1a.Objectives (from AD-416)
Evaluate swine influenza virus (SIV) isolates through in vivo challenge and transmission studies, antigenic characterization with in-house reference sera, and molecular analysis. Assess pathogenesis of selected reassortant swine and avian influenza viruses of mutual interest in pigs. Evaluation of field isolates of swine and avian H2N3 influenza A viruses in the swine host.
1b.Approach (from AD-416)
The ARS' National Animal Disease Center (NADC) will support the proposed USDA-APHIS swine influenza surveillance program by providing additional characterization of emerging swine influenza virus (SIV) isolates identified through the surveillance program. The SIV isolates will be evaluated through in vivo challenge and transmission studies, antigenic characterization with in-house reference sera, and molecular analysis. Molecular analysis may include follow-up sequencing, nucleic acid and amino acid sequence comparisons, as well as computation of evolutionary relationships with reference sequences to construct phylogenetic trees. The in vivo studies will aid in identifying viruses with increased virulence, increased transmissibility, or other phenotypic properties. The molecular analyses will aid in identifying viruses with novel genetic make-up and allow a better understanding of the evolution and epidemiology of whole virus genomes. In addition to the NALN submission criteria, the NADC criteria shall be. 1)Novel subtype;. 2)Novel gene introduction;. 3)Novel antigenic properties; or. 4)Unusual clinical presentation.
Groups of pigs will be inoculated with the appropriate influenza virus strains and doses (to be determined) by the intratracheal or intranasal route. Subsets of animals at selected time points following challenge will be euthanized to determine virus load in the respiratory tract whereas the remaining animals will be further studied to determine the duration of virus shedding and transmission to naive animals introduced a few days after challenge inoculation. Clinical, virologic, and serologic monitoring will be used to assess the outcomes of infection with different viruses to ascertain their potential threat to animal and human health.
In vivo challenge and transmission studies in pigs demonstrated that two 2009 human isolates of pandemic H1N1 (pH1N1) had differing phenotypes in shedding and transmission characteristics. Sequence analysis of the two strains revealed changes in the major surface glycoprotein, hemagglutinin (HA). To evaluate the genetic basis of the phenotypes, reverse genetics technology was utilized to generate reassortant virus clones with the variant HA genes. These studies point to two amino acid changes present in a low percentage of wild type pandemic viruses as being responsible for increased shedding and transmission in swine. A separate study comparing the virulence of 3 swine isolates of pH1N1 was conducted in pigs. The swine isolates behaved similarly to the early human isolate used in comparison and suggests the pH1N1 was well-adapted to swine and humans in 2009 when the pandemic emerged. Sixty-two swine influenza virus (SIV) isolates obtained through the USDA SIV Surveillance System and the USDA, APHIS, National Veterinary Service Laboratory and 15 isolates from University of Minnesota Veterinary Diagnostic Laboratory were prepared and submitted for whole genome sequencing using the Illumina HiSeq platform in collaboration with St. Jude Children's Research Hospital. Analysis of the sequence data is in progress. Evaluation of wild type H2N3 viruses in swine was completed by infecting pigs intranasally with A/swine/Missouri/4296424/2006 or A/chicken/OH/494832/2007 viruses and by placing naive contact pigs on day 2 post-infection with the experimentally infected pigs. Samples were collected from the pigs for virologic and histologic evaluation is currently underway. Progress is monitored via email and conference calls.