero et al., 2005). Among the
sites with frequent haemorrhages are gastro-hepatic and renal
 S. Blome et al. / Virus Research 173 (2013) 122–130
lymphnodes (haemorrhagic enlargement). Following infection
with the Malawi ’83 isolate, these changes are accompanied by
activation of endothelial cells and fibrin deposition in the vessel
lumina. Moreover, increased fenestration and exposure of basal
membrane to the blood stream could be observed (Carrasco et al.,
1997b). Following infection with highly virulent ASFV isolates, acti-
vation of capillary endothelial cells was also accompanied by an
increased fibrinolytic activity and high levels of fibrin monomers
(Villeda et al., 1995).
In early pathology and pathogenesis studies, the haemorrhagic
picture was attributed to direct infection of endothelial cells
(Maurer and Griesemer, 1958). This hypothesis was strengthened
by in vitro infection of endothelial cells (Wilkinson and Wardley,
1978) and demonstration of virus and viral antigen in endothelial
cells of kidney and liver (Fernandez et al., 1992a, 1992b). Later stud-
ies showed that only a minority of endothelial cells was infected in
organs with obvious haemorrhagic pathology, and that only in the
late stages of the disease, infection of endothelial cells was found at
all. At this time, haemorrhagic lesions had been present already for
some days. For this reason, infection of endothelial cells does not
seem to be the primary cause of haemorrhage (Gomez-Villamandos
et al., 1997b; Perez et al., 1994).
It is now an acknowledged opinion that neither vascular
nor lymphoid lesions are directly linked to viral replication in
endothelial cells or lymphocytes (Carrasco et al., 1997a; Gomez-
Villamandos et al., 1995a, 1995c). These findings are in line with the
observations known for several other viral diseases with haemor-
rhagic lesions (Gomez-Villamandos et al., 2003; Lange et al., 2011).
Upon ASFV infection, activated endothelial cells coincide with
ASFV replication in neighbouring monocytes and macrophages.
Thus, stimulation of endothelial cells is likely to result from
cytokine release. Proinflammatory cytokines such as TNF-␣ and IL-
1 are known to stimulate a procoagulant state of the endothelium
and finally activate the coagulation cascade.
4.3. Impairment of platelets and coagulation changes contribute
to evolution of a haemorrhagic syndrome
African swine fever virus infection is accompanied by
thrombocytopenia. This could either be due to impairment of
thrombocytopoiesis or peripheral consumption.
In order to investigate the first option, studies were undertaken
to explore the changes in bone marrow. Here, infection of 25–30%
of bone marrow megakaryocytes could be shown from six days post
infection with the moderately virulent E75 strain (Rodriguez et al.,
1996a, 1996b). This finding was consistent with the appearance of
a marked thrombocytopenia at that time. Thus, impairment and
destruction of platelet precursors could play an important role in
the genesis of thrombocytopenia. Infection of peripheral platelets,
however, was rare. An explanation for this could be that infected
megakaryocytes are destroyed completely in the bone marrow
compartment and do not further maturate into platelets.
In contrast, Perez et al. (1997) could only demonstrate a much
lower percentage of infected megakaryocytes both upon infection
with a highly virulent (Malawi ’83) and a moderately virulent (DR
’78) ASFV isolate. After infection with the highly virulent isolate, a
peak of 9.5% positive cells was reached at day seven and only little
damage was observed. Upon infection with the moderately virulent
strain, less than 1% of megakaryocytes was infected but severe dam-
age was seen together with markedly decreased numbers. These
findings are quite in line with studies conducted by Edwards et al.
(1985) who found that 2–10% of the megakaryocytes were infected
with an ASFV strain of moderate virulence. Furthermore, Edwards
and Dodds (1985) could show in functional studies, i.e. platelet
survival times using cohort labelling with [75 Se] selenmethion-
ine, that despite obvious thrombocytopenia, thrombocytopoiesis
127
was normal during infection as measured by incorporation of the
radionuclides into platelets. Gomez-Villamandos et al. (Gomez-
Villamandos et al., 1997a) also demonstrated that neither vascular
changes nor viral replication in different bone marrow cell popu-
lations gave rise to impaired bone marrow functions. They even
observed an increased haematopoiesis. Thus, it remains debatable
whether impaired thrombocytopoiesis plays a major role in the
pathogenesis of thrombocytopenia in ASF despite the fact that bone
marrow lesions are observed.
Consumption of platelets in the periphery, the second dis-
cussed mechanism leading to thrombocytopenia, could be due to
microthrombi as a correlate for disseminated intravascular coag-
ulation. In acute forms of ASF, fibrin depositions with embedded
platelets are frequently observed (Rodriguez et al., 1996a). Mech-
anisms leading to these microthrombi could include systemic
release of enzymes, cytokines, complement factors, and products
of the arachidonic acid metabolism. These factors are known as
pathogenetic mechanisms in other diseases featuring the same
clinical and pathomorphological correlates (Lange et al., 2011). It
has to be stated that the acute phase of thrombocytopenia coin-
cides with marked viraemia and high levels of immunoglobulin.
Thus, immune mediated microthrombi and aggregates involving
antigen-antibody complexes cannot be ruled out. In conclusion,
it seems likely that both impairment of thrombocytopoiesis and
peripheral consumption play a role in the genesis of thrombocyto-
penia.
Beside the obvious changes in count, both activation and
degranulation of platelets could be observed by Gomez-
Villamandos et al. (1996) after highly and moderately virulent ASFV
infection. This finding coincided with infection and activation of
monocytes/macrophages and probably the secretion of platelet
activating factors. After highly virulent infection, platelet aggrega-
tion and viscous metamorphosis was observed that coincided also
with endothelial lesions. In contrast, these changes were much less
prevalent in animals infected with the moderately virulent ASFV
strain. Virus positive platelets that may contribute to the spreading
of the virus were observed in the final phase of the disease.
In pigs surviving the first weeks of infection, degranulation of
antibody-coated leukocytes led to secondary platelet aggregation
and vasoactive amine release facilitating immune-complex depo-
sition, e.g. in glomeruli (Slauson and Sanchez-Vizcaino, 1981).
Another factor of haemostasis is the coagulation system. Infec-
tion with virulent ASFV isolates leads several coagulation changes
that can be measured through clotting times. In detail, ASFV infec-
tion leads to a prolongation of the activated partial thromboplastin
time (aPTT), prothrombin time (PT), and thrombin clotting time
(TCT) from 4 days post infection onwards. These findings are accom-
panied by the above mentioned acute thrombocytopenia and an
increase in factor VIII-related antigen (Edwards et al., 1984). In
addition, Villeda et al. (1993b) could show that apart from pro-
longation of aPTT and PT, reduced levels of coagulation factors are
present in plasma upon highly virulent ASFV infection. This could be
either attributed to excess of consumption (disseminated intravas-
cular coagulation, DIC) or decreased production. The latter is less
probable as almost normal liver enzyme activity was observed in
the course of the underlying infection. While the aPTT is a marker
for the intrinsic coagulation pathway that is often affected in the
course of DIC syndromes, prolonged PT-values are indicative for an
involvement of the extrinsic coagulation pathway. Taken together,
the observed increases are indicative for a more global activation
of the coagulation system.
Moreover, activation of the fibrinolytic system was observed
(Villeda et al., 1993b). Activation of the coagulation system can
be achieved by expression of tissue factor (coagulation factor III)
by endothelial cells. This phenomenon is observed at least in vitro
upon ASFV infection (Vallee et al., 2001) and could be explained
128 S. Blome et al. / Virus Research 173 (2013) 122–130
by procoagulant mediators released by infected macrophages. In
the respective experiments in porcine aortic endothelial cells,
these findings were accompanied by inhibition of inflammatory
responses. Another possibility is the impairment of degradation of
activated coagulation factors and impaired production of coagula-
tion factors in the liver. While the former seems to be among the
options, the impact of disturbed production seems rather moder-
ate as only few hepatocytes are infected and haemorrhages are seen
much earlier than infection of non-macrophage cells.
Concluding, there is evidence for a DIC as global coagulation
tests show prolonged clotting times, decreased levels of fibrino-
gen are observed, and consumption of coagulation factors can be
seen along with reduced levels of plasminogen (Villeda et al., 1993a,
1993b).
4.4. Additional cytokine and acute phase responses link
pathogenesis and immune response
Different studies on host range and virulence of ASFV demon-
strated that IFN response and its control by viral gene products
play a major role in determining the virulence of ASFV isolates
(Afonso et al., 2004). Attenuated ASFV strains that lack the host
range determinant have been shown to induce myxovirus resis-
tance (Mx) genes that are known to mediate the antiviral response
to several RNA viruses. Recently, it was shown that Mx gene prod-
ucts are also able to inhibit the replication of ASFV in vitro and may
thus also mediate the antiviral state towards ASFV (Netherton et al.,
2009).
An acute phase response has been observed upon highly virulent
ASFV infection as evidenced by increased serum concentrations of
C-reactive protein (CRP), serum amyloid A (SAA), and haptoglobin
starting from day 3 post infection (Sanchez-Cordon et al., 2007).
Acute phase proteins are mainly synthesized by hepatocytes upon
cytokine stimuli, especially proinflammatory mediators such as
TNF-␣, IL-1, and IL-6. Activated Kupffer cells can lead to induction
of an acute phase response. In turn, Kupffer cells can be activated
by proinflammatory cytokines such as TNF-␣ or IL-1 (Gabay and
Kushner, 1999). In the study mentioned above, high serum concen-
trations of SAA and CRP coincided with high serum concentrations
of IL-1 and expression of IL-1 and TNF-␣ by Kupffer cells. The high-
est haptoglobin concentration coincides with the largest number of
IL-6-secreting hepatic macrophages. No impairment of this unspe-
cific immune response could be observed.
In an additional study, Carpintero et al. (2007) demonstrated
high levels of pig major acute phase protein and a decrease
of apolipoprotein A-I is seen upon ASFV infection. In this case,
haptoglobin levels were significantly increased, but only minor dif-
ferences were seen for C-reactive protein.
In addition, it was recently shown that some serological
responses have a direct impact on the pathogenesis: some anti-
bodies show higher titres in animals developing lesions, e.g.
antibodies against the K196R/thymidine kinase, while an effective
immune response is accompanied by antibodies raised against the
A104R/histone like protein (Reis et al., 2007). Moreover, recovery of
swine leucocyte antigen expression during ASFV infection is indica-
tive for an effective immune response and influences viral clearance
(replacement/survival of macrophages in the spleen) (Gonzalez-
Juarrero et al., 1992a).
5. Résumé and gap analysis
The pathogenesis of ASF shares several features with viral
haemorrhagic fevers of humans and animals. Among these are (1)
primary replication in cells of the monocyte/macrophage lineage,
(2) occurrence of cytokine-mediated lesions including apoptosis of
uninfected lymphocytes, (3) activation of endothelial cells and the
coagulation system, and (4) impairment of innate immune func-
tions. Despite these parallels and the possibility to conclude from
research in these related fields, neither virulence factors nor disease
mechanisms are fully understood. Gaps are especially related to the
role of host factors, the differences of ASFV strains concerning their
virulence, mechanisms and implications of chronic disease courses,
and the role of ASF-specific immunity for pathogenesis. The under-
standing of these major points might be the key element for the
development of a first efficacious ASF vaccine.
References
Afonso, C.L., Neilan, J.G., Kutish, G.F., Rock, D.L., 1996. An African swine fever virus
Bc1-2 homolog, 5-HL, suppresses apoptotic cell death. Journal of Virology 70 (7),
4858–4863.
Afonso, C.L., Piccone, M.E., Zaffuto, K.M., Neilan, J., Kutish, G.F., Lu, Z., Balinsky, C.A.,
Gibb, T.R., Bean, T.J., Zsak, L., Rock, D.L., 2004. African swine fever virus multigene
family 360 and 530 genes affect host interferon response. Journal of Virology 78
(4), 1858–1864.
Basta, S., Knoetig, S.M., Spagnuolo-Weaver, M., Allan, G., McCullough, K.C., 1999.
Modulation of monocytic cell activity and virus susceptibility during differenti-
ation into macrophages. Journal of Immunology 162 (7), 3961–3969.
Blome, S., Gabriel, C., Dietze, K., Breithaupt, A., Beer, M., 2012. High virulence of
African swine fever virus caucasus isolate in European wild boars of all ages.
Emerging Infectious Diseases 18 (4), 708.
Brun, A., Rivas, C., Esteban, M., Escribano, J.M., Alonso, C., 1996. African swine fever
virus gene A179L, a viral homologue of bcl-2, protects cells from programmed
cell death. Virology 225 (1), 227–230.
Canals, A., Dominquez, J., Tomillo, J., Babin, M., Alonso, F., 1995. Inhibition of IL-2R
and SLA class II expression on stimulated lymphocytes by a suppressor activity
found in homogenates of African swine fever virus infected cultures. Archives
of Virology 140 (6), 1075–1085.
Carpintero, R., Alonso, C., Pineiro, M., Iturralde, M., Andres, M., Le Potier, M.F., Madec,
F., Alava, M.A., Pineiro, A., Lampreave, F., 2007. Pig major acute-phase protein and
apolipoprotein A-I responses correlate with the clinical course of experimentally
induced African Swine Fever and Aujeszky’s disease. Veterinary Research 38 (5),
741–753.
Carrasco, L., Bautista, M.J., Gomez-Villamandos, J.C., Martin de las Mulas, J., Cha-
con, M.d.L.F., Wilkinson, P.J., Sierra, M.A., 1997a. Development of microscopic
lesions in splenic cords of pigs infected with African swine fever virus. Veterinary
Research 28 (1), 93–99.
Carrasco, L., Chacon, M.d.L.F., Martin de Las Mulas, J., Gomez-Villamandos, J.C., Sierra,
M.A., Villeda, C.J., Wilkinson, P.J., 1997b. Ultrastructural changes related to the
lymph node haemorrhages in acute African swine fever. Research in Veterinary
Science 62 (3), 199–204.
Carrasco, L., de Lara, F.C., Gomez-Villamandos, J.C., Bautista, M.J., Villeda, C.J., Wilkin-
son, P.J., Sierra, M.A., 1996a. The pathogenic role of pulmonary intravascular
macrophages in acute African swine fever. Research in Veterinary Science 61
(3), 193–198.
Carrasco, L., de Lara, F.C., Martin de las Mulas, J., Gomez-Villamandos, J.C., Hervas, J.,
Wilkinson, P.J., Sierra, M.A., 1996b. Virus association with lymphocytes in acute
African swine fever. Veterinary Research 27 (3), 305–312.
Carrasco, L., de Lara, F.C., Martin de las Mulas, J., Gomez-Villamandos, J.C., Perez, J.,
Wilkinson, P.J., Sierra, M.A., 1996c. Apoptosis in lymph nodes in acute African
swine fever. Journal of Comparative Pathology 115 (4), 415–428.
Carrasco, L., Fernandez, A., Gomez Villamandos, J.C., Mozos, E., Mendez, A., Jover,
A., 1992. Kupffer cells and PIMs in acute experimental African swine fever.
Histology and Histopathology 7 (3), 421–425.
Carrasco, L., Gomez-Villamandos, J.C., Bautista, M.J., Martin de las Mulas, J., Villeda,
C.J., Wilkinson, P.J., Sierra, M.A., 1996d. In vivo replication of African swine fever
virus (Malawi ’83) in neutrophils. Veterinary Research 27 (1), 55–62.
Carrasco, L., Nunez, A., Salguero, F.J., Diaz San Segundo, F., Sanchez-Cordon, P.,
Gomez-Villamandos, J.C., Sierra, M.A., 2002. African swine fever: Expression of
interleukin-1 alpha and tumour necrosis factor-alpha by pulmonary intravas-
cular macrophages. Journal of Comparative Pathology 126 (2–3), 194–201.
Chacon, M.R., Almazan, F., Nogal, M.L., Vinuela, E., Rodriguez, J.F., 1995. The African
swine fever virus IAP homolog is a late structural polypeptide. Virology 214 (2),
670–674.
Chamorro, S., Revilla, C., Alvarez, B., Alonso, F., Ezquerra, A., Dominguez, J., 2005.
Phenotypic and functional heterogeneity of porcine blood monocytes and its
relation with maturation. Immunology 114 (1), 63–71.
Detray, D.E., Scott, G.R., 1957. Blood changes in swine with African swine fever.
American Journal of Veterinary Research 18 (68), 484–490.
Edwards, J.F., Dodds, W.J., 1985. Platelet and fibrinogen kinetics in healthy and
African swine fever-affected swine: [75 Se]selenomethionine-labeling study.
American Journal of Veterinary Research 46 (1), 181–184.
Edwards, J.F., Dodds, W.J., Slauson, D.O., 1984. Coagulation changes in African
swine fever virus infection. American Journal of Veterinary Research 45 (11),
2414–2420.
Edwards, J.F., Dodds, W.J., Slauson, D.O., 1985. Megakaryocytic infection and throm-
bocytopenia in African swine fever. Veterinary Pathology 22 (2), 171–176.
 S. Blome et al. / Virus Research 173 (2013) 122–130
Ekue, N.F., Wilkinson, P.J., Wardley, R.C., 1989. Infection of pigs with the Cameroon
isolate (Cam/82) of African swine fever virus. Journal of Comparative Pathology
100 (2), 145–154.
European Food Safety Authority (EFSA), 2009. Scientific report on African Swine
Fever submitted to EFSA prepared by Sánchez-Vizcaíno, J.M., Martínez-López,
B., Martínez-Avilés, M., Martins, C., Boinas, F., Vial, L., Michaud, V., Jori, F., Etter,
E., Albina, E. and Roger, F., 1-141.
Fernandez, A., Perez, J., Carrasco, L., Bautista, M.J., Sanchez-Vizcaino, J.M., Sierra, M.A.,
1992a. Distribution of ASFV antigens in pig tissues experimentally infected with
two different Spanish virus isolates. Zentralblatt fur Veterinarmedizinreine B 39
(6), 393–402.
Fernandez, A., Perez, J., Carrasco, L., Sierra, M.A., Sanchez-Vizcaino, M., Jover, A.,
1992b. Detection of African swine fever viral antigens in paraffin-embedded tis-
