PENGANTAR VIROLOGI

Dr. H. Yuwono
Departemen Mikrobiologi FK Unsri
Palembang
 Virus
• Jasad renik penyebab infeksi (parasit obligat
intrasel) dg ukuran 20-300 nm, dan hanya
mengandung satu jenis asam nukleat
(DNA/RNA) sebagai genomnya.
• Ada yang memiliki selubung dan ada yg tak
berselubung (envelope) di bagian luarnya.
• Yg memiliki selubung tidak tahan terhadap
ether.
 Karakteristik
• Replikasi (perbanyakan diri) hanya dalam sel hidup
• Genome (pembawa sifat) berupa DNA atau RNA

Klasifikasi virus,berdasarkan beberapa sifatnya :

• Jenis asam nukleat :DNA/RNA, rantai tunggal ata ganda
• Ukuran & bentuk : jenis simetri,jlh kapsomer,dan adanya selubung
• Kandungan enzim tertentu : polimerase,neuraminidase
• Kepekaan terhadap ether
• Sifat imunologik
• Cara penularan (transmisi virus)
• Inang ,tropisme sel dan jaringan
• Patologi dan pembentukan badan inklusi
• Simptom penyakit.
 Virus DNA
Tanpa selubung
1.parvovirus
2.Papova (papilloma,polyoma,vacuolating ) virus.
3.adenovirus
Berselubung
1.herpesvirus
2.hepatitis B virus
3.poxvirus
 Virus RNA
1. Tidak berselubung :
a.Picorna
b.REO
2. Berselubung :
a.toga viru
b.arena
c.Corona
d.Retro
e.Bunya
f.Orthomyxo
g.Paramyxo
h.Rhabdo
3. Campuran : ARBO virus
 Identifikasi
• Pembiakan virus :
• Hidup dalam media yang mengandung sel hidup ,yaitu

• 1.telur ayam berembryo

• a. membrana chorioallantoic
• terjadi pembentukan plaque/pocks formation
• contoh : herpes, variola & vaccinia.
• b. pembentukan hemaglutinin
• virus infuenza (orthomyxovirus)
• c. multiplikasi virus
• virus polio tipe 2
• d. embryo mati : virus encephalitis
 Virion Structure
Lipid Envelope Nucleic Acid

Protein
Capsid

Virion
Associated
Spike
Polymerase
Projections
 Virion Components
• Protein
– Structural proteins
– Membrane proteins
– Receptor recognition
– Enzymes
• Genomic nucleic Acid
– DNA
– RNA
• Lipid envelope
– Plasma membrane – Paramyxoviruses
– Nuclear membrane – Herpes viruses
– Golgi membrane - Bunyaviruses
 Virus replication
Maturation
Release

Virion Budding
attachment
to cellular Insertion of virus
receptors proteins into
Genomic membrane Virion
nucleic acid Assembly
Uncoating synthesis
Newly synthesised
virus proteins

Replication of Protein
Genomic Synthesis
nucleic acid mRNA synthesis
 Examples of virus receptors

Virus Cellular receptor

Sialic acid containing glycoproteins or
Influenza virus
glycolipids

Rhinovirus: ICAM-1
Picornavirus
Poliovirus: CD155
HIV CD4
 HIV Receptor Interaction
Fusion Peptide
CD4 Binding CCR5 Binding Exposure

gp120
gp41

V3
CD4

CCR5
 Virus replication
Maturation
Release

Virion Budding
attachment
to cellular Insertion of virus
receptors proteins into
Genomic membrane Virion
nucleic acid Assembly
Uncoating synthesis
Newly synthesised
virus proteins

Replication of Protein
Genomic Synthesis
nucleic acid mRNA synthesis
 Influenza virus particle

