SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY
Location: Virus and Prion Research Unit
Title: Swine Influenza Virus: Emerging Understandings
Submitted to: Swine Disease Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: October 2, 2010
Publication Date: November 4, 2010
Citation: Vincent, A.L., Lorusso, A., Lager, K.M., Gauger, P.C., Gramer, M.R., Ciacci-Zanella, J.R. 2010. Swine influenza virus: emerging understandings. In: Proceedings of 18th Annual Swine Disease Conference for Swine Practitioners, November 4-5, 2010, Ames, IA. p. 57-62.
Introduction: In March-April 2009, a novel pandemic H1N1 emerged in the human population in North America . 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 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 are similar to 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 [2-3]. Other than sporadic transmission to humans [4-5], swine influenza A viruses of the 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. The emergence of the 2009 pandemic virus brought a heightened awareness to the evolution and epidemiology of influenza A viruses in swine and presents a new era of challenges and opportunities for understanding and controlling influenza in pigs.
The hemagglutinin (HA) and the neuraminidase (NA) proteins encoded by gene segments 4 and 6, respectively, play a key role in the influenza life cycle and represent the primary targets of the host humoral immune response . Both HA and NA undergo two types of variation called antigenic drift and antigenic shift. Antigenic drift involves minor changes in the HA and NA genes due to polymerase errors during replication, whereas antigenic shift involves major changes in these molecules resulting from replacement of the entire gene segment as a consequence of reassortment in the event that two (or more) unique viruses infect the same cell. Based upon the HA and NA proteins, 16 HA and 9 NA subtypes, naturally paired in different combinations, have been identified thus far . Only a limited number of subtypes have been established in mammals. Only viruses of H1, H2, H3, N1 and N2  subtypes have circulated widely in the human population and only H1, H3, N1 and N2 subtypes have been consistently isolated from pigs .
Swine influenza was first recognized in pigs in the Midwestern U.S. in 1918 as a respiratory disease that coincided with the human pandemic known as the Spanish flu. Since then, it has become an important disease to the swine industry throughout the world. The first influenza virus was isolated in 1930 by Shope  and was demonstrated to cause respiratory disease in swine that was similar to human influenza. This strain was subsequently recognized belonging to the H1N1 lineage of influenza virus, and swine were utilized in the following years as a model to study influenza pathogenesis in a natural host. From the first characterization of swine influenza virus until the late 1990s, the classical swine lineage H1N1 (cH1N1) was relatively stable at the genetic and antigenic levels in U.S. swine.
North American triple reassortant swine viruses: The epidemiology of influenza in pigs dramatically changed after 1997-1998. In 1998, a severe influenza-like disease was observed in pigs in North Carolina with additional outbreaks in swine herds in Minnesota, Iowa, and Texas. The causative agents for these outbreaks were identified as influenza A viruses of the H3N2 subtype. Genetic analysis of these H3N2 viruses demonstrated the presence of two different genotypes. However, only triple reassortants containing gene segments from the classical swine virus (NP, M, NS), human virus (PB1, HA, NA). and avian virus (PB2, PA)  became successfully established in the pig population. By the end of 1999, viruses antigenically and genetically related to the triple reassortant lineage were widespread in the U.S. swine population . Genetic and antigenic evaluation of H3N2 swine influenza isolates since 1998 [13-14] indicate at least three introductions of human H3 subtype viruses became established in swine, leading to phylogenetic clusters I, II and III. The cluster III viruses became dominant in North America  and continued to evolve into cluster IV . The human lineage PB1, avian lineage PB2 and PA and swine lineage NP, M, and NS found in contemporary swine influenza viruses are referred to as the triple reassortant internal gene (TRIG) constellation .
After their emergence, the H3N2 viruses reassorted with cH1N1 swine influenza viruses. The H1N1 viruses containing the HA and NA from the cH1N1 virus and the TRIG from triple reassortant H3N2 viruses were referred as reassortant H1N1 (rH1N1) and the viruses containing the HA from the classical swine virus and the NA and TRIG from the triple reassortant H3N2 virus are H1N2 viruses [13, 18]. Reassortant H1 viruses are endemic with the H3N2 viruses and co-circulate in most major swine producing regions of the U.S. and Canada. Since 1998, the vast majority of the characterized swine viruses from the U.S. and Canada contain the TRIG, regardless of subtype.
Since 2005, H1N1 and H1N2 viruses with the HA gene derived from human viruses have spread across the U.S. in swine herds forming the delta-cluster H1 . The HAs from the human-like (hu) swine H1 viruses are genetically and antigenically distinct from classical swine lineage and derivatives. However, their TRIG genes are similar to those found in the TRIG cassette of the contemporary swine triple reassortant viruses. The HA from the delta-cluster viruses were shown to have most likely emerged from two separate introductions of human seasonal HA from H1N2 and H1N1 viruses and are differentiated phylogenetically by two distinct sub-clusters, delta1 and delta2, respectively (Lorusso unpublished). Viruses belonging to the delta-cluster were shown to be paired either with a N1 or N2 gene consistently of human lineage and not of swine lineage N1. Since their identification, delta1-subcluster triple reassortant viruses had an N2 gene preference whereas delta2-subcluster viruses had an N1 preference. Limited HI cross-reactivity was demonstrated between the delta1 and delta2 viruses thus supporting the sub-clusters defined in the phylogenetic analysis.
To represent the evolution of the currently circulating H1 viruses, a cluster classification has been proposed. Viruses from the classical H1N1 lineage-HA acquired from the TRIG cassette evolved to form alpha-, beta-, and gamma-clusters based on the genetic makeup of the HA gene; whereas H1 subtypes strains with HA genes most similar to human seasonal H1 viruses form the delta-cluster . All four HA gene cluster types can be found with neuraminidase genes of either the N1 or N2 subtype. In order to study the evolution and the antigenic relationships among the H1 swine influenza virus subtypes, we recently analyzed twelve different isolates, selected from the University of Minnesota Veterinary Diagnostic Laboratory (UMVDL) diagnostic case database (Lorusso, unpublished). The viruses were isolated from outbreaks of respiratory disease in pigs from diagnostic cases submitted to the UMVDL in 2008 and are representative of each of the postulated four H1 clusters. All gene segments were sequenced and analyzed, and antigenic changes were measured for all twelve viruses using the hemagglutination inhibition (HI) assay and mapped by antigenic cartography. All 2008 H1 viruses contained the North American TRIG. Furthermore, variation was demonstrated in the 6 genes that make up the TRIG, but no HA cluster-specific patterns were detected among the genes composing the TRIG constellation.
To evaluate the 2008 viruses at the antigenic level, homologous and heterologous hemagglutination inhibition (HI) assays were performed with a panel of the 2008 and previously generated H1 swine antisera. The viruses representi