﻿Short
Communication
Pathogenicity of highly pathogenic avian H5N1
influenza A viruses isolated from humans between
2003 and 2008 in northern Vietnam
Quynh Mai Le,1
Mutsumi Ito,2
Yukiko Muramoto,2,3
Phuong Vu Mai Hoang,1
Cuong Duc Vuong,1
Yuko Sakai-Tagawa,2
Maki Kiso,2
Makoto Ozawa,4,5
Ryo Takano2,3
and Yoshihiro Kawaoka2,3,4,5,6
Correspondence
Yoshihiro Kawaoka
kawaoka@ims.u-tokyo.ac.jp
Received 7 March 2010
Accepted 28 June 2010
1
National Institute of Hygiene and Epidemiology (NIHE), Hanoi, Vietnam
2
Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science,
University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
3
ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency,
Saitama 332-0012, Japan
4
International Research Center for Infectious Diseases, Institute of Medical Science,
University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
5
Department of Pathobiological Sciences, School of Veterinary Medicine,
University of Wisconsin-Madison, Madison, WI 53706, USA
6
Division of Zoonosis, Department of Microbiology and Infectious Diseases,
Graduate School of Medicine, Kobe University, Kobe 650-0017, Japan
Vietnam is one of the countries most affected by highly pathogenic H5N1 influenza A viruses.
To evaluate the potential pathogenicity in mammals of H5N1 viruses isolated from humans in
Vietnam, we determined the sequences of all eight genes of 22 human isolates collected between
2003 and 2008 and compared their virulence in mice. The isolates were classified into clade 1
and clade 2.3.4 and differed in pathogenicity for mice. Whilst lysine at position 627 of PB2
(PB2-627K) is a critical virulence determinant for clade 2.3.4 viruses, asparagine at position 701
of PB2 and other unknown virulence determinants appear to be involved in the high pathogenicity
of clade 1 viruses, warranting further studies to determine the factors responsible for the high
virulence of H5N1 viruses in mammals.
Since late 2003, highly pathogenic H5N1 avian influenza
viruses have spread among poultry and wild birds in Asia,
Africa and Europe (WHO, 2010b). These highly patho-
genic H5N1 viruses have caused not only outbreaks in
birds, but also several hundred human infections. As of 8
June 2010, .495 humans around the world have been
infected and approximately 60 % of these infections have
been fatal (WHO, 2010b). Upon acquisition of transmis-
sibility among humans, H5N1 viruses will cause a severe
pandemic.
Vietnam is one of the countries most affected by highly
pathogenic H5N1 viruses. Since 2003, H5N1 virus out-
breaks in poultry have been reported in more than 45 of
the 64 Vietnamese provinces (OIE, 2010). Nationwide
vaccination programmes, which began in 2005, may have
contributed, in part, to the reduction in outbreaks among
poultry in 2006. However, H5N1 viruses re-emerged and
outbreaks in poultry have again been reported since 2007.
Since late 2003, when the first human infection was
reported, Vietnam has seen many H5N1 virus patients; 119
cases have been reported to date. The fatality rate remains
high: 45 % (93 patients with 42 fatal cases) for 2003­2005
and 65 % (26 patients with 17 fatal cases) for 2007­2010
(WHO, 2010b).
H5N1 viruses isolated from patients vary in pathogenicity,
as measured in a mouse model; some replicate systemically
with lethal outcomes, whereas others do not (Gao et al.,
1999; Maines et al., 2005). Using this animal model,
determinants of virulence in mammals for H5N1 viruses
have been identified. High haemagglutinin (HA) cleava-
bility conferred by the presence of a series of basic amino
The GenBank/EMBL/DDBJ accession numbers for the sequences
obtained in this study are HM114446­HM114621.
Three supplementary figures and a supplementary table are available
with the online version of this paper.
