Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010-2011

Abstract

Swine influenza virus (SIV) H3N2 with triple reassorted internal genes (TRIG) has been enzootic in Unites States since 1998. Transmission of the 2009 pandemic H1N1 (pH1N1) virus to pigs in the United States was followed by reassortment with endemic SIV, resulting in reassorted viruses that include novel H3N2 genotypes (rH3N2p). Between July and December 2011, 12 cases of human infections with swine-lineage H3N2 viruses containing the pandemic matrix (pM) gene [A(H3N2)v] were detected. Whole-genome analysis of H3N2 viruses isolated from pigs from 2009 to 2011 sequenced in this study and other available H3N2 sequences showed six different rH3N2p genotypes present in the U.S. swine population since 2009. The presence of the pM gene was a common feature among all rH3N2p genotypes, but no specific genotype appeared to predominate in the swine population. We compared the pathogenic, transmission, genetic, and antigenic properties of a human A(H3N2)v isolate and two swine H3N2 isolates, H3N2-TRIG and rH3N2p. Our in vivo study detected no increased virulence in A(H3N2)v or rH3N2p viruses compared to endemic H3N2-TRIG virus. Antibodies to cluster IV H3N2-TRIG and rH3N2p viruses had reduced cross-reactivity to A(H3N2)v compared to other cluster IV H3N2-TRIG and rH3N2p viruses. Genetic analysis of the hemagglutinin gene indicated that although rH3N2p and A(H3N2)v are related to cluster IV of H3N2-TRIG, some recent rH3N2p isolates appeared to be forming a separate cluster along with the human isolates of A(H3N2)v. Continued monitoring of these H3N2 viruses is necessary to evaluate the evolution and potential loss of population immunity in swine and humans.

Fig 1

Lineages of each gene segment from different H3N2 genotypes identified through gene sequencing, BLAST, phylogenetic analysis, and previous data. Lists of the originating laboratories and references for the sequences from this study and GenBank database are shown in the far right column. Superscript letters: a, triple reassorted internal gene cassette (TRIG) identified in endemic swine H3N2 virus circulating in North American swine population since 1998; b, virus isolates used as inocula in the swine pathogenesis and transmission study [A/IN/08/2011, A/IN is A(H3N2)v; A/Sw/IL/A01201606/2011, Sw/IL is rH3N2p; and A/Sw/PA/62170-1/2010, Sw/PA is H3N2-TRIG]; c, isolates obtained through USDA-SIV surveillance system that were genetically characterized in this study reported here. *, Partial sequencing. Abbreviations: PB2, polymerase basic 2; PB1, polymerase basic 1; PA, polymerase acidic; HA, hemagglutinin; NP, nucleoprotein; NA, neuraminidase; M, matrix; NS, nonstructural.

Fig 2

Phylogenetic tree of HA gene generated using the MEGA 4.0 program applying the neighbor-joining algorithm. Tree topology was supported by bootstrap analysis with 1,000 replicates. The isolates used for pathogenesis and transmission study are underlined. The black dots indicate isolates sequenced in the present study. The triangles indicate reassortant H3N2 viruses containing the pandemic matrix (pM) gene. Open or gray color triangles indicate swine (rH3N2p) or human [A(H3N2)v] isolates, respectively.

Fig 3

Alignment of amino acid sequences of the HA1 protein of H3N2 viruses from viral inocula used for experimental infection in pigs and from nasal shedding of two principal and contact pigs per group. Sequences were compared to H3N2, A/Sw/TX/4199-1/1998 (Sw/TX), an H3 cluster I prototype (42). Sw/Ont is A/Sw/Ontario/33853/2005, an H3 cluster IV prototype and representative of older seasonal swine H3N2-TRIG (25). A/IN is A/IN/08/2011, a swine A(H3N2)v virus isolated from a human case in July 2011. Sw/PA is A/Sw/PA/62170-1/2010, a representative of the contemporary swine H3N2-TRIG virus in the North American swine population. Sw/IL is A/Sw/IL/A01201606/2011, another swine reassortant H3N2 (rH3N2p) with pandemic M, NP, NS, PB1, and PA genes. Dots represent consensus sequences compared to reference strain (Sw/TX). Amino acids in the open boxes represent antigenic sites of H3 (47). *, Receptor binding sites (RBS) identified previously. Letters in open boxes represents substitutions. Predicted putative N-glycosylation sites are underlined.

Fig 4

Mean TCID₅₀ log₁₀ virus titers ± the standard error of the mean from nasal swabs of principal pigs at 1, 3, and 5 dpi (A) and contact pigs at 1, 3, 5 and 7 dpc (B). The number of virus positive pigs is indicated at the top of each column. Significant differences (P ≤ 0.05) between groups were compared within the same collection dates.