2009 Annual Report
1a.Objectives (from AD-416)
The objective of the grant is to better define the genetic and antigenic properties of currently circulating H1 subtype swine influenza viruses (SIV). The proposed work will be published and provide a reference for all future descriptions of H1 SIV.
1b.Approach (from AD-416)
Swine influenza viruses isolated from cases of swine respiratory disease outbreaks at the University of Minnesota Veterinary Diagnostic Laboratory (MVDL) during 2007-2008 will be selected based on representation from each of the current H1 genetic clusters using partial HA gene sequence. The isolates (approximately 12) will be transferred to USDA/ARS-NADC and USDA/APHIS-NVSL for the experimental work. The viruses will be propagated for use as antigen in the HI assay and for vaccine preparations. Additionally, the propagated viruses will be used for sequencing of the HA and NA genes. Inactivated virus preparations will be given to two pigs per isolate to produce immune-serum. Immune-sera will be tested with each of the virus antigens in all combinations in the HI assay. In addition, previously generated reference H1 anti-sera and virus will be included in the cross-HI assays (~11 virus isolates and 22 sera) . Geometric mean reciprocal titers will be calculated for each pair of sera, and comparisons will be made to identify greater than 4-fold differences in titers to evaluate sero-cross-reactivity between H1 isolates. Full length sequencing of the HA and NA genes will be conducted for each of the 10 new isolates. Sequences from viruses used to generate the previously evaluated immune-sera will be included as well as sequences in publicly available databases. The sequences will be analyzed and phylogenetic trees will be produced.
Pigs were immunized with 12 swine influenze virus (SIV) isolates from 2008 and immune-sera were generated. Cross-HI assays have been completed and data analyzed. Virus and anti-sera were grouped into 5 antigenic clusters with significant loss in cross-reactivity between clusters. The serologic data suggests current coverage available by inactivated commercial vaccines is inadequate against all circulating variants. Updated vaccines or vaccine platforms with improved cross-reactivity are needed to control SIV. The 12 isolates from 2008 were sequenced using a pyrosequencer with substantial coverage of each of the viral genomes. Phylogenetic analysis of the genomic sequences demonstrated that the viruses represented the 4 phylogenetic clusters of H1 SIV. The 6 internal genes from all viruses were demonstrated to be of the North American triple reassortant SIV lineage. None of the 2008 isolates were closely related to the 2009 pandemic H1N1 virus.