Journal of General Virology (2010), 91, 2485­2490 DOI 10.1099/vir.0.021659-0
021659 G 2010 SGM Printed in Great Britain 2485
acids at the cleavage site (Hatta et al., 2001) is a critical
determinant for virus systemic infection and high lethality
not only in birds, but also in mice. The amino acid at
position 627 of the PB2 protein (PB2-627), which is a host-
range determinant (Subbarao et al., 1993; Naffakh et al.,
2000), is a virulence determinant in mice (Hatta et al.,
2001). In addition, PB2-627K is important for virus
replication in the upper respiratory tract of mice,
suggesting that the amino acid residue at position 627 of
the PB2 protein could facilitate person-to-person trans-
mission of H5N1 viruses (Hatta et al., 2007). Asparagine at
position 701 of the PB2 protein (PB2-701N) is also a
genetic marker of high virulence for H5N1 viruses in
mammals, conferring efficient replication in mammalian
cells (Gabriel et al., 2005; Li et al., 2005). In addition, four
amino acid residues at the C terminus of the NS1 protein
and serine at position 66 of the PB1-F2 protein (PB1-F2-
66S) also contribute to high pathogenicity of H5N1 viruses
in mice (Conenello et al., 2007; Jackson et al., 2008).
In this study, to examine the pathogenicity in mammals of
H5N1 viruses isolated from humans in northern Vietnam
between 2003 and 2008, we infected mice with these H5N1
viruses and compared their virulence. We then analysed the
genome sequences of these viruses to identify potential
determinants of virulence in mammals.
Madin­Darby canine kidney (MDCK) cells and an MDCK
cell line overexpressing the human b-galactoside a-2,6-
sialyltransferase I gene (MDCK-ST6GalI) (Hatakeyama
et al., 2005) were maintained in minimal essential medium
(MEM) containing 5 % newborn calf serum at 37 uC in 5 %
CO2. Nasal swabs, pharyngeal swabs and tracheal aspirates
were collected from avian H5N1 influenza virus-infected
patients in northern Vietnam and were sent to the NIHE in
Vietnam (Dinh et al., 2006). For H5N1 virus isolation,
clinical specimens were inoculated onto MDCK cells in
MEM containing 0.3 % BSA and incubated at 37 uC.
MDCK-ST6GalI cells were used only for UT31203A virus,
as it could not be isolated in MDCK cells. For UT31244II
and UT31244III viruses, 10-day-old embryonated chicken
eggs at 35 uC were used, as they were could not be isolated
in MDCK or MDCK-ST6GalI cells. Stock viruses were
propagated in MDCK cells at 37 uC, except for UT31244II
Table 1. Human H5N1 viruses analysed in this study
Virus strain Abbreviation Date of
collection
Province of
collection*
Clinical
outcome
Collection site
of specimen
Passage
historyD
A/Vietnam/UT3028/2003d UT3028 Dec 2003 Ha Nam Died Trachea C2
A/Vietnam/UT3028II/2003d UT3028II Dec 2003 Ha Nam Died Trachea C2
A/Vietnam/UT3030/2003 UT3030 Dec 2003 Nam Dinh Died Trachea C2
A/Vietnam/UT3035/2003 UT3035 Dec 2003 Bac Giang Recovered Nose C2
A/Vietnam/UT3040/2004§ UT3040 Jan 2004 Bac Ninh Died Pharynx C2
A/Vietnam/UT3040II/2004§ UT3040II Jan 2004 Bac Ninh Died Trachea C2
A/Vietnam/UT3047III/2004 UT3047III Jan 2004 Thai Binh Died Pharynx C2
A/Vietnam/UT3062/2004 UT3062 Jan 2004 Bac Giang Died Pharynx C2
A/Vietnam/UT30259/2004 UT30259 Jul 2004 Ha Tay Died Trachea C2
