Multiple-reassortment events have taken place in swine since the H3N2 virus with TRIG emerged in the North American swine population in 1998 (24
). These H3N2-TRIG viruses consist of HA, NA, and PB1 genes of human virus lineage, the M, NP, and NS genes of swine virus lineage, and the PA and PB2 genes of avian virus lineage. The H3N2-TRIG virus spread widely in swine and reassorted with the classical swine H1N1 (cH1N1) virus, generating a new subtype, H1N2, and reassortant H1N1 (rH1N1) viruses, both containing the six internal genes of the TRIG cassette. Subsequently, two reverse zoonotic events in 2003 and 2005 introduced H1N2 and H1N1 viruses with human virus lineage HA and NA genes into the SIV gene pool (16
). These H1 viruses reassorted with endemic SIV circulating in swine at that time, but retained the TRIG cassette (see review in reference 39
). The emergence of the pandemic H1N1 (pH1N1) virus in 2009 in the human population, followed by further reverse zoonotic events, introduced yet another H1N1 virus with a new gene constellation into the North American swine population (44
). The pH1N1 virus had five gene segments (PB2, PB1, PA, NP, and NS) genetically related to the TRIG cassette, as well as an HA genetically related to the γ-H1 cluster found in the North American swine lineage viruses, while the M and NA genes were from Eurasian lineage (avian-like) swine influenza viruses (10
). Reports of pH1N1 reassortment with endemic H1 SIV resulting in different SIV genotypes detected in swine have been documented around the world (9
). In contrast to the increasing genetic diversity and evolution detected in the H1 subtype in recent years, swine H3N2 viruses have not evolved further into additional distinct genotypes beyond the HA cluster IV. Since the introduction of the H3N2-TRIG in 1998 until 2009, all descendant H3N2 viruses have demonstrated the human-like surface HA and NA genes with a similar TRIG cassette, suggesting that this combination of gene segments favored optimal adaptation to and transmission between swine hosts. A previous publication showed that nine H3N2 viruses obtained from Minnesota in 2006-2007 maintained the same human (HA, NA, and PB1), avian (PB2 and PA), and swine (NP, M, and NS) virus genes that were demonstrated in earlier strains (19
), confirming limited reassortment events had taken place at the time. However, the appearance of pH1N1 in swine led to reassortment between pH1N1 and endemic H3N2-TRIG viruses in pigs.
A recent study identified nine reassortant pH1N1/endemic swine H1 and H3 influenza viruses collected during an active surveillance project conducted in 2009-2010 (9
). Seven genotypes were detected among the three subtypes: H1N1 (one isolate), H3N2 (one isolate), and H1N2 (five isolates). All genotypes consistently contained the pM gene. The single reassortant H3N2 virus was collected in 2009 from pigs in Minnesota and contained pandemic M, NP, and PA genes. Sequence data from the USDA-NAHLN Swine Influenza Surveillance System in the present study, including recently published data (20
) (), revealed five different genotypes of reassortant pH1N1/swine H3N2 viruses that differ from the 2009 isolate reported by Ducatez et al. (9
). Reassortant H3N2 viruses collected from all studies mentioned here and publicly available data in GenBank were found from six different states. The number of gene segments in the reassortant H3N2 viruses that were derived from pH1N1 differed from one to five segments. Phylogenetic analysis of all gene segments indicated that reassortment possibly occurred as multiple events since no individual gene cluster can be rooted to a single virus. Similar to what was reported previously, our analysis of the published sequences and viruses submitted through the USDA-NAHLN Swine Influenza Surveillance System did not find a single genotype that was predominant in the swine population. However, the presence of the pM gene was consistent among all of the H3N2 reassortants detected in this and previous studies (9
). These data suggest that pM from the pH1N1/Eurasian swine lineage may be necessary for the genetic fitness of subsequent rH3N2p reassortants in the swine host.
Swine H3N2-TRIG and H1 subtypes have thus far caused only sporadic human cases despite their wide circulation in the swine population for over a decade. In the United States, 41 swine-origin influenza cases in humans have been reported since 1990 to the present with limited human-to-human transmission (3
). However, the pandemic H1N1 2009 virus, which had the ability to infect humans through aerosol transmission, spread rapidly in the human population around the world and was able to spill back into pigs and spread widely among naive pig populations. Although genetic evolution was apparent in all gene segments, the major genetic differences between pH1N1 and swine γ cluster H1N1-TRIG viruses are the NA and M genes. Since July 2011, there have been reports of 12 cases of human infection with A(H3N2)v, which are viruses with seven genes from the swine-lineage H3N2-TRIG and the pM gene. These 12 cases were identified in five states: Indiana (n
= 2), Iowa (n
= 3), Maine (n
= 2), Pennsylvania (n
= 3), and West Virginia (n
= 2) (3
). All of these cases demonstrated influenza-like clinical signs, and three of the infected people required hospitalization. Eleven of the twelve cases were children. Three cases in Iowa, as well as two cases in West Virginia, had no documented history of direct or indirect swine exposure, suggesting limited human-to-human transmission of A(H3N2)v viruses (3
). The number of individuals infected with A(H3N2)v virus observed in 2011 was a concern and thus prompted the experimental pathogenesis and transmission pig study reported here to determine the efficiency of this virus in spreading in the swine population.
