Evolutionary characterization of the six internal genes of H5N1 human influenza A virus

Yasuaki Hiromoto1, Yoshinao Yamazaki1, Tatsunobu Fukushima1, Takehiko Saito1, Stephen E. Lindstrom1, Katsuhiko Omoe1, Reiko Nerome1, Wilina Lim2, Shigeo Sugita3 and Kuniaki Nerome1

Department of Virology I, National Institute of Infectious Diseases, 23-1, Toyama 1-chome, Shinjuku-ku, Tokyo 162-8640, Japan1
Government Virus Unit, Queen Mary Hospital, Department of Health, Hong Kong, China2
Epizootic Research Station, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Kokubunji-machi, Shimotsuga, Tochigi 329-04, Japan3

Author for correspondence: Kuniaki Nerome. Fax +81 3 5285 1155. e-mail knerome{at}nih.go.jp


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
The entire nucleotide sequences of all six internal genes of six human H5N1 influenza A viruses isolated in Hong Kong in 1997 were analysed in detail from a phylogenetic point of view and compared with the evolutionary patterns of the haemagglutinin and neuraminidase genes. Despite being isolated within a single year in the same geographical location, human H5N1 viruses were characterized by a variety of amino acid substitutions in the ribonucleoprotein complex [PB2, PB1, PA and nucleoprotein (NP)] as well as the matrix (M) proteins 1 and 2 and nonstructural (NS) proteins 1 and 2. The presence of previously reported amino acid sequences specific for human strains was confirmed in the PB2, PA, NP and M2 proteins. Nucleotide and amino acid sequence identities of the six internal genes of H5N1 viruses examined here were separated into at least two variant groups. In agreement with the above result, phylogenetic trees of the six internal genes of human H5N1 viruses were generally composed of two minor clades. Additionally, variable dendrogram topologies suggested that reassortment among viruses contributed further to the genetic variability of these viruses. As a result, it became clear that human H5N1 viruses are characterized by divergent gene constellations, suggesting the possible occurrence of genetic reassortment between viruses of the two evolutionary lineages.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Influenza A viruses have caused several human pandemics, such as the Spanish (H1N1) influenza pandemic of 1918, the Asian (H2N2) influenza pandemic of 1957 and the Hong Kong (H3N2) influenza pandemic of 1968 (Murphy & Webster, 1996 ). Pandemic strains with a novel haemagglutinin (HA) or neuraminidase (NA) may predominate for several decades, before being superseded by the next pandemic virus. In fact, H1N1 influenza virus, which caused over 20 million deaths throughout the world (Crosby, 1976 ), and its descendants prevailed during a 38 year period from 1918 to 1956. Thirty-two years have passed since the last pandemic strain, H3N2, appeared in humans, in 1968.

It has been proposed that the emergence in humans of pandemic strains with a novel HA or NA is associated with genetic reassortment between human and avian viruses and that swine may play an important role in generating reassortants between distinct viruses that originate from the former two hosts (Murphy & Webster, 1996 ). It was shown that RNA segments of H2N2 and H3N2 viruses were derived from those of previously circulating human and avian strains (Kawaoka et al., 1989 ; Scholtissek et al., 1978 ). Subsequent nucleotide sequence study revealed that the PB1 genes of viruses from the 1957 Asian pandemic were derived from an avian virus (Kawaoka et al., 1989 ). In addition, many H3N2 viruses isolated from pigs are antigenically similar to early and recent human H3N2 viruses (Nerome et al., 1995 ; Shu et al., 1994 ). As well as reassortants having swine H1 HA and human N2 NA glycoproteins (Nerome et al., 1983 , 1985 ; Ouchi et al., 1996 ), genetic reassortments involving internal genes of swine H3N2 and H1N1 viruses have occurred repeatedly in strains isolated recently from pigs (Nerome et al., 1995 ; Shu et al., 1994 ). This evidence suggests that future pandemic strains may also emerge after genetic reassortment between human and avian viruses in pigs.

