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Journal of Virology, April 1999, p. 3366-3374, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Rapid Evolution of H5N1 Influenza Viruses in
Chickens in Hong Kong
Nan Nan
Zhou,1,2
Kennedy F.
Shortridge,3
Eric C. J.
Claas,4
Scott L.
Krauss,1 and
Robert G.
Webster1,*
Department of Virology and Molecular Biology,
St. Jude Children's Research Hospital, Memphis, Tennessee
381051; Department of Microbiology, The
University of Hong Kong, Queen Mary Hospital, Hong
Kong,3 and Department of Microbiology,
Jiangxi Medical College, Nanchang,2
People's Republic of China; and Department of Virology,
Leiden University Medical Center, Leiden, The
Netherlands4
Received 9 October 1998/Accepted 4 January 1999
 |
ABSTRACT |
The H5N1 avian influenza virus that killed 6 of 18 persons infected
in Hong Kong in 1997 was transmitted directly from poultry to humans.
Viral isolates from this outbreak may provide molecular clues to
zoonotic transfer. Here we demonstrate that the H5N1 viruses
circulating in poultry comprised two distinguishable
phylogenetic lineages in all genes that were in very rapid
evolution. When introduced into new hosts, influenza viruses usually
undergo rapid alteration of their surface glycoproteins, especially in
the hemagglutinin (HA). Surprisingly, these H5N1 isolates had a large
proportion of amino acid changes in all gene products except in the HA.
These viruses maybe reassortants each of whose HA gene is well adapted to domestic poultry while the rest of the genome arises from a different source. The consensus amino acid sequences of "internal" virion proteins reveal amino acids previously found in human
strains. These human-specific amino acids may be important factors
in zoonotic transmission.
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INTRODUCTION |
At irregular intervals, influenza
viruses that contain avian virus-derived genes cause pandemics that
vary in severity from serious (Asian influenza [1957] and Hong
Kong influenza [1968]) (39) to catastrophic (Spanish
influenza [1918]). The molecular events that permit an avian
influenza virus to transfer to and replicate in humans are largely
unknown, as the precursor viruses have heretofore not been
available for study. The H5N1 influenza viruses that infected 18 humans
in Hong Kong in 1997 and caused the deaths of six (13, 35,
41) had been isolated 2 months earlier from chickens with
lethal influenza (10). Thus, the putative progenitor
virus was available for molecular analysis. There was no convincing
evidence of human-to-human transmission; instead, the available
evidence indicated that each case had been caused by independent
transmission of virus to humans from birds in poultry markets.
Avian influenza viruses have a receptor specificity unlike that
of human strains; they bind preferentially to terminal
SA
2,3-galactose determinants, whereas human strains
preferentially bind to terminal SA2,6-galactose sequences
(20, 25). The direct transmission of H5N1 viruses from
chickens to humans in Hong Kong indicates that receptor specificity is
not a definitive host restriction factor and that an intermediate host
(17, 29) is not necessarily required for the first stage of
transmission to humans. Previous studies indicate that the viral
nucleoprotein (NP) and PB2 and M2 proteins are important in determining
host range (2, 8, 28, 34), but the specific domains that
affect host range involved have not been identified.
Circumstantial evidence has indicated that avian influenza viruses can
directly infect humans (3, 5, 9, 31, 38, 42). In 1983, a
highly lethal influenza outbreak in chickens in Pennsylvania was
caused by an avian influenza virus of the H5 subtype;efforts to isolate
the virus from poultry workers during that outbreak were
unsuccessful (4). The H5N1 viruses isolated from humans in
Hong Kong represent the first known direct transmission of avian
influenza virus that has caused severe respiratory disease and death in
humans. The present study characterized the genomes of H5N1 avian
influenza viruses isolated from poultry markets in Hong Kong and
compared them with virus isolated from the index human case. We
established that a rapidly evolving reassortant influenza virus was
present in Hong Kong poultry markets, and we propose that the presence
of specific amino acids typical of human influenza viruses permitted
direct transmission to humans.
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MATERIALS AND METHODS |
Viruses.
The viruses used in this study are listed in
Table 1 (Table 1 also lists
abbreviations used below for some viruses). The viruses were isolated
in chicken embryos as described previously (30) and
were grown in chicken embryos. Since these viruses are lethal for
chicken embryos, the replication time was optimized and virus was
harvested just before the death of the embryos. All viruses were
handled in a biosafety level 3+ facility approved for use by the U.S.
