Receptor-binding profiles of H7 subtype influenza viruses in different host species.

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galß1-4(6-O-HSO(3))GlcNAc and Neu5Acα2-3Galß1-4(Fucα1-3)(6-O-HSO(3))GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galß1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry.

Different incubation temperatures affect viral polymerase activity and yields of low-pathogenic avian influenza viruses in embryonated chicken eggs.

Various incubation conditions (35°C-38°C, 2-7 days) have been used in surveillance studies of the prevalence of avian influenza viruses in wild birds. Here, we studied viral polymerase activity and virus growth kinetics of low-pathogenic avian influenza viruses (LPAIVs) isolated from field samples [A/duck/Hong Kong/365/1978 (H4N6) and A/duck/Nanchang/2-0480/2000 (H9N2)] during incubation at different temperatures (35°C, 37°C, and 39°C) in the allantoic cavity of 10-day-old embryonated chicken eggs (ECE). The higher incubation temperatures (37°C and 39°C) resulted in a significantly higher rate of virus growth, which is most likely a result of increased viral polymerase activity (20%-60%), than was observed at 35°C, and as much as a 100% greater virus yield (as measured by hemagglutination assay) was observed two days after inoculation. Our findings revealed that the optimal activity of the viral polymerase complex, resulting in the highest yield of LPAIV field isolates, could be obtained by incubation for two days in ECE at 37°C and 39°C.

Matrix gene of influenza a viruses isolated from wild aquatic birds: ecology and emergence of influenza a viruses.

Wild aquatic birds are the primary reservoir of influenza A viruses, but little is known about the viruses' gene pool in wild birds. Therefore, we investigated the ecology and emergence of influenza viruses by conducting phylogenetic analysis of 70 matrix (M) genes of influenza viruses isolated from shorebirds and gulls in the Delaware Bay region and from ducks in Alberta, Canada, during >18 years of surveillance. In our analysis, we included 61 published M genes of isolates from various hosts. We showed that M genes of Canadian duck viruses and those of shorebird and gull viruses in the Delaware Bay shared ancestors with the M genes of North American poultry viruses. We found that North American and Eurasian avian-like lineages are divided into sublineages, indicating that multiple branches of virus evolution may be maintained in wild aquatic birds. The presence of non-H13 gull viruses in the gull-like lineage and of H13 gull viruses in other avian lineages suggested that gulls' M genes do not preferentially associate with the H13 subtype or segregate into a distinct lineage. Some North American avian influenza viruses contained M genes closely related to those of Eurasian avian viruses. Therefore, there may be interregional mixing of the two clades. Reassortment of shorebird M and HA genes was evident, but there was no correlation among the HA or NA subtype, M gene sequence, and isolation time. Overall, these results support the hypothesis that influenza viruses in wild waterfowl contain distinguishable lineages of M genes.

Reassortment and interspecies transmission of North American H6N2 influenza viruses.

H6N2 influenza viruses were isolated from California chickens in 2000 and 2001. Here we report the characterization of these H6N2 viruses, one of the few descriptions of non-H5, non-H7 subtype influenza viruses in this host. The H6N2 viruses were nonpathogenic in experimentally infected chickens and could be divided into three genotypes. All three genotypes of virus had similar surface glycoproteins and all contained an 18 amino acid deletion in the neuraminidase, a characteristic of other chicken influenza viruses. Differences were apparent, however, in the complement of replicative protein genes between the genotypes. The presence of multiple H6N2 genotypes suggests that independent transmission and/or reassortment events may have taken place between aquatic bird and chicken influenza viruses.

Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice.

The continual threat posed by newly emerging influenza virus strains is demonstrated by the recent outbreak of H5N1 influenza virus in Hong Kong. Currently, immunization against influenza virus infection is fairly adequate, but it is imperative that improved vaccines are developed that can protect against a variety of strains and be generated rapidly. Since humoral immunity is ineffective against serologically distinct viruses, one strategy would be to develop vaccines that emphasize cellular immunity. Here we report the successful protection of C57BL/6 mice from a lethal A/HK/156/97 (HK156) infection by immunizing first with an H9N2 isolate, A/Quail/HK/G1/97 (QHKG1), that harbours internal genes 98% homologous to HK156. This strategy also protected mice that are deficient in antibody production, indicating that the immunity is T-cell-mediated. In the course of these studies, we generated a highly pathogenic H5N1 reassortant which implicated NP and PB2 as having an important contribution to pathogenesis when present with a highly cleavable H5. These results provide the first demonstration that protective cell-mediated immunity can be established against the highly virulent HK156 virus and have important implications for the development of novel strategies for the prevention and treatment of HK156 infection and the design of future influenza vaccines.