The first isolation of H5N1 influenza virus was from a 3-year-old boy who died of Reye’s syndrome on May 21, 1997 (de Jong et al., 1997 ; Subbarao et al., 1998 ). Subsequently, it was reported that the cause of death appeared to be related to multiple complications due to influenza virus infection, i.e. bilateral pneumonia, Reye’s syndrome, haemophagocytosis, renal failure and coagulopathy (Yuen et al., 1998 ). Genomic and evolutionary analyses of complete or partial nucleotide sequences showed that all RNA segments of this virus were homologous with those of avian influenza viruses, suggesting direct infection of humans by an avian virus (Suarez et al., 1998 ; Subbarao et al., 1998 ). Seven months later, another 17 patients were recognized as having been infected with avian H5N1 viruses in Hong Kong during the two months November and December 1997, and a total of six deaths were confirmed. In Hong Kong, an outbreak of highly pathogenic H5N1 virus infections of chickens preceded the death of the 3-year-old child, suggesting a close relationship between the chicken outbreak and human infections; this was confirmed recently by studies by Claas et al. (1998) , Suarez et al. (1998) , Shortridge et al. (1998) and Zhou et al. (1999) . The isolation of avian H5 viruses from humans, together with the isolation of avian H7 influenza viruses from human cases of conjunctivitis (Banks et al., 1998 ), demonstrate the potential risk of direct introduction of novel influenza A subtypes into humans from avian sources.

The evolutionary pathways of the HA and NA genes of H5N1 influenza A viruses isolated from humans and chickens in Hong Kong suggested the separation of these HA genes into two subgroups (Bender et al., 1999 ; Suarez et al., 1998 ). However, the remaining internal genes of human H5N1 viruses have not been analysed fully from an evolutionary point of view. In this report, all six internal genes of six human H5N1 viruses were analysed in relation to the evolutionary pathways of the genes encoding the two surface glycoproteins.


   Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Viruses.
Six human H5N1 viruses used in this study were grown either in MDCK cells (M) or 10-day-old embryonated hen’s eggs (E). Passage histories, with X representing unknown passage numbers, are indicated in parentheses with strain abbreviations: A/Hong Kong/156/97 (HK156, MXE3), A/Hong Kong/481/97 (HK481, MXE2), A/Hong Kong/482/97 (HK482, MXE3), A/Hong Kong/483/97 (HK483, MXE3), A/Hong Kong/485/97 (HK485, MXE1) and A/Hong Kong/486/97 (HK486, MXE2).

{blacksquare} Nucleotide sequences.
Viral RNAs were extracted as described previously (Chomczynski & Sacchi, 1987 ). Each RNA segment was amplified in overlapping cassettes by RT–PCR. RT–PCR was done according to a slightly modified method by using a commercial kit (RT–PCR kit AMV version 2.1, Takara). Briefly, first-strand cDNA was synthesized by mixing 9·5 µl RNA with 1 µl 9-mer random primer (50 µM) and 9·5 µl reverse transcription mixture [4 µl 25 mM MgCl2, 2 µl 10x RT–PCR buffer, 2 µl 10 mM dNTPs, 1 µl avian myeloblastosis virus reverse transcriptase and 0·5 µl RNase inhibitor]. This mixture was first incubated at 30 °C for 10 min, followed by 42 °C for 60 min and 99 °C for 5 min. The resulting cDNA was used in subsequent PCR amplifications of overlapping cassettes covering the entire coding region of each gene. RT–PCR products were purified with the QIAquick gel extraction kit (Qiagen) and were sequenced directly by using the PRISM Ready Reaction dye deoxy terminator cycle sequencing kit with AmpliTaq DNA polymerase FS (Perkin-Elmer Applied Biosystems) on a model 377 automatic sequencer (Perkin-Elmer Applied Biosystems). Synthetic oligonucleotide DNA primer sequences employed in PCR and in sequencing reactions for determination of nucleotide sequences of the entire protein coding regions of the PB2, PB1, PA, NP, M and NS genes are available from the authors upon request. GenBank accession numbers of all nucleotide sequence data obtained in the present study, as well as previously reported nucleotide sequences of A/chicken/Hong Kong/220/98 (ckHK220) and A/chicken/Hong Kong/258/98 (ckHK258), are listed in Table 1.


