Available online at www.sciencedirect.

com

Virus Research 131 (2008) 299303

Short communication

F gene recombination between genotype II and VII

Newcastle disease virus
Zhuoming Qin a,1 , Lei Sun b,1 , Baochen Ma c , Zhizhong Cui c ,
Yiping Zhu b , Yoshihiro Kitamura d , Wenjun Liu b,d,
a Institute of Poultry Science, Shandong Academy of Agricultural Science, Jinan 250100, China
Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
c College of Animal Science and Technology, Shandong Agriculture University, Taian 271018, China
d ChinaJapan Joint Laboratory of Molecular Immunology and Molecular Microbiology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101, China
b

Received 27 May 2007; received in revised form 29 September 2007; accepted 2 October 2007
Available online 19 November 2007

Abstract
A velogenic Newcastle disease virus (NDV) strain, designated as SRZ03, was isolated from an egg layer flock with NDV vaccine immunization
failure in China in 2003. Recombination was found in the F gene of SRZ03. Complete genome sequences analysis indicated that the N-terminal of
SRZ03 F gene originated from a genotype II NDV strain, whereas the C-terminal of F gene and the rest of the genes originated from a prevalent
velogenic genotype VII NDV strain. It provides us valuable information for understanding the recombination of nonsegmented negative-sense
RNA viruses.
2007 Elsevier B.V. All rights reserved.
Keywords: Newcastle disease virus; Recombination; F gene

Newcastle disease (ND) is an acute and highly contagious

disease caused by Newcastle disease virus (NDV), a member
of the avian paramyxovirus serotype-1 (APMV-1) (Alexander,
1991). NDV belongs to the genus Avulavirus in the family
Paramyxoviridae (Mayo, 2002a,b). ND is widespread in the
world, especially in China and has caused severe economic
losses in the poultry industry. All APMV-1 isolates are categorized as three pathotypes depending on the severity of disease
caused, i.e., velogenic, mesogenic, and lentogenic (Beard and
Hanson, 1984). The NDV strains display great genetic diversity.
Using phylogenetic analysis, at least eight genotypes (with several subtypes) of NDV were described (Ballagi-Pordany et al.,
1996; Herczeg et al., 1999; Kwon et al., 2003; Liu et al., 2003;
Lomniczi et al., 1998).

Corresponding author at: Center for Molecular Virology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Tel.: +86 10 64807497; fax: +86 10 64807503.
E-mail address: liuwj@im.ac.cn (W. Liu).
1 These authors contributed equally to this work.
0168-1702/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.virusres.2007.10.001

The genome of NDV comprises a nonsegmented singlestranded (ss) negative-sense RNA of approximately 15.2 kb
that codes for nucleocapsid proteins (NP), phosphoprotein
(P), matrix (M) protein, fusion (F) protein, haemagglutininneuraminidase (HN) and an RNA dependent RNA polymerase
(L), respectively, from the 3-terminus to the 5-terminus on the
genome sense RNA (Millar et al., 1988). Because of the high
rates of mutation and replication and large population sizes,
RNA viruses evolve rapidly (Domingo and Holland, 1997).
However, another evolutionary process, recombination, is a
potentially important means of generating more genetic diversity in RNA viruses (Lai, 1992; Worobey and Holmes, 1999). In
general, negative-sense RNA viruses, especially nonsegmented
negative-sense RNA viruses, have lower rates of recombination
than positive-sense RNA viruses. Recombinations in negativesense RNA viruses, such as ambisense arenavirus (Archer and
Rico-Hesse, 2002; Charrel et al., 2001), hantavirus (Klempa et
al., 2003; Sibold et al., 1999; Sironen et al., 2001), influenza
A virus (Gibbs et al., 2001), measles virus (Schierup et al.,
2005), respiratory syncytial virus (Spann et al., 2003) and Newcastle disease virus (HN gene) (Chare et al., 2003) had been

