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Genetic reassortment of influenza A viruses through cross-species transmission contributes to the generation of pandemic influenza viruses. To provide information on the ecology of influenza viruses, we have been conducting a global surveillance of zoonotic influenza and establishing an influenza virus library. Of 4580 influenza virus strains in the library, 3891 have been isolated from over 70 different bird species. The remaining 689 strains were isolated from humans, pigs, horses, seal, whale, and the environment. Phylogenetic analyses of the HA genes of the library isolates demonstrate that the library strains are distributed to all major known clusters of the H1, H2 and H3 subtypes of HA genes that are prevalent in humans. Since past pandemic influenza viruses are most likely genetic reassortants of zoonotic and seasonal influenza viruses, a vast collection of influenza A virus strains from various hosts should be useful for vaccine preparation and diagnosis for future pandemics.

When human influenza viruses are isolated and passaged in chicken embryos, variants with amino acid substitutions around the receptor binding site of hemagglutinin (HA) are selected; however, the mechanisms that underlie this phenomenon have yet to be elucidated. Here, we analyzed the receptor structures that contributed to propagation of egg-passaged human H1N1 viruses. The analysis included seasonal and 2009 pandemic strains, both of which have amino acid substitutions of HA found in strains isolated or passaged in eggs. These viruses exhibited high binding to sulfated glycans containing NeuAcα2-3Gal. In MDCK cells overexpressing the sulfotransferase that synthesize Galβ1-4(SO3--6)GlcNAc, production of human H1N1 viruses was increased up to 90-fold. Furthermore, these sulfated glycans were expressed on the allantoic and amniotic membranes of chicken embryos. These results suggest that 6-sulfo sialyl Lewis X and/or NeuAcα2-3Galβ1-4(SO3--6)GlcNAc are involved in efficient propagation of human H1N1 viruses in chicken embryos.

Vaccination-primed immunity in poultry has been suggested for selection of antigenically drifted highly pathogenic avian influenza viruses (HPAIVs). In this study, we performed two consecutive passage studies of an H5N1 HPAIV in vaccinated chickens, namely, study-I and study-II, to select antigenic variants under immune pressure from the vaccination. In study-I, nine consecutive passages of a wild-type H5N1 HPAIV were carried out in chickens vaccinated with the homologous challenge strain. Antigenically drifted variants with mutations at position 179 in the hemagglutinin (HA) were selected after three passages. Similarly, in study-II, a vaccination-mediated antigenic variant isolated in study-I was used as the vaccine and challenge strain to confirm further antigenic drift after updating the vaccine; after the third passage, additional antigenic variants with a mutation at position 256 in the HA were selected. Thus, our study demonstrated the contribution of vaccination in the selection of antigenic variants of H5 HPAIVs in chickens.

H7N9 highly and low pathogenic avian influenza viruses (HPAIV and LPAIV, respectively) have been isolated from duck meat products that were brought illegally into Japan by flight passengers in their hand luggage. These H7N9 virus isolates were phylogenetically closely related to those prevailing in China. Antigenic analysis revealed that the hemagglutinin of the H7N9 HPAIV isolate was slightly different from those of the H7N9 LPAIV and older H7 strains. These meat products contaminated with AIVs repeatedly brought into Japan lead to increased risks of poultry and public health. Continuous border disease control based on the detection and culling of infected poultry and meat products is, thus, essential for the prevention of introduction and spread of AIVs.

Influenza viruses recognize sialoglycans as receptors. Although viruses isolated form chickens preferentially bind to sialic acid α2,3 galactose (SAα2,3Gal) glycans as do those of ducks, chickens were not experimentally infected with viruses isolated from ducks. A chicken influenza virus, A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR) bound to fucose-branched SAα2,3Gal glycans, whereas the binding towards linear SAα2,3Gal glycans was weak. On the epithelial cells of the upper respiratory tracts of chickens, fucose-branched SAα2,3Gal glycans were detected, but not linear SAα2,3Gal glycans. The growth of Ck/IBR in MDCK-FUT cells, which were genetically prepared to express fucose-branched SAα2,3Gal glycans, was significantly higher than that in the parental MDCK cells. The present results indicate that fucose-branched SAα2,3Gal glycans existing on the epithelial cells lining the upper respiratory tracts of chickens are critical for recognition by Ck/IBR.