Department of Agriculture; the staff wore fitted HEPA-filtered
masks and took prophylactic rimantadine.
Sequencing and analysis.
The viruses sequenced in this study
are listed in Table 1. Viral RNA was extracted from infected
allantoic fluid with the RNeasy extraction kit (Qiagen, Santa
Clara, Calif.) and amplified by reverse transcription-PCR
(32). PCR products were purified with the QIAquick PCR
purification kit (Qiagen), sequenced with synthetic
oligonucleotides by using rhodamine Dye-Terminator Cycle Sequencing Ready Reaction kits with AmpliTaq DNA polymerase FS (Perkin-Elmer, Applied Biosystems Inc. Foster City, Calif.), and electrophoresed on model 377 (Perkin-Elmer, Applied Biosystems Inc.) DNA sequencers by the Center for Biotechnology at St. Jude Children's Research Hospital.
Analysis of sequence data was performed with Wisconsin Package version
9.1-Unix (Genetics Computer Group, Madison, Wis.) and
GeneDoc
version 2.3.000 (
24) software. Phylogenetic analysis
was
done with PHYLIP version 3.57C software (
14) to implement
a
neighbor-joining
algorithm.
Analysis of nucleotide and amino acid changes in avian influenza
viruses.
A defined region was sequenced for each of six viral
genes (the genes for polymerases [PA, PB1, and PB2],
nucleoprotein [NP], hemagglutinin [HA], and neuraminidase [NA])
in groups of two to six related viruses, and the resulting
nucleotide and amino acid sequences were compared within
groups (see Table 3). A matrix analysis was performed with GeneDoc
version 2.3.000 (24) to quantitate the nucleotide and amino
acid changes in each gene of each isolate, and the values were averaged
for each group. The percentage of nucleotide changes that resulted in
amino acid changes was calculated for each gene of each virus in
a group, and the values were averaged.
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RESULTS |
Phylogenetic analysis of H5N1 isolates from poultry in
Hong Kong.
Preliminary studies of the index human virus from
Hong Kong and of the initial chicken isolate showed two distinct groups based on phylogenetic analysis of the HA (33). We now
establish that the eight genes of the H5N1 viruses isolated from
poultry were closely related but comprised two phylogenetic sublineages.
Genetic homology among the H5N1 isolates varied from 97.9 to 100%
(Table
2). The viruses that were most
homologous with the
individual gene segments of H5N1 viruses from Hong
Kong were from
the Eurasian lineage. Despite the limited number of gene
sequences
available for the polymerase (PB2, PB1, and PA) genes in
GenBank,
the most homologous viruses were from Asia. The PB1, NA, M,
and
NS genes were most related to influenza viruses from China. The
largest quantity of information available is for the NP gene,
and the
virus with greatest NP homology was A/Mallard/Astrakhan/244/82
(H14N6),
a virus isolated from Central Asia. For the NA gene,
the closest
related virus was one isolated from an unidentified
migrating
aquatic bird in Hong Kong (ABHK603-98). Sequence information
for
additional Eurasian viruses is needed before any projection
can be made
as to the origins of other gene segments.
Despite the high degree of homology of the Hong Kong H5N1
viruses, phylogenetic analysis showed that there were two
subgroups
of each gene. Figure
1 shows
the phylogenetic subgroups based
on HA1. In one group, most viruses
possessed a carbohydrate side
chain at residue 154 of HA1; the other
group, with one exception,
lacked the carbohydrate. The viruses of both
subgroups possessed
a series of basic amino acids (RERRRKKR) at the
connecting peptide
between HA1 and HA2 and were highly pathogenic in
chickens (Table
1). The sequences encoding the receptor binding sites
were identical
in the two groups and indicated binding to terminal
SA
2,3-galactose
determinants, as was described for the
index human virus (
10,
35).
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FIG. 1.
Phylogenetic trees of the HA1 genes of H5 influenza A
viruses. (A) Evolutionary relationship of the H5N1 Hong Kong viruses to
various H5 viruses isolated previously in North America and Eurasia.
