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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY Title: Human-Like H1 (Delta-Cluster) Swine Influenza Virus (SIV) Can Efficiently Replicate, Transmit, Cause Lung Pathology and Induce Humoral Immune Responses in the Swine Host

Authors
item Ciacci-Zanella, Janice -
item Vincent, Amy
item Zanella, Eraldo -
item Brockmeier, Susan

Submitted to: Pig Veterinary Society International Congress Proceedings
Publication Type: Proceedings
Publication Acceptance Date: March 30, 2010
Publication Date: July 18, 2010
Citation: Ciacci Zanella, J.R., Vincent, A.L., Zanella, E.L., Brockmeier, S. 2010. Human-Like H1 (Delta-Cluster) Swine Influenza Virus (SIV) Can Efficiently Replicate, Transmit, Cause Lung Pathology and Induce Humoral Immune Responses in the Swine Host. In: Proceedings of the International Pig Veterinary Society Congress, July 18-21, 2010, Vancouver, Canada. p. 262.

Technical Abstract: Introduction. Genomic characterization of recently identified H1 influenza A viruses demonstrated the viruses were triple reassortants with an internal gene constellation similar to contemporary U.S. swine influenza virus (SIV) but hemagglutinin (HA) and neuraminidase (NA) most similar to human seasonal influenza virus lineages (1, 2). The HA gene of these viruses form the delta-cluster in phylogenetic analyses of HA genes from North American SIV. PB1-F2 is an influenza A virus protein encoded on gene segment 2 and described to be a virulence factor in other host species, but its role in swine is currently unknown (3). The delta-cluster H1 SIV was demonstrated to contain segment 2 encoding PB1-F2 proteins with and without premature truncations (3). The objective of this work was to study the pathogenesis and transmission of delta-cluster H1 SIV in swine. Materials and methods. Three delta-cluster H1 SIV grown in MDCK cells were used in this study. Two isolates, A/sw/MN/07002083/07 H1N1 (MN07) and A/sw/IL/00685/05 H1N1 (IL05), were chosen due the absence of the full-length coding sequence for PB1-F2, whereas A/sw/TX/01976/08 H1N2 (TX08) contains the full-length PB1-F2 sequence. Forty-nine pigs were divided in seven groups composed of 3 primary inoculated pig groups with each delta-cluster virus, three contact groups and one uninfected control group. Primary pigs (n=10) were infected intratracheally with SIV and naive contact pigs (n=5) were co-mingled after 48 hours to be infected via natural transmission routes. Nasal swabs were taken on 0, 3, 5, 7 and 9 days post infection (dpi) or days post contact (dpc) to evaluate nasal shedding. Oral fluids were also collected on days 3-10, 14, 18 and 20 dpi as described previously (4). Postmortem samples including serum, bronchoalveolar lavage fluid (BALF), lung, trachea and nasal swabs were collected at necropsy at days 5 and 21. Non-challenged age-matched negative control pigs were necropsied at 5 dpi (n=5 pigs). Serologic assays included the hemagglutination inhibition (HI) (5) assay and the Avian Influenza MultiS-Screen ELISA (6). In addition to homologous HI assays, 21 dpi and 19 dpc serum were evaluated against the studied viruses (TX08, MN07 and IL05) as well as a previously generated H1 serum panel in heterologous HI assays. Real-time PCR assays were performed for detection of SIV (oral fluids) and of PRRSV, PCV2 and Mycoplasma hyopneumoniae nucleic acids in BALF using in house assays (3). Results and discussion. All pigs were seronegative for specific antibodies to SIV by the HI assay and by ELISA prior to the start of the study. Pigs were also negative for PRRSV, PCV2 and M. hyopneumoniae nucleic acids in BALF at necropsy. All viruses induced SIV illness; however, clinical signs and macroscopic lesions induced by MN07 were reduced in severity as compared to TX08 group pigs in this study. MN07 infected pigs had lower virus titers in the lung and nasal swabs (day 5) than the TX08 and IL05 groups. However, on day 7 dpi all of the nasal swab samples from MN07 and IL05 inoculated pigs were negative. In comparison, the TX08 inoculated group had 80% of the pigs shedding virus in nasal swab samples on 7 dpi, with average titers of 10**1.2 TCID50/ml. In contrast to TX08, only contact pigs were still shedding on 7 dpc in MN07 (10%) and IL05 (20%) groups. These data demonstrate that all viruses were able to replicate and be shed, however TX08 was more efficient in nasal shedding with higher titers and an extended duration of shedding. Oral fluids samples also showed lower however longer viral titers for MN07 (4-9 dpi) and IL05 (3-8 dpi) in comparison to TX08. Pigs in this study mounted a more robust humoral response to MN07 and IL-05 delta-cluster H1N1 compared to the TX08. This was statistically significant for HI titers at days 14 and 21. Cross-HI using heterologous virus demonstrated little cross-reactivity between the TX08 anti-sera and the MN07 and IL05 delta-cluster H1 viruses. However, there was cross-reactivity between the MN07 virus and IL05 anti-sera. This is consistent with phylogenetic evidence of 2 sub-clusters of delta-H1 SIV. This study indicates that the delta-cluster H1 SIV can efficiently replicate, transmit, cause lung pathology and induce humoral immune responses in the swine host. However, the kinetics of virus replication, pathogenesis, as well as host humoral immune response differed between the 3 viruses. Two sub-clusters of the delta-cluster SIV appear to have emerged simultaneously with limited serologic cross-reactivity between them. What role (if any) the full-length PB1-F2 present in TX08 played in the higher nasal shedding of virus remains to be determined. References 1. Olsen, C. W. 2002. Vir Research 85(2):199-210. 2. Vincent, A.L., et al. 2008. Adv Vir Res 72:127-54. 3. Vincent, A.L., et al. 2009. Vir Genes 39:176-185. 4. Prickett, J. 2008. JVDI 20:156-163. 5. Palmer D.F., et al.1975. Immun Series no 6:51-52. 6. Ciacci-Zanella, J. R., et al.2010 JVDI. 22(1):3-9.

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