Avian influenza viruses possess hemagglutinin (HA) which preferentially bind to the sialic acid α2,3-galactose sialyloligosaccharides (SAα2,3Gal) receptor. In contrast, human influenza viruses bind to sialic acid α2,6-galactose sialyloligosaccharides (SAα2,6Gal). The A/Hong Kong/68 (H3N2) virus preferentially binds to SAα2,6Gal, although its HA gene was derived from an avian influenza virus strain. To elucidate the mechanisms behind acquisition of binding specificity for the human-type receptor, the avian influenza virus, A/duck/Hokkaido/5/77 (H3N2), which carries the HA with SAα2,3Gal receptor specificity, was consecutively passaged in pigs. Viruses that preferentially bind to the SAα2,6Gal receptor were predominantly recovered from the nasal swabs of pigs after three passages. The present results indicate that avian influenza viruses can acquire the potential to infect humans after multiple infections in a pig population. Intensive surveillance of swine influenza is, thus, important for the preparedness for the future pandemics.

H5N1 highly pathogenic avian influenza (HPAI) viruses were isolated from dead wild waterfowl at Khunt, Erkhel, Doityn Tsagaan, Doroo, and Ganga Lakes in Mongolia in July 2005, May 2006, May 2009, July 2009, and May 2010, respectively. The isolates in 2005 and 2006 were classified into genetic clade 2.2, and those in 2009 and 2010 into clade 2.3.2. A/whooper swan/Mongolia/6/2009 (H5N1) experimentally infected ducks and replicated systemically with higher mortality than that of the isolates in 2005 and 2006. Intensive surveillance of avian influenza in migratory waterfowl flying from their nesting lakes in Siberia to Mongolia in every autumn indicate that HPAI viruses have not perpetuated at their nesting lakes until 2009. The present results demonstrate that wild waterfowl were sporadically infected with H5N1 HPAI viruses prevailing in domestic poultry in the southern Asia and died in Mongolia on the way back to their northern territory in spring.

The outbreak of H7N9 low pathogenic avian influenza viruses in China has attracted attention to H7 influenza virus infection in humans. Since we have shown that the pathogenicity of H1N1 and H5N1 influenza viruses in macaques was almost the same as that in humans, we compared the pathogenicities of H7 avian influenza viruses in cynomolgus macaques via intranasal and conjunctival inoculation, which mimics natural infection in humans. H7N9 virus, as well as H7N7 highly pathogenic avian influenza virus, showed more efficient replication and higher pathogenicity in macaques than did H7N1 and H7N3 highly pathogenic avian influenza viruses. These results are different from pathogenicity in chickens as reported previously. Therefore, our results obtained in macaques help to estimate the pathogenicity of H7 avian influenza viruses in humans.

We propose a rational approach to the design of live virus vaccines against influenza infection by alteration of the influenza A virus M2 protein, which is responsible for ion channel activity. Previously we demonstrated that a mutant A/WSN/33 (H1N1) influenza virus with defective M2 ion channel activity did not show appreciable growth defects in cell culture, although its growth was attenuated in mice (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, 2001, J. Virol. 75, 5656–5662). Here, we show that this M2 ion channel defective mutant virus, the M2del29-31, protected mice against challenge with lethal doses of influenza virus, indicating the potential of incorporating this M2 alteration in a live influenza vaccine as one of the attenuating mutations.

Despite their uniform ability to bind to oligosaccharide-containing terminal sialic acids, influenza A viruses show differences in receptor specificity. To test whether agglutination of erythrocytes from different animal species could be used to assess the receptor specificity of influenza A viruses, we determined the agglutinating activities of a range of virus strains, including those with known receptor specificities, using erythrocytes from seven animal species. All equine and avian viruses, including those known to recognize N-acetyl and N-glycolyl sialic acid linked to galactose by the alpha2,3 linkage (NeuAc alpha2,3Gal and NeuGc alpha2,3Gal), agglutinated erythrocytes from all of the animal species tested (chickens, ducks, guinea pigs, humans, sheep, horses, and cows). The human viruses, including those known to preferentially recognize NeuAc alpha2,6Gal, agglutinated all but the horse and cow erythrocytes. Fluorescence-activated cell sorting analysis of erythrocytes using linkage-specific lectins [Sambucus nigra agglutinin for sialic acid (SA) alpha2,6Gal and Maackia amurensis agglutinin for SA alpha2,3Gal] showed that both cow and horse erythrocytes contain a large amount of SA alpha2,3Gal-, but virtually no SA2,6Gal-specific lectin-reactive oligosaccharides on the cell surface, while human and chicken erythrocytes contained both types of oligosaccharides. Considering that the majority (93%) of sialic acid in horse and cow erythrocytes is of the N-glycolyl type, our results suggest that viruses able to agglutinate these erythrocytes (i.e., avian and equine viruses) recognize NeuGc alpha2,3Gal. These findings also show that agglutinating assays with erythrocytes from different animal species would be useful in characterizing the receptor specificity of influenza A viruses.