The tree was generated by using the neighbor-joining algorithm in
PHYLIP version 3.57c software (14) and is rooted to
A/Pintail/Praimoric/625/76 (H2N2). Horizontal lines are proportional to
the numbers of nucleotide substitutions between branch points. (B) One
hundred-replication bootstrap resampling of the H5 Hong Kong viruses
from panel A; the tree is rooted to A/Turkey/England/50-92/91. The
number at each branch point indicates the percent probability of the
accuracy of the resulting topology. Asterisks indicate the presence of
a potential glycosylation site at amino acid 154 of HA. Abbreviations:
Ty/Ont/66, A/Turkey/Ontario/7732/66 (H5N9); Ck/PA/83,
A/Chicken/Pennsylvania/1/83 (H5N2); Ck/Chiap/97,
A/Chicken/Chiapis/15224/97 (H5N2); Ck/Pue/94, CPUE1-94; Ck/Que/95,
CQUE95; Ck/PA/93, A/Chicken/Pennsylvania/13609/93 (H5N2); RT/DEL/93,
A/Ruddy turnstone/Delaware/244/93 (H5N2); Dk/MN/81, DMN81;
Gull/PA/83, GPA83; Ty/Ram/73, TRAM73; Ck/Scot/59, A/Chicken/Scotland/59
(H5N1); Tern/South Africa/61, A/Tern/South Africa/61 (H5N1); Ty/Eng/91,
A/Turkey/England/50-92/91 (H5N1); Dk/Ire/83, A/Duck/Ireland/113/83
(H5N8); Ty/Ire/83, A/Turkey/Ireland/1378/83 (H5N8).
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The NAs of the H5N1 Hong Kong influenza viruses isolated from poultry
all had a 19-amino-acid deletion in the stalk (Fig.
2). Three glycosylation sites in the N1
protein are removed by
this deletion. Partial sequencing of NA
genes from 13 additional
poultry H5N1 viruses confirmed that the
deletion is present in
all of the H5N1 viruses. This deletion
consists of 4% of the NA
molecule and was detectable by
electrophoresis of PCR products
of the NA gene. To check the difference
in the sialic acid binding
site and antigenic sites between the NA of
HK156-97 and the poultry
H5N1 viruses, the NA sequences of the Hong
Kong viruses were aligned
with the N2 protein of A/Tokyo/3/67, which
has been defined by
structural analysis (
11). The alignment
was made as described
by Colman et al. (
12) (Fig.
2).
According to the alignment,
all amino acids in the sialic acid binding
site of N2 are conserved
in the H5N1 Hong Kong viruses. No difference
in antigenic determinants
was observed for HK156-97 and the poultry
H5N1 viruses. There
are three potential glycosylation sites conserved
in the head
region of NA of A/Tokyo/3/67 (N2), A/Parrot/Ulster/77 (N1),
and
the H5N1 Hong Kong viruses (N1). The H5N1 viruses belonging to
group 1 possessed an additional potential glycosylation site at
residue
I204T in the head region.
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FIG. 2.
Alignment of NA amino acid sequences of H5N1 influenza
viruses from Hong Kong. Amino acids in the open boxes are potential
glycosylation sites, those in the dark background are enzyme activity
sites, and those in the light background are antigenic sites. Dashes
represent deletions; dots represent the gaps resulting from aligning
the N1 proteins with N2. par/uls/73, A/Parrot/Ulster/73 (H7N1);
Tokyo/3/67, A/Tokyo/3/67 (H2N2).
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Phylogenetic analysis of the other seven gene segments of the
H5N1 isolates from poultry confirmed that they each belong to
the
Eurasian avian lineage. For conservation of space, only the
terminal branches of the phylogenetic trees of the NA and
internal
genes are shown in Fig.
3. Each
of the genes of H5N1 poultry viruses
from Hong Kong can be divided into
the two subgroups given above
for the HA. However, the human index
virus, HK156-97, and the
early chicken isolate CHK258-97 are
intermediate between the two
groups. Viruses from both subgroups caused
severe disease in humans
(
6,
33,
41).
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FIG. 3.
Terminal branches of the phylogenetic trees of the NA
and internal genes of H5N1 influenza viruses from poultry in Hong Kong.
The phylogenetic trees were generated as described in the legend to
Fig. 1B. The viruses underlined belong to group 1, the viruses
italicized belong to group 2, and the viruses in the boxes are
intermediates. BUDHOK, A/Budgerigar/Hokkaido/1/77 (H4N6); SWHK82,
A/Swine/Hong Kong/126/82 (H3N2); DKHOK80, A/Duck/Hokkaido/8/80 (H3N8);
MALASTRA82, A/Mallard/Astrakhan/244/82 (H14N6).
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The rate of evolution of the HA gene is disproportionately
low.