300

Z. Qin et al. / Virus Research 131 (2008) 299303

reported, but the rates of recombination in negative-sense RNA

viruses were much lower than those of the mutation. Here we first
reported the recombination in the F gene of a mutative velogenic
Newcastle disease virus (NDV) strain.
An ND outbreak occurred in a 67-day-old egg layer flock
which had been vaccinated with the NDV vaccine (La Sota) at 7-,
14- and 40-day-old in Shandong province of China in 2003. The
infected layer showed typical clinical signs and lesions of NDV,
and classical neural clinical signs were observed at the latter
stage. The morbidity and mortality were 100% and 30%, respectively. Tissue specimens (lung, trachea, spleen, and kidney) were
got from the infected layers and virus was isolated in 10-day-old
specific pathogen free (SPF) embryonated chicken eggs by standard procedures (Alexander, 1989). As a result, an NDV strain
named SRZ03 was isolated. Virus was plaque-purified three
times on primary chicken embryo fibroblasts and then grown
in 10-day-old SPF chicken embryonated eggs. The allantoic fluids were harvested and stored at 70 C. Allantoic fluids were
used for RNA extraction, mean death time (MDT), intracerebral
pathogenicity index (ICPI) and intravenous pathogenicity index

(IVPI) test (Alexander, 1989; Collins et al., 1994). The results

showed that SRZ03 was a wild velogenic strain with the MDT
value of 51 h, the ICPI value of 2.0 and the IVPI value of 2.79.
Primers were designed and synthesized on the bases of
published sequences of NDV strains (La Sota, B1, and clone
30, with GenBank accession numbers AF077761, NC002617,
and NDVY18898, respectively) (Zou et al., 2005). Reverse
transcription-polymerase chain reaction (RT-PCR) was used to
amplify the internal fragments of SRZ03 and ligation-anchored
PCR (LA-PCR) was used to amplify the genomic termini (Zou
et al., 2005). Viral RNA was extracted from 200 l of infectious allantoic fluid using Trizol reagents (GIBCO-BRL), and
reverse transcription was performed using the RT primer: 5 ACGGGTAGAA-3 . The PCR products were purified with a
QIAquick PCR purification kit (Qiagen, Valencia, California)
according to the manufacturers instructions. Purified PCR products were sequenced by Amersham ET Dye terminator kit and
analyzed by ABI 3730 DNA sequencer (Perkin-Elmer Applied
Biosystems, Foster City, CA, USA). The data was analyzed
using the MegAlign software (DNAStar 5.01) and phyloge-

Fig. 1. The most likely break points for recombination in the F gene of SRZ03. The break point region (genomic position 44944515) and break point (genomic
position 5143) were indicated by an overstriking line and an arrow, respectively. Nucleotide sequences (genomic positions 43514650, 49515250) around break
points of SRZ03 were shown. The F gene of SRZ03 contained 1782nt at the genomic position 45046285 and its ORF contained 1662nt at the genomic position
45506211. Nucleotides different from the majority sequences on top were denoted by the single letter code, and identical nucleotides were indicated by a ().

Z. Qin et al. / Virus Research 131 (2008) 299303

301

Fig. 2. Phylogenetic trees for different regions of F gene showing the movement of strain SRZ03 (boxed). Trees were generated with the MEGA program (Version
3.1) by using neighbor-joining analysis. Bootstrap values were shown for the relevant nodes. Numbers below branches indicate bootstrap value percentages from
1000 replicates.

netic trees were drawn with the MEGA 3.1 program by using
neighbor-joining analysis.
The whole genome nucleotide sequences of SRZ03 (GenBank accession number EU167540) were aligned and compared
with those of other available NDV isolates which belong to genotype II or VII (Fig. 1). As a result, two most likely break points
were determined in SRZ03. One was at the non-coding region
(genomic nucleotide position 44944515) between the open
reading frame (ORF) of M gene (genomic nucleotide position
32624390) and ORF of F gene (genomic nucleotide position
45506211), but we could not identify the exact point. The
other most likely break point was at the genomic nucleotide
position 5143. The phylogenetic tree based on the N-terminal
(genomic nucleotide position 45045142) of F gene showed that
SRZ03 fell into the cluster of genotype II NDV strains, while
the tree based on the C-terminal (genomic nucleotide position
51436285) of F gene showed that SRZ03 fell into the cluster of genotype VII NDV strains (Fig. 2). Obviously, there was
a recombination happened in the F gene of SRZ03. In addition, the nucleotide sequences of N-terminal and C-terminal
of SRZ03 F gene were compared respectively with those of
other 16 NDV isolates which belong to genotypes II, VII or
IX and the identities were shown in Table 1. The N-terminal
of SRZ03 F gene had higher degree of identities with those