A/Vietnam/HN30262IIIM3/2004 HN30262IIIM3 Aug 2004 Ha Tay Died Trachea C4
A/Vietnam/UT30408III/2005 UT30408III Feb 2005 Thai Binh Recovered Pharynx C2
A/Vietnam/UT30850/2005 UT30850 Oct 2005 Ha Noi Died Trachea C2
A/Vietnam/UT31203A/2007 UT31203A May 2007 Vinh Phuc Recovered Pharynx M1C1
A/Vietnam/UT31239/2007 UT31239 Jun 2007 Thanh Hoa Recovered Nose C2
A/Vietnam/UT31244II/2007|| UT31244II Jun 2007 Ha Nam Died Pharynx E2
A/Vietnam/UT31244III/2007|| UT31244III Jun 2007 Ha Nam Died Pharynx E2
A/Vietnam/UT31312II/2007 UT31312II Jul 2007 Ha Tay Died Trachea C2
A/Vietnam/HN31388M1/2007 HN31388M1 Dec 2007 Son La Died Trachea C2
A/Vietnam/UT31394II/2008 UT31394II Jan 2008 Tuyen Quang Died Trachea C2
A/Vietnam/UT31412II/2008 UT31412II Feb 2008 Hai Duong Died Trachea C2
A/Vietnam/UT31413II/2008 UT31413II Feb 2008 Ninh Binh Died Trachea C2
A/Vietnam/HN31432M/2008 HN31432M Feb 2008 Phu Tho Died Pharynx C2
*See location map (Supplementary Fig. S1).
DC, MDCK cells; M, MDCK-ST6GalI cells; E, eggs. The number indicates the number of passages.
dA/Vietnam/UT3028/2003 and A/Vietnam/UT3028II/2003 were isolated from the same individual, but A/Vietnam/UT3028II/2003 was isolated
1 day later than A/Vietnam/UT3028/2003.
§A/Vietnam/UT3040/2004 and A/Vietnam/UT3040II/2004 were isolated from the same individual, but A/Vietnam/UT3040II/2004 was isolated
1 day later than A/Vietnam/UT3040/2004.
||A/Vietnam/UT31244II/2007 and A/Vietnam/UT31244III/2007 were isolated from the same individual, but A/Vietnam/UT31244III/2007 was
isolated 10 days later than A/Vietnam/UT31244II/2007.
Q. M. Le and others
2486 Journal of General Virology 91
Fig. 1. Phylogenetic relationships among the HA genes of H5N1 viruses isolated from patients in Vietnam. Numbers at branch
nodes indicate neighbour-joining bootstrap values. Analysis was based on nt 77­1672 of the HA gene. The HA gene tree was
rooted to A/goose/Guangdong/1/96. Viruses analysed in this study are shown in red. Bar, 0.01 nucleotide substitutions per
site. Abbreviations: BHG, bar-headed goose; Ck, chicken; Dk, duck; Gs, goose; HC, house crow; MDk, Muscovy duck; Qa,
quail; Tk, turkey; TS, tree sparrow; WSw, whooper swan.
Virulence of H5N1 influenza human isolates in Vietnam
http://vir.sgmjournals.org 2487
and UT31244III, which were propagated in eggs at 35 uC
and stored at 280 uC.
Viral RNAs were extracted with ISOGEN (Nippon Gene)
or a viral RNA mini kit (Qiagen) according to the
manufacturers' instructions. Extracted RNAs were
reverse-transcribed with SuperScript III reverse transcrip-
tase (Invitrogen) and an oligonucleotide complementary to
the 12 nt sequence at the 39 end of the viral RNA and
amplified by PCR with Pfu-ultra (Stratagene) or Phusion
(Finnzymes) high-fidelity DNA polymerase and primers
specific for each segment of the H5N1 influenza viruses.
Primer sequences are available upon request. The PCR
products were cloned into the pCR-Blunt II-TOPO vector
(Invitrogen). At least three clones for each sample were
sequenced by using a BigDye Terminator version 3.1 Cycle
Sequencing kit on an ABI PRISM 3100 Genetic Analyzer
(Applied Biosystems). The GenBank accession numbers for
the nucleotide sequences obtained in this study are
HM114446­HM114621.