Influenza Virus Haemagglutinin

Particle Spike
 Influenza virus entry
Plasma Membrane
Endosome
Pore
H+ Complex

1 vRNPs
2
Virus
Attachment Coated pit
H+ 3
M1

Extracellular Intracellular Nuclear

Envelope
 Influenza A virus Infection
• 15 HA and 9 NA subtypes identified.
• Three major pandemics in the last century:
– 1918 - H1N1
– 1957 - H2N2
– 1968 - H3N2
• The HA protein evolves at a rate of 3.6 x 103 nucleotide
changes/nucleotide site/year.
• Avian influenza virus infects in the gastrointestinal tract of
birds, although pathogenic strains spread beyond these sites.
• Human influenza virus infects at the respiratory tract and
causes the systemic infections through abnormal cytokine
responses.
 Virus replication
Maturation
Release

Virion Budding
attachment
to cellular Insertion of virus
receptors proteins into
Genomic membrane Virion
nucleic acid Assembly
Uncoating synthesis
Newly synthesised
virus proteins

Replication of Protein
Genomic Synthesis
nucleic acid mRNA synthesis
 Baltimore Classification of Viruses
Group Genome Replication Example
1 dsDNA dsDNA mRNA Herpes simplex
virus

2 ssDNA ssDNA dsDNA mRNA Parvovirus

3 dsRNA dsRNA mRNA Reovirus

4 +ve ssRNA dsRNA +ve ssRNA [Acts as mRNA] Enterovirus

5 -ve ssRNA dsRNA -ve ssRNA mRNA Influenza A

virus

6 ssRNA ssRNA dsDNA mRNA Retrovirus

(e.g. HIV)

7 Nicked dsDNA nicked dsDNA intact dsDNA mRNA Hepatitis B

virus
RNA
 Virus Groups
1 dsDNA dsDNA mRNA Herpes simplex
virus

• Some members possess large DNA genomes encoding a range of enzymes

involved in nucleic acid synthesis.
• Depending on virus group viruses show temporal regulation of protein
synthesis.

2 ssDNA ssDNA dsDNA mRNA Parvovirus

• Small DNA genomes with limited coding capacity.

• Some members of this group are dependant upon other viruses for their
replication.
 Virus Groups
• Viruses possessing RNA genomes all encode an RNA-
dependant RNA polymerase.
• RNA viruses show a higher mutation rate compared to DNA
viruses.

3 dsRNA dsRNA mRNA Reovirus

• Segmented genomes.
• Transcribes mRNA from the dsRNA genome without prior protein
synthesis using a virion associated RNA-polymerase
• Early phase of mRNA synthesis is monocistronic mRNA molecules.
 Virus Groups
4 +ve ssRNA dsRNA +ve ssRNA [Acts as mRNA] Enterovirus

• “Positive” RNA viruses - Genome RNA is of the same sense as mRNA and
can be infectious.
• First stage in replication is the translation of the genome RNA with the
production of the virus polymerase.

5 -ve ssRNA dsRNA -ve ssRNA mRNA Influenza A

virus

• “Negative” RNA viruses – Genome RNA is complementary to mRNA.

• Virion-associated RNA-polymerase and first stage in replication is mRNA
transcription.
 Virus Groups
6 ssRNA ssRNA dsDNA mRNA Retrovirus
(e.g. HIV)

• Unique among RNA viruses in that they induce tumours.

• Characteristic feature is their ability to produce a DNA copy of the genome
RNA using a virion associated Reverse Transcriptase.
• DNA copy integrates into the cellular genome.

7 Nicked dsDNA nicked dsDNA intact dsDNA mRNA Hepatitis B

virus
RNA

• Circular DNA genome - double stranded with a nick in one strand.

• The nick is repaired at an early stage in the virus replication cycle.
• The virus encodes RNA polymerase with a reverse transcriptase
activity which produces a RNA intermediate from which the genome
DNA can be copied.
 Virus replication
Maturation
Release

Virion Budding
attachment
to cellular Insertion of virus
receptors proteins into
Genomic membrane Virion
nucleic acid Assembly
Uncoating synthesis
Newly synthesised
virus proteins

Replication of Protein
Genomic Synthesis
nucleic acid mRNA synthesis
 Virus replication
Maturation
Release

Virion Budding
attachment
to cellular Insertion of virus
receptors proteins into
Genomic membrane Virion
nucleic acid Assembly
Uncoating synthesis
Newly synthesised
virus proteins