of genotype II NDVs (96.699.8%) than with those of genotype VII NDVs (84.084.9%), while the C-terminal of SRZ03
F gene had higher degree of identities with those of genotype
VII NDVs (93.599.9%) than with those of genotype II NDVs
(80.682.5%).
As shown in Fig. 2a, there were two subclusters in genotype
II NDVs. The Texas48-like NDV strains (Texas48, AQI-ND025
and SRZ03) which had a polybasic-F0 cleavage site motif typical
for velogenic NDV strain fell into one subcluster. The vaccine
strains (La sota, B1 and V4) which had a motif typical for lentogenic NDV strain fell into the other subcluster. The N-terminal
of SRZ03 F gene had higher degree of similarity with those of
Texas48-like viruses (>99%) than with those of vaccine strains
(9697%) (Table 1). Two different explanations might elucidate
the origin of the N-terminal of SRZ03 F gene. One explanation
was that it originated from the Texas48-like genotype II NDV
in waterfowl. Texas48-like viruses exist indeed in commercial
waterfowls in China, although few of them have been found.
The strains of SD 6 04 Go and SD 1 04 Go were both Texas48like genotype II NDVs isolated from apparently healthy goose
flocks in Shandong province of China in 2004. So we speculated
that the Texas48-like viruses might have being in waterfowls in
nature for very long time with no clinical sign. In China, chickens
and waterfowls are housed closely in some farming villages, pro-

Table 1
Newcastle disease virus strains used in this study and nucleotide identity against F gene of SRZ03
Strains

Location

Hosts

Year

Virulence

F gene accession no.

Genotypea

Identity F1-592b

Identity F593-1662b

SRZ03
La Sota
B1
V4
Bingham
AQI-ND026
Texas48
SD 6 04 Go
SD 1 04 Go
GM
ZJ1
SF02
NA-1
SCL03
WHZ03
Taiwan95
F48E9

China
USA
USA
China
USA
China
USA
China
China
China
China
China
China
China
China
Taiwan
China

Layer
Chicken
Fowl
c
Mixed species

Fowl
Goose
Goose

Goose
Goose
Goose
Broiler
Layer
Chicken
Chicken

2003
1946
1947

1948
2004
2004

2000
2002

2003
2003
1995
1948

Velogenic
Lentogenic
Lentogenic
Lentogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic
Velogenic

EU167540
AJ629062
AF309418
AY225110
A03663
DQ060053
M24698
DQ682446
DQ682441
DQ486859
AF431744
AF473851
DQ659677
DQ363533
DQ363530
NDU62620
AY508514

II
II
II
II
II
II
II
II
II
VII
VII
VII
VII
VII
VII
VII
IX

97.0
96.6
96.8
99.3
99.7
99.7
99.8
99.8
81.6
82.5
81.8
81.6
81.8
81.8
80.6
86.0

84.0
84.6
84.0
84.9
84.8
84.8
84.8
84.7
97.6
98.1
98.5
97.8
99.9
99.8
93.5
86.8

Based on nucleotides at position 1374 of NDV F gene (nucleotide number starts at the ATG start codon of F gene).
Nucleotide identity (%) against N-terminal (position 1592) or C-terminal (position 5931662) of SRZ03 F gene (Nucleotide number starts at the ATG start
codon of F gene).
c Not available.
b

302

Z. Qin et al. / Virus Research 131 (2008) 299303

viding the opportunities for interspecies transmission of viruses.