When an influenza virus is first introduced into a new host,
it usually undergoes a period of rapid evolutionary change (18, 27). Nucleotide changes that result in amino acid changes are usually most numerous in the HA, due to immunological selection, and
are less numerous in the internal genes. Table
3 shows the number of nucleotide changes,
the number of amino acid changes, and the percentage of changes in six
of the gene sequences of H5N1 viruses. Where possible, we compared
sequence information with that for H5N2 influenza viruses that
had apparently emerged from wild aquatic birds and then
spread through the chicken population of Mexico and become highly
pathogenic (16). We also used the available but limited
sequence information on influenza viruses from aquatic birds for
comparison. In the HA gene of H5N1 poultry viruses, only 5.9% of the
nucleotide changes produced amino acid changes, which was an order of
magnitude less than the percentage of similar changes in the PA gene
(Table 3). The NA gene and the genes comprising the replication complex
(the genes for PB2, PB1, PA, and NP) all had a higher percentage of
nonsilent nucleotide substitutions than did the HA gene.
In H5N1 viruses isolated from poultry in Hong Kong, the percentage of
coding changes in the HA gene (5.9%) was an order of
magnitude less
than that found in H5N2 virus from chickens in
Mexico (57.3%). This
finding indicates a surprisingly low rate
of evolution of the HA gene.
Only the HA gene of influenza viruses
from aquatic birds had a
comparably low proportion of coding changes
(H5 from duck,
15.1%). After transmission to humans the H5 HA
gene evolved
rapidly, which is expected in a new host. The rates
of coding
changes in the NA and other genes (those for PB2, PB1,
PA, and NP) in
H5 viruses from Hong Kong and Mexico were higher
than those found in
influenza viruses from aquatic birds. This
rapid rate of change is
typical of a virus in a new
host.
These studies indicate that the HA gene of the H5N1 influenza viruses
isolated from poultry in Hong Kong is optimally adapted,
while the
other gene segments are evolving rapidly. After transmission
to humans
the HA genes shows the expected high rate of
change.
Correlation between host range and amino acids in PA, PB2, NP, and
M2 gene products.
Previous studies have indicated that the NP,
PB2, and M2 gene products are important in determining the host
range of influenza viruses (8, 28, 34). We therefore
compared the amino acid sequences of these proteins in the Hong
Kong poultry H5N1 isolates with those of other viruses.
We also compared the sequences of the PA proteins.
When available sequence information for the avian and human
influenza virus proteins was compared, certain amino acids appeared
to
be specific to avian or human viruses. In the case of M2
proteins,
10 amino acids have previously been found almost exclusively
in
human influenza viruses; we found 3 of these amino acids in the
M2
protein of H5N1 viruses from poultry in Hong Kong (Fig.
4).
In each of the poultry isolates, the
amino acids at M2 residues
16, 28, and 55 were G, V, and F,
respectively, while in 20 other
avian influenza viruses these amino
acids were E, I, and L (A/Chicken/Hong
Kong/14/76 also had an F at
position 55). Thus, three amino acids
in M2 of the chicken H5N1
influenza viruses are highly related
to the sequence in human
influenza viruses. Among the other internal
gene products (PB2, PB1,
PA, NP, M1, and NS) of the H5N1 viruses
from Hong Kong,
"human-like" amino acids were detected in PB2
(S199, T661,
and R702), PA (N409), and NP (M136) (Table
4). No
human-like substitutions were
detected in the PB1, M1, or NS proteins.
Thus, the H5N1 viruses
isolated from poultry in Hong Kong possess
several amino residues
characteristically found in human strains.
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FIG. 4.
Alignment of amino acids of the M2 proteins of human and
avian influenza A viruses. The open boxes indicate amino acids found
almost exclusively in human influenza viruses. The solid boxes indicate
human-like amino acids that are present in avian viruses. Asterisks
indicate avian-like amino acids that are present in human viruses.