sues by use of immunohistologic methods and polyclonal antibodies. American
Journal of Veterinary Research 53 (8), 1462–1467.
Fernandez, A., Perez, J., Martin de las Mulas, J., Carrasco, L., Dominguez, J., Sierra, M.A.,
1992c. Localization of African swine fever viral antigen, swine IgM, IgG and C1q
in lung and liver tissues of experimentally infected pigs. Journal of Comparative
Pathology 107 (1), 81–90.
Fernandez de Marco, M., Salguero, F.J., Bautista, M.J., Nunez, A., Sanchez-Cordon, P.J.,
Gomez-Villamandos, J.C., 2007. An immunohistochemical study of the tonsils
in pigs with acute African swine fever virus infection. Research in Veterinary
Science 83 (2), 198–203.
Gabay, C., Kushner, I., 1999. Acute-phase proteins and other systemic responses to
inflammation. New England Journal of Medicine 340 (6), 448–454.
Gabriel, C., Blome, S., Malogolovkin, A., Parilov, S., Kolbasov, D., Teifke, J.P., Beer, M.,
2011. Characterization of african Swine Fever virus caucasus isolate in European
wild boars. Emerging Infectious Diseases 17 (12), 2342–2345.
Galindo, I., Hernaez, B., Diaz-Gil, G., Escribano, J.M., Alonso, C., 2008. A179L, a viral
Bcl-2 homologue, targets the core Bcl-2 apoptotic machinery and its upstream
BH3 activators with selective binding restrictions for Bid and Noxa. Virology 375
(2), 561–572.
Gil, S., Sepulveda, N., Albina, E., Leitao, A., Martins, C., 2008. The low-virulent African
swine fever virus (ASFV/NH/P68) induces enhanced expression and production
of relevant regulatory cytokines (IFNalpha, TNFalpha and IL12p40) on porcine
macrophages in comparison to the highly virulent ASFV/L60. Archives of Virol-
ogy 153 (10), 1845–1854.
Gomez-Villamandos, J.C., Bautista, M.J., Carrasco, L., Caballero, M.J., Hervas, J., Villeda,
C.J., Wilkinson, P.J., Sierra, M.A., 1997a. African swine fever virus infection of bone
marrow: lesions and pathogenesis. Veterinary Pathology 34 (2), 97–107.
Gomez-Villamandos, J.C., Bautista, M.J., Hervas, J., Carrasco, L., de Lara, F.C., Perez,
J., Wilkinson, P.J., Sierra, M.A., 1996. Subcellular changes in platelets in acute
and subacute African swine fever. Journal of Comparative Pathology 115 (4),
327–341.
Gomez-Villamandos, J.C., Carrasco, L., Bautista, M.J., Sierra, M.A., Quezada, M., Her-
vas, J., Chacon Mde, L., Ruiz-Villamor, E., Salguero, F.J., Sonchez-Cordon, P.J.,
Romanini, S., Nunez, A., Mekonen, T., Mendez, A., Jover, A., 2003. African swine
fever and classical swine fever: a review of the pathogenesis. Deutsche Tier-
arztliche Wochenschrift 110 (4), 165–169.
Gomez-Villamandos, J.C., Hervas, J., Mendez, A., Carrasco, L., Martin de las Mulas,
J., Villeda, C.J., Wilkinson, P.J., Sierra, M.A., 1995a. Experimental African swine
fever: apoptosis of lymphocytes and virus replication in other cells. Journal of
General Virology 76 (Pt 9), 2399–2405.
Gomez-Villamandos, J.C., Hervas, J., Mendez, A., Carrasco, L., Villeda, C.J., Sierra, M.A.,
Wilkonson, P.J., 1995b. A pathological study of the perisinusoidal unit of the liver
in acute African swine fever. Research in Veterinary Science 59 (2), 146–151.
Gomez-Villamandos, J.C., Hervas, J., Mendez, A., Carrasco, L., Villeda, C.J., Wilkinson,
P.J., Sierra, M.A., 1995c. Pathological changes in the renal interstitial capillaries
of pigs inoculated with two different strains of African swine fever virus. Journal
of Comparative Pathology 112 (3), 283–298.
Gomez-Villamandos, J.C., Hervas, J., Mendez, A., Carrasco, L., Villeda, C.J., Wilkinson,
P.J., Sierra, M.A., 1995d. Ultrastructural study of the renal tubular system in acute
experimental African swine fever: virus replication in glomerular mesangial
cells and in the collecting ducts. Archives of Virology 140 (3), 581–589.
Gomez-Villamandos, J.C., Hervas, J., Moreno, C., Carrasco, L., Bautista, M.J., Caballero,
J.M., Wilkinson, P.J., Sierra, M.A., 1997b. Subcellular changes in the tonsils of
pigs infected with acute African swine fever virus. Veterinary Research 28 (2),
179–189.
Gomez del Moral, M., Ortuno, E., Fernandez-Zapatero, P., Alonso, F., Alonso, C.,
Ezquerra, A., Dominguez, J., 1999. African swine fever virus infection induces
tumor necrosis factor alpha production: implications in pathogenesis. Journal
of Virology 73 (3), 2173–2180.
Gonzalez-Juarrero, M., Lunney, J.K., Sanchez-Vizcaino, J.M., Mebus, C., 1992a. Mod-
ulation of splenic macrophages, and swine leukocyte antigen (SLA) and viral
antigen expression following African swine fever virus (ASFV) inoculation.
Archives of Virology 123 (1-2), 145–156.
Gonzalez-Juarrero, M., Mebus, C.A., Pan, R., Revilla, Y., Alonso, J.M., Lunney,
J.K., 1992b. Swine leukocyte antigen and macrophage marker expression on
both African swine fever virus-infected and non-infected primary porcine
macrophage cultures. Veterinary Immunology and Immunopathology 32 (3-4),
243–259.
Gonzalez, S., Mendoza, C., Sanchez-Vizcaino, J.M., Alonso, F., 1990. Inhibitory effect of
African swine fever virus on lectin-dependent swine lymphocyte proliferation.
Veterinary Immunology and Immunopathology 26 (1), 71–80.
Gregg, D.A., Mebus, C.A., Schlafer, D.H., 1995. Early infection of interdigitating den-
dritic cells in the pig lymph node with African swine fever viruses of high
129
and low virulence: immunohistochemical and ultrastructural studies. Journal
of Veterinary Diagnostic Investigation 7 (1), 23–30.
Greig, A., 1972. Pathogenesis of African swine fever in pigs naturally exposed to the
disease. Journal of Comparative Pathology 82 (1), 73–79.
Greig, A., Plowright, W., 1970. The excretion of two virulent strains of African swine
fever virus by domestic pigs. Journal of Hygiene 68 (4), 673–682.
Haresnape, J.M., Wilkinson, P.J., Mellor, P.S., 1988. Isolation of African swine fever
virus from ticks of the Ornithodoros moubata complex (Ixodoidea: Argasidae)
collected within the African swine fever enzootic area of Malawi. Epidemiology
and Infection 101 (1), 173–185.
Hernaez, B., Diaz-Gil, G., Garcia-Gallo, M., Ignacio Quetglas, J., Rodriguez-Crespo,
I., Dixon, L., Escribano, J.M., Alonso, C., 2004a. The African swine fever virus
dynein-binding protein p54 induces infected cell apoptosis. FEBS Letters 569
(1-3), 224–228.
Hernaez, B., Escribano, J.M., Alonso, C., 2004b. Switching on and off the cell death
cascade: African swine fever virus apoptosis regulation. Progress in Molecular
and Subcellular Biology 36, 57–69.
Hervas, J., Gomez-Villamandos, J.C., Mendez, A., Carrasco, L., Perez, J., Wilkinson, P.J.,
Sierra, M.A., 1996a. Structural and ultrastructural study of glomerular changes
in African swine fever. Journal of Comparative Pathology 115 (1), 61–75.
Hervas, J., Gomez-Villamandos, J.C., Mendez, A., Carrasco, L., Sierra, M.A., 1996b.
The lesional changes and pathogenesis in the kidney in African swine fever.
Veterinary Research Communications 20 (3), 285–299.
Heuschele, W.P., 1967. Studies on the pathogenesis of African swine fever. I. Quan-
titative studies on the sequential development of virus in pig tissues. Archiv fur
die gesamte Virusforschung 21 (3), 349–356.
Hurtado, C., Granja, A.G., Bustos, M.J., Nogal, M.L., Gonzalez de Buitrago, G., de
Yebenes, V.G., Salas, M.L., Revilla, Y., Carrascosa, A.L., 2004. The C-type lectin
homologue gene (EP153R) of African swine fever virus inhibits apoptosis both
in virus infection and in heterologous expression. Virology 326 (1), 160–170.
Kleiboeker, S.B., 2002. Swine fever: classical swine fever and African swine fever.
The Veterinary clinics of North America. Food Animal Practice 18 (3), 431–451.
Kleiboeker, S.B., Scoles, G.A., 2001. Pathogenesis of African swine fever virus in
Ornithodoros ticks. Animal Health Research Reviews/Conference of Research
Workers in Animal Diseases 2 (2), 121–128.
Knudsen, R.C., Genovesi, E.V., 1987. In vivo and in vitro effects of moderately vir-
ulent African swine fever virus on mitogenesis of pig lymphocytes. Veterinary
Immunology and Immunopathology 15 (4), 323–336.
Lange, A., Blome, S., Moennig, V., Greiser-Wilke, I., 2011. Pathogenesis of classi-
cal swine fever–similarities to viral haemorrhagic fevers: a review. Berl Munch
Tierarztl Wochenschr 124 (1-2), 36–47.
Malmquist, W.A., Hay, D., 1960. Hemadsorption and cytopathic effect produced
by African Swine Fever virus in swine bone marrow and buffy coat cultures.
American Journal of Veterinary Research 21, 104–108.
Maurer, F.D., Griesemer, R.A., 1958. The pathology of African swine fever; a com-
parison with hog cholera. American Journal of Veterinary Research 19 (72),
517–539.
McVicar, J.W., Mebus, C.A., Becker, H.N., Belden, R.C., Gibbs, E.P., 1981. Induced
African swine fever in feral pigs. Journal of the American Veterinary Medical
Association 179 (5), 441–446.
Mebus, C.A., 1987. Pathobiology and pathogenesis. In: Becker, Y. (Ed.), African Swine
Fever. Martinus Nijhoff, Boston, pp. 21–30.
Mebus, C.A., 1988. African swine fever. Advances in Virus Research 35, 251–269.
Murtaugh, M.P., Baarsch, M.J., Zhou, Y., Scamurra, R.W., Lin, G., 1996. Inflam-
matory cytokines in animal health and disease. Veterinary Immunology and
Immunopathology 54 (1-4), 45–55.
Netherton, C.L., Simpson, J., Haller, O., Wileman, T.E., Takamatsu, H.H., Monaghan, P.,
Taylor, G., 2009. Inhibition of a large double-stranded DNA virus by MxA protein.
Journal of Virology 83 (5), 2310–2320.
Nogal, M.L., Gonzalez de Buitrago, G., Rodriguez, C., Cubelos, B., Carrascosa, A.L., Salas,
M.L., Revilla, Y., 2001. African swine fever virus IAP homologue inhibits caspase
activation and promotes cell survival in mammalian cells. Journal of Virology 75
(6), 2535–2543.
Oura, C.A., Powell, P.P., Parkhouse, R.M., 1998a. African swine fever: a disease char-
acterized by apoptosis. Journal of General Virology 79 (Pt 6), 1427–1438.
Oura, C.A., Powell, P.P., Parkhouse, R.M., 1998b. Detection of African swine fever
virus in infected pig tissues by immunocytochemistry and in sity hybridisation.
Journal of Virological Methods 72 (2), 205–217.
Penrith, M.L., Thomson, G.R., Bastos, A.D., Phiri, O.C., Lubisi, B.A., Du Plessis, E.C.,
Macome, F., Pinto, F., Botha, B., Esterhuysen, J., 2004a. An investigation into nat-
ural resistance to African swine fever in domestic pigs from an endemic area in
southern Africa. Revue Scientifique et Technique 23 (3), 965–977.
Penrith, M.L., Thomson, G.R., Bastos, A.D.S., 2004b. African swine fever. In: Coet-
zer, J.A.W., Tustin, R.C. (Eds.), Infectious Diseases of Livestock with Special
Reference to Southern Africa. , 2nd ed. Oxford University Press, Cape Town,
pp. 1087–1119.
Penrith, M.L., Vosloo, W., 2009. Review of African swine fever: transmission, spread
and control. Journal of the South African Veterinary Association 80 (2), 58–62.
Perez, J., Bautista, M.J., Rodriguez, F., Wilkinson, P.J., Sierra, M.A., Martin de las Mulas,
J., 1997. Double-labelling immunohistochemical study of megakaryocytes in
African swine fever. Veterinary Record 141 (15), 386–390.
Perez, J., Fernandez, A.I., Sierra, M.A., Herraez, P., Fernandez, A., Martin de las Mulas,
J., 1998. Serological and immunohistochemical study of African swine fever in
wild boar in Spain. Veterinary Record 143 (5), 136–139.
Perez, J., Rodriguez, F., Fernandez, A., Martin de las Mulas, J., Gomez-Villamandos, J.C.,
Sierra, M.A., 1994. Detection of African swine fever virus protein VP73 in tissues
130 S. Blome et al. / Virus Research 173 (2013) 122–130
of experimentally and naturally infected pigs. Journal of Veterinary Diagnostic
Investigation 6 (3), 360–365.
Plowright, W., Thomson, G.R., Neser, J.A., 1994. African swine fever. In: Coetzer,
J.A.W., Thomson, G.R., Tustin, R.C. (Eds.), Infectious Diseases of Livestock with
Special Reference to Southern Africa. Cape Town Oxford University Press, pp.
567–599.
Portugal, R., Leitao, A., Martins, C., 2009. Characterization of African swine fever
virus IAP homologue expression in porcine macrophages infected with different
virulence isolates. Veterinary Microbiology 139 (1-2), 140–146.
Quintero, J.C., Wesley, R.D., Whyard, T.C., Gregg, D., Mebus, C.A., 1986. In vitro and
in vivo association of African swine fever virus with swine erythrocytes. Amer-
ican Journal of Veterinary Research 47 (5), 1125–1131.
Ramiro-Ibanez, F., Escribano, J.M., Alonso, C., 1995. Application of a monoclonal anti-
body recognizing protein p30 to detect African swine fever virus-infected cells
in peripheral blood. Journal of Virological Methods 55 (3), 339–345.
Ramiro-Ibanez, F., Ortega, A., Brun, A., Escribano, J.M., Alonso, C., 1996. Apoptosis: a
mechanism of cell killing and lymphoid organ impairment during acute African
swine fever virus infection. Journal of General Virology 77 (Pt 9), 2209–2219.
Ramiro-Ibanez, F., Ortega, A., Ruiz-Gonzalvo, F., Escribano, J.M., Alonso, C., 1997.
Modulation of immune cell populations and activation markers in the patho-
genesis of African swine fever virus infection. Virus Research 47 (1), 31–40.
Reis, A.L., Parkhouse, R.M., Penedos, A.R., Martins, C., Leitao, A., 2007. Systematic
analysis of longitudinal serological responses of pigs infected experimentally
with African swine fever virus. Journal of General Virology 88 (Pt 9), 2426–2434.
Revilla, Y., Cebrian, A., Baixeras, E., Martinez, C., Vinuela, E., Salas, M.L., 1997. Inhibi-
tion of apoptosis by the African swine fever virus Bcl-2 homologue: role of the
BH1 domain. Virology 228 (2), 400–404.
Rodriguez, C.I., Nogal, M.L., Carrascosa, A.L., Salas, M.L., Fresno, M., Revilla, Y., 2002.
African swine fever virus IAP-like protein induces the activation of nuclear factor
kappa B. Journal of Virology 76 (8), 3936–3942.
Rodriguez, F., Fernandez, A., Martin de las Mulas, J.P., Sierra, M.A., Jover, A., 1996a.
African swine fever: morphopathology of a viral haemorrhagic disease. Veteri-
nary Record 139 (11), 249–254.
Rodriguez, F., Martin de las Mulas, J., Herraez, P., Sanchez Vizcaino, J.M., Fer-
nandez, A., 1996b. Immunohistopathological study of African swine fever
(strain E-75)-infected bone marrow. Journal of Comparative Pathology 114 (4),
399–406.
Salguero, F.J., Ruiz-Villamor, E., Bautista, M.J., Sanchez-Cordon, P.J., Carrasco, L.,
Gomez-Villamandos, J.C., 2002. Changes in macrophages in spleen and lymph
nodes during acute African swine fever: expression of cytokines. Veterinary
Immunology and Immunopathology 90 (1-2), 11–22.
Salguero, F.J., Sanchez-Cordon, P.J., Nunez, A., Fernandez de Marco, M., Gomez-
Villamandos, J.C., 2005. Proinflammatory cytokines induce lymphocyte
apoptosis in acute African swine fever infection. Journal of Comparative Pathol-
ogy 132 (4), 289–302.
Sanchez-Cordon, P.J., Ceron, J.J., Nunez, A., Martinez-Subiela, S., Pedrera, M., Romero-
Trevejo, J.L., Garrido, M.R., Gomez-Villamandos, J.C., 2007. Serum concentrations
of C-reactive protein, serum amyloid A, and haptoglobin in pigs inoculated with
African swine fever or classical swine fever viruses. American Journal of Veteri-
nary Research 68 (7), 772–777.
Sanchez-Cordon, P.J., Romero-Trevejo, J.L., Pedrera, M., Sanchez-Vizcaino, J.M.,
Bautista, M.J., Gomez-Villamandos, J.C., 2008. Role of hepatic macrophages dur-
ing the viral haemorrhagic fever induced by African Swine Fever Virus. Histology
and Histopathology 23 (6), 683–691.
Sanchez-Torres, C., Gomez-Puertas, P., Gomez-del-Moral, M., Alonso, F., Escrib-
ano, J.M., Ezquerra, A., Dominguez, J., 2003. Expression of porcine CD163 on
monocytes/macrophages correlates with permissiveness to African swine fever
infection. Archives of Virology 148 (12), 2307–2323.
Sanchez, E.G., Quintas, A., Perez-Nunez, D., Nogal, M., Barroso, S., Carrascosa, A.L.,
Revilla, Y., 2012. African swine fever virus uses macropinocytosis to enter host
cells. PLoS Pathogens 8 (6), e1002754.
Sierra, M.A., Carrasco, L., Gomez-Villamandos, J.C., Martin de las Mulas, J., Mendez, A.,
Jover, A., 1990. Pulmonary intravascular macrophages in lungs of pigs inoculated
with African swine fever virus of differing virulence. Journal of Comparative
Pathology 102 (3), 323–334.
Sierra, M.A., Gomez-Villamandos, J.C., Carrasco, L., Fernandez, A., Mozos, E., Jover, A.,
1991. In vivo study of hemadsorption in African swine fever virus infected cells.
Veterinary Pathology 28 (2), 178–181.
Sierra, M.A., Quezada, M., Fernandez, A., Carrasco, L., Gomez-Villamandos, J.C., Mar-
tin de las Mulas, J., Sanchez-Vizcaino, J.M., 1989. Experimental African swine
fever: evidence of the virus in interstitial tissues of the kidney. Veterinary Pathol-
ogy 26 (2), 173–176.
Slauson, D.O., Sanchez-Vizcaino, J.M., 1981. Leukocyte-dependent platelet vasoac-
tive amine release and immune complex deposition in African swine fever.
Veterinary Pathology 18 (6), 813–826.
Takamatsu, H., Martins, C., Escribano, J.M., Alonso, C., Dixon, L.K., Salas, M.L., Revilla,
Y., 2011. Asfarviridae. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J.
(Eds.), Virus Taxonomy. Ninth Report of the ICTV. Elsevier, Oxford, pp. 153–162.
Vallee, I., Tait, S.W., Powell, P.P., 2001. African swine fever virus infection of porcine
aortic endothelial cells leads to inhibition of inflammatory responses, activation
of the thrombotic state, and apoptosis. Journal of Virology 75 (21), 10372–10382.
Villeda, C.J., Gomez-Villamandos, J.C., Williams, S.M., Hervas, J., Wilkinson, P.J., Vin-
uela, E., 1995. The role of fibrinolysis in the pathogenesis of the haemorrhagic
syndrome produced by virulent isolates of African swine fever virus. Thrombosis
and Haemostasis 73 (1), 112–117.
Villeda, C.J., Williams, S.M., Wilkinson, P.J., Vinuela, E., 1993a. Consumption coagu-
lopathy associated with shock in acute African swine fever. Archives of Virology
133 (3-4), 467–475.
Villeda, C.J., Williams, S.M., Wilkinson, P.J., Vinuela, E., 1993b. Haemostatic abnor-
malities in African swine fever a comparison of two virus strains of different
virulence (Dominican Republic ‘78 and Malta ‘78). Archives of Virology 130 (1-2),
71–83.
Wardley, R.C., 1982. Effect of African swine fever on lymphocyte mitogenesis.
Immunology 46 (1), 215–220.
Wardley, R.C., Wilkinson, P.J., 1977. The association of African swine fever virus with
blood components of infected pigs. Archives of Virology 55 (4), 327–334.
Wilkinson, P.J., Wardley, R.C., 1978. The replication of African swine fever virus in
pig endothelial cells. British Veterinary Journal 134 (3), 280–282.
Zsak, L., Neilan, J.G., 2002. Regulation of apoptosis in African swine fever virus-
infected macrophages. The Scientific World Journal 2, 1186–1195.
 Virus Research 178 (2013) 328–339

