Progress 01/01/05 to 12/31/05
Outputs
Using the 12-plasmid reverse genetics system, we have completed re-creation of the parental A/Swine/Ontario/01911/99, as well as mutant viruses with targeted mutations at amino acid 226, 228 or 226 + 228 in the HA gene of this virus. These mutations create viruses that contain signature amino acids for SA2,3Gal receptor preference rather than the SA2,6Gal receptor preference amino acid signatures present in the parental virus. The parental and mutant viruses have been partially assessed for their receptor binding characteristics in a series of receptor binding assays, but we awaiting back-ordered reagents to complete those assays. We have completed experiments to assess the relative infectivity of the parent and mutant viruses in primary swine respiratory epithelial cells and found that the mutation at aa226 strongly impacts infectivity in these swine cells.
Impacts
The information we gain through this research will advance our understanding of the genetic factors that control interspecies transmission of influenza viruses and the pathogenesis of influenza in pigs, specifically the impact of different receptor specificities on influenza virus pathogenesis in pigs.
Publications
- Landolt, G., A.I. Karasin, B. Milde, S. Carman, and C.W. Olsen. 2005. The role of hemagglutinin residues 226 and 288 in receptor specificity and growth characteristics of an H4N6 avian influenza virus in primary porcine respiratory epithelial cells, in: Proceedings of the 86th Annual Meeting of the Conference of Research Workers in Animal Diseases (Abstract 190).
- Gambaryan, A., A.I. Karasin, A. Tuzikov, A. Chinarev, M. Matrosovich, N. Bovin, C.W. Olsen and A.I. Klimov. 2005. Receptor-binding properties of swine influenza viruses isolated in MDCK cells. Virus Research 114:15-22.
- Karasin, A.I., S. Carman, and C.W. Olsen. 2006. Identification of human H1N2 and human-swine reassortant H1N2 and H1N1 influenza A viruses among pigs in Ontario, Canada, 2003-2005. Journal of Clinical Microbiology 44:1123-1126.
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Progress 09/15/02 to 09/14/05
Outputs
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.
Impacts
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.
Publications
- Batemann, A. et al. 2007. In preparation.
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Progress 01/01/04 to 12/31/04
Outputs
Using the 12-plasmid reverse genetics system, we have completed re-creation of the parental A/Swine/Ontario/01911/99, as well as mutant viruses with targeted mutations at amino acids 226, 228 or 226 + 228 in the HA gene of this virus. These mutations create viruses that contain signature amino acids for SA2,3Gal receptor preference rather than the SA2,6Gal receptor preference amino acid signatures present in the parental virus. The parental and mutant viruses are now being assessed for their receptor binding characteristics in a series of complementary receptor binding assays, and for their relative infectivity in primary respiratory epithelial cells derived from different hosts and expressing different relative levels of SA2,3Gal and SA2,6Gal receptors.
Impacts
The information we gain through this research will advance our understanding of the genetic factors that control interspecies transmission of influenza viruses and the pathogenesis of influenza in pigs, specifically the impact of different receptor specificities on influenza virus pathogenesis in pigs.
Publications
- Olsen, C. 2004. Public health implications of influenza in animals, in: Swiss Society for Microbiology 63rd Annual Assembly, Lugano, Programme, abstract M9, p. 37.
- Olsen, C.W., G.A. Landolt, and A.I. Karasin. 2004. The emergence of novel influenza viruses among pigs in North America due to interspecies transmission and reassortment. Int. Congress Series 1263:196-199.
- Olsen, C.W. 2004. The emergence and evolution of influenza viruses: a global concern for animal and human health (Invited Keynote Address). In Proceedings of the 85th Annual Meeting of the Conference of Research Workers in Animal Diseases, Abstract 213.
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Progress 01/01/03 to 12/31/03
Outputs
We have created a molecular clone of A/Swine/Ontario/01911/99 by reverse genetics and are completing additional reverse genetics work to engineer specific amino acid mutations into the hemagglutinin gene that will allow us to evaluate receptor specificity. The phenotype of the different reverse-genetics created viruses will then be tested in a newly-developed primary respiratory epithelial cell culture system (see WIS04468 CRIS report), and ultimately in pigs.
Impacts
The information we gain through this research will advance our understanding of the genetic factors that control interspecies transmission of influenza viruses and the pathogenesis of influenza in pigs, specifically the impact of different receptor specificities on influenza virus pathogenesis in pigs.
Publications
- Olsen, C.W. 2002. Emergence of novel strains of swine influenza virus in North America, pp. 37-43, in: A. Morilla, K.-J. Yoon and J.J. Zimmerman (eds.) Trends in Emerging Viral Infections of Swine. Iowa State University Press, Ames, IA.
- Olsen, C.W. 2003. The emergence of novel influenza viruses among pigs in North America due to interspecies transmission and reassortment. In: Conference Program and Abstracts of the Optios for the Control of Influenza V Meeting, abstract W09-1.
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Progress 09/15/02 to 12/31/02
Outputs
Regarding Objective 1 ("define the hemagglutinin [HA] sequences that control receptor binding properties of H4 influenza viruses"), we have completed the viral RNA sequencing and are working to create our molecular clone of A/Swine/Ontario/01911/99 by reverse genetics. We are also poised to begin selection of receptor variants of A/Swine/Ontario/01911/99. Regarding Objective 2 ("compare the replication kinetics and pathogenic potential of H4N6 viruses of different receptor specificities and gene constellations in pigs"), this work cannot begin until completion of Objective 1.
Impacts
Influenza virus infection of pigs is both an economically important animal disease and a human public health concern. The information we gain through this research will advance our understanding of both the pathogenesis of influenza in pigs and the genetic factors that impact interspecies transmission of influenza viruses.
Publications
- Olsen, C., S. Swenson, G. Landolt, D. Hutto, A. Karasin, S. Carman and B. Schmitt. 2002. Experimental infection of pigs with an avian H4N6 influenza virus isolated from pigs in Canada in 1999, in: Abstracts of Papers Presented at the 83rd Annual Meeting of the Conference of Research Workers in Animal Disease (Abstract 106P).
- Olsen, C.W. 2002. Swine influenza - a world of change, in: Proceedings of the Indiana Veterinary Medical Association Annual Meeting 2003.
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