Abbreviations for avian viruses: Ck/Bres/02, A/Chicken/Brescia/19/02
(H7N7); Fpv/Dob/27, A/FPV/Dobson/27 (H7N7); Fpv/Ros/34,
A/FPV/Rostock/34 (H7N1); Fpv/Wey/34, A/FPV/Weybridge (H7N7);
Shear/Aus/72, A/Shearwater/Australia/1/72 (H6N5); Ck/HK/14/76,
A/Chicken/Hong Kong/14/76 (H1N1); Dk/HK/193/77, A/Duck/Hong Kong/193/77
(H1N2); Budg/Hok/77, A/Budgerigar/Hokkaido/1/77 (H4N6); Dk/Bav/77,
A/Duck/Bavaria/2/77 (H1N1); Mal/NY/78, A/Mallard/New York/6750/78
(H2N2); Gull/MD/78, A/Gull/Maryland/1824/78 (H13N6); Gull/MA/80,
A/Gull/Massachusetts/26/80 (H13N6); Ty/MN/80, A/Turkey/Minnesota/833/80
(H4N2); Ty/MN/81, A/Turkey/Minnesota/166/81 (H1N1); Ck/PA/83,
A/Chicken/Pennsylvania/1370/83 (H5N2); Ck/Vic/85,
A/Chicken/Victoria/1/85 (H7N7); Oys/Ger/87, A/Oyster catcher/Germany/87
(H1N1); Dk/Nan/92, A/Duck/Nanchang/1749/92 (H11N2). Abbreviations for
human viruses: wsn/33, A/WSN/33 (H1N1); PR/8/34, A/Puerto Rico/8/34
(H1N1); FM/47, A/Fort Monmouth/1/47 (H1N1); FW/50, A/Fort Warren/1/50
(H1N1); Sing/57, A/Singapore/1/57 (H2N2); Lening/57, A/Leningrad/134/57
(H2N2); Ann/60, A/Ann Arbor/6/60 (H2N2); Korea/68, A/Korea/426/68
(H2N2); Aichi/68, A/Aichi/2/68 (H3N2); USSR/70, A/USSR/90/70 (H1N1);
Udorn/72, A/Udorn/72 (H3N2); PCham/73, A/Port Chalmers/1/73 (H3N2);
USSR/77, A/USSR/90/77 (H1N1); Bangk/79, A/Bangkok/1/79 (H3N2); Mem/88,
A/Memphis/8/88 (H3N2); Guangd/89, A/Guangdong/39/89 (H3N2).
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DISCUSSION |
Sequence analysis of each of the gene segments of H5N1 viruses
isolated from poultry in Hong Kong established that of two distinguishable groups of the viruses cocirculated in domestic poultry.
The origin of these two groups is unresolved. They may have originated
through geographical separation of the original reassortant H5N1
possessing a chicken-adapted H5 gene and seven genes from a
still-unidentified Eurasian avian influenza virus. Separation may have
occurred in different parts of Asia providing poultry to Hong Kong or
in different avian species in Hong Kong. Since the original chicken
H5N1 isolate (CHK258-97) is intermediate between the two lineages (Fig.
1 and 3), the two lineages may be a recent evolutionary
development that occurred in different markets in Hong Kong. Each gene
segment of the two groups of viruses encoded amino acids that
distinguish the two groups (results not shown). However, the facts that
both groups infected humans and caused similar disease patterns
(6, 33, 41) suggest that these amino acids are not directly
involved in host range transfer.
The receptor specificity of influenza virus HA usually depends on the
host species from which the virus is isolated. However, it was found
that the H5N1 isolate from a human (HK156-97) has the same sequence in
the receptor binding site of HA as found in poultry H5N1 viruses
(10, 19, 33). This finding indicates that an avian virus can
be transmitted to humans without a change in its receptor binding
properties. However, this does not mean that avian viruses can be
transmitted to humans as effectively as human virus. The difference in
host cell receptor specificity may have played a role in restricting
transmission of these H5N1 viruses among humans. Although a large human
population in Hong Kong may have had contact with the infected
chickens, only 18 cases were found to be positive by virus isolation.
Serological studies with humans are still in progress, but the
available evidence indicates little if any human-to-human transmission.
When influenza virus is introduced into a new host, the proportion of
nucleotide changes that result in amino acid changes is usually highest
in the HA gene. In the H5N1 influenza viruses isolated from Hong Kong
poultry markets, this was not the case, the highest percentage of
coding changes that altered amino acids was approximately six times
higher in the PA gene than in the HA gene. The emergence of highly
pathogenic H5 influenza viruses in chickens in Mexico represents
the most comparable case for which information is available. Those
isolates showed a large proportion of amino acid changes in the
HA1 protein (57.3%) and a lower proportion in the products of the
internal genes. This reversal of the pattern of coding changes suggests
that the HA gene is better adapted to chickens than are the other
genes. The Hong Kong H5N1 viruses may be reassortants that acquired the
HA gene from an H5 virus that is well adapted to domestic poultry, while the other seven genes may have come from another source, such
as wild aquatic birds. The HA could possibly have come from A/Goose/Guangdong/1/96 (H5N1), a virus that was highly lethal in geese
(40). Guangdong Province lies adjacent to Hong Kong and
provides much of Hong Kong's domestic poultry. The other seven genes
are of Eurasian avian origin, but their source is undetermined. Additional sequence information from Eurasian influenza viruses should elucidate the origin of these gene segments.