Contents lists available at ScienceDirect

Virus Research
journal homepage: www.elsevier.com/locate/virusres

Pathogenesis of highly virulent African swine fever virus in domestic

pigs exposed via intraoropharyngeal, intranasopharyngeal, and
intramuscular inoculation, and by direct contact with infected pigs
Erin B. Howey a,b , Vivian O’Donnell a,c , Helena C. de Carvalho Ferreira a,b ,
Manuel V. Borca a , Jonathan Arzt a,∗
a
Plum Island Animal Disease Center, Foreign Animal Disease Research Unit, Agricultural Research Service, United States Department of Agriculture,
Greenport, NY 11944, USA
b
Oak Ridge Institute for Science and Education, PIADC Research Participation Program, Oak Ridge, TN 37831, USA
c
Department of Pathobiology and Veterinary Science, University of Connecticut at Storrs, Storrs, CT 06269, USA

a r t i c l e i n f o a b s t r a c t

Article history: To investigate the pathogenesis of African swine fever virus (ASFV), domestic pigs (n = 18) were chal-
Received 9 July 2013 lenged with a range (102 –106 50% hemadsorbing doses (HAD50 )) of the highly virulent ASFV-Malawi
Received in revised form strain by inoculation via the intraoropharyngeal (IOP), intranasopharyngeal (INP), or intramuscular (IM)
13 September 2013
routes. A subsequent contact challenge experiment was performed in which six IOP-inoculated donor
Accepted 13 September 2013
pigs were allowed to have direct contact (DC) with six naïve pigs for exposure times that varied from 24 to
Available online 26 September 2013
72 h. All challenge routes resulted in clinical progression and postmortem lesions similar to those previ-
ously described in experimental and natural infection. The onset of clinical signs occurred between 1 and 7
Keywords:
African swine fever
days post inoculation (dpi) and included pyrexia with variable progression to obtundation, hematochezia,
ASFV melena, moribundity and death with a duration of 4–11 days. Viremia was first detected between 4 and
Transmission 5 dpi in all inoculation groups whereas ASFV shedding from the nasal cavity and tonsil was first detected
Challenge at 3–9 dpi. IM and DC were the most consistent modes of infection, with 12/12 (100%) of pigs challenged
Pathogenesis by these routes becoming infected. Several clinical and virological parameters were significantly differ-
Domestic pig ent between IM and DC groups indicating dissimilarity between these modes of infection. Amongst the
simulated natural routes, INP inoculation resulted in the most consistent progression of disease across
the widest range of doses whilst preserving simulation of natural exposure and therefore may provide a
superior system for pathogenesis and vaccine efficacy investigation.
Published by Elsevier B.V.

1. Introduction Georgia which has subsequently led to spread to Russia, Armenia,

African swine fever (ASF) is a highly contagious, often fatal,
transboundary disease of domestic and wild suids caused by ASF
virus (ASFV) (Tulman et al., 2009). It is the sole member of the
Asfarviridae family and is a large, double stranded DNA virus with
a 170–190 kb genome. Due to the lack of a commercial or experi-
mental vaccine or treatment, large economical losses are associated
with ASFV outbreaks (Sánchez-Vizcaíno et al., 2012). Such an event
occurred in 2007, when ASFV was introduced into the Republic of
∗ Corresponding author at: Foreign Animal Disease Research Unit, USDA/ARS
Plum Island Animal Disease Center, P.O. Box 848, Greenport, NY 11944, USA. Tel.:
+1 631 323 3336; fax: +1 631 323 3006.
E-mail addresses: Erin.Howey@ars.usda.gov (E.B. Howey),
Vivian.ODonnell@ars.usda.gov (V. O’Donnell), Helena.Ferreira@ars.usda.gov
(H.C. de Carvalho Ferreira), Manuel.Borca@ars.usda.gov (M.V. Borca),
Jonathan.Arzt@ars.usda.gov (J. Arzt).
Azerbaijan, Ukraine, and most recently to Belarus (WAHID, 2013),
with the range of the virus progressively extending west toward
Europe (Costard et al., 2013).
In domestic swine, it is generally accepted that under natu-
ral conditions, ASFV is primarily transmitted via direct contact
with excreted viral particles through nuzzling and/or ingestion
(Sánchez-Vizcaíno et al., 2012; Wardley et al., 1983). However, the
detailed mechanism of this mode of transmission has yet to be
explicitly defined. Arthropod-borne transmission via Ornithodoros
spp. ticks, which is a relevant means of transmission of ASFV within
the wild suid population and from wild suids to domestic swine, is
a minor method of transmission between domestic pigs (Arzt et al.,
2010; Jori et al., 2013; Penrith, 2009).
An extensive portion of in vivo ASF research has utilized the
intramuscular (IM) route of inoculation (Ballester et al., 2010;
Carrasco et al., 1996, 1997; Childerstone et al., 1998; Fernández
de Marco et al., 2007; Gómez-Villamandos et al., 1995a, 1995b,

0168-1702/$ – see front matter. Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.virusres.2013.09.024
 E.B. Howey et al. / Virus Research 178 (2013) 328–339
1997a, 1997b, 1998; Oura et al., 1998; Pan and Hess, 1984; Salguero
et al., 2004). This method results in highly reproducible clinical
disease. However, since IM inoculation does not result in viral
interaction with the oral and upper respiratory mucosal surfaces,
it bypasses many of the innate defense mechanisms the virus
would normally encounter through natural infection. Therefore,
it may be a suboptimal inoculation method to examine the early
pathogenesis and previremic stages of disease. Similarly, these
limitations make IM challenge, an unnatural model for vaccine
assessment.
The conventional pathogenesis of ASF has been investigated
in vivo using simulated natural routes of inoculation including
intranasal (IN) (de Carvalho Ferreira et al., 2012, 2013a; Greig,
1972; Greig and Plowright, 1970; Heuschele, 1967; Plowright et al.,
1968), intraoral (IO) (Boulanger et al., 1967; Colgrove et al., 1969),
combined oronasal (ON) (Boinas et al., 2004; Hamdy and Dardiri,
1984; McVicar, 1984; Mebus et al., 1978; Mebus and Dardiri, 1979,
1980) and direct contact/aerosol (DC) (de Carvalho Ferreira et al.,
2013b; Wilkinson and Donaldson, 1977; Wilkinson et al., 1977,
1981). Within all of the studies noted, the main route of virus
detection was through virus isolation and was usually performed
only on the blood, serum or a limited number of tissues. Few
have focused on virus detection within the secretions (de Carvalho
Ferreira et al., 2012; Greig and Plowright, 1970; McVicar, 1984;
Wilkinson et al., 1983; Ekue et al., 1989). While these reports have
contributed to current understanding of the viral dynamics and
pathogenesis of ASF, they fail to capture some specific information
which may contribute to develop the next generation of rationally
designed countermeasures. Specifically, confirmation of the pri-
mary site(s) of infection and elucidation of virus–host interactions
during early pathogenesis would facilitate the process of develop-
ing and validating effective prophylaxis. Furthermore, variability
in methodologies including virus strain, dose, and route of chal-
lenge has confounded comparison across studies. The importance
of standardizing experimental models including the description
and interpretation of clinical and pathologic findings in experi-
mental ASF has recently been emphasized (Galindo-Cardiel et al.,
2013). The goal of such standardization is to facilitate compari-
son of studies across research groups for the purpose of advancing
ASF vaccine development whilst avoiding redundancy of research
activities.
In the current study, we compared four routes of challenge of
domestic pigs with ASFV in order to characterize route- and dose-
dependent viral dynamics and the transmission of ASFV via direct
contact. This was done to determine how different means of expo-
sure/inoculation alter the pathogenesis of ASFV in swine and to
determine which route most closely simulates natural infection
with optimum experimental reproducibility.
2. Materials and methods
2.1. Virus
The highly pathogenic ASFV isolate Malawi Lil-20/1 was orig-
inally isolated from Ornithodoros sp. ticks during a field outbreak
in Malawi in 1983 and obtained from L. Dixon (Institute of Ani-
mal Health, Pirbright Laboratory, Woking, Surrey, United Kingdom)
(Borca et al., 1998). The stock virus was passaged once in primary
porcine macrophages derived from heparinized swine blood. The
tissue culture supernatant was then stored as 108 50% hemadsorb-
ing dose (HAD50 ) aliquots at −70 ◦ C.
The inoculum was prepared by initially diluting stock virus 10-
fold in Dulbeco’s Modified Eagle’s Medium (DMEM) (Gibco; Invit-
rogen, NY) containing 1% antibiotic/antimycotic (10,000 units/mL
of penicillin, 10,000 ␮g/mL of streptomycin, and 25 ␮g/mL of
Fungizone® ) (Gibco; Invitrogen, NY) and subsequently performing
329
additional dilution to achieve the desired viral dose in 2 ml of
media.
2.2. Animals
Conventional castrated male Yorkshire pigs from a herd that
is demonstrated to be free of porcine reproductive and respira-
tory syndrome virus were used for all experiments. Animals were
dewormed and vaccinated for common porcine pathogens includ-
ing porcine circovirus 2 prior to arrival at our facility. Pigs were
approximately 3–4 months old and 22–27 kg upon arrival. All
animal procedures were performed following Protocol 225-10-R
approved by the Plum Island Animal Disease Center Institutional
Animal Care and Use Committee (IACUC), which ensured ethi-
cal and humane treatment of experimental animals. The animal
experiments were carried out in BSL-3 Ag isolation rooms at Plum
Island Animal Disease Center. Animals were kept within the hous-
ing facilities 7 days prior to the start of the experiments to allow
for acclimation to the new environment. All animals were fed daily
and had unlimited access to water.
All animals except for donors in the DC study were allowed to
survive until terminal disease. Severely moribund animals were
humanely euthanized intravenously with 85.8 mg/kg of sodium
pentobarbital. Animals without clinical disease were humanely
euthanized at the end of the study as described above.
2.3. Study design
2.3.1. Direct inoculation studies
Experiments were performed to compare three routes of direct
inoculation. Eighteen pigs were used in these experiments. Six pigs
were assigned to each of the three inoculation route groupings:
intramuscular (IM), intranasopharyngeal (INP) and intraoropha-
ryngeal (IOP). Inoculation route groupings were further subdivided
into three inoculation dosage groups: low dose 102 HAD50 (n = 2),
mid dose 104 HAD50 (n = 2) and high dose 106 HAD50 (n = 2). IM-
inoculated pigs received a 2 mL injection of inoculum within the
right semimembranosus muscle. Pigs assigned to INP inoculation
were sedated with an intramuscular injection of telazol (3 mg/kg),
ketamine (8 mg/kg), xylazine (4 mg/kg), placed in sternal recum-
bency and 2 mL inoculum was instilled through the nares into the
nasopharynx via a 15 gauge silicon catheter (Abbott Laboratories,
IL). Pigs assigned to IOP inoculation received 2 mL of the inoculum
perorally via syringe with an attached 6 in. sterile metal cannula.
Animals were sedated, placed in dorsal recumbency and 2 mL of the
inoculum was deposited in the ventral portion of the soft palate and
palatine tonsil.
2.3.2. Direct contact (DC) transmission studies
Additional experiments were performed to characterize ASFV
transmission from IOP-inoculated pigs and viral dynamics in pigs
challenged by direct contact. Donor (n = 6) and naïve pigs (n = 6)
were initially housed in two separate rooms. Donor pigs were inoc-
ulated via the intraoropharyngeal method as described above with
2 mL of inoculum at a dose of 105 HAD50 of the same ASFV Malawi
Lil-20/1 described above. Upon detection of pyrexia (rectal tem-
perature greater than or equal to 40 ◦ C) in at least 4 donor pigs (6
days post inoculation (dpi)), contact pigs were transferred into the
donor room and allowed to comingle with inoculated pigs. Feed
was withheld for approximately 4 h to encourage contact between
pigs. After 24 h (1 day post contact (dpc)), two contact animals were
removed and isolated into a separate room and two donor animals
were euthanized to maintain the donor/naïve pig ratio. This pro-
cess was repeated at 48 and 72 h (2 and 3 dpc). Contact-exposed
animals were then monitored for onset and progression of clinical
disease.
330 E.B. Howey et al. / Virus Research 178 (2013) 328–339

Table 1
Clinical signs and scoring value for ASF.

Characteristic Score

Behavior and mentation 0 Normal, alert, responsive

1 Mildly obtunded. Slightly reduced liveliness, stands up unassisted, resists restraint or rectal thermometer
2 Obtunded. Reluctant to stand but will do so when assisted; decreased resistance to restraint or rectal thermometer
3 Intermittent ataxia, disorientation, can still stand/walk or will not stand/walk even when assisted, still conscious
4 Moribund. Nonambulatory, unconscious/nonresponsive

Neurologic signs 0 Normal

2 Unambiguous neurologic signs (e.g. convulsions, seizures)

Defecation 0 Normal to mildly soft stools

1 Profuse watery diarrhea, ±mild hematochezia or melena
2 Severe to marked hematochezia/melena

Body temperaturea 0 38–40 ◦ C

1 Temperature greater than or equal to 40 ◦ C at any point of study
2 Temperature greater than or equal to 40 ◦ C for at least 2 subsequent days
3 Temperature greater than or equal to 41 ◦ C
4 Temperature less than 38 ◦ C
a
Temperature scores are never reduced.

2.4. Clinical evaluation
Incidence proportion of ASF was calculated for each dose/route
combination as [(number of successfully infected pigs for that dose
and route) ÷ (all pigs subjected to that dose and route)]. For all stud-
ies, clinical evaluations were performed on all animals until death
or termination of the experiment. Clinical signs used to charac-
terize progression of ASF are listed in Table 1. These clinical signs
were assigned a numerical value based on severity and significance.
The sum of the scores of all clinical signs present for each pig was
recorded daily.
2.5. Sample collection and processing
animals will appear in a separate manuscript (Howey et al., in
progress).
2.6. Virus isolation and titration
Blood, serum, nasal swabs, tonsil scrapings, urine (postmortem
only) and tissues from infected pigs were screened for ASFV
by virus isolation and titration (VI) on primary porcine blood
macrophage cell cultures prepared from defibrinated swine blood
as previously described (Zsak et al., 2005). Presence of virus was
determined by identification of infected cells by rosette formation
(hemadsorption) and titers expressed as doses calculated by the
Spearman–Karber method (Mahy and Kangro, 1996).

Sample collection including whole blood with EDTA, clotted
blood for the collection of serum, swab specimens from the nasal
cavity and superficial scrapings from the tonsil of the soft palate
were performed on the day of inoculation at 0 days post inocu-
lation (dpi) followed by sample collection every other day. Pigs
were manually restrained for sample collection. Whole blood and
serum were collected via the jugular vein. Tonsil scrapings were
collected with a sterile spoon by gently scraping along the surface
of the tonsil of the soft palate, exfoliating superficial epithelial cells
while making sure to not disrupt the mucosa. Nasal swabs were
collected bilaterally using sterile cotton swabs extended just cau-
dal to the alar fold. Following collection, nasal swabs and tonsil
scrapings were immediately immersed in 1 mL of DMEM with 5%
antibiotic/antimycotic; then, all samples were transferred on ice
to the laboratory for processing. All samples were transferred to
cryovials and stored at −70 ◦ C until they were analyzed by virus
isolation (VI).
Necropsies were performed as soon as possible following
euthanasia or natural death. Tissues collected during postmortem
examination included: tonsil of the soft palate, lingual tonsil, nasal
tonsil, dorsal soft palate (rostral and caudal), dorsal nasophar-
ynx, caudal nasal turbinate, epiglottis, trachea, bronchial mucosa,
lung, thymus, liver, spleen, kidney, adrenal gland, thyroid, pan-
creas, small intestine, large intestine, bone marrow, and lymph
nodes including: retropharyngeal, submandibular, hilar, gastro-
hepatic, renal, inguinal and popliteal. Urine was also taken via direct
aspiration from the urinary bladder at the time of necropsy. Tis-
sue samples collected were placed in cryovials as described above
and cryomolds, embedded in Optimal Cutting Temperature (OCT)
medium (Tissue-Tek O.C.T. compound, Sakura Finetek, CA) and then
frozen in liquid nitrogen. Detailed description of tissues from these
2.7. Immunomicroscopy
Microscopic localization of the virus in tissues positive
for ASFV via VI was performed via immunohistochemistry
(IHC). Immunofluorescence (IFA) was used to detect virus in
combination with other cellular markers in order to further
determine/characterize phenotypes of infected cells. All primary
antibodies were extensively tested prior to this study in sections of
tissue from ASFV infected or non-infected pigs, using IHC, to ensure
specificity.
OCT-embedded tissue samples for IHC and IFA were cryosec-
tioned onto electrostatically charged glass slides and fixed for
10 min in acetone at −20 ◦ C. Slides were blocked for 2 h at 20 ◦ C
with Phosphate buffered saline with Tween (PBST) containing 6%
mixed serum and 2% powdered non-fat milk. A mouse monoclonal
primary antibody 1D9, targeting the ASFV VP30 (Cuesta-Geijo et al.,
2012; Galindo et al., 2012) (kindly provided by Javier Dominguez
Juncal, Departamento de Biotecnología, Instituto Nacional de Inves-
tigación y Tecnología Agraria y Alimentaria (INIA)), was diluted
1:250 in blocking buffer and applied to tissue sections that were
then incubated for 20 h at 4 ◦ C.
For IHC, specific anti-ASFV immunoreactivity was detected
using a commercial micropolymer alkaline phosphatase detection
system (Mach 3 AP kit; Biocare, CA) as per manufacturers’ recom-
mendation with an alkaline phosphatase substrate (Vector Red;
Vector Laboratories, CA). Slides were counterstained with Gill’s
hematoxylin and cover slipped using routine methods. A duplicate
serial section of each tissue screened was treated with a mouse
monoclonal anti-foot and mouth disease virus (FMDV) antibody
(10GA4, targeting FMDV O1-Brugge (Arzt et al., 2009)) serving as a
negative control.
 E.B. Howey et al. / Virus Research 178 (2013) 328–339 331

Table 2
Clinical characteristics of pigs infected with ASFV by different routes and dosages.

