Molecular Changes h5n1

Journal of Medical Virology 66:107±114 (2002)
Molecular Changes Associated With the

Transmission of Avian In¯uenza A H5N1 and H9N2
Viruses to Humans
M. Shaw,1* L. Cooper,1 X. Xu,1 W. Thompson,1 S. Krauss,2 Y. Guan,3 N. Zhou,2 A. Klimov,1 N. Cox,1
R. Webster,2 W. Lim,4 K. Shortridge,3 and K. Subbarao1
1
In¯uenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention,
Atlanta, Georgia
2
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee
3
Department of Microbiology, The University of Hong Kong, University Pathology Building, Queen Mary Hospital,
Hong Kong SAR, China
4
Government Virus Unit, Queen Mary Hospital, Hong Kong SAR, China
In order to identify molecular changes associated INTRODUCTION

with the transmission of avian in¯uenza A H5N1
The isolation of an H5N1 in¯uenza A virus from a
and H9N2 viruses to humans, the internal genes
fatally ill patient in the Hong Kong SAR, China, in May
from these viruses were compared to sequences
of 1997 was a suf®ciently remarkable event to generate
from other avian and human in¯uenza A isolates.
speculation concerning the pandemic potential of these
Phylogenetically, each of the internal genes of all
viruses [CDC, 1997; de Jong et al., 1997]. Partial
sixteen of the human H5N1 and both of the H9N2
molecular analysis of this isolate, A/Hong Kong/156/97
isolates were closely related to one another and
(H5N1), indicated that all eight genes were derived
fell into a distinct clade separate from clades
from avian in¯uenza viruses [Claas et al., 1998; Sub-
formed by the same genes of other avian and
barao et al., 1998]. There was no indication as to what
human viruses. All six internal genes were most
allowed this particular strain to make the interspecies
closely related to those of avian isolates circulat-
leap from an avian host to a child, and in the absence of
ing in Asia, indicating that reassortment with
additional infections, it was initially assumed that the
human strains had not occurred for any of these
case was an isolated incident.
18 isolates. Amino acids previously identi®ed as
In November and December of the same year, 17
host-speci®c residues were predominantly avian
additional cases of febrile respiratory illness in Hong
in the human isolates although most of the
Kong were con®rmed to be caused by H5N1 viruses on
proteins also contained residues observed pre-
the basis of virus isolation (®fteen cases) or serology
viously only in sequences of human in¯uenza
(two cases). Five of these later victims died [CDC, 1998;
viruses. For the majority of the nonglycoprotein
Yuen et al., 1998]. Multiple serious infections due to a
genes, three distinct subgroups could be distin-
virus previously observed to be pathogenic only in birds
guished on bootstrap analyses of the nucleotide
led to concern that the virus could cause a pandemic in
sequences, suggesting multiple introductions of
a human population that was seronegative for H5 HA.
avian virus strains capable of infecting humans.
The outbreak ended after all chickens and other poultry
The shared nonglycoprotein gene constellations
in Hong Kong were destroyed [Shortridge et al., 2000]
of the human H5N1 and H9N2 isolates and
but questions remain as to why these viruses were able
their detection in avian isolates only since 1997
to infect humans and cause serious disease.
when the ®rst human infections were detected
suggest that this particular gene combination
may confer the ability to infect humans and cause
Grant sponsor: National Institute of Allergy and Infectious
disease. J. Med. Virol. 66:107±114, 2002. Diseases; Grant number: Public Health Research Contract
{
Published 2002 Wiley-Liss, Inc. AI95357; Grant sponsor: Cancer Center Support; Grant number:
CORE CA-21765; Grant sponsor: American Lebanese Associated
KEY WORDS: avian in¯uenza virus; phyloge- Charities.
netics; interspecies transmis- *Correspondence to: M. Shaw, In¯uenza Branch, Mailstop G16,
Centers for Disease Control and Prevention, 1600 Clifton Road
sion; host range NE, Atlanta GA 30333. E-mail: mws2@cdc.gov
Accepted 14 May 2001
Published 2002 WILEY-LISS, INC. {This article is a