View this table:
[in this window]
[in a new window]
 
Table 1. GenBank accession numbers of nucleotide sequences of H5N1 influenza A viruses determined in the present study

 
{blacksquare} Phylogenetic analysis
All genes examined in the present study were analysed phylogenetically by the neighbour-joining method (Nei & Gojobori, 1986 ; Saitou & Nei, 1987 ). To determine the robustness of the trees, the probabilities of the internal branches were estimated by 500 bootstrap replications (Felsenstein, 1985 ). Dendrograms were also constructed by the maximum-likelihood method by using the PHYLIP software version 3.57c (Felsenstein, 1995 ) to evaluate the consistency of the tree topologies.


   Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Comparison of nucleotide and amino acid variabilities in the PB2, PB1, PA and NP genes
Pairwise comparisons of nucleotide and predicted amino acid sequences of the genes encoding the proteins of the ribonucleoprotein complex [PB2, PB1, PA and nucleoprotein (NP)] revealed a number of differences among strains, which are summarized in Table 2. For instance, when compared with that of the earliest human isolate, HK156, the PB2 protein sequences contained between four and eight amino acid differences (0·5–1·1%), of which three conserved substitutions were observed in viruses of each sublineage (Table 2). As shown in Table 3, HK482 and HK486 viruses contained substitutions at positions 355 (K->Q), 675 (L->I) and 683 (T->A) relative to HK156, while HK481, HK483 and HK485 viruses varied at residues 199 (S->K), 318 (R->K) and 508 (Q->R). HK483 and HK485 viruses possessed an additional substitution, at position 627 (E->K).


View this table:
[in this window]
[in a new window]
 
Table 2. Nucleotide and amino acid sequence differences among the internal genes of human H5N1 influenza viruses

 

View this table:
[in this window]
[in a new window]
 
Table 3. Predicted amino acid differences observed among human and chicken H5N1 viruses isolated in Hong Kong

 
Less variation was observed in the PB1 proteins, which differed by two to five amino acids (0·3–0·7%). Only two conserved changes were demonstrated in HK482 and HK486, at positions 171 (M->V) and 198 (K->R), although the PB1 protein of HK483 shared two amino acid differences with that of a chicken isolate (ckHK220), at positions 397 (V->I) and 653 (K->R). Interestingly, the PA proteins were shown to have the highest and the lowest amino acid variation among the three polymerase proteins. While the sequences of HK156, HK481, HK482 and HK486 demonstrated complete identity, that of HK483 contained nine amino acid differences. Also, five conserved changes were identified in the PA proteins of HK483 and HK485 viruses, at residues 127 (V->I), 336 (L->M), 409 (N->S), 497 (R->K) and 505 (V->I).

Through alignment of predicted NP sequences, it was apparent that those of Hong Kong H5N1 viruses were relatively conserved, with only two amino acid differences characterizing those of HK483 and HK485 viruses, at positions 319 (K->N) and 423 (V->A), when compared with HK156 NP. However, HK481 varied by eight residues, supporting the apparent genetic divergence of this gene. From pairwise comparisons of nucleotide and amino acid sequences, it became apparent that the PB2, PB1, PA and NP genes of the six strains were separated into at least two groups, represented by HK156 and HK483. Also, it was evident that the above four genes of HK156 were more similar to those of HK482 and HK486 than to those of HK483, HK485 and HK481. However, the nucleotide and predicted amino acid sequences of the PB2 and NP genes of HK481 were characterized by their greater variation from those of the HK156-like and HK483-like strains. For instance, the PB2 gene of HK481 was distinguishable from those of HK156 and HK483 by 30 and 27 nucleotide differences, respectively, resulting in eight and six amino acid differences.