Group No. infected/total no. Total days survival (±SD) Pyrexia Clinical signs

Days to onset (±SD) Days to onset (±SD) Maximum clinical score (±SD)

IM
Overall 6/6 8.5 (±1.8)c 5.2 (±1.2)c 4.5 (±1.2)c 6.3 (±1.4)c
102 (n = 2) 2/2 10.5 (±0.3) 5.5 (±0.7) 5.5 (±0.7) 7.0 (±1.4)
104 (n = 2) 2/2 8.0 (±1.4) 5.5 (±0.7) 5.0 (±0.0) 6.5 (±2.1)
106 (n = 2) 2/2 7.0 (±0.0) 4.5 (±2.1) 3.0 (±0.0) 5.5 (±0.7)

INP
Overall 5/6 10.3 (±1.9)c 5.0 (±2.7)c 2.6 (±2.5)c 6.2 (±2.8)c
102 (n = 2) 1/2 11.0 (±1.4) 7.0 (±0.0)a 7.0 (±0.0)a 9.0 (±0.0)a
104 (n = 2) 2/2 11.0 (±1.4) 7.0 (±0.0) 1.5 (±0.7) 5.0 (±1.4)
106 (n = 2) 2/2 9.0 (±2.8) 2.0 (±0.0) 1.5 (±0.7) 6.0 (±4.2)

IOP
Overall 4/6 10.5 (±2.4)c 5.8 (±1.3)c 4.3 (±2.4)c 4.8 (±4.0)c
102 (n = 2) 0/2 13.0 (±0.0) – – –
104 (n = 2) 2/2 10.0 (±2.8) 6.5 (±0.7) 3.5 (±3.5) 6.5 (±5.0)
106 (n = 2) 2/2 8.5 (±0.7) 5.0 (±1.4) 5.0 (±1.4) 3.0 (±0.0)

DC
Overall 6/6 10.3 (±1.2)c 5. (±1.8)c 5.0 (±1.9)c 6.8 (±2.0)c
1 dpc (n = 2) 2/2 10.5 (±0.7) 5.0 (±0.0) 4.5 (±0.7) 7.5 (±0.7)
2 dpc (n = 2) 2/2 10.5 (±2.1) 3.5 (±2.1) 3.5 (±2.1) 8.0 (±1.4)
3 dpc (n = 2) 2/2 10.0 (±1.4) 7.0 (±0.0) 7.0 (±0.0) 5.0 (±2.8)

IOP donor
Overall 4/6 –b 3.6 (±0.6) 4.0 (±1.7) 4.0 (±2.5)

–, not detected; SD, standard deviation.

a
One animal affected within group.
b
Survival not calculated due to predetermined euthanasia.
c
No statistical difference between group overall values (IM, INP, IOP and DC).

Multichannel IFA was performed similarly as described for IHC,
except ASFV antigen detection was performed in combination with
primary antibodies with specificity for host cellular markers includ-
ing pancytokeratin (180059, Zymed, Invitrogen, NY) for epithelium,
CD163 and CD172a for macrophages (MCA 2311, and MCA2312GA,
respectively AbD Serotec, UK). After incubation of primary antibod-
ies for 20 h at 4 ◦ C, isotype-specific secondary antibodies labeled
with fluorescent dyes (AF 350, 488, 594, and 647, AlexaFluor;
Molecular Probes Inc., OR) were applied and slides were incubated
for 1 h at 37 ◦ C. Images were obtained using a Nikon Eclipse 90i
multichannel epi-fluorescent microscope equipped with 350, 488,
594, and 647 excitation filter cubes and digital camera.
2.8. Statistical analyses
All statistical analyses were performed using R statistical pro-
gram (R core Team, 2013)), employing different R packages
(explained in detail below). Statistical significance was considered
as p < 0.05. All data reported herein is not statistically significant
unless explicitly stated.
2.8.1. Survival analysis
Duration of survival for each pig was calculated as the total
number of days from inoculation/exposure to natural death or
euthanasia due to moribundity. Survival functions were calculated
for pigs belonging to the IM, INP, IOP and DC groups and com-
pared through a Cox proportional hazard regression model using
the package “Survival” for R software (Therneau, 2013). Multiple
comparisons were made: (1) between inoculation/infection routes
(IM, INP, IOP and DC), (2) within inoculated groups (IM, INP and
IOP) comparing type of inoculation route and dose, (3) within each
dose (comparing different inoculation routes), (4) within each route
(comparing doses), and (5) within the DC pigs (comparing expo-
sure length). Survival was not calculated for donor pigs (IOP donor
group) from the contact experiment since these pigs were eutha-
nized at predetermined times regardless of clinical disease.
2.8.2. Clinical signs, viremia and shedding
For all clinical and virological parameters examined, the
mean ± standard deviation (SD) for each infection group/dose com-
bination was calculated using data values collected per individual
animal per time point. The effect of the inoculation/infection route
used to establish infection was compared across groups (both
within the direct inoculation studies and between direct inoc-
ulation and contact studies) for the outcome variables present
in Tables 2 and 3. These comparisons were made using regres-
sion analysis, using a linear model, considering the 4 types of
inoculation/infection route as categorical explanatory variables:
intramuscular (IM), intranasopharyngeal (INP), intraoropharyngeal
(IOP) and direct contact (DC). A linear model was also used to assess
the effects of different exposure levels applied in the pigs infected
by direct contact, with exposure as explanatory variable (with 3
exposure levels – 1 day, 2 days and 3 days) and variables present
in Tables 2 and 3 as outcome. Linear model assumptions were con-
firmed through visual inspection of the residual plots. The statistical
power of these experiments was analyzed retrospectively using the
package “pwr” (Champely, 2012). The study did not have enough
power to test for possible interactions of type of inoculation route
and dose used for inoculation. The power of the analysis using only
the explanatory variable type of inoculation/infection route (deter-
mining differences between inoculation/infection groups) varied
from 0.58 to 0.31, depending on the number of missing values.
3. Results
3.1. Direct inoculation studies
3.1.1. Inoculation and infectivity
Successful infection of individual pigs was determined by the
presence of characteristic clinical signs combined with detection
of ASFV within the blood components (viremia). In the IM group,
all animals regardless of dose were successfully infected (2/2 high
dose, 2/2 mid dose, and 2/2 low dose (100% incidence)). In the INP
332 E.B. Howey et al. / Virus Research 178 (2013) 328–339

Table 3
Virologic parameters of pigs infected with ASFV by different routes and dosages.

Group No. infected/ Viremia Shedding

total no.
Days to onset Duration Maximum Days to onset of Maximum Days to onset of Maximum
(±SD) titer tonsil shedding titer (±SD)b nasal shedding titer (±SD)b
(±SD) (±SD)
(±SD) (±SD)b,c,g

IM
Overall 6/6 4.3 (±1.6)e 5.2 (±1.3)e 6.5 (±0.6)c 8.0 (±1.2)e 2.4 (±0.4)e 9.0 (±0.0)a,e 2.1 (±0.0)a,e
102 (n = 2) 2/2 6.0 (±1.4) 5.5 (±0.7) 6.1 (±0.0)c 9.0 (±0.0)a 2.8 (±0.0)a 9.0 (±0.0)a 2.1 (±0.0)a
104 (n = 2) 2/2 4.0 (±1.4) 5.0 (±2.8) 6.4 (±0.9)c 8.0 (±1.4) 2.4 (±0.5) – –
106 (n = 2) 2/2 3.0 (±0.0) 5.0 (±0.0) 6.9 (±0.2)c 7.0 (±0.0)a 2.1 (±0.0)a – –

INP
Overall 5/6 5.4 (±1.1)e 5.6 (±1.7)e 7.0 (±1.2) 7.5 (±1.7)e 3.3 (±1.0)e 7.7 (±3.5)e 4.2 (±1.7)e,f
102 (n = 2) 1/2 5.0 (±0.0)a 6.0 (±0.0)a 5.8 (±0.0)a 9.0 (±0.0)a 2.8 (±0.0)a – –
104 (n = 2) 2/2 5.0 (±1.4) 7.0 (±0.0) 8.3 (±0.0)c 6.0 (±0.0) 4.0 (±0.7) 6.0 (±2.8) 5.2 (±0.5)
106 (n = 2) 2/2 6.0 (±1.4) 4.0 (±1.4) 6.3 (±0.4) 9.0 (±0.0)a 2.3 (±0.0)a 11.0 (±0.0)a 2.3 (±0.0)a

IOP
Overall 4/6 5.0 (±1.2)e 5.3 (±1.7)e 8.5 (±0.1) 5.5 (±1.9)e,f 5.3 (±1.7)f 6.5 (±1.0)e 5.2 (±1.1)f
102 (n = 2) 0/2 – – – – – – –
104 (n = 2) 2/2 6.0 (±0.0) 5.0 (±2.8) 8.6 (±0.0) 7.0 (±1.4) 4.4 (±0.5) 7.0 (±1.4) 4.7 (±0.2)
106 (n = 2) 2/2 4.0 (±0.0) 5.5 (±0.7) 8.4 (±0.2) 4.0 (±0.0) 6.2 (±2.3) 6.0 (±0.0) 5.8 (±1.4)

DC
Overall 6/6 5.2 (±1.5)e 6.2 (±1.7)e 8.8 (±0.3) 3.5 (±2.4)f 5.2 (±0.8)f 7.7 (±0.6)e 4.7 (±0.1)f
1 dpc (n = 2) 2/2 4.0 (±1.4) 7.5 (±2.1) 8.7 (±0.2) 1.5 (±0.7) 4.7 (±0.5) 8.0 (±0.0) 4.6 (±0.4)
2 dpc (n = 2) 2/2 5.0 (±1.4) 6.5 (±0.7) 8.8 (±0.4) 4.0 (±2.8) 5.8 (±0.0) 7.5 (±0.7) 4.7 (±0.2)
3 dpc (n = 2) 2/2 6.5 (±0.7) 4.5 (±0.7) 8.9 (±0.5) 5.0 (±2.8) 5.2 (±1.2) 7.5 (±0.7) 4.8 (±0.0)

IOP donor
Overall 4/6 4.8 (±2.2) –d 7.4 (±3.2) 2.8 (±1.5) 4.1 (±1.2) 6.5 (±1.3) 4.6 (±1.3)

–, not detected; SD, standard deviation.

a
One animal affected within group.
b
Log10 HAD50 /mL.
c
All titers derived from whole blood except ‘c’ below (serum derived).
d
Duration of viremia not calculated due to predetermined euthanasia.
e
Group overall values (IM, INP, IOP and DC) with a common superscript are not significantly different (p > 0.05).
f
Group overall values (IM, INP, IOP and DC) with a common superscript are not significantly different (p > 0.05).
g
No statistical differences were calculated for this group.

and IOP groups, incidence was 100% within the mid (2/2) and high
dose (2/2) groups; however amongst low dose animals, 1/2 pigs
in the INP group and the entire (0/2) IOP group did not become
infected (low dose incidence = 50%, 0% respectively).
3.1.2. Survival
The IM-inoculated pigs had the shortest overall survival (mean
8.5 ± 1.8 dpi, n = 6) (Table 2, Figs. 1A and 2), however, no statisti-
cally significant differences were found between the IM, INP and
IOP groups in the Cox proportional hazard analysis. IM-inoculated
pigs also had the shortest survival following onset of pyrexia (mean
4.3 ± 1.9 days, n = 6). Mean survival after pyrexia was longest within
the low dose group (6 ± 0.0 days, n = 2) and equal within the mid
and high dose groups (3.5 ± 2.1 days, n = 2 per group). Amongst
the INP-inoculated pigs, overall mean survival was 10.3 ± 1.9 dpi
with 5.5 ± 1.8 days survival following the onset of pyrexia. Sur-
vival was shortest within the high dose group (9.0 ± 2.8 days,
n = 2) compared to the mid and low dose groups (11.0 ± 1.4 days,
n = 2 per group). Overall survival within the intraoropharyngeal
group was the longest compared to all other routes with a mean
of 10.5 ± 2.4 dpi (n = 6). Survival following onset of pyrexia was
4.5 ± 2.1 days; however, the number of days to onset of pyrexia
was the longest (5.8 ± 1.3 dpi).
The dose used for inoculation had a significant effect on the pig’s
survival, with pigs inoculated with the low dose (102 HAD50 ASFV)
having a significantly longer survival (p = 0.02) than pigs inoculated
with a higher dose (106 HAD50 ASFV). However, when groups were
compared within the same dose (i.e. across route), there were no
statistical differences between the different inoculation routes.
3.1.3. Clinical signs
For all route-dose combinations, the most consistent and com-
monly earliest detected clinical sign was pyrexia ranging from 40
to 41.9 ◦ C. Diarrhea, hematochezia and melena were common in
all groups during the late stages of infection, except the contact
experiment donors. Less consistent clinical signs included obtunda-
tion, oculonasal discharge, coughing, and dermal hyperemia which
were often transient and variable throughout all groups. Termi-
nal stage ASF was characterized by moribundity including a rapid
and substantial decrease in body temperature, lack of response to
stimulus, inability to stand or ambulate, and occasionally, seizure-
like behavior. There were no statistically significant differences
(p > 0.05) between the IM, INP, IOP and DC routes for total days of
survival, days to onset of pyrexia and clinical signs, and maximum
clinical score (Table 2).
Amongst the IM-inoculated pigs, the onset of clinical signs
was shortest within the high dose group (Table 2, Fig. 2) includ-
ing pyrexia (4.5 ± 2.1 days) and obtundation (6.0 ± 0.0 days)
which occurred in all pigs (n = 6). Melena/hematochezia was
observed in one animal from the mid and high dose groups,
with the shortest onset (5.0 ± 0.0 days) within the high dose.
The overall highest mean clinical score was 6.3 ± 1.4 with the
highest mean clinical score observed within the low dose group
(n = 2). The IM-inoculated pigs, had significantly earlier onset of
obtundation and onset of melena/hematochezia (p < 0.05) in com-
parison to every group, or in comparison to the DC and INP routes
individually.
The lowest overall number of days to the onset of clinical
signs was amongst the INP-inoculated pigs (mean 2.6 ± 2.5 dpi, 5/6
 E.B. Howey et al. / Virus Research 178 (2013) 328–339
Fig. 1. Descriptive survival analyses for ASFV-exposed pigs relating dose/exposure length to survival duration within the direct inoculation studies (A) and direct contact
study (B).
pigs). However, onset within the low dose group was substantially
longer (7 days) than the mid and high dose groups (1.5 ± 0.7 days)
(Fig. 2D–F). The lowest overall number of days to onset of pyrexia
was amongst the INP-inoculated pigs (mean 5.0 ± 2.7 dpi, n = 2)
with the high dose group having the shortest onset (2.0 ± 0.0 days,
n = 2) compared to the low (1/2 pigs) and mid dose (2/2 pigs) groups
(7.0 ± 0.0 days). Obtundation and melena/hematochezia occurred
in 4 out of 6 pigs with respective means to onset of 9.5 ± 1.0 and
10.0 ± 0.8 days. The mean maximum clinical score amongst the
INP-inoculated pigs was 6.2 ±;2.8 with the highest clinical score
occurring within the low dose group (9.0 ± 0.0).
The mean maximum clinical score for the IOP groups was
4.8 ± 4.0 (4/6 pigs) with the lowest clinical score found within
the high dose group. Clinical signs consistent with ASF were
not observed in pigs from the low dose group. Onset of pyrexia
occurred within the mid and high dose groups at 6.5 ± 0.7 and
5.0 ± 1.4 days respectively (n = 2 per group). Obtundation and
melena/hematochezia were detected only in one mid dose IOP-
inoculated animal starting at 10 and 11 dpi respectively.
3.1.4. Viremia and ASFV shedding
The IM-inoculated pigs (n = 6) had the shortest overall period
to onset of viremia (mean 4.3 ± 1.6 dpi) and number of days of
viremia (5.2 ± 1.3 days) (Table 3). Amongst the IM-inoculated pigs,
the low dose group had both the longest time before onset and
longest duration of viremia (Fig. 2A, Table 3). The highest mean
titer of ASFV in serum was detected within the high dose group
(6.9 ± 0.2 log10 HAD50 /mL). Onset to detection of ASFV from the
tonsil was shortest within the high dose group. Nasal shedding
was only detected in 1 pig within the low dose IM group which
occurred at 9 dpi. There was an overall mean of 4.0 ± 1.6 days from
onset of viremia to detection of ASFV in secretions. IM-inoculated
333
pigs had significantly different results in comparison with pigs
infected by DC. Specifically these pigs had longer days to onset
of tonsil shedding, and lower maximum ASFV titers in tonsil and
nasal swabs (Table 3).
The overall number of days to onset of viremia was the longest
in the INP groups at 5.4 ± 1.1 dpi (5/6 pigs). The number of days of
viremia averaged 5.6 ± 1.7 days with the high dose group having
the shortest length of viremia (Fig. 2C, Table 3). The overall mean
maximum serum/whole blood titer was 7.0 ± 1.2 log10 HAD50 /mL.
Tonsillar ASFV was detected in 4 out of 6 INP-inoculated ani-
mals with a mean onset of 7.5 ± 1.7 days and a max titer of
3.3 ± 1.0 log10 HAD50 /mL. Nasal shedding was detected in 3 out
of 6 animals with a mean onset of 7.7 ± 3.5 dpi and a max
titer of 4.2 ± 1.7 log10 HAD50 /mL. The mean difference between
the onset of viremia and onset of ASFV detection in secretions
was 1.5 ± 1.9 days. In comparison to DC-exposed pigs, the INP-
inoculated pigs had significantly longer days to onset of tonsil
shedding (for INP), and significantly lower maximum titers in
tonsil.
Within the IOP groups, overall onset of viremia was detected at
5.0 ± 1.2 dpi with overall duration of viremia for 5.3 ± 1.7 days for 4
out of 6 pigs inoculated (Table 3). The high dose group had both the
shortest onset to viremia and shortest duration. The mean maxi-
mum titer from whole blood was 8.5 ± 0.1 log10 HAD50 /mL. Tonsil
and nasal shedding was detected within all infected animals (4/6)
with a mean onset of shedding at 5.5 ± 1.9 and 6.5 ± 1.0 dpi respec-
tively. The mean maximum titers for tonsil and nasal swabs for
IOP-inoculated pigs were 5.3 ± 1.7 and 5.2 ± 1.1 log10 HAD50 /mL.
The period between onset of viremia and viral shedding was the
shortest of all routes at 0.5 ± 1.2 days. There were no statistical sig-
nificant differences in any of the outcome variables between the
IOP and the DC groups.
334 E.B. Howey et al. / Virus Research 178 (2013) 328–339

Fig. 2. Relationship between clinical score and virus shedding in pigs inoculated IM (A–C), INP (D–F), and IOP (G–I) with ASFV Malawi at 102 , 104 and 106 HAD50 . Lines
indicating viral titer in whole blood, serum, tonsil swabs and nasal swabs are expressed on the right Y-axis. The daily clinical score (bars) are expressed on the left Y-axis.