US Government work and, as such, is in the public
domain in the United States of America.
DOI 10.1002/jmv.2118
108 Shaw et al.
Phylogenetic analyses of a broader sample of avian examined, abbreviations used for them in the text, and
in¯uenza viruses isolated from live-poultry markets in the portion of the six internal genes examined. All
Hong Kong in December of 1997 showed that the H5N1 and H9N2 in¯uenza A viruses were handled un-
internal genes of the human and avian in¯uenza H5N1 der BL3 containment conditions.
isolates were most closely related to a single H9N2
quail in¯uenza A virus isolated in the area [Guan et al., RT-PCR and Nucleotide Sequencing
1999]. In March of 1999, two human cases of in¯uenza
A virus infection in Hong Kong were determined to Virion RNA extracted using the RNeasy RNA extrac-
be caused by avian-like in¯uenza H9N2 viruses tion kit and manufacturer's protocol (Qiagen, Chats-
[Peiris et al., 1999; Lin et al., 2000], and an additional worth, CA) was used for PCR ampli®cation. cDNA
nine probable human cases of H9N2 in¯uenza were synthesis and PCR ampli®cation of the coding region of
reported from Guangdong Province, China [Guo et al., the six internal genes were carried out in one or two
1999; Chen et al., 2000]. Studies of sera from blood parts [Klimov et al., 1992], using gene speci®c primer
donors and poultry workers in Hong Kong suggested sets (primer sequences available on request).
that other unrecognized H9N2 infections may have PCR-derived dsDNA was used as a template for
occurred [Peiris et al., 1999; Eick et al., 2000]. While the automated sequencing on an Applied Biosystem 373A
H9N2 infections caused relatively mild illness not automated DNA sequencer using cycle sequencing dye
associated with complications [Peiris et al., 1999], these terminator chemistry (Perkin-Elmer, Foster City, CA)
incidents represent similar, unusual transmissions of or on a Visible Genetics GeneBlaster system using
avian in¯uenza viruses to humans. Cy5.5 and/or Cy5.0-labeled primers or dye-terminators
Since the studies on the initial human H5N1 isolate (Visible Genetics, Inc., Toronto, ON). The sequences of
gave no speci®c indication of changes that might explain the primers used for these reactions are available upon
its ability to infect humans, a more extensive genetic request. GenBank accession numbers for the sequences
analysis of the avian and human H5N1 and H9N2 obtained from this study are shown in Table I.
viruses was undertaken in an attempt to better under-
stand their relationship to cocirculating avian viruses Phylogenetic and
that had not demonstrated an ability to cause disease in Statistical Analyses
humans. The detailed analyses that are described in Nucleotide sequences were analyzed using version
this report show that the viruses formed a distinct clade 8.0 of the sequence analysis software package of the
for each nonglycoprotein gene that was most closely University of Wisconsin at Madison, Genetic Computer
related to previously characterized avian strains and Group [Devereaux et al., 1984]. Version 3.5 of the Phy-
unrelated to human H3N2 strains circulating concur- logeny Inference Package [Felsenstein, 1989] was used
rently in Hong Kong. The larger sample size of the to estimate phylogenies and calculate bootstrap values
present analysis allowed the detection of conserved from the nucleotide sequences.
amino acids de®ning the clade containing the human
isolates.
RESULTS
MATERIALS AND METHODS The open reading frames of 9 of the 16 human H5N1
in¯uenza virus isolates and both human H9N2 isolates
Viruses
were sequenced in their entirety (Table I). While se-
In¯uenza A (H5N1 and H9N2) viruses examined in quences for the nonglycoprotein genes of A/HK/481/97
this study were isolated from nasopharyngeal swabs or through A/HK/486/97 had been reported previously
tracheal aspirates collected from patients with in¯u- [Hiromoto et al., 2000], the present analyses have
enza-like illness in Hong Kong. Viruses were sent to the utilized independently derived sequences determined
WHO Collaborating Center for Reference and Research for viruses of equivalent passage history in order to
on In¯uenza at the CDC by the National In¯uenza minimize variation due to laboratory growth. Variation
Center of Hong Kong, SAR, China. The viruses were from these previously determined sequences was mini-
isolated and identi®ed during normal or enhanced mal (99.063±100% identity at the nucleotide level) and
surveillance of in¯uenza activity. Madin Darby canine had no effect on the observed phylogenetic relationships.
kidney (MDCK) cells were used for virus isolation and For comparison, the nonglycoprotein genes from two
propagation. All virus genes were sequenced at equiva- human H3N2 viruses isolated in Hong Kong during the
lent passage levels. Fifteen of the sixteen H5N1 viruses H5N1 outbreak were included in the analysis [Cooper
were isolated from patients during November and and Subbarao, 2000]. In agreement with results re-
December 1997, and A/HK/156/97 was isolated in May ported earlier for the initial in¯uenza A (H5N1) infec-
1997. The two human H9N2 isolates were obtained in tion and virus isolation [Subbarao et al., 1998], the
March 1999. The nonglycoprotein genes from two sequences of all genes examined in all of the H5N1 and
human H3N2 viruses isolated during surveillance of H9N2 isolates presented in this study were clearly of
local in¯uenza activity while the H5N1 outbreak was avian origin, with greater than 90% nucleotide simi-
ongoing, were sequenced in their entirety for compar- larity when compared to avian virus sequences avail-
ison. Table I lists the strain designations of the viruses able in GenBank as of March, 2001.
TABLE I. Human In¯uenza Viruses Analyzed*
I Isolate Isolation date PB2 PB1 PA NP M NS

