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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY Title: Research Updates: Experimental Evaluation of 2009 Pandemic A/H1N1 in Pigs

item Vincent, Amy
item Lager, Kelly
item Harland, Michelle
item Lorusso, Alessio
item Ciacci-Zanella, Janice -
item Zanella, Eraldo -
item Kehrli Jr, Marcus
item Gauger, Philip -
item Janke, Bruce -
item Klimov, Alexander -

Submitted to: American Association of Swine Veterinarians Annual Meeting
Publication Type: Proceedings
Publication Acceptance Date: January 5, 2010
Publication Date: March 6, 2010
Citation: Vincent, A.L., Lager, K.M., Harland, M.L., Lorusso, A., Ciacci-Zanella, J.R., Zanella, E.L., Kehrli, Jr., M.E., Gauger, P.C., Janke, B.H., Klimov, A. 2010. Research Updates: Experimental Evaluation of 2009 Pandemic A/H1N1 in Pigs. In: Proceedings of the 41st Annual Meeting of the American Association of Swine Veterinarians, March 6-9, 2010, Omaha, Nebraska. p. 495-497.

Technical Abstract: Introduction: In March 2009, a novel pandemic A/H1N1 emerged in the human population in North America (2). The gene constellation of the emerging virus was demonstrated to be a combination of genes from swine influenza A viruses (SIV) of North American and Eurasian lineages that had never before been identified in swine or other species. The emergent A/H1N1 quickly spread in the human population and the outbreak reached pandemic level 6 as declared by the World Health Organization on June 11, 2009. Although the 8 gene segments of the novel virus have lineage with available sequences of corresponding genes from SIV from North America and Eurasia, no closely related ancestral SIV with this gene combination has been identified in North America or elsewhere in the world (10,11). Other than sporadic transmission to humans (6,9), swine influenza A viruses of the classical H1N1 subtype historically have been distinct from avian and other mammalian H1N1 influenza viruses in characteristics of host specificity, serologic cross-reactivity, and/or nucleotide sequence. However, spillover of human pandemic viruses to swine occurred following the Spanish flu pandemic of 1918 (H1N1) and the Hong Kong flu pandemic of 1968 (H3N2), and spillover of human seasonal viruses occurred in 1997 (H3N2) and 2003 (H1N1). In North America, multiple subtypes of endemic SIV (H3N2, H1N1, and H1N2) with a triple reassortant internal gene (TRIG) constellation derived from swine, avian and human influenza viruses co-circulate in most major swine producing regions of the U.S. and Canada since 1997-98 [reviewed in (14)]. Additionally, introduction of H1N1 and H1N2 viruses with the HA and NA genes originating from contemporary human seasonal influenza A viruses (hu-like H1) that are genetically and antigenically distinct from the classical swine H1 lineage were reported in pigs in Canada (4). Since 2005, hu-like H1N1 and H1N2 viruses have emerged in swine herds across the U.S. as human-swine reassortants possessing the TRIG (13). Four phylogenetic clusters (alpha, beta, gamma, and delta) of H1 SIV are now endemic in U.S. swine (13,15). However, to date, Eurasian lineage SIVs have not been reported in the U.S., thus the potential impact of transmission of the pandemic H1N1 virus to the U.S. pig population is unknown. This presentation will summarize a series of studies conducted at the USDA-ARS National Animal Disease Center evaluating the pandemic 2009 A/H1N1 in swine. Materials and methods. A/California/04/2009 (CA/09), A/New York/18/2009 (NY/09), and A/Mexico/4108/2009 (MX/09) received from the Centers for Disease Control and Prevention (CDC) were propagated in Madin-Darby Canine Kidney (MDCK) cells for use in the studies described below. Cross-bred pigs from a herd free of SIV and porcine reproductive and respiratory syndrome virus (PRRSV) were housed in ABSL3 containment and cared for in compliance with the Institutional Animal Care and Use Committee of the National Animal Disease Center. For pathogenesis studies (Studies 1 and 2), pigs were challenged at approximately 4-5 weeks of age. In Study 2, naïve contact pigs were placed with primary challenged pigs to evaluate transmission to direct and indirect contacts. To evaluate vaccine efficacy (Study 3), pigs were vaccinated with 2 mL of each vaccine by the intramuscular route at approximately 4 weeks of age, boosted at 7 weeks of age, and challenged at 10 weeks of age. Pigs were inoculated intra-tracheally with 2 mL of 1 x 10**5 50% tissue