3.2. Direct contact experiment
3.2.1. Inoculation and infectivity
For the IOP-inoculated donor pigs from the contact experiment,
the overall incidence of ASFV infection was 66% (4/6 pigs), identical
to the incidence in IOP-inoculated animals from the direct inocula-
tion studies. Within DC groups, all animals, regardless of exposure
time, were successfully infected (incidence = 100%) (Table 2).
3.2.2. Survival
All donor animals were euthanized between 7 and 9 dpi in accor-
dance with the predetermined scheme to maintain 1:1 ratio with
contact pigs. Naïve pigs were exposed to IOP-inoculated donors for
1, 2, or 3 days. Overall mean survival for all three exposure groups
(n = 6) was 10.3 ± 1.2 dpc with the longest onset of survival follow-
ing onset of pyrexia (5.9 ± 2.0 days) compared to direct infection
routes (Table 2, Fig. 1B). Animals exposed to donors for 3 dpc had the
shortest overall survival compared to the 1 dpc and 2 dpc-exposure
groups. However, there were no statistical differences in the sur-
vival according to the Cox proportional analysis between different
exposure durations.
3.2.3. Clinical signs
Among the IOP-inoculated donors, the mean number of days to
onset of pyrexia was 3.6 ± 0.6 dpi. The mean duration to onset of
clinical signs was 4.0 ± 1.7 dpi (Table 2). Two animals were pyrexic
for 1–2 days but their temperature returned to within normal limits
before euthanasia. These two pigs did not become viremic nor was
shedding detected. One pig was not pyrexic by the time of euthana-
sia, but was viremic and shed virus nasally on the day of euthanasia
(8 dpi). Four out of six pigs were obtunded with a mean onset of
4.0 ± 2.5 dpi. Donor animals did not have melena or hematochezia.
Amongst the DC-exposed pigs, onset of clinical signs began with
pyrexia that was detected first within the 2 dpc-exposure group
(3.5 ± 2.1 dpc, n = 2) followed by the 1 dpc-exposure (4.5 ± 0.7 dpc,
n = 2) and 3 dpc-exposure (7.0 ± 0.0 dpc, n = 2) groups. Obtundation
 E.B. Howey et al. / Virus Research 178 (2013) 328–339 335

Fig. 3. Relationship between clinical score and virus shedding in donor pigs inoculated IOP (A) with ASFV Malawi at 105 HAD50 and pigs exposed via Direct Contact (DC) for
24 h (B), 48 h (C) and 72 h (D). Lines indicating viral titer in whole blood, serum, tonsil swabs and nasal swabs are expressed on the right Y-axis. The daily clinical score (bars)
are expressed on the left Y-axis.

was detected within 5 out of 6 pigs with a mean onset of lesion amongst pigs was enlarged and often hemorrhagic lymph
10.6 ± 1.1 dpc. Melena/hematochezia was detected in 2 of 6 pigs nodes. The most severe lymphadenomegaly commonly involved
with a mean onset of 11.5 ± 0.7 dpc. The DC-exposed pigs had the the gastrohepatic lymph nodes. Splenomegaly ranged from mild
highest overall maximum clinical score (6.8 ± 2.0); with the highest to severe enlargement. Renal lesions varied from mild cortical
score in the 2 dpc group followed by the 1 dpc and 3 dpc groups. petechia to renomegaly with diffuse hemorrhage and conges-
tion.
Histologically, lymphoid organs of all animals examined had
3.2.4. Viremia and ASFV shedding
perifollicular regions expanded by cellular debris, extensive
The mean detection of onset of viremia occurred within 4 out of
hemorrhage, and distinct fragmented nuclear remnants, most con-
6 of the donor group pigs at 4.8 ± 2.2 dpi with the mean maximum
sistent with lymphocyte apoptosis (Fig. 4A and B). Lymphoid
whole blood titer of 7.4 ± 3.2 log10 HAD50 /mL. The onset of tonsil
follicles were still identifiable but less distinct compared to normal
shedding was detected prior to viremia in 2 pigs. Onset of tonsil and
lymphoid architecture in naïve pigs.
nasal shedding was detected at 2.8 ± 1.5 and 6.5 ± 1.3 dpi respec-
tively. Maximum titers for tonsil and nasal swabs were 4.1 ± 1.2
and 4.6 ± 1.3 log10 HAD50 /mL respectively (Fig. 3A, Table 3). The dif-
3.4. Immunohistochemical localization of ASFV antigens
ference between onset of viremia and shedding averaged 1.3 ± 0.6
days.
Examination of the distribution of virus and its association
Amongst contact-exposed pigs, mean onset of viremia
with specific cellular markers was performed on tissues collected
was 5.2 ± 1.5 dpc and was detected first within the 1 dpc-
from pigs of each inoculation route. Within lymphoid tissues
exposure group (4.0 ± 1.4 dpc, n = 2) followed by the 2 dpc
of pigs with fulminant ASF, there was extensive cell-associated
and 3 dpc-exposure groups (5.0 ± 1.4 and 6.5 ± 0.7 dpc, n = 2
labeling with the anti-ASFV-VP30 antibody. Localization was pre-
per group). The overall mean highest whole blood titer was
dominately within interfollicular regions with few immunopositive
8.8 ± 0.3 log10 HAD50 /mL, which was the highest amongst all routes
cells within follicles (Fig. 4C). Within the tonsil of the soft
(Table 3). Tonsillar detection of ASFV occurred prior to viremia in 4
palate, labeling was most prominent surrounding tonsillar crypts,
pigs. The mean onset of tonsil and nasal shedding was detected at
with few labeled cells within the tonsillar crypt epithelium.
3.5 ± 2.4 and 7.7 ± 0.6 dpc respectively, with mean maximum titers
This VP30-positive labeling did not colocalize with pancytoker-
of 5.2 ± 0.8 and 4.7 ± 0.1 log10 HAD50 /mL. The difference between
atin.
onset of viremia and shedding averaged 1.7 ± 1.7 days.
Within all positive tissues, immunoreactive cells were mor-
phologically consistent with large mononuclear cells (interpreted
3.3. Postmortem lesions as macrophages). Combined multichannel immunofluorescence
demonstrated that cells immunopositive for ASFV were also com-
All four routes produced comparable gross and histologic monly positive for monocyte/macrophage cellular markers such as
lesions in pigs at terminal stages of disease. The most consistent CD163 (Fig. 4D–G) and CD172a (not shown).
336 E.B. Howey et al. / Virus Research 178 (2013) 328–339

Fig. 4. Histopathology of lesions from pigs infected with ASFV-Malawi. Palatine tonsil, animal #38, INP ASFV-Malawi 102 HAD50 /mL; Lymphoid tissues including tonsil of
the soft palate (A and B), lymph nodes, and spleen displayed loss of lymphocytes and karyorrhectic debris. Scale bars: (A) 500 ␮m, (B) 100 ␮m immunohistochemical staining
(C) via Alkaline phosphatase polymer kit with anti-ASFV VP30 (1D9). Positive labeling extensively throughout interfollicular regions. Scale bar 500 ␮m. Inset (c), region of
interest at higher magnification. Multichannel immunofluorescence (D–G). (D) Low magnification three channel image containing anti-ASFV (red), pancytokeratin (green),
and macrophage marker CD163 (aqua). Scale bar 500 ␮m. (E) Three channel image containing Anti-ASFV (red), leukocyte marker macrophage marker CD163 (aqua) and
nuclear staining with DAPI (blue). (F) Three channel image containing anti-ASFV (red), pancytokeratin (green), and nuclear staining with DAPI (blue). (G) Four channel merged
image with colocalization of anti-ASFV (red) and leukocyte marker macrophage marker CD163 (aqua), pancytokeratin (green), and nuclear staining with DAPI (blue). Scale
bar (E–G) 100 ␮m.