Replication of Protein
Genomic Synthesis
nucleic acid mRNA synthesis
 Cytopathic Effect (cpe)
Adenovirus Herpes virus
 Specimens for viral diagnosis

From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Table 51-1.
Growth of virus on embryonated eggs

Davis, Duylbecco, Eisen, Ginsberg “Microbiology” 4th ed, J.B. Lippincott 1990, Fig. 48-1
Primary cell culture

+ enzymes

time
Subculture

enzymes

time
 Cell culture

Growth of cells in culture. A primary culture is defined as the original plating of cells from a tissue, grown to a confluent monolayer, without subculturing. A
cell strain (solid line) is defined as a euploid population of cells subcultivated more than once in vitro, lacking the property of indefinite serial passage. Cell
strains ultimately undergo degeneration and death, also called crisis or senescence. A cell line (dashed line) is an aneuploid population of cells that can be
grown in culture indefinitely. Spontaneous transformation or alteration of a cell strain to an immortal cell line can occur at any time during cultivation of the
cell strain. The time in culture and corresponding number of subcultivations or passages are shown on the abscissas. The ordinate shows the total number of
cells that would accumulate if all were retained in culture. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott
Williams & Wilkins, Philadelphia Fig. 2.3)
 Cultured cells
• Primary
– Heterogeneous – many cell types
– Closest to animal
– Technical hassle
• Diploid cell strain
– Relatively homogeneous – fewer cell types
– Further from animal
– Technically less hassle
• Continuous cell line
– Immortal
– Most homogeneous
– Genetically weird – furthest from animal
– Hassle free
– Suspension or monolayer
 Transformation
• Immortalization
• Loss of contact inhibition
• Anchorage independence
– Growth in soft agar
– Growth in suspension
• Tumor formation in athymic (nude) mice
 Cultured cell morphologies

Epithelial-like Fibroblast like

(human lung carcinoma, A549) (baby hamster kidney, BHK)
Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.4
CPE: Measles on human lung carcinoma (A549)

Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.5
 CPE: vaccinia on monkey kidney (BSC40)

Low multiplicity of infection (moi) High moi, 48 hr

single plaque
Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.5
Hemadsorbtion

add red blood cells

 Hemadsorption

Hemadsorption of erythrocytes to cells infected with influenza viruses, mumps virus, parainfluenza viruses, or togaviruses. These viruses
express a hemagglutinin on their surfaces, which bind erythrocytes of selected animal species. (From Medical Microbiology, 5 th ed., Murray,
Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-5.)
 Cytolology: CPE

Syncytium formation by measles virus. Multinucleated giant cell (arrow) visible in a histologic section of lung biopsy tissue
from a measles virus-induced giant cell pneumonia in an immunocompromised child. (From Medical Microbiology, 5 th ed.,
Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-1.)
 CPE: inclusion bodies
neuron

Negri
bodies

Negri
bodies

Immunohistochemical staining of intra-cytoplasmic viral inclusions in the neuron of a human rabies patient. (Fields Vriology (2007) 5th edition,
Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 39.9)
 Plaque assay: method

1:100

1:10 1:10 1:10 1:10 1:10

virus

serial dilution

10-2 10-3 10-4 10-5 10-6 10-7

plate 1 ml

plaques

(100,000) (10,000) (1000) 100 10 1

Titer = 1 x 107 pfu/ml

Plaque assay

Titer = 2.4 x 108 pfu/ml

Transformation
loss of contact inhibition
 Focus formation by transforming viruses

Focus assay. Monolayers of the NIH3T3 mouse fibroblast cell line were infected with Maloney murine sarcoma virus. The top two panels show
photomicrographs of uninfected cells (left) and a single virus-induced focus (right). The bottom two panels show stained dishes of uninfected (left) and infected
(right) cells. Foci are clearly visible as darker areas on the infected dish. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters
Kluwer/Lippincott Williams & Wilkins, Philadelphia Fig. 2.7)
 Endpoint titration
dilution
10-5 10-6 10-7 10-8