Comparison of amino acid variation in the M1, M2, NS1 and NS2 proteins
Deduced M1 amino acid sequences of the human Hong Kong H5N1 viruses investigated here were found to contain limited amino acid variation when compared with that of HK156 (Table 3). In fact, only one amino acid difference was characteristic of HK482 and HK486, at position 15 (I->V). Similarly, variation in the M2 proteins was limited to two amino acid changes in the cellular carboxyl domain of viruses HK482 and HK486. The NS1 protein revealed a total of four conserved amino acid changes, which were observed in HK483 and HK485 viruses at positions 2 (D->N), 91 (T->A), 189 (D->N) and 213 (P->L). In a similar manner to the HA, NP and PB2 proteins of HK481, the NS1 protein of this virus showed the most variation, with seven amino acid changes. Likewise, the NS2 proteins of HK483 and HK485 viruses were found to contain three conserved amino acid differences (2·5%), at residues 2 (D->N), 31 (M->I) and 56 (H->Y). Unlike the M1 gene, the M2, NS1 and NS2 genes could also be divided into two groups, with the above three genes of HK156, HK482 and HK486 showing higher nucleotide and amino acid variation than those of HK483, HK485 and HK481. It became apparent that the number of amino acid differences in the M1 and M2 genes was considerably lower than that in the PB2, PB1, PA, NP and NS genes. On the other hand, the NS1 gene of HK156 was more similar to those of HK482 and HK486 than to those of HK483, HK485 and HK481. Also, it was evident that the NS2 genes of HK483 and HK485 were slightly distinguishable from HK156, HK482, HK486 and HK481, even at the level of amino acid sequence.

Evolutionary analysis of the PB2, PB1, PA and NP genes
Even though the PB2 genes of influenza A viruses have evolved into lineages represented by human, swine, equine and avian viruses, that of the Hong Kong H5N1 viruses apparently belonged to an avian virus group (Fig. 1a). In fact, the PB2 genes of H5N1 viruses branched from that of a virus similar to budgerigar/Hokkaido/1/77, forming a lineage independent of other avian viruses, which divided further into minor branch clusters. Indeed, the first group, containing HK156, HK482 and HK486, appeared to be separated from HK483 and HK485, which formed a second minor clade together with one chicken virus (ckHK220). In addition, the PB2 gene of HK481 was slightly distinct from these two branch clusters (indicated by an arrow).




View larger version (70K):
[in this window]
[in a new window]
 
Fig. 1. Phylogenetic trees of the PB2 (a), PB1 (b), PA (c), NP (d), M (e) and NS (f) genes of influenza A viruses including those of Hong Kong H5N1 influenza viruses isolated from humans and chickens. Each tree was constructed by the neighbour-joining method (Nei & Gojobori, 1986 ; Saitou & Nei, 1987 ). Internal branching probabilities were determined by bootstrap analysis using 500 replications and are indicated as percentages at each branch point. Dendrograms for these genes were also constructed by the maximum-likelihood method (data not shown), which demonstrated terminal branch clustering of H5N1 viruses identical to those shown. Abbreviations: ck, chicken; ty, turkey; dk, duck; eq, equine; sw, swine; fpv, fowl plaque virus. The first and second minor branch groups are indicated as (i) and (ii). Arrows indicate viral genes the evolutionary locations of which diverged slightly from those of the two sublineages (i, ii).

 
A number of human, non-human mammalian and avian viruses were included in the evolutionary tree of the PB1 gene (Fig. 1b), which demonstrated that the PB1 genes of five human H5N1 isolates were apparently quite similar, as they were included in the first branch cluster (labelled i). In contrast, the PB1 gene of HK483 was distinguishable from those of the remaining human viruses and was rather more similar to that of ckHK220. Genetic separation into two minor clades containing HK156-like and HK483-like strains was supported by bootstrap probabilities of 90% (i) and 100% (ii), respectively.

The dendrogram of the PA genes of influenza A viruses constructed in the present study demonstrated evolution into human, swine, equine and avian lineages and characterized a new divergent lineage containing the genes of the Hong Kong H5N1 viruses, which were apparently most similar to genes of avian viruses (Fig. 1c). Also, the H5N1 virus gene lineage appeared to have divided from a putative ancestor common to swine and avian viruses. Similar to the PB1 gene, the PA genes of HK156 and HK483 viruses were separated into two distinct minor clades (i and ii). The evolutionary locations of HK481, HK482 and HK485 were closest to that of HK156, suggesting that the PA genes of HK483 and HK485 were distinguishable from those of other human H5 viruses belonging to the first minor clade (i).