4. Discussion clarify and/or challenge previous conclusions derived from a collec-

tion of pathogenesis studies whose work is not directly comparable
The goal of the investigation described herein was to evaluate due to variability of virus strain, dose, inoculation technique, and
dose- and route-dependent effects on the clinical course of ASF and detection methods. In addition, this work contributes to the estab-
viral dynamics of ASFV within domestic pigs. This work serves to lishment of challenge model systems for ASF which closely simulate
 E.B. Howey et al. / Virus Research 178 (2013) 328–339
natural infection. The goals of the model systems were to achieve
modes of challenging pigs that were (1) consistent with previ-
ous accounts of ASF, (2) consistent across experimental subjects,
(3) compatible with the current understanding of natural routes
of direct pig-to-pig transmission, (4) allowed control of dose and
timing of challenge, and (5) provided natural engagement of host
immune defenses. Recent studies have led toward standardization
of clinical scoring systems for ASF (de Carvalho Ferreira et al., 2012;
Galindo-Cardiel et al., 2013), and the current work demonstrates
adaptation of these systems where appropriate.
Four routes of challenge (IM, IOP, INP, DC) resulted in a sim-
ilar clinical course of ASF that was consistent with previously
described field cases (Ayoade and Adeyemi, 2003; Detray, 1963;
Sánchez Botija, 1982) and experimental models (Boulanger et al.,
1967; Colgrove et al., 1969; Greig, 1972; Greig and Plowright,
1970; Heuschele, 1967; McVicar, 1984; Mebus et al., 1978; Mebus
and Dardiri, 1979, 1980; Plowright et al., 1968; Wilkinson and
Donaldson, 1977; Wilkinson et al., 1977, 1980). Clinical signs did
not vary substantially according to route of inoculation. The great-
est consistency was achieved similarly by IM and DC challenge
which both had 100% incidence of infection. However, these sys-
tems suffer from unnatural delivery of virus (IM) and inability to
precisely control dose and timing of challenge (DC). All pigs chal-
lenged via DC became infected with ASFV regardless of length of
exposure, thereby demonstrating that only 24 h of exposure to
shedding animals is required for infection with ASFV. This finding
is consistent with the previous works that demonstrated trans-
mission after 24 h (Greig and Plowright, 1970) or 6 h (Ekue et al.,
1989) of exposure to donor pigs. However, other studies have
concluded that the quantity of virus shed 24 h following pyrexia
was inadequate to ensure infection in pigs exposed via direct con-
tact (Heuschele, 1967; Plowright et al., 1968). INP inoculation had
higher incidence whilst maintaining a natural exposure route and
allowing control of dose and timing of challenge. Additionally, the
shortest time to onset of clinical signs was observed within the
INP groups, however this difference was not statistically signifi-
cant.
Precise determination of pig infectious dose 50% ([PID50 ], i.e. the
minimum dose necessary to infected 50% of pigs) for the distinct
inoculation routes was not possible due to logistical constraints
which dictated some aspects of experimental design. However, the
data herein suggests that the required dose of ASFV to establish
infection is lowest for IM, intermediate for INP, and highest for IOP
delivery of ASFV to domestic swine. Specifically, low dose inocula-
tion (102 HAD50 ) incidence proportions were 100% (IM), 50% (INP),
and 0% (IOP). Yet, at 104 HAD50 all routes had 100% incidence of
infection. This is consistent with previous studies that have demon-
strated low ASFV IM dose (lower than 105 TCID50 ) (Pan and Hess,
1984; McVicar, 1984) and high IOP dose (PID50 higher than 103
TCID50 ) (de Carvalho Ferreira et al., 2012; Greig, 1972; McVicar,
1984). Additionally, Maurer et al. demonstrated that a minimal
infectious dose of 105 HAD50 was necessary to infect pigs orally
with ASFV (Maurer and Griesemer, 1958). The precise manner by
which specific routes of inoculation of ASFV correlate with vari-
able PID50 via distinct pathogenesis mechanisms remains to be
elucidated.
The route-specific clinical and virological characteristics
described herein are likely to be the result of distinct route-
determined, unique pathogenesis events in early ASFV infection.
Pigs inoculated IM had the shortest mean survival duration; how-
ever the difference was not statistically significant. Shortest onset
to viremia amongst IM-inoculated pigs was statistically significant.
This may be due to more direct access to the vascular system asso-
ciated with deposition within the muscle as compared to contact
with intact mucosa as occurred with simulated natural challenge
routes. Furthermore, the finding of several statistically significant
337
differences in the viremia and shedding parameters between the
intramuscular and the direct contact route suggests that the intra-
muscular route is a poor simulator of natural infection. As a result,
inoculation via IM should be avoided in experimental studies try-
ing to mimic a natural course of disease. Contrastingly, both the
IOP and INP inoculated pigs had comparable (i.e. not significantly
different) results compared to the DC exposed pigs suggesting that
IOP and INP are more likely to closely simulate natural infection.
The implied superior infectivity of ASFV via INP inoculation as
compared to IOP was reflected in slightly higher incidence pro-
portion and delayed onset of pyrexia and longer survival time
seen among INP-inoculated pigs in comparison with all success-
fully IOP-inoculated pigs (including donors in DC experiment).
However, IOP led to significantly higher shedding titers in the
tonsil compared with INP inoculation. This effect may have been
due to anatomic and physiological factors currently under inves-
tigation in our laboratory. IOP inoculation directly deposits the
inoculum along the tonsil of the soft palate. While this tonsil has
been documented as an early site of viral replication (Heuschele,
1967; Plowright et al., 1968), to our knowledge, specific virus–host
interactions in ASFV primary infection in vivo have never been
explicitly described. Thus, the lower incidence of infection by
the IOP route compared to INP-inoculation could be explained
by lack of establishment of systemic disease subsequent to pri-
mary infection within the tonsil of the soft palate and other
oropharyngeal tissues. Since low-dose INP-inoculated pigs had a
higher incidence of successful infection than IOP, the nasophar-
ynx may provide more permissive conditions for establishing
systemic ASFV infection. INP inoculation targets nasopharyngeal
tissues, including nasal tonsil, dorsal soft palate and walls/roof
of the nasopharynx. The data presented in this study suggest
that sites targeted by INP inoculation, or other tissues in the
respiratory tract, may provide important portals for ASFV infec-
tion.
Increased inoculation dose of ASFV was associated with a sig-
nificant decrease in survival duration of pigs. Animals inoculated
with 106 HAD50 , regardless of route, had a decreased mean number
of days to onset of clinical signs (pyrexia, obtundation, hema-
tochezia/melena) and survival compared to the mid and low dosage
groups. The number of days to onset of viremia and shedding was
decreased compared to mid and low doses as well, suggesting that
a higher viral dose resulted in overwhelming primary replication
and faster systemic dissemination of the virus. Paradoxically, the
high-dose pigs had lower mean maximum clinical scores because
of the rapidity of progression to death before the full severity of dis-
ease had occurred. Thus, maximum clinical score does not (in itself)
describe the severity of disease but must be evaluated in context
with duration of survival, virological characteristics, and pathologic
findings.
During the clinical phase of infection, ASFV was variably
detected in whole blood, serum and tonsil and nasal swabs.
Amongst all challenge routes, viremia and shedding commonly
either coincided with the onset of pyrexia or it occurred 2 days
before or after pyrexia, which is in agreement with other studies
(Greig and Plowright, 1970; McVicar, 1984). The DC-exposed pigs
had the highest mean ASFV titer within whole blood. Throughout
the course of this study whole blood samples had titers of almost 2
log10 higher than serum samples. This is consistent with previous
studies that has described greater number of mature virus particles
associated with erythrocytes and within circulating monocytes in
whole blood, in comparison to free virions within serum (Genovesi
et al., 1988; Wardley and Wilkinson, 1977).
Detection of ASFV within secretions (tonsil scraping and nasal
swabs) varied across infection routes and dosage groups. ASFV
titers in tonsil scrapings and nasal swabs were generally quite sim-
ilar, suggesting that virus was released from both regions and/or
338 E.B. Howey et al. / Virus Research 178 (2013) 328–339
virus quantities equilibrate between nasal and oral secretions. One
noteworthy exception to this trend was amongst DC-exposed pigs
wherein ASFV was consistently detected in tonsil scrapings (for 1–6
days) prior to detection in nasal swabs. Across all three inocula-
tion routes, initial detection of ASFV shedding occurred at the same
time, or subsequent to, virus detection in whole blood and serum.
However within the contact challenge study, ASFV was detected
in tonsil scraping samples 1–4 days prior to detection in blood of
IOP-inoculated donors and DC-exposed pigs. This indicates that,
under these distinct conditions, it was uniquely possible to detect
local viral replication and shedding within the oral cavity prior to
systemic dissemination. This is consistent with previous studies in
which ASFV could be detected within oral and pharyngeal swabs
1–2 days prior to viremia and pyrexia (Greig and Plowright, 1970)
or as early as 24 h post inoculation (hpi) in lymphoid and respiratory
tract tissues (Heuschele, 1967; Plowright et al., 1968).
Gross and histologic changes in pigs in the late stages of disease
were similar regardless of route of infection and were consis-
tent with postmortem findings described in field and experimental
cases of ASF. Systemic hemorrhages affecting the kidney and heart
and pulmonary congestion and edema are similar to previously
described lesions (Detray, 1963; Mebus, 1988). Amongst individ-
ual pigs, severity of lesions varied from mild to severe based upon
recently published recommendations for standardization of patho-
logic findings for ASF (Galindo-Cardiel et al., 2013). The hallmark
depletion and apoptosis of lymphocytes throughout the lymphoid
tissue with subsequent infiltration of macrophages commonly
described for ASF (Gómez-Villamandos et al., 2003) was observed
histologically within this study. Detailed pathological description
will be forthcoming in a separate manuscript (Howey et al., in
preparation).
Immunohistochemical visualization of ASFV antigen was
demonstrated in tonsil of the soft palate of pigs in terminal stages
of ASF, further confirming the presence and characterizing the dis-
tribution of virus within tissues. Immunoreactive cells were large
mononuclear cells and colocalization with monocyte/macrophage
cellular antigen suggested that the predominate infected cell type
is of monocytic origin. Colocalization of ASFV with CD163 supports
previous suggestions that this molecule may serve as an important
ASFV receptor (Alonso et al., 2013; Sánchez-Torres et al., 2003).
Additionally, this is consistent with numerous reports indicat-
ing monocytotropism of ASFV (Detray, 1963; Tulman et al., 2009;
Wardley and Wilkinson, 1978).
5. Conclusions
The current study has demonstrated that four modes of chal-
lenging pigs with ASFV result in similar ASF syndromes with slight
variations. The IM route may be advantageous for the study of late
stage ASF events; however several significant differences from DC
indicated this is not an optimal system to simulate natural infection.
DC would be preferable when simulating a natural manner of trans-
mission was utmost priority; however this system is expensive due
to the inclusion of additional (donor) animals in each experiment
and does not allow control of dose quantity and timing. The two
simulated natural manners of inoculation, IOP and INP, allowed
precise control of timing and dose of ASFV delivery and had similar
incidence of successful infection of pigs. Overall, the slightly lower
minimum infectious dose and slightly higher incidence of infec-
tion subsequent to INP inoculation in these experiments suggest
that INP may be the preferred route of inoculation for pathogenesis
and vaccine challenge studies of pigs with ASFV. Further validation
of these techniques with larger sample sizes would contribute to
stronger conclusions regarding the differences between the infec-
tion systems described herein.
Conflict of interests
The authors do not have any financial or personal conflict of
interests.
Acknowledgements
EH planned and carried out the live animal experiments, virus
isolation, immunomicroscopy and drafted the manuscript. VO pro-
vided expertise in conceptual design, as well as optimization and
troubleshooting of techniques. HF performed the statistical analysis
and helped with manuscript revisions. MB consulted and con-
tributed on all aspects of study design and implementation; also
provided critical review of manuscript. JA conceived the study,
oversaw the implementation of experiments, helped draft and
revised the manuscript. All authors read and approved the final
manuscript.
We wish to thank Meghan Tucker, George Smoliga and Ethan
Hartwig for superb technical support. Luis Rodriguez and Carolina
Stenfeldt are acknowledged for critical review of the manuscript.
Juan Pacheco supported data manipulation and consulted regard-
ing VI technique. In addition, we would like to thank the staff of the
Animal Resource Branch for their support in collecting data during
animal experiments.
This research was funded, in part, by Agricultural Research
Service–Current Research Information System project No. 1940-
32000-056-00D. Additional funds came from an interagency
agreement with the Science and Technology Directorate of the U.S.
Department of Homeland Security under Award Number HSHQPM-
12-X-00005. Erin Howey’s position is funded through the Plum
Island Animal Disease Research Participation Program adminis-
tered by the Oak Ridge Institute for Science and Education through
an interagency agreement between the U.S. Department of Energy
and the U.S. Department of Agriculture.
References
Alonso, C., Galindo, I., Cuesta-Geijo, M.A., Cabezas, M., Hernaez, B., Muñoz-Moreno,
R., 2013. African swine fever virus–cell interactions: from virus entry to cell
survival. Virus Res. 173 (1), 42–57.
Arzt, J., Gregg, D.A., Clavijo, A., Rodriguez, L.L., 2009. Optimization of immuno-
histochemical and fluorescent antibody techniques for localization of
foot-and-mouth disease virus in animal tissues. J. Vet. Diagn. Invest. 21 (6),
779–792.
Arzt, J., White, W.R., Thomsen, B.V., Brown, C.C., 2010. Agricultural diseases on the
move early in the third millennium. Vet. Pathol. 47 (1), 15–27.
Ayoade, G., Adeyemi, I., 2003. African swine fever: an overview. Rev. Élev. Méd. Vét.
Pays Trop. 56 (3–4), 129–134.
Ballester, M., Galindo-Cardiel, I., Gallardo, C., Argilaguet, J.M., Segalés, J., Rodríguez,
J.M., Rodríguez, F., 2010. Intranuclear detection of African swine fever virus DNA
in several cell types from formalin-fixed and paraffin-embedded tissues using a
new in situ hybridisation protocol. J. Virol. Methods 168 (1–2), 38–43.
Boinas, F.S., Hutchings, G.H., Dixon, L.K., Wilkinson, P.J., 2004. Characterization
of pathogenic and non-pathogenic African swine fever virus isolates from
Ornithodoros erraticus inhabiting pig premises in Portugal. J. Gen. Virol. 85 (8),
2177–2187.
Borca, M.V., Carrillo, C., Zsak, L., Laegreid, W.W., Kutish, G.F., Neilan, J.G., Burrage,
T.G., Rock, D.L., 1998. Deletion of a CD2-like gene, 8-DR, from African swine
fever virus affects viral infection in domestic swine. J. Virol. 72 (4), 2881–2889.
Boulanger, P., Bannister, G.L., Gray, D.P., Ruckerbauer, G.M., Willis, N.G., 1967. African
swine fever. II. Detection of the virus in swine tissues by means of the modified
direct complement-fixation test. Can. J. Comp. Med. Vet. Sci. 31 (1), 7–11.
Carrasco, L., Chacon, M.d.L.F., Martin de Las Mulas, J., Gómez-Villamandos, J.C., Sierra,
M.A., Villeda, C.J., Wilkinson, P.J., 1997. Ultrastructural changes related to the
lymph node haemorrhages in acute African swine fever. Res. Vet. Sci. 62 (3),
199–204.
Carrasco, L., de Lara, F.C., Martín de las Mulas, J., Gómez-Villamandos, J.C., Hervás, J.,
Wilkinson, P.J., Sierra, M.A., 1996. Virus association with lymphocytes in acute
African swine fever. Vet. Res. 27 (3), 305–312.
Champely, S., 2012. pwr: Basic functions for power analysis. R package version 1.1.1.
http://CRAN.R-project.org/package=pwr
Childerstone, A., Takamatsu, H., Yang, H., Denyer, M., Parkhouse, R.M., 1998. Modu-
lation of T cell and monocyte function in the spleen following infection of pigs
with African swine fever virus. Vet. Immunol. Immunopathol. 62 (4), 281–296.
 E.B. Howey et al. / Virus Research 178 (2013) 328–339
Colgrove, G.S., Haelterman, E.O., Coggins, L., 1969. Pathogenesis of African swine
fever in young pigs. Am. J. Vet. Res. 30 (8), 1343–1359.
Costard, S., Mur, L., Lubroth, J., Sánchez-Vizcaíno, J.M., Pfeiffer, D.U., 2013. Epidemi-
ology of African swine fever virus. Virus Res. 173 (1), 191–197.
Cuesta-Geijo, M.A., Galindo, I., Hernáez, B., Quetglas, J.I., Dalmau-Mena, I., Alonso,
C., 2012. Endosomal maturation, Rab7 GTPase and phosphoinositides in African
swine fever virus entry. PLoS ONE 7 (11), e48853.
de Carvalho Ferreira, H.C., Weesendorp, E., Elbers, A.R., Bouma, A., Quak, S., Stege-
man, J.A., Loeffen, W.L., 2012. African swine fever virus excretion patterns in
persistently infected animals: a quantitative approach. Vet. Microbiol. 160 (3–4),
327–340.
de Carvalho Ferreira, H.C., Weesendorp, E., Quak, S., Stegeman, J.A., Loeffen, W.L.,
2013a. Quantification of airborne African swine fever virus after experimental
infection? Vet. Microbiol. 165 (3–4), 243–251.
de Carvalho Ferreira, H.C., Backer, J.A., Weesendorp, E., Klinkenberg, D., Stegeman,
J.A., Loeffen, W.L., 2013b. Transmission rate of African swine fever virus under
experimental conditions. Vet. Microbiol. 165 (3–4), 296–304.
Detray, D.E., 1963. African swine fever. Adv. Vet. Sci. Comp. Med. 19, 299–333.
Ekue, N.F., Wilkinson, P.J., Wardley, R.C., 1989. Infection of pigs with the Cameroon
isolate (Cam/82) of African swine fever virus. J. Comp. Pathol. 100 (2), 145–154.
Fernández de Marco, M., Salguero, F.J., Bautista, M.J., Núñez, A., Sánchez-Cordón, P.J.,
Gómez-Villamandos, J.C., 2007. An immunohistochemical study of the tonsils in
pigs with acute African swine fever virus infection. Res. Vet. Sci. 83 (2), 198–203.
Galindo-Cardiel, I., Ballester, M., Solanes, D., Nofrarías, M., López-Soria, S., Argilaguet,
J.M., Lacasta, A., Accensi, F., Rodríguez, F., Segalés, J., 2013. Standardization of
pathological investigations in the framework of experimental ASFV infections.
Virus Res. 173 (1), 180–190.
Galindo, I., Hernáez, B., Muñoz-Moreno, R., Cuesta-Geijo, M.A., Dalmau-Mena, I.,
Alonso, C., 2012. The ATF6 branch of unfolded protein response and apopto-
sis are activated to promote African swine fever virus infection. Cell Death Dis.
3 (7), e341.
Genovesi, E.V., Knudsen, R.C., Whyard, T.C., Mebus, C.A., 1988. Moderately viru-
lent African swine fever virus infection: blood cell changes and infective virus
distribution among blood components. Am. J. Vet. Res. 49 (3), 338.
Gómez-Villamandos, J.C., Bautista, M.J., Carrasco, L., Caballero, M.J., Hervás, J., Villeda,
C.J., Wilkinson, P.J., Sierra, M.A., 1997a. African swine fever virus infection of bone
marrow: lesions and pathogenesis. Vet. Pathol. 34 (2), 97–107.
Gómez-Villamandos, J.C., Bautista, M.J., Carrasco, L., Chacón-Manrique de Lara, F.,
Hervás, J., Wilkinson, P.J., Sierra, M.A., 1998. Thrombocytopenia associated with
apoptotic megakaryocytes in a viral haemorrhagic syndrome induced by a mod-
erately virulent strain of African swine fever virus. J. Comp. Pathol. 118 (1),
1–13.
Gómez-Villamandos, J.C., Carrasco, L., Bautista, M.J., Sierra, M.A., Quezada, M.,
Hervás, J., Chacón, M.de L., Ruiz-Villamor, E., Salguero, F.J., Sónchez-Cordón, P.J.,
Romanini, S., Núñez, A., Mekonen, T., Méndez, A., Jover, A., 2003. African swine
fever and classical swine fever: a review of the pathogenesis. Dtsch. Tierarztl.
Wochenschr. 110 (4), 165–169.
Gómez-Villamandos, J.C., Hervás, J., Méndez, A., Carrasco, L., Martín de las Mulas,
J., Villeda, C.J., Wilkinson, P.J., Sierra, M.A., 1995a. Experimental African swine
fever: apoptosis of lymphocytes and virus replication in other cells. J. Gen. Virol.
76 (Pt. 9), 2399–2405.
Gómez-Villamandos, J.C., Hervás, J., Méndez, A., Carrasco, L., Villeda, C.J., Wilkinson,
P.J., Sierra, M.A., 1995b. Ultrastructural study of the renal tubular system in acute
experimental African swine fever: virus replication in glomerular mesangial
cells and in the collecting ducts. Arch. Virol. 140 (3), 581–589.
Gómez-Villamandos, J.C., Hervás, J., Moreno, C., Carrasco, L., Bautista, M.J., Caballero,
J.M., Wilkinson, P.J., Sierra, M.A., 1997b. Subcellular changes in the tonsils of pigs
infected with acute African swine fever virus. Vet. Res. 28 (2), 179–189.
Greig, A., 1972. Pathogenesis of African swine fever in pigs naturally exposed to the
disease. J. Comp. Pathol. 82 (1), 73–79.
Greig, A., Plowright, W., 1970. The excretion of two virulent strains of African swine
fever virus by domestic pigs. J. Hyg. (Lond.) 68 (4), 673–682.
Hamdy, F.M., Dardiri, A.H., 1984. Clinical and immunologic responses of pigs to
African swine fever virus isolated from the Western Hemisphere. Am. J. Vet.
Res. 45 (4), 711.
Heuschele, W.P., 1967. Studies on the pathogenesis of African swine fever. I. Quan-
titative studies on the sequential development of virus in pig tissues. Arch.
Gesamte Virusforsch. 21 (3), 349–356.
http://www.oie.int/wahis 2/public/wahid.php/Wahidhome/Home (accessed
3.09.13). Jori, F., Vial, L., Penrith, M.L., Perez-Sanchez, R., Etter, E., Albina, E.,
339
Michaud, V., Roger, F., 2013. Review of the sylvatic cycle of African swine fever
in sub-Saharan Africa and the Indian Ocean. Virus Res. 173 (1), 212–227.
Mahy, B.W.J., Kangro, H.O. (Eds.), 1996. Virology Methods Manual. Academic Press,
San Diego.
Maurer, F.D., Griesemer, R.A., 1958. The pathology of African swine fever; a compar-
ison with hog cholera. Am. J. Vet. Res. 19 (72), 517–539.
McVicar, J.W., 1984. Quantitative aspects of the transmission of African swine fever.
Am. J. Vet. Res. 45 (8), 1535–1541.
Mebus, C.A., Dardiri, A.H., 1979. Additional characteristics of disease caused by the
African swine fever viruses isolated from Brazil and the Dominican Republic.
Proc. Annu. Meet. U. S. Anim. Health Assoc. (83), 227–239.
Mebus, C.A., Dardiri, A.H., 1980. Western hemisphere isolates of African swine fever
virus: asymptomatic carriers and resistance to challenge inoculation. Am. J. Vet.
Res. 41 (11), 1867–1869.
Mebus, C.A., Dardiri, A.H., Hamdy, F.M., Ferris, D.H., Hess, W.R., Callis, J.J., 1978.
Some characteristics of African swine fever viruses isolated from Brazil and
the Dominican Republic. Proc. Annu. Meet. U. S. Anim. Health Assoc. (82),
232–236.
Mebus, C.A., 1988. African swine fever. Adv. Virus Res. 35, 251–269.
Oura, C.A., Powell, P.P., Parkhouse, R.M., 1998. Detection of African swine fever virus
in infected pig tissues by immunocytochemistry and in situ hybridisation. J.
Virol. Methods 72 (2), 205–217.
Pan, I.C., Hess, W.R., 1984. Virulence in African swine fever: its measurement and
implications. Am. J. Vet. Res. 45 (2), 361–366.
Penrith, M.L., 2009. African swine fever. Onderstepoort J. Vet. Res. 76 (1), 91–95.
Plowright, W., Parker, J., Staple, R.F., 1968. The growth of a virulent strain of African
swine fever virus in domestic pigs. J. Hyg. (Lond.) 66 (1), 117–134.
R Core Team, 2013. R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna, Austria
http://www.R-project.org/
Salguero, F.J., Sánchez-Cordón, P.J., Sierra, M.A., Jover, A., Núñez, A., Gómez-
Villamandos, J.C., 2004. Apoptosis of thymocytes in experimental African Swine
Fever virus infection. Histol. Histopathol. 19 (1), 77–84.
Sánchez-Torres, C., Gómez-Puertas, P., Gómez-del-Moral, M., Alonso, F., Escrib-
ano, J.M., Ezquerra, A., Domínguez, J., 2003. Expression of porcine CD163 on
monocytes/macrophages correlates with permissiveness to African swine fever
infection. Arch. Virol. 148 (12), 2307–2323.
Sánchez-Vizcaíno, J.M., Mur, L., Martínez-López, B., 2012. African swine fever: an
epidemiological update. Transbound Emerg. Dis. 59 (s1), 27–35.
Sánchez Botija, C., 1982. African Swine Fever. New developments. Rev. Sci. Tech. Off.
Int. Epiz. 1 (4), 1065–1094.
Tulman, E.R., Delhon, G.A., Ku, B.K., Rock, D.L., 2009. African swine fever virus. Curr.
Top. Microbiol. Immunol. 328, 43–87.
Therneau, T., 2013. A Package for Survival Analysis in S. R package version 2.37-4.
http://CRAN.R-project.org/package=survival
Wardley, R.C., de, M.A.C., Black, D.N., de Castro Portugal, F.L., Enjuanes, L., Hess, W.R.,
Mebus, C., Ordas, A., Rutili, D., Sánchez-Vizcaíno, J., Vigário, J.D., Wilkinson, P.J.,
Moura Nunes, J.F., Thomson, G., 1983. African swine fever virus. Brief review.
Arch. Virol. 76 (2), 73–90.
Wardley, R.C., Wilkinson, P.J., 1978. The growth of virulent African swine fever virus
in pig monocytes and macrophages. J. Gen. Virol. 38 (1), 183–186.
Wardley, R.C., Wilkinson, P.J., 1977. The association of African swine fever virus with
blood components of infected pigs. Arch. Virol. 55 (4), 327–334.
Wilkinson, P.J., Donaldson, A.I., 1977. Transmission studies with African swine fever
virus. The early distribution of virus in pigs infected by airborne virus. J. Comp.
Pathol. 87 (3), 497–501.
Wilkinson, P.J., Donaldson, A.I., Greig, A., Bruce, W., 1977. Transmission studies with
African swine fever virus. Infections of pigs by airborne virus. J. Comp. Pathol.
87 (3), 487–495.
Wilkinson, P.J., Lawman, M.J., Johnston, R.S., 1980. African swine fever in Malta, 1978.
Vet. Rec. 106 (5), 94–97.
Wilkinson, P.J., Wardley, R.C., Williams, S.M., 1981. African swine fever virus
(Malta/78) in pigs. J. Comp. Pathol. 91 (2), 277–284.
Wilkinson, P.J., Wardley, R.C., Williams, S., 1983. Studies in pigs infected with African
swine fever virus (Malta/78). In: Wilkinson, P.J. (Ed.), CEC/FAO Expert Consulta-
tion on African Swine Fever Research. September 1981, Sardinia.
Zsak, L., Borca, M.V., Risatti, G.R., Zsak, A., French, R.A., Lu, Z., Kutish, G.F., Neilan,
J.G., Callahan, J.D., Nelson, W.M., Rock, D.L., 2005. Preclinical diagnosis of African
swine fever in contact-exposed swine by a real-time PCR assay. J. Clin. Microbiol.
43 (1), 112–119.
 viruses
Review
African Swine Fever Virus: A Review
Inmaculada Galindo and Covadonga Alonso *
Dpto. de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA),
Ctra. de la Coruña km 7.5, 28040 Madrid, Spain; galindo@inia.es
* Correspondence: calonso@inia.es

Academic Editors: Linda Dixon and Simon Graham

Received: 10 April 2017; Accepted: 4 May 2017; Published: 10 May 2017

Abstract: African swine fever (ASF) is a highly contagious viral disease of swine which causes high
mortality, approaching 100%, in domestic pigs. ASF is caused by a large, double stranded DNA virus,
ASF virus (ASFV), which replicates predominantly in the cytoplasm of macrophages and is the only
member of the Asfarviridae family, genus Asfivirus. The natural hosts of this virus include wild suids
and arthropod vectors of the Ornithodoros genus. The infection of ASFV in its reservoir hosts is usually
asymptomatic and develops a persistent infection. In contrast, infection of domestic pigs leads to a
lethal hemorrhagic fever for which there is no effective vaccine. Identification of ASFV genes involved
in virulence and the characterization of mechanisms used by the virus to evade the immune response
of the host are recognized as critical steps in the development of a vaccine. Moreover, the interplay of
the viral products with host pathways, which are relevant for virus replication, provides the basic
information needed for the identification of potential targets for the development of intervention
strategies against this disease.

Keywords: African swine fever virus; ASFV; virus entry; endocytosis; endosomal pathway; host cell
targets; cellular responses; ER stress; apoptosis; autophagy; A179L

1. Introduction
African swine fever (ASF) is a viral disease of swine that leads to a high mortality in domestic
pigs while being asymptomatic in the natural suid reservoir hosts [1–3]. It causes important economic
losses that are unavoidable in the absence of an effective vaccine and the available methods of disease
control are the quarantine of the affected area and the slaughter of the infected animals [4]. ASF is
caused by the ASF virus (ASFV), a double-stranded DNA virus with a complex molecular structure.
It is the only member of the Asfarviridae family [5] and the only DNA virus transmitted by arthropods,
soft ticks of the Ornithodoros genus [3,6]. Soft ticks (Ornithodoros moubata) are involved in the sylvatic
transmission cycle of the virus in Africa and O. erraticus in Europe. The wild boar that suffers an acute
disease similar to the domestic pig appears to be relevant in the transmission cycle in Europe.
The disease caused by this virus was first identified in Kenya in the 1920s [7]. Then, it was
confined to Africa until it spread to Europe in the middle of the last century, and later to South America
and the Caribbean. The disease was eradicated from Europe (except of Sardinia) at the 1990s via
drastic control and eradication programs. However, in 2007, the disease spread again out of Africa
into the Caucasus, especially Georgia, and in 2014 it reached the eastern territory of the European
Union. The latest reports of the disease include an increasing list of EU countries, Poland and the three
Baltic republics [8,9] and very recently Moldova [10]. Due to the absence of vaccines with protective
efficacy, ASF represents a serious threat to all European countries. The epidemiological complexity
of ASF has been clearly demonstrated in eastern and southern Africa, where genetic characterization
of ASFV based on sequence variation in the C-terminal region of the B646L gene encoding the major
capsid protein p72, revealed the presence of 22 genotypes [11,12]. Recently, a new genotype, genotype

Viruses 2017, 9, 103; doi:10.3390/v9050103 www.mdpi.com/journal/viruses

Viruses 2017, 9, 103 2 of 10

XXIII, that shares a common ancestor with genotypes IX and X, which comprise isolates circulating
in Eastern African countries and the Republic of Congo, has been described [13]. This review paper
summarizes the current state of knowledge about ASFV.

2. African Swine Fever Virus

ASFV is a large, enveloped virus with icosahedral morphology and an average diameter of 200 nm.
The viral genome consists of a single molecule of linear, covalently close-ended, double stranded DNA.
The genomes of different isolates vary in length between 170 and 190 Kbp and encode between 151
and 167 open reading frames. ASFV replication cycle is mainly cytoplasmic, but the nucleus is also a
site of viral DNA synthesis at early times [14,15]. The disassembly of the lamina network close to the
sites where the viral genome starts its replication and the redistribution of several nuclear proteins
suggests the existence of sophisticated mechanisms to regulate the nuclear machinery during viral
infection [16].
Transcription of viral genes is strongly regulated. Four classes of mRNAs have been identified
by their distinctive accumulation kinetics—including immediate–early, early, intermediate, and late
transcripts. Immediate–early and early genes are expressed before the onset of DNA replication,
whereas intermediate and late genes are expressed afterwards. The presence of intermediate genes
suggests a cascade model for the regulation of ASFV gene expression [17,18]. Enzymes required
for DNA replication are expressed immediately after virus entry into the cytoplasm from partially
uncoated core particles and using enzymes and other factors packaged in virus particles [17–20].
Virus morphogenesis takes place in the viral factories where the main late phase of DNA replication
also occurs.