Phylogenetic analysis of the sequence data was performed
with CLUSTAL W software, which relies on neighbour-
joining methods to generate phylogenetic trees. Estimates
of the phylogenies were calculated by performing 100
neighbour-joining bootstrap replicates.
To determine the 50 % mouse lethal dose (MLD50), groups
(n54 per group) of 6-week-old female BALB/c mice (Japan
SLC) were anaesthetized with sevoflurane and infected
intranasally with 50 ml of serial 10-fold dilutions of viruses,
thereby creating doses ranging from 100
to 105
p.f.u. Mice
were monitored daily for clinical signs of infection for
14 days post-infection. MLD50 values were calculated by
using the method of Reed & Muench (1938).
We sequenced the entire genomes of 22 H5N1 influenza
viruses isolated from patients between December 2003 and
February 2008 in northern Vietnam. The dates and
locations of the virus isolations are summarized in Table
1 and Supplementary Fig. S1 (available in JGV Online). To
understand the evolution of H5N1 influenza viruses in
Vietnam, we performed phylogenetic analysis of the HA
genes of these 22 strains in addition to other available
sequences of H5N1 viruses isolated in Vietnam. According
to the recent nomenclature system for highly pathogenic
H5N1 viral HA genes, the viruses isolated from poultry in
Vietnam were classified into seven different subclades:
clades 0, 1, 2.3.2, 2.3.4, 3, 5 and 7, as reported previously
(Wan et al., 2008; WHO, 2008, 2010a). The human H5N1
isolates studied here belonged to only two clades: the
viruses isolated between 2003 and early 2005 were clade 1
and those isolated since late 2005 were clade 2.3.4 (Fig. 1).
Next, we investigated the phylogenetic relationships of the
other viral genes. With the exception of the neuraminidase
(NA) gene, the phylogenetic trees of seven gene segments
showed similar evolutionary relationships to that of the HA
gene (Supplementary Fig. S2, available in JGV Online). For
the NA gene, although the phylogenetic relationship was
similar to that of the HA gene, the clade 2.3.4 viruses were
separated into two sublineages. These results indicate that
the 22 human isolates analysed here belonged to two
genetic groups: 11 viruses were in clade 1, whereas nine
viruses were in clade 2.3.4, with UT30850 and HN31432M
being slightly different from the others in their NA genes.
To evaluate the pathogenicity of these human isolates in
mammals, BALB/c mice were infected intranasally with
these viruses and MLD50 values were determined. As
shown in Table 2, the human isolates differed in virulence,
ranging in their MLD50 from .105
p.f.u. for UT31413II to
0.46 p.f.u. for UT31239. Both virulent and avirulent viruses
were found in clade 1 and clade 2.3.4, and did not correlate
with clinical outcomes (Tables 1 and 2).
We then correlated the results of the mouse pathogenicity
data with the sequence information (Table 2). All isolates
examined here contained polybasic amino acids at the
cleavage site of the HA protein, which are necessary for the
high virulence of H5N1 viruses in mice (Hatta et al., 2007).
No isolate possessed glutamic acid at position 92 of the
NS1 protein (NS1-92E), which is associated with high
virulence of these viruses in pigs (Seo et al., 2002).
Similarly, no isolates possessed PB1-F2-66S, which is
another marker for high virulence of H5N1 viruses in
mice. The PB1-F2 protein of isolates UT31244II and
UT31244III possessed an 11 aa deletion at the C terminus.
Although all of the isolates in clade 1 except for UT3035
possessed either PB2-627K or PB2-701N, which are
responsible for high pathogenicity in mice, some viruses
(UT3028, UT3028II, UT3030, UT3040II, UT3047III and
UT30408III) showed relatively low virulence for mice.