As shown in the evolutionary tree for the NP genes of influenza A viruses (Fig. 1d), this gene could be classified into at least three large branch groups. The NP genes of the six Hong Kong H5N1 viruses located at the top of dendrogram could be separated further into two minor clades (i and ii). It was also evident that the NP gene of HK481 was different from those of other human H5N1 viruses, based on the bootstrap value of 88% (indicated by an arrow). Furthermore, it was apparent that the NP genes of chicken H5N1 viruses belonged to the second minor lineage (ii).

Evolutionary analysis of the M and NS genes
Evolutionary analysis of the M genes of previously reported influenza A viruses and those of the six H5N1 viruses revealed the divergence of the M gene into several lineages (Fig. 1e). Even though this evolutionary profile of the M gene was largely compatible with that reported previously (Ito et al., 1991 ), our tree characterized further the location of the M genes from six human and one chicken H5N1 isolates from Hong Kong, which were distinguishable from other avian viruses. In contrast to the analysis of the other gene segments, the M gene of HK156 was essentially closest to that of HK483, being located in branch cluster (i). The second minor clade (ii) was composed of the M genes of two human H5N1 strains, HK482 and HK486.

The smallest RNA segment, which encodes the nonstructural NS1 and NS2 proteins, has evolved into three lineages (Fig. 1f). A sublineage consisting of the NS genes of the Hong Kong H5N1 viruses apparently diverged from a putative ancestor common to those of avian and avian/swine strains. In fact, the NS genes of the H5N1 viruses were most similar to that of the avian strain oystercatcher/Germany/87. Like the PB2 and NP genes of these viruses, the NS1 genes were separated into two groups, including HK156, HK482 and HK486 (i) and HK483 and HK485 (ii). The NS gene of HK481 was located somewhat distant from these two minor lineages. The probability of this branch location was confirmed by a bootstrap value of 77%.

Correlation between amino acid changes in the PB2, PA, NP and M2 proteins and host range
Variation in the internal PB2, NP and M2 proteins has been reported to correlate with the host range of influenza virus (Buckler-White & Murphy, 1986 ; Murphy et al., 1989 ; Scholtissek et al., 1985 ; Subbarao et al., 1993 ). In a previous report, the amino acid sequences of these proteins in the Hong Kong poultry H5N1 viruses have been compared with those of other avian and human viruses (Zhou et al., 1999 ). Therefore, we also compared the amino acid changes in the PB2, PA, NP and M2 gene products of the Hong Kong H5N1 viruses (Table 4). As a result, the six strains were found to contain three amino acid residues characteristic of the PB2 protein of human viruses at positions 199 (S), 661 (T) and 667 (I). Additionally, two strains contained three residues, 627 (K), 661 (T) and 667 (I), while one strain possessed two amino acid residues, 661 (T) and 667 (I), characteristic of the PB2 protein of human isolates. The PA proteins of four strains were also shown to contain one human-specific amino acid residue, at position 409 (N), and the NP proteins of all strains possessed an amino acid specific for human isolates at position 136 (M). It was of particular interest that the M2 protein of all strains demonstrated human-specific amino acids at positions 16 (G), 28 (V) and 55 (F).


View this table:
[in this window]
[in a new window]
 
Table 4. Comparison of amino acids found in the internal proteins of H5N1 Hong Kong viruses with those of avian and human viruses

 
Gene constellations
On the basis of the branching profiles of the phylogenetic trees, the gene constellations of six human H5N1 viruses are illustrated in Table 5. Although all genes of HK156 belonged to the first minor group (i), only the M gene of HK483 was derived from the first minor clade, while the remaining seven RNA segments of HK483 were located in the second minor branch cluster (ii). The gene constellation of HK482 appeared to be similar to that of HK486, with six genes (PB2, PB1, PA, HA, NP and NS) located in the first minor lineage (i) and the remaining two genes (NA and M) belonging to the second minor lineage (ii). Although five genes of HK481 were grouped into the first minor branch, the PB2, NP and NS genes appeared to be slightly divergent from those of other isolates. With the exception of the PB1 gene, the gene constellation of HK485 was identical to that of HK483. As a result, the eight RNA segments of human H5N1 viruses appear to consist of two minor evolutionary lineages.