3. Virion Structure
The ASFV particle has an icosahedral morphology composed of several concentric domains: the
internal core formed by the central genome contains the nucleoid, which is coated by a thick protein
layer named core shell; an inner lipid envelope surrounding the core; and finally the capsid, which is
the outermost layer of the intracellular virions [21]. The extracellular virions possess an additional
external envelope that is obtained when the virus buds out through the plasma membrane [22].
However, the importance of this envelope is unclear as it is not required for infectivity [23].

4. Viral Entry Mechanisms

The ASFV infectious cycle starts with the viral adsorption and entry into the host cell. Early
studies on ASFV entry characterized this event as a low pH- and temperature-dependent process
consistent with saturable and specific receptor-mediated endocytosis in Vero cells and porcine
macrophages [24–27]. However, the receptor(s) for the virus still remain unknown. The limited
cell tropism of ASFV suggests that a macrophage-specific receptor is required for infection. Successful
infection of porcine macrophages and monocytes by ASFV correlates to the expression of the CD163
scavenger receptor, a hallmark of macrophage maturation. It was previously suggested as a potential
virus receptor, as monoclonal antibodies against this molecule were able to block infection of primary
alveolar macrophages [28]. However, more recent studies have demonstrated that CD163 is not
necessary for infection with the Georgia 2007/1 virus isolates. Gene-edited pigs possessing a complete
knockout of CD163 produced using the CRISPR/Cas9 system showed no differences in clinical
signs, mortality, pathology, or viremia [29]. One conclusion from these studies was that CD163 may
be necessary but insufficient for infection, suggesting that other macrophage surface proteins may
participate in the infection process.
While there is support for receptor-dependent mechanisms of viral entry, such as
clathrin-mediated dynamin-dependent endocytosis [30,31], there is also evidence that ASFV exploits
other mechanisms, such as phagocytosis [32] and macropinocytosis [33,34]. Also, cholesterol is required
Viruses 2017, 9, 103 3 of 10

for a successful
Viruses 2017, 9, 103 entry. These mechanisms occur both in the macrophage target cell and in Vero3cells of 10
using viral isolates adapted to this cell line.
In addition,
addition, some
someASFV
ASFVproteins
proteinsare
areinvolved
involvedinin
thethe entry
entry mechanism
mechanism such
such as p30,
as p30, important
important for
for viral
viral internalization,
internalization, while
while other
other proteins
proteins suchsuch as and
as p12 p12 p54
and have
p54 have
been been identified
identified as potential
as potential viral
viral attachment
attachment proteins
proteins [35–37].
[35–37].

5. ASFV Enters the Endosomal Pathway

ASFV infection by either pathway of entry should finally reach the endocytic pathway pathway [38]. Once
the virus has entered the endocytic pathway, it it must
must passpass through
through different
different endosome
endosome populations
populations
to achieve a successful infection (Figure 1). Endocytic pathway maturation is carefully orchestrated
by proteins and lipids
lipids that
that are
arerecruited
recruitedto tothe
theendosomal
endosomalmembrane.
membrane.Rab Rab GTPase
GTPase protein
protein family
family is
is
thethe major
major regulator
regulator of of
thethe endosomal
endosomal maturation
maturation pathway,
pathway, where
where each
each member
member of of
thethe
RabRab family
family is
is specifically
specifically located
located to atodifferent
a different endosomal
endosomal compartment
compartment [39].[39]. Incoming
Incoming viruses
viruses are found
are found in
in early
early endosomes (EE) labeled with Rab5 and EEA1 markers from very few
endosomes (EE) labeled with Rab5 and EEA1 markers from very few minutes after adsorption. In minutes after adsorption.
In fact,
fact, complete
complete encapsidated
encapsidated virions
virions are only
are only found found
at theatlevel
the level
of EEofbut
EEnot
butinnot
otherin mature
other mature
acidic
acidic compartments
compartments [38].inhibition
[38]. The The inhibition of endosomal
of endosomal acidification
acidification with bafilomycin
with bafilomycin A1 prevents
A1 prevents viral
viral decapsidation
decapsidation and and
onlyonlyunderunder
thisthis condition
condition it is
it is possibletotoobserve
possible observecomplete
complete viruses
viruses inside
multivesicular endosomes positive for CD63 CD63 andand latelate endosomes
endosomes expressing
expressing Rab7.
Rab7. In normal
conditions, late endosomes harbor harbor only
only viral
viral cores
cores lacking
lacking thethe capsid
capsid protein
protein [38].
[38].

Figure 1.1. ASFV

ASFVenters the the
enters host host
cell through a complex
cell through processprocess
a complex involving dynamin-
involving and clathrin-
dynamin- and
mediated endocytosis
clathrin-mediated and macropinocytosis.
endocytosis Only few
and macropinocytosis. Onlyseconds later, later,
few seconds ASFVASFV
progresses through
progresses the
through
endocytic
the pathway
endocytic andand
pathway reaches
reachesmature
mature endosomal
endosomalcompartments
compartmentswherewhereviral
viral decapsidation
decapsidation and
envelope with
fusion of the inner viral envelope with the
the endosomal
endosomal membrane
membrane occurs.
occurs. Newly synthesized virions
exocytosis budding at the plasma
are assembled in the viral factory and will exit the cell either by exocytosis
membrane or through the formation
formation of of apoptotic
apoptotic bodies.
bodies.

Viral decapsidation
Viral decapsidation occurs
occurs at
at the
the acidic
acidic intraluminal
intraluminal pH
pH in
in mature
mature endosomal
endosomal compartments
compartments
between 30
between 30 and
and 45
45 min
min post
post infection
infection (mpi).
(mpi). Mature
Mature endosomal
endosomal compartments
compartments are
are multivesicular
multivesicular
bodies expressing CD63 that are characterized by the presence of intraluminal vesicles and also, late
endosomes expressing Rab7. Dependence on the endosomal maturation for sequential viral
uncoating and penetration has been also described for other viruses [40,41]. Once decapsidated, virus
particles expose the inner envelope which allows their interaction and subsequent fusion of this viral
Viruses 2017, 9, 103 4 of 10

bodies expressing CD63 that are characterized by the presence of intraluminal vesicles and also, late
endosomes expressing Rab7. Dependence on the endosomal maturation for sequential viral uncoating
and penetration has been also described for other viruses [40,41]. Once decapsidated, virus particles
expose the inner envelope which allows their interaction and subsequent fusion of this viral membrane
with the limiting membrane of the endosomes and naked cores can be released into cytosol in order
to start replication. This process is strongly dependent on the cholesterol efflux at the endosomal
membrane. In fact, blocking cholesterol transport at this level causes retention of virions inside
endosomes, inhibiting infection progression [42]. The inner envelope viral protein pE248R is also
involved in viral fusion. This protein shares sequence similarity with some members of the poxviral
entry/fusion complex [34].
Other inhibitors of endosome maturation such as wortmannin, a phosphatidylinositol 3
(PI3)-kinase inhibitor that blocks early endosome fusion, and nocodazole, an inhibitor that disturbs
microtubule-dependent endosomal transport also prevent ASFV infection [38].

6. ASFV Gene Expression and DNA Replication

Incoming ASFV virions should reach their replication site in the perinuclear area close to the
microtubule organizing center (MTOC) [43]. Immediate early and early genes are expressed before the
onset of DNA replication. Both DNA chains are alternatives used as the coding strand. This is possible
due to the action of several enzymes involved in viral transcription that are packed in the viral core.
Following DNA replication, the transcription of intermediate and late genes begins. ASFV commits
approximately 20% of its genome to encode genes involved in the transcription and modification of its
mRNAs. This transcriptional machinery gives to ASFV a relative independence from its host and an
accurate positional and temporal control of its gene expression [44]. The existence of a nuclear stage
in the replication of ASFV DNA has been determined by in situ hybridization and autoradiography
in thin sections of infected cells, although the precise role of the nucleus in viral replication remains
unclear. Sedimentation analysis of replicating viral DNA in alkaline sucrose gradients has shown
that small DNA fragments are pulse-labeled in the nucleus at early times in the replication of the
viral DNA, whereas larger molecules are synthesized in the cytoplasm at later times. The replicative
intermediates that are synthesized both in the nucleus and cytoplasm of ASFV-infected cells consist
of head to head concatemers. It is possible that the nucleus may provide small transcripts or other
factors required for priming virus replication or that an early stage of virus DNA replication should
take place [15].

7. Formation of the Viral Factory

Microtubules are required for the transport of the virus to perinuclear area, where replication takes
place. Integrity of microtubules is required for the formation of viral factories [45] and virus particles
are found associated to stabilized microtubules at entry [46]. Also, nocodazole, which interferes with
the polymerization of microtubule filaments, prevents the correct formation of factories [43,47–49].
Structural protein p54 interacts with the dynein motor protein during virus infection and could
constitute a molecular mechanism for microtubule-mediated virus transport [43].
The viral factory, localized in the cytoplasm close to the nucleus, can be described as a single
and large perinuclear area at the MTOC. On this site, viral proteins and DNA are accumulated and
newly synthesized virions are assembled. A cage made of intermediate filament vimentin surrounds
the viral factory likely to prevent the sensing of viral components into the cytoplasm and concentrate
structural proteins at sites of assembly [50]. There are many features shared between aggresomes
and VFs. However, neither HDAC6 nor Bag3 are required for factory formation, suggesting that
aggresomes and viral factories are not the same structures [51].
Formation of viral replication sites depends on several cellular determinants. For example, Rho
GTPase inhibitors produce an abnormal viral factory size with the accumulation of envelope precursors
and immature virions [46]. This specialized site at the MTOC, contains viral DNA, most of the viral
Viruses 2017, 9, 103 5 of 10

proteins, immature and mature virions, and also abundant virus-induced membranes. Viral factories
contain precursors that develop into icosahedral intermediates by the assembly of the icosahedral
capsid and the core shell domain. The last step of virion morphogenesis will be the encapsidation
of DNA giving rise to mature virions. Finally, the newly formed virus leaves the factory and is
transported to the cell surface by kinesin where it is released by budding [50]. Extracellular virus is
covered by an additional external envelope that is acquired during this budding process [22].

8. ER Stress and Unfolded Protein Response

The virus modifies and interacts with cellular pathways in response to infection. The endoplasmic
reticulum (ER) is an essential organelle for ASFV replication and maturation and a large number of
viral proteins are synthesized in infected cells and accumulated in the ER during the viral life cycle.
This process can trigger ER stress and the unfolded protein response (UPR) of the host cell as the
induction of caspase 12 indicates [52]. Viruses have evolved various mechanisms to counteract these
cellular responses that would limit or inhibit viral replication. This response is a regulatory program
that upregulates a large number of genes, such as ER chaperones and ER-associated degradation
(ERAD) components, which increase the folding capacity of the ER. ASFV induces the upregulation of
the chaperones calnexin and calreticulin, but not ERp57, PDI [52], or BiP/Grp78 [52,53]. Moreover,
ASFV induces selectively the transcription factor 6 (ATF6) signaling pathway of the UPR, but not the
protein kinase-like ER kinase (PERK) or the inositol-requiring enzyme 1 (IRE1) pathways. Thus, the
capacity of ASFV to regulate the UPR signaling cascade may prevent the effects that are detrimental to
the infection, while maintaining those that are beneficial. Importantly, viral protein DP71L is involved
in ATF4 downregulation and in CHOP inhibition [54]. DP71L, homolog of the neurovirulence factor
ICP34.5 of HSV-1 and the cellular gene GADD34, binds to catalytic subunit of protein phosphatase
1 (PP1) and causes the dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 α),
thereby preventing the inhibition of protein synthesis produced by ER stress and the UPR [55].

9. ASFV and Apoptosis

ER stress after ASFV infection is reflected by the activation of caspase 12, which follows similar
temporal dynamics to the activation of mitochondrial caspase 9 and effector caspase 3 [52]. Apoptosis
represents an important innate cellular mechanism to prevent virus infection, and many viruses
have developed strategies for inhibiting or delaying this cellular response in turn [56]. Thus, ASFV
A179L gene encodes an homolog of antiapoptotic Bcl-2 protein to prolong host cell survival until
the replication of the viral genome is completed [57]. This viral Bcl-2 is expressed both at early and
late times after infection and inhibits the action of several pro-apoptotic BH3-only proteins, known
to be rapid inducers of apoptosis, such as activated Bid, BimL, BimS, BimEL, Bad, Bmf, Bik, Puma,
and DP5 [58]. Another ASFV gene, A224L, encodes a member of the family of apoptosis inhibitors
known as IAP proteins and is able to inhibit caspase activation and to promote cell survival [59,60].
Viral IAP not only blocks caspase-3 activation but also activates NF-κB [61]. It is interesting that the
virus encodes an IκB-like molecule (A238L) that interferes with NF-κB activation [62]. A238L and
A224L are expressed at different times during ASFV infection, this suggests that ASFV requires a
low NF-κB activity at early times of infection to avoid immune responses but a higher activity at
late times, probably to prevent apoptosis as the cellular systems are abused [61]. At very late stages
of ASFV infection, infected cells undergo apoptosis [63] and show the characteristic morphological
changes of programmed cell death, including typical membrane blebbing of the infected cell that led
to the formation of numerous vesicles containing virus [45] and this could be an efficient system for
virus spread.

10. ASFV and Autophagy

A179L, the viral Bcl2 homolog of African swine fever virus, not only interacts with pro-apoptotic
Bcl2 family proteins to inhibit apoptosis but also inhibits autophagy by interacting with Beclin1
Viruses 2017, 9, 103 6 of 10

through its BH3 homology domain. ASFV is armed to counteract elimination by autophagy as other
DNA viruses. An example of that is the HSV-1 ICP34.5 protein, which inhibits autophagy by targeting
Beclin1 [64]. ASFV encodes a protein homologous to ICP34.5, which exerts other functions. ASFV
DP71L inhibits the ER stress response activating PP1/protein phosphatase 1 [55]. However, in contrast
to HSV-1 ICP34.5, it does not interact with Beclin1 [65].
In fact, ASFV infection does not induce LC3 activation or autophagosome formation in Vero
cells [65]. Autophagy is a relevant cellular defense mechanism that allows the orderly degradation
and recycling of cellular components. Autophagy eliminates intracellular pathogens and has a crucial
role for innate and adaptive immune responses. Some DNA viruses, such as ASFV and HSV-1, have
developed strategies to keep this cellular response under control to prevent the degradation of newly
assembled virions. In contrast, most RNA viruses have been reported to induce autophagy in infected
cells, and in several cases autophagy may enhance viral replication [66].

11. ASFV Egress

The mature particle is transported from the virus factories to the cell surface through a
microtubule-mediated mechanism [48] depending on the motor protein conventional kinesin [67] and
on the capsid protein pE120R [68]. Once on the cell surface, particles exit the host cell by budding at
the membrane, acquiring an additional envelope [22]. Both intracellular and extracellular viruses are
infectious but structurally and antigenically different [68], and this could have important implications
in the host immune response against ASFV.

12. Potential Vaccines and Antivirals

Previous attempts to develop vaccines against ASFV have failed to induce protective immunity.
Currently, there are several reports of the protection elicited by experimental vaccines based in live
attenuated ASFV (LAV) containing single or double gene deletions in the genome [69] and other
different approaches, including DNA vaccines [70,71]. Some of them might turn into successful
vaccine candidates. Nowadays, a vaccine to be used in the field should meet a number of requirements.
Any potential ASFV vaccine should include markers for differentiation between infected or vaccinated
animals that allows for DIVA diagnostics. Also, vaccine production could not be possible because
of the lack of a cell line supporting the replication of attenuated vaccine viruses without modifying
the virus virulence. At present, wild boars were found to be major players of disease spread in both
the Baltics and Poland. Hence, a live vaccine given as bait for these animals could be crucial to limit
disease spread. In the current scenario, a vaccine would make possible to eradicate the disease together
with infection surveillance by diagnostics. Available diagnostic methods allow both virus detection
and also detection of antibodies for identification of survivors and asymptomatic carriers [72].
Until an effective vaccine is developed, the possibility of using antivirals remains. Antiviral
strategies have been extensively applied in human infections but can be used in animal health.
Antivirals are useful for an early control of virus spread after an outbreak. In addition, the
combination of antivirals with vaccination protocols could be applied to elicit the immune response
required for effective protection against the disease. Here, we have reviewed potential molecular
targets to be considered as targets for antivirals. Antivirals against ASFV described include
resveratrol and oxyresveratrol [73], microalgae [74], cholesterol lowering drugs [46] or inhibitors
of cholesterol transport [42], antitumoral lauryl-gallate [75], anticonvulsivant valproic acid [75],
dynamin inhibitors [38], fluoroquinolones [76], serine protease inhibitors [52], specific peptides [77],
and miscelanea [32]. These could be eventually applied in a quick response to reduce susceptible
animals in order to create ‘safe areas’ around the outbreaks and thus control the spread of the infection.