We obtained an A(H3N2)v (A/IN) virus isolated from the first human case in July 2011 and compared its pathogenesis and transmission efficiency to a swine H3N2-TRIG (Sw/PA), as well as to a reassortant swine H3N2 virus with pandemic M, NS, NP, PA, and PB1 genes (Sw/IL) in the natural swine host. It was unknown whether the presence of genes from the 2009 pandemic H1N1 viruses in these reassortant H3N2p viruses (rH3N2p) would lead to a different phenotypic behavior compared to H3N2-TRIG-infected pigs. Our results demonstrated the ability of all three tested H3N2 viruses to infect, cause pneumonia and histopathologic lung lesions, and transmit to contact pigs. Clinical signs in all groups were typical of SIV-associated disease reported previously by our and other groups with no increase in clinical illness or pathogenesis detected in any A(H3N2)v or rH3N2p virus-infected pigs. In fact, the degree of macroscopic and microscopic lung lesions and the virus titers in nasal swabs and BALF were lower in both of the reassortant virus-infected groups than in the H3N2-TRIG-infected group. It appeared that the novel A(H3N2)v virus isolated from a human was the least pathogenic, and the swine rH3N2p virus (with pandemic M, NS, NP, PA, and PB1 genes) replicated to the lowest titers of the three viruses. Overall, all three viruses maintained the ability to transmit from the inoculated to contact pigs (indicated by seroconversion). The kinetics of virus shedding in the A(H3N2)v contact pigs were delayed compared to the H3N2-TRIG group; however, it was intermediate to the kinetics of the rH3N2p virus. Considering that the surface glycoproteins, HA and NA, are relatively similar, the differences in shedding may be due to different gene constellations found in the three viruses. Future studies utilizing reverse genetics to swap individual genes of the reassortant viruses are needed to investigate the role of the pM or other genes in pathogenesis and transmission in vivo.
The Ducatez et.al. (9
) study that identified the nine reassortant pH1N1/endemic swine influenza viruses mentioned above also assessed virus replication and pathogenicity of the reassortant H3N2-pM, -NP, and -PA in comparison to pH1N1 and endemic H3N2-TRIG viruses using a ferret model. The H3N2 reassortant virus was shown to cause only mild clinical signs in ferrets, suggesting no enhancement of virulence properties had occurred through reassortment. The study did not evaluate transmission efficiency and thus cannot interpret whether pM and/or these particular gene combinations altered the transmission phenotype in ferrets. A recent study in guinea pigs used reverse genetics to separately swap the M or NA genes of pH1N1 virus into PR8, a representative of historic human H1N1 (6
). Wild-type PR8 replicated and transmitted poorly in the guinea pig model, but the presence of the pM gene in the virus resulted in an increased transmission rate of 62.5% compared to the wild-type virus. Although the exact mechanism contributing to the increased aerosol transmission in guinea pigs was not determined, the study indicated that the pM gene of Eurasian swine virus lineage contributed a selective advantage for viral transmission in the guinea pig model. This finding correlates with the epidemiology of the emerging reassortant H3N2 viruses where the different genotypes all contain the pM gene. However, although the pM may be required for successful reassortment and/or fitness of new gene combinations, acquisition of pH1N1 genes does not appear to confer an advantage over previously swine-adapted H3N2 in the swine host.
The surface hemagglutinin (HA) glycoprotein of influenza viruses are known to play a major role in influenza virus cross-species transmission since it contains the viral receptor binding sites (RBS). Compatibility between RBS on the HA protein to the corresponding receptor expressed on the cells of the host species is required for successful transmission (14
). Human and swine influenza viruses preferentially bind to sialic acid (SA) attached to galactose in an SAα-2,6 linkage on the host epithelial cells. The majority of the upper (nasal passage and trachea) and lower (bronchi and alveoli) respiratory tract of humans and pigs have SAα-2,6 receptors. The receptors preferred by avian influenza viruses, SAα-2,3 can be found on the nonciliated cuboidal bronchiolar cells in the lower respiratory tracts of humans and pigs but are also found in swine trachea (15
). As a result, pigs (and humans) can potentially be infected by both mammalian-adapted and avian influenza viruses for generating new reassortant influenza viruses.