View this table:
[in this window]
[in a new window]
 
Table 5. Phylogenetic differentiation of RNA segments of H5N1 viruses isolated from humans

 

   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Human H5N1 influenza A viruses are separated into two antigenic groups on the basis of haemagglutination-inhibition tests (Bender et al., 1999 ; Shortridge et al., 1998 ). In agreement with the above reports, we also confirmed the presence of two antigenic groups among human H5N1 influenza A viruses on the basis of haemagglutination-inhibition tests with mouse immune sera as well as plaque-neutralization tests with post-infection ferret anti-sera (data not shown). Evolutionary analysis further indicated that the HA and NA genes are divided into two minor evolutionary clades (Bender et al., 1999 ; Suarez et al., 1998 ). Through a series of evolutionary analyses, the six internal genes of human H5N1 viruses were also found to have diverged generally into two distinguishable evolutionary groups. All RNA segments were, therefore, confirmed to have divided into essentially two minor evolutionary lineages, including HK156-like (i) and HK483- or HK482-like (ii) strains. However, the phylogenetic locations of the PB2, NP and NS genes of HK481 were somewhat divergent from those of the above two groups. It has been reported before that phylogenetic analysis of the internal genes of H5N1 viruses isolated from poultry in Hong Kong also revealed divergence into two minor evolutionary lineages (Zhou et al., 1999 ). Thus, genetic analysis of H5N1 viruses isolated from birds and humans in Hong Kong suggests that these viruses are inherently highly variable and prone to rapid change.

Coupled with previous evidence (Bender et al., 1999 ; Suarez et al., 1998 ), it is apparent that variation among human H5N1 viruses could be observed in all eight RNA segments and that subsequent amino acid differences occurred not only in the surface HA protein, but also in the internal proteins of these viruses. In fact, a number of amino acid changes were observed in all internal proteins of Hong Kong poultry H5N1 viruses (Zhou et al., 1999 ). Moreover, human and poultry H5N1 viruses (Zhou et al., 1999 ) possessed human-like amino acids in the NP, PB2 and M2 proteins that correlated with host-determinant factors. Considering the short time span within which human H5N1 viruses were isolated in a local region, it was particularly intriguing that the high nucleotide and amino acid variation among the internal genes of H5N1 viruses was actually more pronounced than that observed among epidemic human H3N2 influenza viruses isolated in different years (Lindstrom et al., 1998 ).

It is still uncertain whether variable gene constellations contribute to changes in biological properties or virulence of these viruses. Ten human H5N1 viruses have been reported to be separated into two distinguishable virulent groups in mice, including HK156-like (high-pathogenetic group) and HK481-like (low-pathogenetic group) strains, which did not appear to correlate with variation of the HA gene (Gao et al., 1999 ). The presence of multiple basic amino acids between the HA1 and HA2 molecules has been reported to be an important factor that contributes to virulence of avian influenza viruses in chickens and other poultry (Horimoto et al., 1995 ; Kawaoka et al., 1987 ). However, it was shown that the multiple basic amino acids between the HA1 and HA2 molecules of the high- and low-virulent strains were identical to one another (Gao et al., 1999 ). Accordingly, the possibility cannot be excluded that other viral proteins may influence the virulence of H5N1 virus. Phylogenetic analysis of the internal genes of human H5N1 isolates revealed that, with the exception of the earliest human isolate, HK156, the evolutionary clustering of virus genes seemed to be congruent with the biological grouping of viruses based on virulence in a mouse model. These results suggest that variable gene constellations and amino acid substitutions in the NA and internal proteins may contribute to changes in the virulence of H5N1 viruses. Having considered the above evidence, further study of the virulence and transmission of H5N1 virus in relation to specific genetic changes, in addition to continued monitoring of emerging influenza viruses in southern China, should be undertaken in more detail from different points of view.