A chimeric gene encoding a fusion protein consisting of the DNA-binding domain of the immediate-early (IE) protein of pseudorabies virus (PRV) and a tail-truncated Vmw65 of herpes simplex virus 1, lacking the transcription activation domain, was constructed. The chimeric gene product inhibited transcription from the PRV IE promoter in a transient expression assay. A HeLa cell line stably transformed with the chimeric gene showed remarkable resistance to PRV infection. In the transformed cells infected with PRV, transcription of the PRV IE gene was repressed, indicating that the resistance of the cells to PRV infection was due to interference with IE gene transcription by the fusion protein.

The addition of oligosaccharide side chains to influenza virus hemagglutinin (HA) is believed to facilitate viral escape from immune pressure in the human population. To determine the implicit potentials for acquisition of N-linked glycosylation, we analyzed the genetic background of 16 subtypes of avian influenza virus, some of which may be potential pandemic viruses in the future. We found a significant difference among HA subtypes in their genomic sequences to produce N-glycosylation sites. Notably, recently circulating avian influenza viruses of the H5 and H9 subtypes may have rather greater capacities to undergo mutations associated with glycosylation of HA than past pandemic viruses. We hypothesize that influenza viruses maintained in natural reservoirs could have different potentials for sustained circulation, depending on their HA subtypes, if introduced into the human population.

A benign Newcastle disease virus (NDV) recently became highly virulent during replication in domestic chickens. It is still unclear whether NDVs circulating among wild waterfowl also have the potential to become highly pathogenic (velogenic) in chickens. To demonstrate experimentally the generation of velogenic NDV from a nonpathogenic waterfowl isolate, we passaged an avirulent goose isolate in chickens. After nine consecutive passages by air-sac inoculation, followed by five passages in chick brain, the virus became highly virulent in chickens, producing a 100% mortality rate, and demonstrating typical velogenic properties in pathogenicity tests. Sequence analysis at the fusion protein cleavage site showed that the original isolate contained the typical avirulent type sequence, E-R-Q-E-R/L, which progressed incrementally to a typical virulent type, K-R-Q-K-R/F, during repeated passage in chickens. These results demonstrate that avirulent viruses, maintained in wild waterfowl in nature and bearing the consensus avirulent type sequence, have the potential to become velogenic after transmission to and circulation in chicken populations. The results also suggest that chickens provide a mechanism for the selection of virulent viruses from an avirulent background.

A chimeric gene encoding a fusion protein consisting of the DNA-binding domain of the immediate-early (IE) protein of pseudorabies virus (PRV) and a tail-truncated VP16 of herpes simplex virus 1, lacking the transcription activation domain, has been shown to repress transcription of the PRV IE gene, resulting in the inhibition of PRV growth in vitro. To assess the antiviral potential of the fusion protein in vivo, transgenic mice containing the chimeric gene under the control of the virus- and interferon-inducible Mx 1 promoter were generated. A transgenic mouse line showed marked resistance to PRV infection when the mice were challenged intranasally with PRV. Inhibition of PRV replication was also observed in monolayers of embryonic cells prepared from the transgenic mice. In the cells infected with PRV, transcription of the PRV IE gene was repressed. The present results indicate that the chimeric gene is able to exert a significant antiviral effect against PRV infection in vivo.

Filoviruses, including Ebola virus, are cytotoxic. To investigate the role of the Ebola virus glycoprotein (GP) in this cytopathic effect, we transiently expressed the GP in human kidney 293T cells. Expression of wild-type GP, but not the secretory form of the molecule lacking a membrane anchor, induced rounding and detachment of the cells, as did a chimeric GP containing its ectodomain and influenza virus hemagglutinin transmembrane–cytoplasmic domain. These results indicate that the GP ectodomain and its anchorage to the membrane are required for GP-induced morphologic changes in host cells. Since cell rounding and detachment could be associated with reduced levels of cell adhesion molecules, we also studied the expression of integrins, which are major molecules for adhesion to extracellular matrices, and found that the β1 integrin group is downregulated by the GP. This result was further extended by experiments in which anti-β1 monoclonal antibodies or purified integrins inhibited the infectivity of vesicular stomatitis virus pseudotyped with the GP. We suggest that integrins, especially the β1 group, might interact with the GP and perhaps be involved in Ebola virus entry into cells.