A/HK/156/97 (H5N1) 05/97 100 (AF036363) 100 (AF036362) 100 (AF036361) 100 (AF036359) 100 (AF036358) 100 (AF036360)
A/HK481/97 (H5N1)a 11/97 100 (AF258837) 100 (AF258818) 100 (AF257193) 100 (AF255744) 100 (AF255365) 100 (AF256178)
A/HK/482/97 (H5N1)a 11/97 100 (AF258838) 100 (AF258819) 100 (AF257194) 100 (AF255745) 100 (AF255366) 100 (AF256179)
A/HK/485/97 (H5N1)a 12/97 14 (AF258847) 14 (AF258828) 14 (AF257203) 70 (AF255754 66 (AF255375 37 (AF256189)
and AF255755) and AF255376)
Human H5N1 and H9N2 In¯uenza Viruses
A/HK/486/97 (H5N1)a 12/97 100 (AF258840) 100 (AF258821) 100 (AF257196) 100 (AF255747) 100 (AF255368) 100 (AF256181)
A/HK/488/97 (H5N1) 12/97 14 (AF258848) 14 (AF258829) 14 (AF257204) 64 (AF255756 67 (AF255377 43 (AF256190)
and AF255757) and AF255378)
A/HK/491/97 (H5N1) 12/97 14 (AF258849) 13 (AF258830) 15 (AF257205) 62 (AF255758 71 (AF255379 38 (AF256191)
and AF255759) and AF255380)
A/HK/503/97 (H5N1) 12/97 13 (AF258850) 17 (AF258831) 15 (AF257206) 54 (AF255760 35 (AF255381) 37 (AF256192)
and AF255761)
and AF255763)
and AF255765)
and AF255767)
*Values represent percent ORF sequenced followed by (GenBank accession number).
a
Independently derived sequences for these viruses have been reported by Hiromoto et al. [2000] as noted in the text.
109
110 Shaw et al.
Nucleotide homologies between the human 1997 Phylogenetic Relationships of