There was the possibility that the Texas48-like NDVs in waterfowls transmitted to chickens directly. The other explanation
was that it originated from the avirulent vaccine strains, such as
La Sota and B1, because these vaccines have being heavily used
in China to control ND in chickens and waterfowls. But one
question is that the recombinant SRZ03 had an 112 RRQKRF117
motif in the N-terminal of F gene which was clearly different
from 112 GRQGRL117 in La Sota and B1. It had been reported
that avirulent viruses, maintained in waterfowl in nature and
bearing the consensus avirulent type sequence, had the potential to become velogenic after transmission to and circulation
in chicken populations. Chicken can select highly pathogenic
derivatives from nonpathogenic precursors (Shengqing et al.,
2002). This report might answer this question to some extent.
In China, velogenic genotype VII NDV strains were responsible for most ND outbreaks in chickens and circulated
predominantly in recent years (Liu et al., 2007; Wang et al.,
2006). The C-terminal of F gene and the rest of the genes of
SRZ03 had high degree of similarity with those of the genotype VII NDV strains. Therefore, it was more likely that the
C-terminal of F gene and the test of the genes of SRZ03 originated from one of the prevalent genotype VII NDV strains in
chickens.
ND has been epidemic in chicken flocks, and vaccines have
been widely used in China to control this disease. Avian flocks
are inoculated regularly with the avirulent and attenuated vaccines, such as La Sota and B1, which belonged to genotype II
NDV, but ND outbreaks still occur frequently and immunization
failure is common in some area. Recombination is an important
parameter to be considered in vaccination with live, attenuated
viruses. The recombination in NDV might be a potential reason
for immunization failure.
This is the first case to report the recombination in the F gene
of NDV, which provide us valuable information for understanding the recombination of nonsegmented negative-strand RNA
viruses. On the other hand, this finding will raise questions concerning phylogenetic studies using partial F gene sequences. The
wide prevalence of ND, the closed or mixed feeding of chickens
and waterfowls, and the heavy use of NDV vaccines all provided
the ideal environments for NDV recombination. Whether this
recombination could affect the antigenicity and pathogenicity
of NDV, and whether recombinant viruses have serious consequences on the vaccine efficacy need to be further determined.
They potentially could have deleterious effects. Therefore, NDV
live vaccine strains should be carefully used to prevent their
conversion to virulent strains.
Acknowledgements
This study was funded by Project for Science and Technology
Development of Shandong Province (030317), the Ministry of
Science and Technology of China program (2006BAD06A04),
National Basic Research Program (973 Program) of China
(2005CB523002), Natural Science Foundation of Shandong
Province (031020101) and Hundreds of Talents Program of Chinese Academy of Sciences. We thank Shandong Agriculture

University for invaluable contributions for this study. We also

thank Huai-ying Xu, Ye-feng He and You-ling Wang for their
help in sample collections.
References
Alexander, D.J., 1989. Newcastle disease. In: Purchase, H.G., Arp, L.H., Domermuth, C.H., Perason, J.E. (Eds.), A Laboratory Manual for the Isolation and
Identification of Avian Pathogens. American Association of Avian Pathologists, Kenner Square, pp. 114120.
Alexander, D.J., 1991. Newcastle disease and other paramyxovirus infections.
In: Calnek, B.W., Barnes, H.J., Beard, C.W., Reid, W.M., Yoder Jr., H.W.
(Eds.), Diseases of Poultry. Iowa State University Press, Ames, pp. 496519.
Archer, A.M., Rico-Hesse, R., 2002. High genetic divergence and recombination
in Arenaviruses from the Americas. Virology 304, 274281.
Ballagi-Pordany, A., Wehmann, E., Herczeg, J., Belak, S., Lomniczi, B., 1996.
Identification and grouping of Newcastle disease virus strains by restriction
site analysis of a region from the F gene. Arch. Virol. 141, 243261.
Beard, C.W., Hanson, R.P., 1984. Newcastle disease. In: Hofstad, M.S., Barnes,
H.J., Calnek, B.W., Reid, W.M., Yonder, H.W. (Eds.), Diseases of Poultry.
Iowa State University Press, Ames, pp. 425470.
Chare, E.R., Gould, E.A., Holmes, E.C., 2003. Phylogenetic analysis reveals a
low rate of homologous recombination in negative-sense RNA viruses. J.
Gen. Virol. 84, 26912703.
Charrel, R.N., de, L.X., Fulhorst, C.F., 2001. The Whitewater Arroyo virus:
natural evidence for genetic recombination among Tacaribe serocomplex
viruses (family Arenaviridae). Virology 283, 161166.
Collins, M.S., Strong, I., Alexander, D.J., 1994. Evaluation of the molecular basis
of pathogenicity of the variant Newcastle disease viruses termed pigeon
PMV-1 viruses. Arch. Virol. 134, 403411.
Domingo, E., Holland, J.J., 1997. RNA virus mutations and fitness for survival.
Annu. Rev. Microbiol. 51, 151178.
Gibbs, M.J., Armstrong, J.S., Gibbs, A.J., 2001. Recombination in the hemagglutinin gene of the 1918 Spanish flu. Science 293, 18421845.
Herczeg, J., Wehmann, E., Bragg, R.R., Travassos Dias, P.M., Hadjiev, G.,
Werner, O., Lomniczi, B., 1999. Two novel genetic groups (VIIb and VIII)
responsible for recent Newcastle disease outbreaks in Southern Africa, one
(VIIb) of which reached Southern Europe. Arch. Virol. 144, 20872099.
Klempa, B., Schmidt, H.A., Ulrich, R., Kaluz, S., Labuda, M., Meisel, H., Hjelle,
B., Kruger, D.H., 2003. Genetic interaction between distinct Dobrava hantavirus subtypes in Apodemus agrarius and A. avicollis in nature. J. Virol.
77, 804809.
Kwon, H.J., Cho, S.H., Ahn, Y.J., Seo, S.H., Choi, K.S., Kim, S.J., 2003. Molecular epidemiology of Newcastle disease in Republic of Korea. Vet. Microbiol.
95, 3948.
Lai, M.M., 1992. RNA recombination in animal and plant viruses. Microbiol.
Rev. 56, 6179.
Liu, H., Wang, Z., Wu, Y., Zheng, D., Sun, C., Bi, D., Zuo, Y., Xu, T., 2007.
Molecular epidemiological analysis of Newcastle disease virus isolated in
China in 2005. J. Virol. Methods 140, 206211.
Liu, X.F., Wan, H.Q., Ni, X.X., Wu, Y.T., Liu, W.B., 2003. Pathotypical and
genotypical characterization of strains of Newcastle disease virus isolated
from outbreaks in chicken and goose flocks in some regions of China during
19852001. Arch. Virol. 148, 13871403.
Lomniczi, B., Wehmann, E., Herczeg, J., Ballagi-Pordany, A., Kaleta, E.F.,
Werner, O., Meulemans, G., Jorgensen, P.H., Mante, A.P., Gielkens, A.L.,
Capua, I., Damoser, J., 1998. Newcastle disease outbreaks in recent years
in western Europe were caused by an old (VI) and a novel genotype (VII).
Arch. Virol. 143, 4964.
Mayo, M.A., 2002b. Virus taxonomyHouston 2002. Arch. Virol. 147,
10711076.
Mayo, M.A., 2002a. A summary of taxonomic changes recently approved by
ICTV. Arch. Virol. 147, 16551663.
Millar, N.S., Chambers, P., Emmerson, P.T., 1988. Nucleotide sequence of the
fusion and haemagglutinin-neuraminidase glycoprotein genes of Newcastle
disease virus, strain Ulster: molecular basis for variations in pathogenicity
between strains. J. Gen. Virol. 69, 613620.