Amino acids at positions 190, 226, and 228 of the RBS in the HA1 protein were shown to correlate with receptor specificity. In human H3 viruses, amino acid positions D190, L/I226, and S228 correlated with SAα-2,6 receptors, while in swine H3 viruses those positions are D/A190, V226, and S228 (27
). All three isolates—A/IN A(H3N2)v, Sw/PA H3N2-TRIG, and Sw/IL rH3N2p—were able to infect and transmit to contact pigs. The RBS of both A/IN and Sw/PA viruses displayed the sequence of the reported SAα-2,6 receptor preference, while Sw/IL contained an amino acid S228G change. G228 is more typically found in the HA1 protein of avian influenza viruses with a preference for SAα-2,3 receptor (40
). This mutation that suggests the a SAα-2,3 receptor preference possibly contributed to the low virus titer in the lungs and a short virus shedding period detected in the principal Sw/IL-infected pigs, as well as the slight delay in the kinetics of virus transmission in the contact pigs. A number of amino acid mutations were also detected in the major antigenic sites A, B, C, and D of the three viruses compared to both the cluster I and IV prototype viruses. The total number of mutations was the lowest in Sw/PA (H3N2-TRIG) and highest in Sw/IL (rH3N2p). These mutations appeared to have affected the antibody binding property, as evidenced by the HI cross-reactivity test where antisera against the historic H3N2 cluster I virus (Sw/TX/98) cross-reacted to Sw/IL 2-fold lower than to Sw/PA. The lack of HI cross-reactivity indicates antigenic drift of the tested viruses from the cluster I virus. HI cross-reactive titers were higher within the cluster IV viruses. Antibodies primed against the swine H3N2-TRIG (Sw/PA) cross-reacted against both A(H3N2)v (A/IN) and rH3N2p (Sw/IL) and an older H3N2 cluster IV. This suggests that the North American swine population should have some level of immunity against the new reassortant pH1N1/H3N2-TRIG viruses if they had been previously exposed or vaccinated with a contemporary cluster IV H3N2 virus. However, the A/IN virus A(H3N2)v had a loss in cross-reactivity against all contemporary and reference virus-antisera, suggesting a potential antigenic drift in this newly emerging phylogenetic node of H3 from the swine-lineage HA. In addition, it was demonstrated that ferret sera primed against H3N2-TRIG viruses isolated from human cases do not cross-react with the seasonal human H3N2 virus incorporated in human influenza vaccines and vice versa (34
). This demonstrates that although the H3N2 viruses currently circulating in swine were originally transmitted from humans in the mid-1990s, they are now antigenically distinct from contemporary human H3N2 viruses. Some subsets of the human population may be vulnerable to both TRIG- and reassortant-H3N2 virus infections. It can be speculated that A(H3N2)v viruses in humans may also diverge separately and distinctly from H3N2-TRIG and rH3N2p viruses found in swine if successfully established in the human population, and thus continued monitoring of H3 viruses in both populations is necessary.
In summary, our data demonstrate that at least six different swine rH3N2p genotypes currently circulate in the North American swine population as the result of genetic reassortment between pH1N1 and contemporary swine H3N2-TRIG viruses. However, all genotypes contained some internal gene segments, as well as the surface glycoprotein genes, HA and NA, that are already found in the swine population, and thus far no specific genotype appears to predominate in the swine population based on diagnostic case submissions and surveillance efforts. The results from our pathogenesis and transmission study with two different H3N2 viruses, rH3N2p and H3N2-TRIG isolated from pigs and A(H3N2)v isolated from a human, detected no major biological differences compared to that observed with the swine H3N2-TRIG virus. Antibodies specific to the H3N2-TRIG virus cross-reacted with both reassortant H3N2 viruses. Genetic analysis of all of the reassortant H3N2-pM viruses with different internal gene combinations were grouped in the same HA gene cluster as the contemporary swine H3N2-TRIG, although the most recent isolates appear to be forming a separate cluster with the human isolates of A(H3N2)v. Despite the fact that some degree of antigenic cross-reactivity is conserved at this point, continued monitoring of this group of H3 viruses is necessary to evaluate population immunity in both swine and humans. Overall, these findings further substantiate the continued genetic instability and evolution of influenza A viruses, that influenza A viruses are readily shared between humans and animals, and the role of pigs in generating reassortant influenza viruses. Thus, it is essential to continue to monitor influenza viruses through whole-genome analysis, antigenic cross-reactivity, and in vivo studies to examine the phenotypic nature of viruses with novel and emerging genetic and/or antigenic combinations.