UT3035, which has neither PB2-627K nor PB2-701N, had
low virulence. These results indicate that although PB2-
627K and PB2-701N contribute to high pathogenicity in
mice, they are not sufficient to confer high virulence in
mice. On the other hand, all of the clade 2.3.4 isolates that
possessed PB2-627K showed high virulence in mice,
whereas those that possessed PB2-627E (avian type) were
of low virulence.
Four amino acid residues (E-S-E-V) at the C terminus of
NS1 are associated with virulence of H5N1 viruses in mice
(Jackson et al., 2008). Among the clade 1 viruses tested,
some (UT3028, UT3028II, UT3030, UT3035, UT3047III
and UT30408III) were avirulent even though they pos-
sessed this motif. Whilst most of the clade 2.3.4 viruses in
this study lacked this sequence motif, some were virulent,
indicating that, like PB2-627K and PB2-701N, there may be
some other sequence motif that can substitute for this NS1
sequence motif. These results suggest that, in addition to
the HA cleavage-site sequence, PB2-627K, PB2-701N, the C
terminus of NS1 and PB1-F2-66S, there are other virulence
determinants that have yet to be discovered.
In this study, we phylogenetically investigated 22 H5N1
viruses isolated from humans in northern Vietnam and
determined their pathogenicity in mice. All of the
Q. M. Le and others
2488 Journal of General Virology 91
Vietnamese human isolates examined in this study belonged
to either clade 1 or clade 2.3.4. Both of these lineages contain
human H5N1 viruses isolated in other Asian countries
besides Vietnam. Although none of these human isolates
from northern Vietnam belonged to clades 2.3.2, 3, 5 or 7, a
limited number of human isolates from China in 2009 were
assigned to clades 2.3.2 and 7 (WHO, 2010a).
We also found differences in pathogenicity for mice
between two strains that were isolated from the same
individual but on different days and are genetically very
closely related to each other: UT3040 showed high
virulence, whereas UT3040II was of low virulence, even
though both strains possessed the human-type PB2-627K.
These two strains differ in their amino acid sequences by
only three residues (one each in PB1, PA and NP;
Supplementary Table S1, available in JGV Online). The
amino acid residues found in PB1 and NP of UT3040II are
specific for this virus among the 22 human isolates
analysed here, implying that these amino acids may
contribute to its attenuated phenotype in mice. Because
glutamic acid at position 142 of the PA of UT3040 was also
found in UT3028II and HN31432M, which were of low
virulence, it remains unclear whether this amino acid
residue in PA contributes to pathogenicity. To determine
whether these mutations in the polymerase complex affect
polymerase activity, the polymerase activity of UT3040 and
UT3040II was assessed by use of a plasmid-based
minigenome assay essentially as described by Ozawa et al.
(2007) (Supplementary Fig. S3, available in JGV Online).
Briefly, human embryonic kidney 293 cells were co-
transfected with plasmids for the expression of the
viral polymerase complex proteins (i.e. PB2, PB1, PA and
NP) and a firefly luciferase-encoding influenza viral
minigenome together with pGL4.74[hRluc/TK] (Promega),
which expresses Renilla luciferase and served as an internal
control. Firefly luciferase activity was measured by using a
dual-luciferase reporter assay system (Promega) according
to the manufacturer's instructions. The viral polymerase
complex from UT3040 exhibited significantly higher activity
than that from UT3040II, suggesting that the difference in
virulence between UT3040 and UT3040II originates from
the difference in their polymerase activities. Reverse-genetics
studies will help to determine the amino acid residues
responsible for the difference in virulence between these
viruses.