H5N1 and the 1999 H9N2 viruses ranged from a low of the Nonglycoprotein Genes
96.7% identity for the NS gene [HK/532/97 (H5N1) vs.
HK/1073/99 (H9N2)] to a high of 99.4% for the NP gene Simpli®ed phylogenetic trees for the six internal
[HK/481/97 (H5N1) vs. HK/1074/99 (H9N2)]. When genes are presented in Figure 1 with each scaled to
compared with the most recent sequences available for show nucleotide changes per 100 nucleotides to indicate
avian in¯uenza viruses, the human H5N1 and H9N2 the relative degrees of variation of the different genes.
viruses were most closely related to H5N1 and H9N2 The dendrogram for each gene includes A/goose/
viruses isolated in live poultry markets in Hong Kong in Guangdong/1/96 (H5N1), the prototype for the avian
1997 [Guan et al., 1999; Shortridge et al., 1998]. lineage from which the H5 HA gene seen in the 1997
Fig. 1. Simpli®ed dendrograms showing the phylogenetic relation- and ck/HK/739/94 H9N2 viruses; (Clade IV) ck/Korea/38349-p96323/
ships of human and avian in¯uenza isolates compared to in¯uenza A 96 and ck/Korea/25232-p96006/96 H9N2 viruses. Accession numbers
viruses from different hosts. Individual viruses included in the for nucleotide sequences other than those cited in Table I are: (NS)
dendrograms are subtype H9N2 unless otherwise noted. The nucleo- AF036360, AF046083, AF098569 through AF098576, AF144307,
tide sequences were compared to sequences available from GenBank AF156472 through AF156485, U49492, and X15282; (M) AF046082,
using the sequence analysis software of the University of Wisconsin AF046090, AF098560 through AF098568, AF144306, AF156458
Genetic Computer Group [Devereaux et al., 1984] and mapped into a through AF156471, M63527, and X53029; (NP) AF028710, AF046-
phylogenetic tree using the Phylogeny Inference Package, version 3.5 084, AF098617 through AF098623, AF144303, AF156402 through
[Felsenstein, 1989]. Branches de®ned by nodes occurring in more than AF156415, D00601, and M30764; (PA) AF046087, AF046095, AF09-
85% of multiple bootstrap replicates are denoted by the heavy lines. 8604 through AF098611, AF144302, AF156444 through AF156457,
Clades de®ning multiple virus isolates denoted by capital letters are: M26078, M26083, and M26084; (PB1) AF046085, AF046094, AF09-
(Clade I) qa/HK/G1/97 (H9N2), teal/HK/w312/97 (H6N1), the human 8590 through AF098598, AF144301, AF156416 through AF156429,
H9N2 isolates from 1999, and the human and avian H5N1 isolates M25924, and M25925; (PB2) AF046086, AF046093, AF098577 through
from the 1997 outbreak in Hong Kong; (Clade II) ck/HK/G9/97, ck/HK/ AF098584, AF144300, AF156430 through AF156443, M27684, M36-
G23/97, and pg/HK/Y233/97 H9N2 viruses; (Clade III) ck/Beijing/1/94 037, and M36046.
Human H5N1 and H9N2 In¯uenza Viruses 111
Hong Kong outbreak most likely originated [Xu et al., TABLE II. Conserved Clade-De®ning Amino Acids
1999]. Representative avian and human viruses, in-
Amino Human
cluding the human A/HK/498/97 (H3N2) virus, are acid Clade I Clade II Clade III Clade IV H3N2
shown for comparison.
A grouping into four distinct phylogenetic clades was NS1
92 F D D D D
reported earlier for the PB1 and PB2 genes of H9N2 202 T A A A A
viruses circulating in eastern Asia from 1992 to 1997 218 Q STOP STOP Q Q
[Guan et al., 1999]. When the additional H5N1 and 223 F A A
H9N2 isolates from humans were included, these four NS2
clades could consistently be distinguished for all six 14 K M M M L
60 N S S S N
nonglycoprotein genes with the same viruses grouping M1
together with greater than 95% certainty in 100 boot- 157 A S S S S
strap analyses. Clade I contained A/quail/HK/G1/97 M2
(H9N2) virus [Guan et al., 1999] and the two human 10 I P P P P
82 N S S S N
H9N2 isolates, A/teal/HK/W312/97 (H6N1) virus [Hoff- NP
mann et al., 2000], and the avian and human H5N1 52 Q Y Y Y Y
viruses from the 1997 outbreak in Hong Kong. For each 136 M L L L M
gene, the association of the human H5N1 and H9N2 371 V M M M M
isolates within the same phylogenetic clade occurred in 373 A T T T N
430 K T T T T
100% of the bootstrap trials and, with the exception of PA
the PB1 and PB2 genes where clades I and II grouped 20 T A A A A
together, were distinct from the other clades. 85 A T T T T
The other three clades shown in Figure 1 consisted 118 T I I I I
318 R K K K K
entirely of avian H9N2 viruses: Clade II was composed 367 M K K K K
of A/ck/HK/G9/97 and two similar viruses (ck/HK/G23/ 387 I V V V I
97 and pg/HK/Y233/97), clade III of A/ck/Beijing/1/94 394 H/Q D D D D
and ck/HK/739/94 viruses, and clade IV contained A/ck/ 400 L P P S L