Quail have emerged as a potential intermediate host in the spread of avian influenza A viruses in poultry in Hong Kong. To better understand this possible role, we tested the replication and transmission in quail of influenza A viruses of all 15 HA subtypes. Quail supported the replication of at least 14 subtypes. Influenza A viruses replicated predominantly in the respiratory tract. Transmission experiments suggested that perpetuation of avian influenza viruses in quail requires adaptation. Swine influenza viruses were isolated from the respiratory tract of quail at low levels. There was no evidence of human influenza A or B virus replication. Interestingly, a human–avian recombinant containing the surface glycoprotein genes of a quail virus and the internal genes of a human virus replicated and transmitted readily in quail; therefore, quail could function as amplifiers of influenza virus reassortants that have the potential to infect humans and/or other mammalian species.

Influenza viruses of the H3N2 subtype similar to Aichi/2/68 and Victoria/3/75 persist in pigs many years after their antigenic counterparts have disappeared from humans (Shortridge et al. (1977). Science 19, 1454-1455). To provide information on the mechanism of conservation of these influenza viruses in pigs, the hemagglutinin (HA) of four isolates from swine derived from Taiwan and Southern China were analyzed antigenically and genetically. The reactivity pattern of these viruses with a panel of monoclonal antibodies indicates that the HAs of these swine viruses were antigenically closely related to duck H3 and early human H3 viruses. Sequence analysis of the H3 genes from three swine viruses revealed that the swine H3 genes are more closely related to the duck genes than to early human H3 virus (A/Aichi/2/68). The degree of sequence homology of these genes is extremely high (more than 96.5%). Furthermore, the deduced amino acid sequence of the three swine HAs at residues 226 to 228 in the proposed receptor-binding site is Gln-Ser-Gly and is common with the majority of avian influenza viruses. These findings indicate that these H3 viruses may have been introduced into pigs from ducks. The HA gene of the fourth swine influenza virus from Southern China was genetically equally related to avian and early human H3 strains although the sequence through the receptor-binding pocket (226-228) was typical of a human H3 virus, suggesting that either this swine HA gene was derived from ducks or an early human H3 virus was introduced into the pig population where the virus accumulated substantial mutations. The present strains revealed genetic heterogeneity of swine H3 influenza viruses in nature.

Monoclonal antibodies to the hemagglutinin (HA) molecule of A/Seal/Mass/1/80 (H7N7) have been prepared and used to establish an operational antigenic map. Four nonoverlapping antigenic areas on the HA of seal influenza viruses were defined. Monoclonal antibodies belonging to two of the groups (III and IV) failed to inhibit hemagglutination of intact virus yet effectively neutralized viral infectivity. These findings could not be explained by differences in affinities and were interpreted in terms of functional differences between the assays. Antibodies that failed to inhibit hemagglutination may bind nearer to the hydrophobic end of the HA molecule and might not block the receptor binding site for erythrocytes. Antibodies to these areas may inhibit infectivity by interference with cell fusion or viral replication. The monoclonal antibodies that failed to inhibit hemagglutination of intact virus nevertheless did inhibit HA activity of purified isolated rosettes of hemagglutinin. The mechanism of inhibition is not resolved but may involve the access of a larger number of antibody molecules to the HA when it is in the form of rosettes compared to when it is located on the viral membrane. The reactivity of seal influenza virus and other H7 viruses from birds with the monoclonal antibodies show that some avian strains possess HA molecules that are closely related to those of seal virus and may therefore be related to viruses that were important in the evolution of this strain. Since HI tests do not necessarily detect all antibodies that neutralize viral infectivity, the question is raised as to whether HI assays alone can be used for determining the efficacy of all influenza virus vaccines.

The hemagglutinins of H3 influenza viruses isolated from migratory ducks on the Pacific flyway in Japan during the period 1977 to 1985 were analyzed antigenically and genetically. Antigenic analysis using monoclonal antibodies to the hemagglutinins of A/Aichi/2/68 (H3N2) and A/duck/Hokkaido/8/80 (H3N8) viruses showed that antigenic drift occurred extensively in human strains, whereas the hemagglutinins of duck viruses were highly conserved. It was also found that the hemagglutinins of duck viruses were antigenically closely related to that of human 1968 H3 prototype strains. Nucleotide sequence analysis of seven duck H3 hemagglutinin genes showed a limited number of changes among the six duck isolates and between these duck isolates and Aichi/68. The deduced amino acid sequence revealed amino acid changes randomly distributed throughout the molecule and not confined to antigenic sites. These findings indicate that the duck virus hemagglutinin genes are conserved in nature and that viruses of different lineages cocirculate.