Z. Qin et al. / Virus Research 131 (2008) 299303

Schierup, M.H., Mordhorst, C.H., Muller, C.P., Christensen, L.S., 2005. Evidence of recombination among early-vaccination era measles virus strains.
BMC Evol. Biol. 5, 52.
Shengqing, Y., Kishida, N., Ito, H., Kida, H., Otsuki, K., Kawaoka, Y., Ito,
T., 2002. Generation of velogenic Newcastle disease viruses from a nonpathogenic waterfowl isolate by passaging in chickens. Virology 301,
206211.
Sibold, C., Meisel, H., Kruger, D.H., Labuda, M., Lysy, J., Kozuch, O., Pejcoch, M., Vaheri, A., Plyusnin, A., 1999. Recombination in Tula hantavirus
evolution: analysis of genetic lineages from Slovakia. J. Virol. 73, 667675.
Sironen, T., Vaheri, A., Plyusnin, A., 2001. Molecular evolution of Puumala
hantavirus. J. Virol. 75, 1180311810.

303

Spann, K.M., Collins, P.L., Teng, M.N., 2003. Genetic recombination during
coinfection of two mutants of human respiratory syncytial virus. J. Virol.
77, 20.
Wang, Z., Liu, H., Xu, J., Bao, J., Zheng, D., Sun, C., Wei, R., Song, C., Chen, J.,
2006. Genotyping of Newcastle disease viruses isolated from 2002 to 2004
in China. Ann. N.Y. Acad. Sci. 1081, 228239.
Worobey, M., Holmes, E.C., 1999. Evolutionary aspects of recombination in
RNA viruses. J. Gen. Virol. 80, 25352543.
Zou, J., Shan, S.H., Yao, N.T., Gong, Z.X., 2005. Complete genome sequence
and biological characterizations of a Novel Goose Paramyxovirus-SF02
isolated in China. Virus Gene 30, 1321.