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Two isolates of H4N6 influenza A viruses were recently recovered from pigs with pneumonia in Canada. These were the first H4N6 subtype viruses ever isolated from naturally infected pigs. Our genetic analyses revealed these to be wholly avian viruses, but despite their avian lineage, preliminary research results have demonstrated that they can spread from pig-to-pig. Furthermore, additional results have shown that they preferentially bind to SAL2,6Gal molecules, which is the receptor type present in human tracheal cells, rather than the SAL2,3Gal receptors utilized by avian viruses. Given this finding and the fact that our swine and human populations are both immunologically naive to H4 viruses, these viruses pose an emerging disease concern for both veterinary and human health.
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Our overall goals are to enhance our basic understanding of influenza virus/receptor interactions and the impact of receptor specificity and other genetic factors on the pathogenesis of H4N6 influenza virus infection in pigs. This research is highly consistent with the USDA's objective to support research that 'provides basic knowledge which allows advances in applied research' and will 'increase knowledge needed to sustain animal health,' and addresses both the emerging disease and genomics NRI strategic issues. Our specific research objectives are to: <OL> <LI> Define the hemagglutinin (HA) gene sequences that control the receptor binding properties of H4 influenza viruses <LI> Compare the replication kinetics and pathogenic potential of H4N6 viruses of different receptor specificities and gene constellations in pigs
NON-TECHNICAL SUMMARY: A new subtype of influenza virus (H4N6) not previously isolated from naturally infected pigs has recently been recovered from pigs in North America. The purpose of this study is to enhance our understanding of influenza virus and receptor interactions, and the impact of receptor specificity and other genetic factors on the pathogenesis of H4N6 influenza virus infection in pigs.
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APPROACH: <BR> Objective 1: We will associate the results of solid-phase receptor binding assays with sequence differences in the HA genes of two H4N6 viruses isolated previously from pigs (A/Swine/Ontario/01911-1, -2/99) (Sw/ONT), as well as in the HA genes of receptor binding variant viruses that we will create by passage in eggs and cell culture in vitro and by reverse genetics engineering. To further address the genetic factors that control replication efficiency of influenza viruses in pigs, we will use reverse genetics to create additional H4N6 viruses with different constellations of influenza virus internal protein genes.<BR> Objective 2: We will infect pigs with the original H4N6 swine isolates and the genetically-defined variant viruses created in Objective 1, determine the kinetics and extents of replication of each virus (based on virus shedding and tissue distribution of viral antigen), and compare the intensity of the clinical disease and gross and microscopic pathologic lesions induced.
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PROGRESS: 2002/09 TO 2005/09<BR>
The overall aim of this project was to enhance our understanding of the pathogenesis of avian influenza A virus infection in pigs. We specifically sought to determine the amino acids (AAs) in the hemagglutinin (HA) gene of an H4N6 wholly avian influenza virus isolated from pigs in 1999 (A/Swine/Ontario/00190/99 [Sw/ONT/99]) that control 2,3- versus 2,6-linked sialic acid (SA) binding, and to determine the impact of 2,3- versus 2,6-linked SA binding preference on the infectivity of this virus in pigs. Using a 12-plasmid reverse genetics system, we re-created the parental virus (rgSw/ONT/99) and subsequently created viruses with site-directed mutations in the H4 HA gene of rgSw/ONT/99 corresponding to one or both of the AAs in H3 viruses (residues 226 and 228 in the H3 numbering scheme) that are known to control SA receptor binding of H3 viruses. We specifically changed each of these residues in the H4 gene, individually and in combination, to AAs known to confer preferential binding of H3 viruses to 2,3-linked SAs. Using biotinylated sialyloligosaccharide receptor analogs and a direct binding assay (employing both SL and SLN forms of 2,3- and 2,6-linked SAs), we demonstrated that the rgSw/ONT/99 virus had the same 2,6-linked SA binding preference as the original natural virus isolate. We then demonstrated that the AA residue in the H4 HA corresponding to AA226 in H3 viruses was, as in H3 viruses, the dominant determinant of 2,3- versus 2,6-linked SA binding preference for rgSw/ONT/99, while the residue corresponding to AA228 had minimal impact. Due to USDA restrictions on in vivo administration of the Canadian-origin H4N6 viruses to pigs, we were not able to directly compare the infectivities of rgSw/ONT/99 and the AA226/AA228 site-directed mutants in pigs as originally planned. However, we examined their infectivities in primary swine respiratory epithelial cells (SRECs) cultured in vitro, using a cell culture system we developed for a different project. Like epithelial cells from tracheal tissues harvested from pigs in vivo, we demonstrated that the SRECs cultured in vitro expressed both 2,3- and 2,6-linked SAs in approximately equal levels, and supported infection and replication of swine influenza viruses. In repeated infection experiments, we showed that the parental Sw/ONT/99 and rgSw/ONT/99 viruses consistently infected nearly all of the SRECs, as did the AA228 mutant. However, the AA226 and dual AA226/228 mutants with preferential binding to 2,3-linked SAs infected a highly statistically significantly lower percentage of SRECs. Thus, although SRECs express both 2,3- and 2,6-linked SAs, it appears that efficient infection of swine respiratory epithelial cells by avian influenza A viruses depends on mutation from an avian-like 2,3-linked SA receptor preference to a mammalian-like 2,6-linked SA receptor preference.
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IMPACT: 2002/09 TO 2005/09<BR>
The information we have gained through this research advances our understanding of the genetic factors that control interspecies transmission of influenza viruses and the pathogenesis of influenza, specifically the impact of different receptor specificities on influenza virus infectivity in pigs.