Table 2. Virulence in BALB/c mice and molecular characterization of H5N1 viruses
Virus strain Clade classification Amino acid MLD50 [log10 (p.f.u.)] Virulence in mice*
PB2 C terminus of NS1
627 701
UT3028 1 K D E-S-E-V 2.3 Low
UT3028II 1 K D E-S-E-V 2.5 Low
UT3030 1 E N E-S-E-V 2.5 Low
UT3035 1 E D E-S-E-V 2.5 Low
UT3040 1 K D 10 aa deletion 0.3 High
UT3040II 1 K D 10 aa deletion 3.3 Low
UT3047III 1 E N E-S-E-V 3.5 Low
UT3062 1 K D E-S-E-V 0.4 High
UT30259 1 K D E-S-E-V 1.3 High
HN30262IIIM3 1 E N E-S-E-I 1.4 High
UT30408III 1 K D E-S-E-V 3.5 Low
UT30850 2.3.49D K D E-S-E-V 1.0 High
UT31203A 2.3.4 K D G-S-E-V 0.2 High
UT31239 2.3.4 K D G-S-E-V 20.3 (0.46 p.f.u.) High
UT31244IId 2.3.4 E D G-S-E-V 4.7 Low
UT31244IIId 2.3.4 E D G-S-E-V 3.3 Low
UT31312II 2.3.4 E D G-S-E-V 4.0 Low
HN31388M1 2.3.4 E7/K1§ D G-S-E-V 2.5 Low
UT31394II 2.3.4 K D G-S-E-V 0.3 High
UT31412II 2.3.4 K D G-S-E-V 0.6 High
UT31413II 2.3.4 E D G-S-E-V .5.0 Low
HN31432M 2.3.49D E D 10 aa deletion 2.9 Low
*Viruses with an MLD50 ,102
p.f.u. were considered to be of high virulence in this study.
DClade 2.3.49 indicates that these isolates differ slightly from the others in the NA gene.
dUT31244II and UT31244III lack 11 aa at the C terminus of the PB1-F2 protein.
§Number of clones possessing K or E of a total of eight clones.
Virulence of H5N1 influenza human isolates in Vietnam
http://vir.sgmjournals.org 2489
Acknowledgements
We thank Susan Watson for editing the manuscript. This work was
supported by ERATO (Japan Science and Technology Agency), a
Grant-in-Aid for Specially Promoted Research, a contract research
fund for the Program of Founding Research Centers for Emerging
and Reemerging Infectious Diseases from the Ministry of Education,
Culture, Sports, Science, and Technology, grants-in-aid from the
Ministry of Health, Labor, and Welfare of Japan, National Institute of
Allergy and Infectious Disease Public Health Service research grants
and the Center for Research on Influenza Pathogenesis (CRIP) funded
by the National Institute of Allergy and Infectious Diseases (contract
HHSN266200700010C).
References
Conenello, G. M., Zamarin, D., Perrone, L. A., Tumpey, T. & Palese, P.
(2007). A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918
influenza A viruses contributes to increased virulence. PLoS Pathog 3,
1414­1421.
Dinh, P. N., Long, H. T., Tien, N. T., Hien, N. T., Mai, L. T. Q., Phong,
L. H., Tuan, L. V., Tan, H. V., Nguyen, N. B. & other authors (2006).
Risk factors for human infection with avian influenza A H5N1,
Vietnam, 2004. Emerg Infect Dis 12, 1841­1847.
Gabriel, G., Dauber, B., Wolff, T., Planz, O., Klenk, H. D. & Stech, J.
(2005). The viral polymerase mediates adaptation of an avian
influenza virus to a mammalian host. Proc Natl Acad Sci U S A
102, 18590­18595.
Gao, P., Watanabe, S., Ito, T., Goto, H., Wells, K., McGregor, M.,
Cooley, A. J. & Kawaoka, Y. (1999). Biological heterogeneity,
including systemic replication in mice, of H5N1 influenza A virus
isolates from humans in Hong Kong. J Virol 73, 3184­3189.
Hatakeyama, S., Sakai-Tagawa, Y., Kiso, M., Goto, H., Kawakami, C.,
Mitamura, K., Sugaya, N., Suzuki, Y. & Kawaoka, Y. (2005).