Acknowledgments: This work was funded by grant AGL2015-69598-R of the Ministerio de Economía, Industria
y Competitividad of Spain.
Conflicts of Interest: The authors declare no conflict of interest.
Viruses 2017, 9, 103 7 of 10

References
1. Parker, J.; Plowright, W.; Pierce, M.A. The epizootiology of african swine fever in Africa. Vet. Rec. 1969, 85,
668–674. [PubMed]
2. Thomson, G.R.; Gainaru, M.D.; van Dellen, A.F. Experimental infection of warthos (Phacochoerus aethiopicus)
with african swine fever virus. Onderstepoort J. Vet. Res. 1980, 47, 19–22. [PubMed]
3. Anderson, E.C.; Hutchings, G.H.; Mukarati, N.; Wilkinson, P.J. African swine fever virus infection of the
bushpig (Potamochoerus porcus) and its significance in the epidemiology of the disease. Vet. Microbiol. 1998,
62, 1–15. [CrossRef]
4. Penrith, M.L.; Vosloo, W. Review of african swine fever: Transmission, spread and control. J. S. Afr. Vet.
Assoc. 2009, 80, 58–62. [CrossRef] [PubMed]
5. Dixon, L.K.; Escribano, J.M.; Martins, C.; Rock, D.L.; Salas, M.L.; Wilkinson, P.J. Asfarviridae. In Virus
Taxonomy. VIIIth Report of the ICTV; Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U., Ball, L.A., Eds.;
Elsevier/Academic Press: London, UK, 2005; pp. 135–143.
6. Kleiboeker, S.B.; Scoles, G.A.; Burrage, T.G.; Sur, J. African swine fever virus replication in the midgut
epithelium is required for infection of ornithodoros ticks. J. Virol. 1999, 73, 8587–8598. [PubMed]
7. Montgomery, R.E. On a form of swine fever occurring in british east Africa (Kenya Colony). J. Comp. Pathol.
Ther. 1921, 34, 159–191. [CrossRef]
8. Wozniakowski, G.; Kozak, E.; Kowalczyk, A.; Lyjak, M.; Pomorska-Mol, M.; Niemczuk, K.; Pejsak, Z. Current
status of african swine fever virus in a population of wild boar in eastern Poland (2014–2015). Arch. Virol.
2016, 161, 189–195. [CrossRef] [PubMed]
9. Pejsak, Z.; Truszczynski, M.; Niemczuk, K.; Kozak, E.; Markowska-Daniel, I. Epidemiology of african swine
fever in Poland since the detection of the first case. Pol. J. Vet. Sci. 2014, 17, 665–672. [CrossRef] [PubMed]
10. OIE-WAHID. World Animal Health Information Database (Wahid) [Database on the Internet]. World
Organisation for Animal Health (OIE), 2017. [Cited World Animal Health Information System (WAHIS)].
Available online: http://www.Oie.Int/wahis/public.Php?Page=home (accessed on 27 March 2017).
11. Boshoff, C.I.; Bastos, A.D.; Gerber, L.J.; Vosloo, W. Genetic characterisation of african swine fever viruses
from outbreaks in southern Africa (1973–1999). Vet. Microbiol. 2007, 121, 45–55. [CrossRef] [PubMed]
12. Bastos, A.D.; Penrith, M.L.; Cruciere, C.; Edrich, J.L.; Hutchings, G.; Roger, F.; Couacy-Hymann, E.;
Thomson, G.R. Genotyping field strains of african swine fever virus by partial p72 gene characterisation.
Arch. Virol. 2003, 148, 693–706. [CrossRef] [PubMed]
13. Achenbach, J.E.; Gallardo, C.; Nieto-Pelegrin, E.; Rivera-Arroyo, B.; Degefa-Negi, T.; Arias, M.; Jenberie, S.;
Mulisa, D.D.; Gizaw, D.; Gelaye, E.; et al. Identification of a new genotype of african swine fever virus in
domestic pigs from Ethiopia. Transbound. Emerg. Dis. 2016. [CrossRef] [PubMed]
14. Garcia-Beato, R.; Salas, M.L.; Vinuela, E.; Salas, J. Role of the host cell nucleus in the replication of African
swine fever virus DNA. Virology 1992, 188, 637–649. [CrossRef]
15. Rojo, G.; Garcia-Beato, R.; Vinuela, E.; Salas, M.L.; Salas, J. Replication of african swine fever virus DNA in
infected cells. Virology 1999, 257, 524–536. [CrossRef] [PubMed]
16. Ballester, M.; Rodriguez-Carino, C.; Perez, M.; Gallardo, C.; Rodriguez, J.M.; Salas, M.L.; Rodriguez, F.
Disruption of nuclear organization during the initial phase of african swine fever virus infection. J. Virol.
2011, 85, 8263–8269. [CrossRef] [PubMed]
17. Almazan, F.; Rodriguez, J.M.; Andres, G.; Perez, R.; Vinuela, E.; Rodriguez, J.F. Transcriptional analysis of
multigene family 110 of african swine fever virus. J. Virol. 1992, 66, 6655–6667. [PubMed]
18. Rodriguez, J.M.; Salas, M.L.; Vinuela, E. Intermediate class of mRNAs in African swine fever virus. J. Virol.
1996, 70, 8584–8589. [PubMed]
19. Almazan, F.; Rodriguez, J.M.; Angulo, A.; Vinuela, E.; Rodriguez, J.F. Transcriptional mapping of a late gene
coding for the p12 attachment protein of African swine fever virus. J. Virol. 1993, 67, 553–556. [PubMed]
20. Salas, M.L.; Kuznar, J.; Vinuela, E. Polyadenylation, methylation, and capping of the RNA synthesized
in vitro by African swine fever virus. Virology 1981, 113, 484–491. [CrossRef]
21. Salas, M.L.; Andres, G. African swine fever virus morphogenesis. Virus Res. 2013, 173, 29–41. [CrossRef]
[PubMed]
22. Breese, S.S., Jr.; de Boer, C.J. Electron microscope observations of African swine fever virus in tissue culture
cells. Virology 1966, 28, 420–428. [CrossRef]
Viruses 2017, 9, 103 8 of 10

23. Schloer, G.M. Polypeptides and structure of African swine fever virus. Virus Res. 1985, 3, 295–310. [CrossRef]
24. Valdeira, M.L.; Geraldes, A. Morphological study on the entry of African swine fever virus into cells. Biol.
Cell 1985, 55, 35–40. [CrossRef] [PubMed]
25. Alcami, A.; Carrascosa, A.L.; Vinuela, E. Interaction of African swine fever virus with macrophages. Virus
Res. 1990, 17, 93–104. [CrossRef]
26. Alcami, A.; Carrascosa, A.L.; Vinuela, E. The entry of African swine fever virus into vero cells. Virology 1989,
171, 68–75. [CrossRef]
27. Alcami, A.; Carrascosa, A.L.; Vinuela, E. Saturable binding sites mediate the entry of African swine fever
virus into vero cells. Virology 1989, 168, 393–398. [CrossRef]
28. Sanchez-Torres, C.; Gomez-Puertas, P.; Gomez-del-Moral, M.; Alonso, F.; Escribano, J.M.; Ezquerra, A.;
Dominguez, J. Expression of porcine cd163 on monocytes/macrophages correlates with permissiveness to
African swine fever infection. Arch. Virol. 2003, 148, 2307–2323. [CrossRef] [PubMed]
29. Popescu, L.; Gaudreault, N.N.; Whitworth, K.M.; Murgia, M.V.; Nietfeld, J.C.; Mileham, A.; Samuel, M.;
Wells, K.D.; Prather, R.S.; Rowland, R.R. Genetically edited pigs lacking cd163 show no resistance following
infection with the African swine fever virus isolate, georgia 2007/1. Virology 2017, 501, 102–106. [CrossRef]
[PubMed]
30. Galindo, I.; Cuesta-Geijo, M.A.; Hlavova, K.; Munoz-Moreno, R.; Barrado-Gil, L.; Dominguez, J.; Alonso, C.
African swine fever virus infects macrophages, the natural host cells, via clathrin and cholesterol-dependent
endocytosis. Virus Res. 2015, 200, 45–55. [CrossRef] [PubMed]
31. Hernaez, B.; Alonso, C. Dynamin and clathrin-dependent endocytosis in African swine fever virus entry.
J. Virol. 2010, 84, 2100–2109. [CrossRef] [PubMed]
32. Basta, S.; Gerber, H.; Schaub, A.; Summerfield, A.; McCullough, K.C. Cellular processes essential for African
swine fever virus to infect and replicate in primary macrophages. Vet. Microbiol. 2010, 140, 9–17. [CrossRef]
[PubMed]
33. Sanchez, E.G.; Quintas, A.; Perez-Nunez, D.; Nogal, M.; Barroso, S.; Carrascosa, A.L.; Revilla, Y. African
swine fever virus uses macropinocytosis to enter host cells. PLoS Pathog. 2012, 8, e1002754. [CrossRef]
[PubMed]
34. Hernaez, B.; Guerra, M.; Salas, M.L.; Andres, G. African swine fever virus undergoes outer envelope
disruption, capsid disassembly and inner envelope fusion before core release from multivesicular endosomes.
PLoS Pathog. 2016, 12, e1005595. [CrossRef] [PubMed]
35. Gomez-Puertas, P.; Rodriguez, F.; Oviedo, J.M.; Brun, A.; Alonso, C.; Escribano, J.M. The african swine fever
virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the
antibody-mediated protective immune response. Virology 1998, 243, 461–471. [CrossRef] [PubMed]
36. Angulo, A.; Vinuela, E.; Alcami, A. Comparison of the sequence of the gene encoding African swine fever
virus attachment protein p12 from field virus isolates and viruses passaged in tissue culture. J. Virol. 1992,
66, 3869–3872. [PubMed]
37. Angulo, A.; Vinuela, E.; Alcami, A. Inhibition of African swine fever virus binding and infectivity by purified
recombinant virus attachment protein p12. J. Virol. 1993, 67, 5463–5471. [PubMed]
38. Cuesta-Geijo, M.A.; Galindo, I.; Hernaez, B.; Quetglas, J.I.; Dalmau-Mena, I.; Alonso, C. Endosomal
maturation, Rab7 GTPase and phosphoinositides in African swine fever virus entry. PLoS ONE 2012,
7, e48853. [CrossRef] [PubMed]
39. Stenmark, H. Rab GTPases as coordinators of vesicle traffic. Nat. Rev. Mol. Cell Biol. 2009, 10, 513–525.
[CrossRef] [PubMed]
40. Lozach, P.Y.; Mancini, R.; Bitto, D.; Meier, R.; Oestereich, L.; Overby, A.K.; Pettersson, R.F.; Helenius, A. Entry
of bunyaviruses into mammalian cells. Cell Host Microbe 2010, 7, 488–499. [CrossRef] [PubMed]
41. Pasqual, G.; Rojek, J.M.; Masin, M.; Chatton, J.Y.; Kunz, S. Old world arenaviruses enter the host cell via the
multivesicular body and depend on the endosomal sorting complex required for transport. PLoS Pathog.
2011, 7, e1002232. [CrossRef]
42. Cuesta-Geijo, M.A.; Chiappi, M.; Galindo, I.; Barrado-Gil, L.; Munoz-Moreno, R.; Carrascosa, J.L.; Alonso, C.
Cholesterol flux is required for endosomal progression of african swine fever virions during the initial
establishment of infection. J. Virol. 2015, 90, 1534–1543. [CrossRef] [PubMed]
Viruses 2017, 9, 103 9 of 10

43. Alonso, C.; Miskin, J.; Hernaez, B.; Fernandez-Zapatero, P.; Soto, L.; Canto, C.; Rodriguez-Crespo, I.; Dixon, L.;
Escribano, J.M. African swine fever virus protein p54 interacts with the microtubular motor complex through
direct binding to light-chain dynein. J. Virol. 2001, 75, 9819–9827. [CrossRef] [PubMed]
44. Rodriguez, J.M.; Salas, M.L. African swine fever virus transcription. Virus Res. 2013, 173, 15–28. [CrossRef]
[PubMed]
45. Hernaez, B.; Escribano, J.M.; Alonso, C. Visualization of the African swine fever virus infection in living cells
by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera. Virology
2006, 350, 1–14. [CrossRef] [PubMed]
46. Quetglas, J.I.; Hernaez, B.; Galindo, I.; Munoz-Moreno, R.; Cuesta-Geijo, M.A.; Alonso, C. Small Rho GTPases
and cholesterol biosynthetic pathway intermediates in African swine fever virus infection. J. Virol. 2012, 86,
1758–1767. [CrossRef] [PubMed]
47. Carvalho, Z.G.; de Matos, A.P.; Rodrigues-Pousada, C. Association of African swine fever virus with the
cytoskeleton. Virus Res. 1988, 11, 175–192. [CrossRef]
48. De Matos, A.P.; Carvalho, Z.G. African swine fever virus interaction with microtubules. Biol. Cell 1993, 78,
229–234. [CrossRef]
49. Heath, C.M.; Windsor, M.; Wileman, T. Aggresomes resemble sites specialized for virus assembly. J. Cell Biol.
2001, 153, 449–455. [CrossRef] [PubMed]
50. Stefanovic, S.; Windsor, M.; Nagata, K.I.; Inagaki, M.; Wileman, T. Vimentin rearrangement during African
swine fever virus infection involves retrograde transport along microtubules and phosphorylation of
vimentin by calcium calmodulin kinase II. J. Virol. 2005, 79, 11766–11775. [CrossRef] [PubMed]
51. Munoz-Moreno, R.; Barrado-Gil, L.; Galindo, I.; Alonso, C. Analysis of HDAC6 and BAG3-aggresome
pathways in African swine fever viral factory formation. Viruses 2015, 7, 1823–1831. [CrossRef] [PubMed]
52. Galindo, I.; Hernaez, B.; Munoz-Moreno, R.; Cuesta-Geijo, M.A.; Dalmau-Mena, I.; Alonso, C. The ATF6
branch of unfolded protein response and apoptosis are activated to promote African swine fever virus
infection. Cell Death Dis. 2012, 3, e341. [CrossRef] [PubMed]
53. Netherton, C.L.; Parsley, J.C.; Wileman, T. African swine fever virus inhibits induction of the stress-induced
proapoptotic transcription factor CHOP/GADD153. J. Virol. 2004, 78, 10825–10828. [CrossRef] [PubMed]
54. Zhang, F.; Moon, A.; Childs, K.; Goodbourn, S.; Dixon, L.K. The African swine fever virus DP71L protein
recruits the protein phosphatase 1 catalytic subunit to dephosphorylate eIF2alpha and inhibits chop induction
but is dispensable for these activities during virus infection. J. Virol. 2010, 84, 10681–10689. [CrossRef]
[PubMed]
55. Rivera, J.; Abrams, C.; Hernaez, B.; Alcazar, A.; Escribano, J.M.; Dixon, L.; Alonso, C. The MyD116 African
swine fever virus homologue interacts with the catalytic subunit of protein phosphatase 1 and activates its
phosphatase activity. J. Virol. 2007, 81, 2923–2929. [CrossRef] [PubMed]
56. Benedict, C.A.; Norris, P.S.; Ware, C.F. To kill or be killed: Viral evasion of apoptosis. Nat. Immunol. 2002, 3,
1013–1018. [CrossRef] [PubMed]
57. Brun, A.; Rivas, C.; Esteban, M.; Escribano, J.M.; Alonso, C. African swine fever virus gene a179l, a viral
homologue of BCL-2, protects cells from programmed cell death. Virology 1996, 225, 227–230. [CrossRef]
[PubMed]
58. Galindo, I.; Hernaez, B.; Diaz-Gil, G.; Escribano, J.M.; Alonso, C. A179l, a viral BCL-2 homologue, targets the
core BCL-2 apoptotic machinery and its upstream BH3 activators with selective binding restrictions for bid
and noxa. Virology 2008, 375, 561–572. [CrossRef] [PubMed]
59. Chacon, M.R.; Almazan, F.; Nogal, M.L.; Vinuela, E.; Rodriguez, J.F. The African swine fever virus IAP
homolog is a late structural polypeptide. Virology 1995, 214, 670–674. [CrossRef] [PubMed]
60. Nogal, M.L.; Gonzalez de Buitrago, G.; Rodriguez, C.; Cubelos, B.; Carrascosa, A.L.; Salas, M.L.; Revilla, Y.
African swine fever virus IAP homologue inhibits caspase activation and promotes cell survival in
mammalian cells. J. Virol. 2001, 75, 2535–2543. [CrossRef] [PubMed]
61. Rodriguez, C.I.; Nogal, M.L.; Carrascosa, A.L.; Salas, M.L.; Fresno, M.; Revilla, Y. African swine fever virus
IAP-like protein induces the activation of nuclear factor kappa B. J. Virol. 2002, 76, 3936–3942. [CrossRef]
[PubMed]
62. Revilla, Y.; Callejo, M.; Rodriguez, J.M.; Culebras, E.; Nogal, M.L.; Salas, M.L.; Vinuela, E.; Fresno, M.
Inhibition of nuclear factor kappa b activation by a virus-encoded I kappa b-like protein. J. Biol. Chem. 1998,
273, 5405–5411. [CrossRef] [PubMed]
Viruses 2017, 9, 103 10 of 10

63. Ramiro-Ibanez, F.; Ortega, A.; Brun, A.; Escribano, J.M.; Alonso, C. Apoptosis: A mechanism of cell killing
and lymphoid organ impairment during acute african swine fever virus infection. J. Gen. Virol. 1996, 77,
2209–2219. [CrossRef] [PubMed]
64. Orvedahl, A.; Alexander, D.; Talloczy, Z.; Sun, Q.; Wei, Y.; Zhang, W.; Burns, D.; Leib, D.A.; Levine, B. HSV-1
ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe 2007, 1, 23–35.
[CrossRef] [PubMed]
65. Hernaez, B.; Cabezas, M.; Munoz-Moreno, R.; Galindo, I.; Cuesta-Geijo, M.A.; Alonso, C. A179l, a new viral
Bcl2 homolog targeting Beclin 1 autophagy related protein. Curr. Mol. Med. 2013, 13, 305–316. [CrossRef]
[PubMed]
66. Klionsky, D.J. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition).
Autophagy 2016, 12, 1–222. [CrossRef] [PubMed]
67. Jouvenet, N.; Monaghan, P.; Way, M.; Wileman, T. Transport of african swine fever virus from assembly
sites to the plasma membrane is dependent on microtubules and conventional kinesin. J. Virol. 2004, 78,
7990–8001. [CrossRef] [PubMed]
68. Andres, G.; Garcia-Escudero, R.; Vinuela, E.; Salas, M.L.; Rodriguez, J.M. African swine fever virus structural
protein pE120R is essential for virus transport from assembly sites to plasma membrane but not for infectivity.
J. Virol. 2001, 75, 6758–6768. [CrossRef] [PubMed]
69. O’Donnell, V.; Risatti, G.R.; Holinka, L.G.; Krug, P.W.; Carlson, J.; Velazquez-Salinas, L.; Azzinaro, P.A.;
Gladue, D.P.; Borca, M.V. Simultaneous deletion of the 9gl and uk genes from the African swine fever virus
georgia 2007 isolate offers increased safety and protection against homologous challenge. J. Virol. 2017, 91,
e01760-16.
70. Lacasta, A.; Ballester, M.; Monteagudo, P.L.; Rodriguez, J.M.; Salas, M.L.; Accensi, F.; Pina-Pedrero, S.;
Bensaid, A.; Argilaguet, J.; Lopez-Soria, S.; et al. Expression library immunization can confer protection
against lethal challenge with african swine fever virus. J. Virol. 2014, 88, 13322–13332. [CrossRef] [PubMed]
71. Reis, A.L.; Abrams, C.C.; Goatley, L.C.; Netherton, C.; Chapman, D.G.; Sanchez-Cordon, P.; Dixon, L.K.
Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces
virulence in domestic pigs and induces a protective response. Vaccine 2016, 34, 4698–4705. [CrossRef]
[PubMed]
72. Cubillos, C.; Gomez-Sebastian, S.; Moreno, N.; Nunez, M.C.; Mulumba-Mfumu, L.K.; Quembo, C.J.; Heath, L.;
Etter, E.M.; Jori, F.; Escribano, J.M.; et al. African swine fever virus serodiagnosis: A general review with a
focus on the analyses of african serum samples. Virus Res. 2013, 173, 159–167. [CrossRef] [PubMed]
73. Galindo, I.; Hernaez, B.; Berna, J.; Fenoll, J.; Cenis, J.L.; Escribano, J.M.; Alonso, C. Comparative inhibitory
activity of the stilbenes resveratrol and oxyresveratrol on african swine fever virus replication. Antivir. Res.
2011, 91, 57–63. [CrossRef] [PubMed]
74. Fabregas, J.; Garcia, D.; Fernandez-Alonso, M.; Rocha, A.I.; Gomez-Puertas, P.; Escribano, J.M.; Otero, A.;
Coll, J.M. In vitro inhibition of the replication of haemorrhagic septicaemia virus (VHSV) and African swine
fever virus (ASFV) by extracts from marine microalgae. Antivir. Res. 1999, 44, 67–73. [CrossRef]
75. Hurtado, C.; Bustos, M.J.; Sabina, P.; Nogal, M.L.; Granja, A.G.; Gonzalez, M.E.; Gonzalez-Porque, P.;
Revilla, Y.; Carrascosa, A.L. Antiviral activity of lauryl gallate against animal viruses. Antivir. Ther. 2008, 13,
909–917. [PubMed]
76. Mottola, C.; Freitas, F.B.; Simoes, M.; Martins, C.; Leitao, A.; Ferreira, F. In vitro antiviral activity of
fluoroquinolones against African swine fever virus. Vet. Microbiol. 2013, 165, 86–94. [CrossRef] [PubMed]
77. Hernaez, B.; Tarrago, T.; Giralt, E.; Escribano, J.M.; Alonso, C. Small peptide inhibitors disrupt a high-affinity
interaction between cytoplasmic dynein and a viral cargo protein. J. Virol. 2010, 84, 10792–10801. [CrossRef]
[PubMed]

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).