When H2 was introduced into humans in 1957 the molecule did change
rapidly. Schafer et al. (27) reported that the earliest stages in the evolution of the human lineage appear to have been under
greater selective pressures than the later branches as judged by their
ratios of coding to noncoding changes. Initially, 1.6 nucleotide
changes resulted in amino acid changes; later, 3.7 nucleotide changes
were required per amino acid change. This was also seen in the case of
European swine virus. When the H1N1 avian-like viruses appeared in
swine in Europe in 1979, the NP genes of these new viruses evolved at a
higher rate than NP genes in human and classic swine viruses over the
period of 1930 to 1988. We do not see more rapid evolutionary changes
in H3 for 1968 because we do not know when the avian HA was transmitted
to humans. Some serological studies suggest that H3 was introduced into
humans several years ahead of isolation of the virus in humans in 1968 (21).
The large proportion of amino acid changes in the NAs and internal gene
products of the Hong Kong H5N1 isolates indicates that these viruses
are evolving rapidly and therefore that chickens are a new host. These
findings support the hypothesis that chickens represent a host
population distinct from aquatic birds. As such, they may play a role
in the evolution of the virus and may be an intermediate host in
zoonotic transmission (19). Surveillance in poultry in Hong
Kong between 1975 and 1985 detected H5 influenza viruses in ducks and
geese but not in chickens (31), again suggesting that this
virus was introduced into chickens relatively recently.
Specific amino acids in the M2 and PB2 proteins that have been
associated with host range variants (8, 22, 28, 34) were not
implicated by these studies. However, it must be noted that the host
range of influenza viruses is a polygenic trait that can vary from
virus to virus. The functional domains of PB2, PB1, PA, and NP that are
associated with RNA synthesis, chain extension, and nuclear
localization are being resolved (7, 23, 37). We found that
the H5N1 Hong Kong viruses contain two human-like amino acids (T661 and
R702) that are located in the functional domain (C-terminal 124 amino
acids) of PB2 responsible for interaction with other polymerase
components (26). The human-like amino acid (M136) in the NP
of H5N1 viruses is located in the RNA binding domain of the protein
(1), and the human-like amino acid (V28) in M2 of the H5N1
viruses is located in the domain serving as an ion channel
(15). Although we do not know the function of these
molecules in determining host range, we speculate that these amino
acids are involved in the proliferation of H5N1 viruses in chickens and
in humans. Identification of these host range-specific amino acids will
provide a starting point for studies aimed at identifying the
functional sites that mediate host range.
Our analysis of available sequence information from GenBank reveals
that a number of influenza viruses isolated from humans contain
avian-specific amino acids. These amino acids were present in the older
human strains (A/WSN/33 [H1N1] and A/Puerto Rico/8/34 [H1N1])
identified as descendants of the virus causing the 1918 Spanish
influenza pandemic and may be remnants of avian influenza viruses that
were transmitted to humans. It is noteworthy that the partial sequences
of the NP and M protein of the 1918 virus (36) contain more
avian-like amino acids than in A/WSN/33. This indicates that the
avian-like amino acids in A/WSN/33 were probably not derived from
passage in chicken eggs.
The information that is now available underscores the wisdom of the
slaughter of 1.6 million domestic fowl in Hong Kong in 1997. The H5N1
virus was evolving rapidly in this host. It had acquired a number of
amino acids that correlate with replication in humans. Eradication of
the chicken population in Hong Kong eliminated the immediate
opportunity for the H5N1 viruses to be transmitted to humans.
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ACKNOWLEDGMENTS |
These studies were supported by Public Health Research Grant
AI29680 and Cancer Center Support (CORE) grant CA-21765 from the
National Institutes of Health and by the American Lebanese Syrian
Associated Charities, the World Health Organization, the Department of
Health of the Hong Kong government, and the Committee on Research and
Conference Grants and the Office of the Vice-Chancellor, The University
of Hong Kong.
We thank Enid M. Bedford and Kimberly Hampton for preparation of the
manuscript and Sharon Naron for editorial assistance.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Virology and Molecular Biology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. Phone: (901) 495-3400. Fax: (901)
523-2622. E-mail: robert.webster{at}stjude.org.
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Abstract
Introduction