The Hong Kong outbreak has taught us many lessons concerning direct infection of humans by avian influenza viruses that demonstrate genetic reassortment, genetic polymorphism and antigenic variation among strains, as well as high pathogenicity in lungs and neurovirulence in mice. However, despite the highly variable nature of H5N1 viruses, both genetically and antigenically, the results of vaccination experiments in mice have suggested that there is a relatively high degree of cross-protective immunity elicited by human H5N1 viruses, as well as by non-pathogenic avian viruses such as A/duck/Singapore/F119-3/97 (H5N3) (Lu et al., 1999 ; and data not shown). A variety of strategies for vaccine production have been undertaken, including production of subunit vaccines by using recombinant baculovirus, preparation of avirulent strains by using reverse genetics and the use of avirulent avian isolates. It is suggested that the time has now come for intensified efforts in the surveillance and control of future pandemic strains by using a wide variety of techniques and strategies.


   Acknowledgments
 
The authors would like to express their sincere gratitude to Dr John Wood, National Institute for Biological Standards and Control, UK, for his constructive comments during the preparation of this manuscript. This research was supported by research grants provided by the Department of Infectious Diseases, Ministry of Health and Welfare, Japan.


   Footnotes
 
The GenBank accession numbers of the sequences reported in this study are AF036358AF036360, AF036362, AF036363, AF084261AF084270, AF084276AF084278, AF084282AF084287, AF115284AF115289 and AF115290AF115295.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Banks, J., Speidel, E. & Alexander, D. J. (1998). Characterisation of an avian influenza A virus isolated from a human – is an intermediate host necessary for the emergence of pandemic influenza viruses?Archives of Virology 143, 781-787.[Medline]

Bender, C., Hall, H., Huang, J., Klimov, A., Cox, N., Hay, A., Gregory, V., Cameron, K., Lim, W. & Subbarao, K. (1999). Characterization of the surface proteins of influenza A (H5N1) viruses isolated from humans in 1997–1998.Virology 254, 115-123.[Medline]

Buckler-White, A. J. & Murphy, B. R. (1986). Nucleotide sequence analysis of the nucleoprotein gene of an avian and a human influenza virus strain identifies two classes of nucleoproteins.Virology 155, 345-355.[Medline]

Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction.Analytical Biochemistry 162, 156-159.[Medline]

Claas, E. C., Osterhaus, A. D., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477; erratum 1292.[Medline]

Crosby, A. W. (1976). Flu and the American expeditionary force. In Epidemic and Peace 1918, pp. 145–170. Westport, CT: Green Wood Press.

de Jong, J. C., Claas, E. C., Osterhaus, A. D., Webster, R. G. & Lim, W. L. (1997). A pandemic warning?Nature 389, 554.[Medline]

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap.Evolution 39, 783-791.

Felsenstein, J. (1995). PHYLIP (Phylogeny Inference Package) version 3.57c. Distributed by the author. Department of Genetics, University of Washington, Seattle, WA, USA.

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.Journal of Virology 73, 3184-3189.[Abstract/Free Full Text]

Horimoto, T., Rivera, E., Pearson, J., Senne, D., Krauss, S., Kawaoka, Y. & Webster, R. G. (1995). Origin and molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in Mexico.Virology 213, 223-230.[Medline]

Ito, T., Gorman, O. T., Kawaoka, Y., Bean, W. J. & Webster, R. G. (1991). Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins.Journal of Virology 65, 5491-5498.[Medline]

Kawaoka, Y., Nestorowicz, A., Alexander, D. J. & Webster, R. G. (1987). Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain. Virology 158, 218–227; erratum 159, 196.[Medline]

Kawaoka, Y., Krauss, S. & Webster, R. G. (1989). Avian-to-human transmission of the PB1 gene of influenza A viruses in the 1957 and 1968 pandemics.Journal of Virology 63, 4603-4608.[Medline]

Lindstrom, S. E., Hiromoto, Y., Nerome, R., Omoe, K., Sugita, S., Yamazaki, Y., Takahashi, T. & Nerome, K. (1998). Phylogenetic analysis of the entire genome of influenza A (H3N2) viruses from Japan: evidence for genetic reassortment of the six internal genes.Journal of Virology 72, 8021-8031.[Abstract/Free Full Text]

Lu, X., Tumpey, T. M., Morken, T., Zaki, S. R., Cox, N. J. & Katz, J. M. (1999). A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans.Journal of Virology 73, 5903-5911.[Abstract/Free Full Text]

Murphy, B. R. & Webster, R. G. (1996). Orthomyxoviruses. In Fields Virology, pp. 1397-1445. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: Lippincott–Raven.