1
5 replicate samplings

2 = infected

3
= uninfected
4

Five replicate wells of cells are infected with one ml of each of four different virus dilutions, incubated, and
scored for infection by looking for CPE. In this example, the final titer is 10 6.3 TCID50 per ml. (TCID = tissue
culture infective dose)
Hemagglutination

RBC
 Hemagglutination test: method
1:8

1:2 1:2 1:2 1:2 1:2

virus

serial dilution

8 16 32 64 128 256
mix with red
blood cells

side view

top view

Titer = 32 HA units/ml
 Hemagglutination assay: influenza virus

Hemagglutination assay. Seven different samples of influenza virus, numbered 1 through 7 at the left, were serially diluted as indicated at the top, mixed
with chicken red blood cells (RBC), and incubated on ice for 1 to 2 hours. Wells in the bottom row contain no virus. Agglutinated RBCs coat wells evenly,
in contrast to nonagglutinated cells, which form a distinct button at the bottom of the well. The HA titer, shown at the right, is the last dilution that
shows complete hemagglutination activity. (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams
& Wilkins, Philadelphia Fig. 2.9)
 Direct particle count
Beads (104/ml)

10 beads => 1 ul
1.5 x 104 virus/ml
15 virus => 1.5 x 104 virus/ml
 Direct particle count

Direct electron microscopic particle count. An electron micrograph of a spray droplet containing 15 latex beads (spheres) and 14 vaccinia virus
particles (slightly smaller, brick-shaped particles). (From Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott
Williams & Wilkins, Philadelphia Fig. 2.8)
 Comparison of quantitative methods

Method Amount (per ml)

Direct electron microscope count 1010 EM particles

Quantal infectivity assay in eggs 109 egg ID50

Quantal infectivity assay by plaque formation 108 pfu

Hemagglutination assay 103 HA units

Fields Vriology (2007) 5th edition, Knipe, DM & Howley, PM, eds, Wolters Kluwer/Lippincott Williams & Wilkins, Philadelphia Table 2-4
Assays for viral proteins and nucleic acids

From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Box 51-4.
Antibody detection

Neutralization, hemagglutination, and

hemagglutination inhibition assays. In the assay
shown, tenfold dilutions of serum were incubated
with virus. Aliquots of the mixture were then added
to cell cultures or erythrocytes. In the absence of
antibody, the virus infected the monolayer (indicated
by CPE) and caused hemagglutination (i.e., formed a
gel-like suspension of erythrocytes). In the presence
of the antibody, infection was blocked
(neutralization), and hemagglutination was inhibited,
allowing the erythrocytes to pellet. The titer of
antibody in the serum was 100. pfu, Plaque-forming
units.From Medical Microbiology, 5th ed., Murray,
Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 51-6.
Antibody detection: western blot

Western blot analysis of HIV antigens

and antibody. HIV protein antigens are
separated by electrophoresis and blotted
onto nitrocellulose paper strips. The strip
is incubated with patient antibody,
washed to remove the unbound
antibody, and then reacted with enzyme-
conjugated antihuman antibody and
chromophoric substrate. Serum from an
HIV-infected person binds and identifies
the major antigenic proteins of HIV. This
data demonstrates the seroconversion of
one HIV-infected individual with sera
collected on day 0 (D0) to day 30 (D30)
compared to a known positive control
(PC) and negative control (NC). (From
Medical Microbiology, 5th ed., Murray,
Rosenthal & Pfaller, Mosby Inc., 2005,
Fig. 51-7. )
 Diagnostic methods for common human viruses

From Schaechter’s Mechanisms of Microbial Disease; 4th ed.; Engleberg, DiRita & Dermody; Lippincott, Williams & Wilkins; 2007; Table 31-3
 Summary
• 4 main clinical diagnostic techniques
– Culture, serology, antigen detection, nucleic acid detection
• Virus culture
– Cultured cell types
– Cytopathic effect
– Not all viruses can be cultured
• Virus quantitation
– Biological
– Physical
• Basic serological techniques