Korea/38349-p96323/96 and A/ck/Korea/25232-p96006/ 547 E D D D D
615 R K K K K
96 viruses as described earlier [Guan et al., 1999]. The 651 S A A A A
other recent H9N2 viruses from east Asia shown in 688 G E E E E
Figure 1 were not consistently associated with the same PB1
clade for each gene suggesting that they might repre- 54 R R K K K
213 T T N N N
sent reassortants between or intermediate variants of 215 K K R R R
the clades. 253 H H Y Y Y
257 A A T T/H T
Residues De®ning Clade I Containing 302 V V I I I
the 1997 H5N1 and 1999 H9N2 637 V V I I I
Viruses From Hong Kong 694 T T N N N
715 M M V V V
Speci®c residues were identi®ed in different non- 757 G G K K K
glycoprotein gene products that distinguished the clade 758 K K STOP STOP STOP
I viruses from the most closely related strains for which PB2
147 M I I/V I/V I
sequence information is available (Table II). One amino 195 N N D D D
acid residue in M1, 2 each in M2 and NS2, 3 in NS1, 4 in 197 N N K R/K K
NP, and 11 in PA could be used to distinguish the clade 299 K K R R R
I viruses. 334 K K S S S
As described above and shown in Figure 1, the PB1 340 R R K K R
381 M M L L L
and PB2 genes from the viruses that formed clades I and 447 Q K W W W
II for the other nonglycoprotein genes grouped to- 524 M M T T T
gether. For PB1, this combined III clade had 11 567 E E D D N
de®ning residues (Arg54, Leu or Val102, Thr213, Lys- 655 A A V V V
667 I I V V I
215, His253, Ala257, Val302, Val637, Thr694, Met715, 717 T T A A A
and Lys758). PB2 had 11 clade-de®ning residues
common to clades I and II (Asn195, Asn197, Lys299,
Lys334, Arg340, Met381, Met524, Glu567, Ala655,
Ile667, and Thr717) and two residues (Met147 and
Gln447) present only in the clade I isolates (Table II). resulting in an additional amino acid at the carboxy-
As shown in Table II, the PB1 genes of the clade I and terminus. A search of sequences available in GenBank
II viruses could also be distinguished from all other indicated that this additional Lysine is unique to
viruses available for comparison because of a change in viruses in these two clades. In contrast, two of the
the usual termination codon at nucleotides 2296±2298 other avian H9N2 viruses shown in Figure 1, dk/HK/
112 Shaw et al.
y439/97 and qa/HK/af157/92, have an additional Glu at isolated in Hong Kong in late 1997 (Table III). Positions
the carboxy terminus. previously considered to be host-speci®c on the basis of
multiple sequence alignments varied within a parti-
Host-Speci®c Residues cular sequence, with some `àvian'' and some ``human''
amino acid markers (Table III). The addition of the two
Table III lists 36 amino acids in the M1, M2, NP, PA,
human H3N2 isolates reveal that two amino acid
and PB2 polypeptides that have been described pre-
positions each in NP (31 and 127) and PA (241 and
viously as residues that are host-speci®c [Okazaki
312) should no longer be considered host-speci®c. Of the
et al., 1989; Gorman et al., 1990; Ito et al., 1991;
remaining 32 amino acids, the sequences of the clade I
Webster et al., 1992; Scholtissek et al., 1993]. Addi-
H5N1 and H9N2 viruses were avian-like at 23 sites,
tional sequences published after these initial studies
human-like at 5 sites, and mixtures of the two at 4
[Suarez et al., 1999; Garcia et al., 1997; Lindstrom et al.,
other sites.
1998] were also included in the analysis to verify
the earlier observations. The deduced amino acid se-
quences of the H5N1 and H9N2 viruses are shown DISCUSSION
compared with those of the most closely related avian
The nonglycoprotein genes of the human H5N1 and
clade and with two human in¯uenza A H3N2 viruses
H9N2 viruses were clearly closely related to each other
and differed from those of other human in¯uenza
TABLE III. Host-Associated Amino Acids in Predicted Virus A viruses. The high degree of similarity between the
Gene Products* avian and human isolates indicates that any selec-
Amino Human tive pressure speci®c to replication in humans was
acid Avian Human Clade I Clade II H3N2a minimal. This is to be expected since the patients most
likely acquired their infections from exposure to in-
M1
137 T A T T A fected poultry and there was no evidence of sustained
M2 human-to-human transmission [Mounts et al., 1999;
16 E G E/G G G Bridges et al., 2000].
20 S/N N S S N The extensive sequence analyses undertaken in this
28 I I/V V V V study led to two clear conclusions: First, for each of the
55 L F F F F
78 Q K Q Q K nonglycoprotein genes, residues were present that dis-
NP tinguished the clade containing the avian-like human