Murphy, B. R., Buckler-White, A. J., London, W. T. & Snyder, M. H. (1989). Characterization of the M protein and nucleoprotein genes of an avian influenza A virus which are involved in host range restriction in monkeys.Vaccine 7, 557-561.[Medline]

Nei, M. & Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions.Molecular Biology and Evolution 3, 418-426.[Abstract]

Nerome, K., Sakamoto, S., Yano, N., Yamamoto, T., Kobayashi, S., Webster, R. G. & Oya, A. (1983). Antigenic characteristics and genome composition of a naturally occurring recombinant influenza virus isolated from a pig in Japan.Journal of General Virology 64, 2611-2620.[Abstract]

Nerome, K., Yoshioka, Y., Sakamoto, S., Yasuhara, H. & Oya, A. (1985). Characterization of a 1980-swine recombinant influenza virus possessing H1 hemagglutinin and N2 neuraminidase similar to that of the earliest Hong Kong (H3N2) virus.Archives of Virology 86, 197-211.[Medline]

Nerome, K., Kanegae, Y., Shortridge, K. F., Sugita, S. & Ishida, M. (1995). Genetic analysis of porcine H3N2 viruses originating in southern China.Journal of General Virology 76, 613-624.[Abstract]

Ouchi, A., Nerome, K., Kanegae, Y., Ishida, M., Nerome, R., Hayashi, K., Hashimoto, T., Kaji, M., Kaji, Y. & Inaba, Y. (1996). Large outbreak of swine influenza in southern Japan caused by reassortant (H1N2) influenza viruses: its epizootic background and characterization of the causative viruses.Journal of General Virology 77, 1751-1759.[Abstract]

Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees.Molecular Biology and Evolution 4, 406-425.[Abstract]

Scholtissek, C., Rohde, W., Von Hoyningen, V. & Rott, R. (1978). On the origin of the human influenza virus subtypes H2N2 and H3N2.Virology 87, 13-20.[Medline]

Scholtissek, C., Burger, H., Kistner, O. & Shortridge, K. F. (1985). The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses.Virology 147, 287-294.[Medline]

Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka, Y., Kodihalli, S., Krauss, S., Markwell, D., Murti, K. G., Norwood, M., Senne, D., Sims, L., Takada, A. & Webster, R. G. (1998). Characterization of avian H5N1 influenza viruses from poultry in Hong Kong.Virology 252, 331-342.[Medline]

Shu, L. L., Lin, Y. P., Wright, S. M., Shortridge, K. F. & Webster, R. G. (1994). Evidence for interspecies transmission and reassortment of influenza A viruses in pigs in southern China.Virology 202, 825-833.[Medline]

Suarez, D. L., Perdue, M. L., Cox, N., Rowe, T., Bender, C., Huang, J. & Swayne, D. E. (1998). Comparisons of highly virulent H5N1 influenza A viruses isolated from humans and chickens from Hong Kong.Journal of Virology 72, 6678-6688.[Abstract/Free Full Text]

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.Journal of Virology 67, 1761-1764.[Abstract]

Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M., Swayne, D., Bender, C., Huang, J., Hemphill, M., Rowe, T., Shaw, M., Xu, X., Fukuda, K. & Cox, N. (1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness.Science 279, 393-396.[Abstract/Free Full Text]

Yuen, K. Y., Chan, P. K., Peiris, M., Tsang, D. N., Que, T. L., Shortridge, K. F., Cheung, P. T., To, W. K., Ho, E. T., Sung, R. & Cheng, A. F. (1998). Clinical features and rapid viral diagnosis of human disease associated with avian influenza A H5N1 virus.Lancet 351, 467-471.[Medline]

Zhou, N. N., Shortridge, K. F., Claas, E. C. J., Krauss, S. L. & Webster, R. G. (1999). Rapid evolution of H5N1 influenza viruses in chickens in Hong Kong.Journal of Virology 73, 3366-3374.[Abstract/Free Full Text]

Received 29 October 1999; accepted 14 January 2000.