culture infectious dose (TCID50) of CA/09 or MX/09 as previously described (15). Pigs were observed twice daily for signs of clinical disease and fever. Nasal swabs were taken and placed into 2 mL minimal essential medium (MEM) on 0, 1, 2, 3, 4, 5 and/or 7 days post infection (dpi) to evaluate nasal virus shedding and stored at 80°C until study completion. In Studies 1 and 2, pigs were humanely euthanized with a lethal dose of pentobarbital (Sleepaway, Fort Dodge Animal Health, Fort Dodge, IA) on 3, 5, or 7 dpi to evaluate lung lesions and viral load in the lung and other selected tissues. Fresh samples were taken from lung, tonsil, inguinal lymph node, liver, spleen, kidney, semitendinosus skeletal muscle (ham), and colon contents (feces) using individual sterile instruments for each tissue. Fresh necropsy samples were stored at -80°C until processed for downstream assays. Additional samples of the same tissues were fixed in 10% buffered formalin and processed by routine methods for histopathologic and immunohistopathologic examination. Immunohistochemical methods for detection of influenza antigen in tissues using monoclonal antibody against type A nucleoprotein were employed as previously described (16). For Study 3, pigs were euthanized at 5 dpi as this was the peak virus titers and pneumonia based on results from Studies 1 and 2. Tissues were tested for virus by a real-time RT-PCR (qRT-PCR) specific for the pandemic H1N1 matrix gene (Lorusso, submitted) and virus isolation on Madin Darby Canine Kidney (MDCK) cells. Briefly, approximately 500 mg of tissue was homogenized in sterile phosphate buffered saline (PBS) with antibiotics using a power homogenizer with sterile generators at 20% w/v. The MagMax Microarray (Ambion) protocol for RNA extraction from tissues was followed using 100 uL of tissue homogenate. The MagMax Viral RNA Isolation (Ambion) kit protocol was used as per manufacturer’s instructions for serum by adding 50 uL to the Mag Max plate for RNA extraction. For virus isolation, 200 uL of the tissue homogenate or serum sample was placed on confluent MDCK cells in 24-well plates. After 48 hrs, 200 uL of cell culture supernatant from each well of the 24-well plate after one freeze and thaw cycle was subsequently passed onto confluent 48 well plates. After 48 hrs, evidence of cytopathic effects was evaluated and presence of virus antigen confirmed by immuno-cytochemical staining (5). At necropsy for all in vivo studies, lungs were removed and evaluated for the percentage of the lung affected with plum colored and well-demarcated consolidated lesions typical of influenza virus infection in pigs. The percentage of the surface affected with pneumonia was visually estimated for each lung lobe, and a total percentage for the entire lung was calculated based on weighted proportions of each lobe to the total lung volume (3). Each lung was then lavaged with 50 mL MEM to obtain bronchoalveolar lavage fluid (BALF). Tissue samples from the trachea and right cardiac lung lobe and other affected lobes were taken and fixed in 10% buffered formalin for histopathologic examination. Tissues were routinely processed and stained with hematoxylin and eosin. Lung sections were given a score from 0-3 to reflect the severity of bronchial epithelial injury using previously described methods (8). Ten-fold serial dilutions in serum-free MEM supplemented with TPCK trypsin and antibiotics were made with each BALF sample and nasal swab filtrate sample. Each dilution was plated in triplicate in 100 uL volumes onto PBS-washed confluent MDCK cells in 96-well plates. Plates were evaluated for CPE between 48-72 hours post infection. At 72 hours, plates were fixed with 4% phosphate-buffered formalin and stained using immunocytochemistry. A TCID50 titer was calculated for each sample using the method of Reed and Muench (7). Serologic cross reactivity between swine antisera and 2009 A/H1N1 was evaluated in conjunction with Study 1. Thirty-eight polyclonal anti-sera from pigs immunized with 19 H1 SIV isolated during 1999-2008 were tested against the viruses in a standard hemagglutination inhibition (HI) assay. Each of the 4 phylogenetic clusters ( alpha, beta, gamma, and delta) (13,15) of endemic North American H1 swine influenza viruses were represented in the panel of sera as previously described (12). The HI assays were then performed with the A/CA/04/2009, A/NY/18/2009, and A/Mexico/4108/2009 viruses as

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