31 R K R R R/K isolates from other avian in¯uenza sequences and may,
33 V I V V I therefore, be considered candidates for further investi-
61 I L I I L
100 R V R R V gation. Second, this analysis forces a re-evaluation of
127 E D E E E the amino acids that were previously considered to be
136 L M M L/M M ``host-speci®c.''
214 R K R R K The amino acid sequences deduced for the internal
283 L P L L P genes showed that all the avian-like viruses isolated
293 R K R R K
313 F Y F F Y from humans in 1997 and 1999 belonged to a single
375 D G/E D D G clade that had characteristic amino acids not found in
PA other avian in¯uenza A virus sequences. The conserved
28 P L P P L amino acids that distinguish clade I viruses are obvious
55 D N D D N
65 S L S F L candidates for further examination to determine their
100 V A V V A effects on host range since viruses from the other avian
241 C Y C C C clades circulating widely in eastern Asia at that time
312 K R K K K were not reported to infect humans. The increased
382 E D E E D surveillance in Hong Kong during and after the 1997
400 Q/T/S L L P L
409 S N N/S S N outbreak would most likely have detected any such
552 T S T T S infections had they occurred.
PB2 Another conclusion arising from the present analysis
44 A S A A S of the nonglycoprotein genes of the human H5N1 and
81 T M T/A I M
199 A S A/S A S H9N2 isolates is that the assignment of particular
271 T A T T A signature amino acids as host-speci®c must be re-
588 A I A A I evaluated. The majority of the predicted gene products
613 V T V V T showed residues previously considered to be avian-
661 A T T T/A T speci®c and human-speci®c in the same molecule. It is
674 A/S T A A T
702 K R K/R K R possible that the presence of these human-associated
residues in otherwise `àvian'' polypeptides is what
*Okazaki et al., 1989; Gorman et al., 1990; Ito et al., 1991; Webster allowed these viruses to infect humans. However,
et al., 1992; Scholtissek et al., 1993; Garcia et al., 1997; Lindstrom
et al., 1998, Suarez et al., 1999. residues previously considered to be `àvian'' were seen
a
HK/498/97 and HK/498/97 (H3N2) viruses. in the NP and PA sequences predicted for the human
Human H5N1 and H9N2 In¯uenza Viruses 113
H3N2 viruses analyzed (Table III). It should be noted ing importance that new in¯uenza outbreaks in birds
that most of the available sequence data for the be monitored closely in order to give warning as early as
nonglycoprotein genes were obtained before 1990. possible when a new gene constellation appears that
While the few sequences available for viruses isolated might allow viruses with different glycoprotein sub-
between 1990 and 1997 [Garcia et al., 1997; Lindstrom types to spread to humans.
et al., 1998; Suarez et al., 1999] are consistent with the
amino acid assignments shown in Table III, it is still ACKNOWLEDGMENTS
possible that at least some of the ``host-range'' substitu-
tions seen in the clade I viruses occurred prior to the We thank Carolyn Bridges, Hector Izurieta, and Keiji
1997 outbreak; their appearance may be only coin- Fukuda from the Epidemiology Section of the In¯uenza
cidental to the human infections. It will be necessary to Branch at CDC, and Paul Saw, K.H. Mak, and the staff
examine more internal gene sequences from both avian of the Hong Kong Department of Health for their
and human isolates in order to determine what affect, if logistical and technical assistance in the acquisition of
any, these residues have on the host range of these specimens. We also thank Sarah Cantrell and Mark
viruses. Hemphill for excellent technical assistance. These
Until the signi®cance of the unique, clade-de®ning studies were supported in part by Public Health
amino acids identi®ed in the nonglycoprotein genes is Research Contract AI95357 from the National Institute
elucidated and the potential effects of the mixture of of Allergy and Infectious Diseases.
human- and avian-speci®c residues in the same poly-
peptide are determined, it is too early to correlate any of
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Molecular Changes h5n1

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Molecular Changes h5n1

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Journal of Medical Virology 66:107±114 (2002)

Molecular Changes Associated With the

In order to identify molecular changes associated INTRODUCTION

Published 2002 WILEY-LISS, INC. {This article is a

I Isolate Isolation date PB2 PB1 PA NP M NS

Nucleotide homologies between the human 1997 Phylogenetic Relationships of

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