Influenza A Subtype H3 Viruses in Feral Swine, United States, 2011–2012

To determine whether, and to what extent, influenza A subtype H3 viruses were present in feral swine in the United States, we conducted serologic and virologic surveillance during October 2011–September 2012. These animals were periodically exposed to and infected with A(H3N2) viruses, suggesting they may threaten human and animal health.

hemagglutinin (HA) and neuraminidase (NA) subtypes were determined at the NVSL using standard hemagglutination-inhibition (HI) and NA inhibition testing procedures.
To maximize the chances of detecting H3 IAV infection in feral swine samples tested, the 22 isolates described above were selected to represent a wide range of antigenically distinct H3 IAVs (Technical Appendix Table). Ten of them represented 2 of the contemporary H3 antigenic clusters in the swine population: H3N2-α and H3N2-β (2). Four human H3N2v isolates were also included, and these isolates antigenically belong to H3N2-β.
A/Perth/16/2009(H3N2) and A/Victoria/361/2011(H3N2), 2 seasonal influenza vaccine strains recommended by the World Health Organization (WHO), represent the antigenic variants predominantly circulating in human population. These viruses do not cross-react with the 10 swine IAVs and 4 H3N2v viruses described in the previous paragraph. An avian origin H3N2 canine influenza virus (CIV) (3) and an equine origin H3N8 CIV (4) were also selected, which do not cross-react with swine H3N2, human H3N2v, or H3N2 seasonal influenza viruses. Except for H3N2 CIV, the 2 H3 avian influenza viruses selected do not cross-react with the other isolates listed in the Table. The selected 2009 H1N1 virus also did not react with any H3 IAVs selected for this study (Technical Appendix Table).
All avian influenza viruses were propagated in MDCK cells (ATCC, Manassas, VA, USA) and then stored at −70°C until analysis.

Serologic Assays
Influenza-specific ELISAs were performed by using IDEXX Influenza A Ab Test (IDEXX, [Westbrook, ME, USA) based on the manufacturer's specifications. HI assays were performed according to the WHO manual on animal influenza diagnosis and surveillances (http://whqlibdoc.who.int/hq/2002/WHO_CDS_CSR_NCS_2002.5.pdf). Before HI tests were conducted, feral swine serum was treated with receptor-destroying enzyme (Denka Seiken Co., Tokyo, Japan) by 1:3 (v/v) at 37°C for 18 h, and then heat inactivated at 55°C for 30 min. Then, the serum was diluted with phosphate-buffered saline (for a final dilution of 1:10), and 22 influenza A viruses were tested by HI assay with 0.5% turkey red blood cells. The HI for the A/Victoria/369/2011(H3N2) testing was performed using 0.5% guinea pig red blood cells.
Microneutralization (MN) assay was performed in MDCK cells. Neutralizing titers were expressed as the reciprocal of the serum dilution that inhibited 50% of the viral growth of 100 tissue culture infectious doses of the virus. The MN titers were determined by HA assay using 0.5% turkey red blood cells as described (2).

Serologic Data Analyses
If the titers of a serum against a specific influenza isolate were >40, the serum was classified as positive to this isolate. Student t tests were performed to test the null hypothesis: there was no significant difference among the HI or MN titers between different groups of IAVs.

Molecular Characterization and Phylogenetic Analyses
The multiple sequence alignments were conducted by using the MUSCLE software package (5). The phylogenetic analyses were performed by using maximum-likelihood by GARLI version (6), and bootstrap resampling analyses were conducted with 1,000 runs using PAUP* 4.0 Beta (7) with a neighbor-joining method, as described (8).