SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY
Location: Virus and Prion Research Unit
Title: Genetic and Antigenic Characterization of 2008 Swine Influenza Viruses from the United States
Submitted to: Pig Veterinary Society International Congress Proceedings
Publication Type: Proceedings
Publication Acceptance Date: March 30, 2010
Publication Date: July 18, 2010
Citation: Vincent, A.L., Harland, M.L., Lorusso, A., Alt, D.P., Bayles, D.O., Swenson, S.L., Gramer, M.R., Lager, K.M., Lewis, N. 2010. Genetic and Antigenic Characterization of 2008 Swine Influenza Viruses from the United States. In: Proceedings of the International Pig Veterinary Society Congress, July 18-21, 2010, Vancouver, Canada. p. 259.
Introduction. Prior to the introduction of the 2009 pandemic H1N1 virus (1) from humans into pigs in the U.S., four phylogenetic clusters of the hemagglutinin (HA) gene from swine influenza viruses (SIV) co-circulated. Viruses from the classical H1N1 SIV lineage mutated over time to form alpha-, beta-, and gamma-clusters. SIV with HA genes most similar to human seasonal H1 viruses emerged in 2003 to form the delta-cluster (2). All four HA cluster gene types can be found with neuraminidase genes of the N1 or N2 subtype. Limited information was available regarding the 6 genes that make up the triple reassortant internal gene (TRIG) cassette in contemporary H1 SIV. In addition, information regarding the antigenic relatedness of the H1 viruses and diagnostic reagent updates were in need due to the dynamic and variable nature of H1 SIV. We characterized 12 H1 isolates from 2008 based on sequencing and phylogenetic analysis of all eight gene segments and based on serologic cross-reactivity in the hemagglutination inhibition (HI) assay.
Materials and methods. Viruses. Swine influenza viruses isolated from outbreaks of respiratory disease in pigs were grown in Mardin-Darby Canine Kidney (MDCK) cells. Virus isolates were: A/SW/NC/02023/2008 H1N1, A/SW/OH/02026/2008 H1N1, A/SW/MO/02060/2008 H1N1, A/SW/IA/02096/2008 H1N1, A/SW/KY/02086/2008 H1N1, A/SW/MN/02011/2008 H1N2, A/SW/MN/02093/2008 H1N1, A/SW/MN/02053/2008 H1N1, A/SW/NE/02013/2008 H1N1, A/SW/NC/02084/2008 H1N1, A/SW/TX/01976/2008 H1N2, and A/SW/IA/02039/2008 H1N2.
Antigenic characterization. Four-week-old cross-bred pigs were obtained from a herd free of influenza virus and antibodies. Two pigs per virus were immunized with inactivated virus combined with commercial adjuvant by the intramuscular route. Two or three vaccinations were given approximately 2-3 weeks apart until homologous HI titers reached = 1:80.
The HI assays were performed with turkey RBC’s using panels of homologous and heterologous viruses and anti-sera with standard techniques. Quantitative analyses of the antigenic properties of swine influenza A (H1) viruses were performed using antigenic cartography, previously used for human and swine influenza A (H3N2) viruses(3-4).
Genetic characterization. Each virus isolate was concentrated and semi-purified on a sucrose cushion followed by library preparation for de novo pyrosequencing on a Genome Sequencer FLX system following the manufacturer’s recommendations with modifications. Briefly, viral RNA was extracted, fragmented, reverse transcribed, and ligated to oligonucleotide adaptors containing multiplex identifier (MID) labels. Pools containing MID-labeled viral cDNA were used to prepare sequencing beads via Roche’s GS-FLX standard chemistry emulsion-based PCR. Prepared beads were loaded into the small 16 regions on a GS-FLX standard chemistry pico-titer plate and sequenced using the GS-FLX LR 70 standard chemistry. Sequencing reads were compared to an influenza database created from > 85,000 influenza sequences extracted from GenBank in Dec. 2008. Using the BLAST results, sequencing reads were filtered, influenza specific reads extracted, and extracted reads were assembled with the Roche GS De Novo Assembler (Newbler) version 2.0 application. Gene segments with large gaps were closed by traditional primer based sequencing using an ABI 3100 genetic analyzer. The sequences were analyzed using SeqMan (DNASTAR). Phylogenetic analyses were conducted using MEGA version 4.
Results. Based on genetic analysis, each of the four previously described phylogenetic clusters of H1 SIV was represented in the 2008 panel. Additionally, it was demonstrated that the delta-cluster HA diverged into sub-clusters delta-1 and delta-2. Serologic cross-reactivity paired with antigenic cartography demonstrated that the phylogenetic clusters are divergent antigenically as well. The genes composing the TRIG were all North American in lineage, although genetic variation was apparent.
Discussion. In 2008, five genetic and antigenic clusters of H1 SIV co-circulated in the U.S. pig population with limited HI cross-reactivity between them. Genetic diversity was demonstrated in all gene segments, but most notably in the HA gene. Gene segments from the 2009 pandemic H1N1 formed clusters separate from North American swine lineage viruses, suggesting this virus was not present in U.S. pigs prior to 2009. The HA variability has important implications for diagnostic testing as well as vaccine development.
Acknowledgments. We thank Brian Pottebaum and Jason Huegel for assistance with animal studies. Funding was provided by USDA/ARS and National Pork Checkoff.
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