Enhanced expression of an a2,6-linked sialic acid on MDCK cells
improves isolation of human influenza viruses and evaluation of their
sensitivity to a neuraminidase inhibitor. J Clin Microbiol 43, 4139­4146.
Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y. (2001). Molecular
basis for high virulence of Hong Kong H5N1 influenza A viruses.
Science 293, 1840­1842.
Hatta, M., Hatta, Y., Kim, J. H., Watanabe, S., Shinya, K., Nguyen, T., Lien,
P. S., Le, Q. M. & Kawaoka, Y. (2007). Growth of H5N1 influenza A
viruses in the upper respiratory tracts of mice. PLoS Pathog 3, 1374­1379.
Jackson, D., Hossain, M. J., Hickman, D., Perez, D. R. & Lamb, R. A.
(2008). A new influenza virus virulence determinant: the NS1 protein
four C-terminal residues modulate pathogenicity. Proc Natl Acad Sci
U S A 105, 4381­4386.
Li, Z., Chen, H., Jiao, P., Deng, G., Tian, G., Li, Y., Hoffmann, E.,
Webster, R. G., Matsuoka, Y. & Yu, K. (2005). Molecular basis of
replication of duck H5N1 influenza viruses in a mammalian mouse
model. J Virol 79, 12058­12064.
Maines, T. R., Lu, X. H., Erb, S. M., Edwards, L., Guarner, J., Greer, P. W.,
Nguyen, D. C., Szretter, K. J., Chen, L. M. & other authors (2005).
Avian influenza (H5N1) viruses isolated from humans in Asia in 2004
exhibit increased virulence in mammals. J Virol 79, 11788­11800.
Naffakh, N., Massin, P., Escriou, N., Crescenzo-Chaigne, B. & van
der Werf, S. (2000). Genetic analysis of the compatibility between
polymerase proteins from human and avian strains of influenza A
viruses. J Gen Virol 81, 1283­1291.
OIE (2010). Update on highly pathogenic avian influenza in animals
(type H5 and H7). Accessed 1 March 2010. http://www.oie.int/
downld/AVIAN%20INFLUENZA/A_AI-Asia.htm
Ozawa, M., Fujii, K., Muramoto, Y., Yamada, S., Yamayoshi, S.,
Takada, A., Goto, H., Horimoto, T. & Kawaoka, Y. (2007).
Contributions of two nuclear localization signals of influenza A virus
nucleoprotein to viral replication. J Virol 81, 30­41.
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty
per cent endpoints. Am J Hyg 27, 493­497.
Seo, S. H., Hoffmann, E. & Webster, R. G. (2002). Lethal H5N1
influenza viruses escape host anti-viral cytokine responses. Nat Med 8,
950­954.
Subbarao, E. K., London, W. & Murphy, B. R. (1993). A single amino
acid in the PB2 gene of influenza A virus is a determinant of host
range. J Virol 67, 1761­1764.
Wan, X. F., Nguyen, T., Davis, C. T., Smith, C. B., Zhao, Z. M., Carrel, M.,
Inui, K., Do, H. T., Mai, D. T. & other authors (2008). Evolution of highly
pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and
2007. PLoS One 3, e3462.
WHO (2008). Towards a unified nomenclature system for highly
pathogenic avian influenza virus (H5N1). Emerg Infect Dis 14, e1.
WHO (2010a). Antigenic and genetic characteristics of influenza
A(H5N1) and influenza A(H9N2) viruses and candidate vaccine viruses
developed for potential use in human vaccines. Accessed 1 March 2010.
http://www.who.int/csr/disease/avian_influenza/guidelines/h5n1virus/
en/index.html
WHO (2010b). Cumulative number of confirmed human cases of
avian influenza A/(H5N1) reported to WHO. Accessed 11 June 2010.
http://www.who.int/csr/disease/avian_influenza/country/cases_table_
2010_06_08/en/index.html
Q. M. Le and others
2490 Journal of General Virology 91
