Triple Reassortant H3N2 Viruses Research Articles

Swine influenza viruses in Northern Vietnam in 2013–2014

Swine are an important intermediate host for emergence of pandemic influenza. Vietnam is the largest swine producer in South East Asia. Systematic virological and serological surveillance of swine influenza viruses was carried out in Northern Vietnam from May 2013 to June 2014 with monthly sampling of pigs in local and large collective slaughterhouses and in a live pig market. Influenza A seroprevalence in the local slaughterhouses and in the large collective slaughterhouse was 48.7% and 29.1%, respectively. Seventy-seven influenza A viruses were isolated, all from the large collective slaughterhouse. Genetic analysis revealed six virus genotypes including H1N1 2009 pandemic (H1N1pdm09) viruses, H1N2 with H1 of human origin, H3N2 and H1N1pdm09 reassortants, and triple-reassortant H3N2 viruses. Phylogenetic analysis of swine and human H1N1pdm09 viruses showed evidence of repeated spill-over from humans to swine rather than the establishment of H1N1pdm09 as long-term distinct lineage in swine. Surveillance at the large collective slaughterhouse proved to be the most efficient, cost-effective, and sustainable method of surveillance for swine influenza viruses in Vietnam.

Genetic characterization of swine influenza viruses isolated in Japan between 2009 and 2012

Eleven swine influenza viruses (SIVs) isolated from pigs in Japanese institutions between 2009 and 2012 were genetically characterized. Seven H1N1 were shown to have originated from A(H1N1)pdm09 viruses. Two H1N2 viruses contained H1 and N2 genes of Japanese H1N2 SIV origin together with internal genes of A(H1N1)pdm09 viruses. Two H3N2 viruses isolated during animal quarantine were identified as triple reassortant H3N2 viruses maintained among pigs in North America. This study shows that A(H1N1)pdm09 viruses and their reassortant strains are already present in domestic pigs in Japan and that novel SIVs are possibly being imported from abroad.

Molecular and Antigenic Characterization of Reassortant H3N2 Viruses from Turkeys with a Unique Constellation of Pandemic H1N1 Internal Genes

Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production. The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage. However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA, nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95% while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C, D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro.

Pathobiology of triple reassortant H3N2 influenza viruses in breeder turkeys and its potential implication for vaccine studies in turkeys

Triple reassortant (TR) H3N2 influenza viruses have been isolated from turkeys in the United States since 2003. These TR H3N2 virus infections have been associated with drastic declines in egg production in breeder turkeys although co-infection with multiple agents could have been responsible for exacerbating the clinical signs. In this study, we experimentally confirmed that TR H3N2 influenza virus alone can cause drastic reduction/complete cessation of egg production and pathology of the reproductive tract in 26-week-old breeder turkeys. We confirmed high levels of virus replication and abundant distribution of avian specific α2,3 sialic acid-galactose receptors in the oviduct of these turkeys. Although 2–6-week-old turkeys are routinely used for pathogenicity and vaccine protection studies, the low levels of viral shedding and asymptomatic infections in this age group often pose difficulty in interpretation of results. Our study shows that breeder turkeys should be used to assess the potential pathogenicity of TR H3N2 viruses and the viral titers and pathology of the oviduct as well as egg production data can be good measures of protection following in vivo challenge in vaccine efficacy studies.

Genetic Evolution of Swine Influenza A (H3N2) Viruses in China from 1970 to 2006

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.

Interspecies and intraspecies transmission of triple reassortant H3N2 influenza A viruses

The triple reassortant H3N2 viruses were isolated for the first time from pigs in 1998 and are known to be endemic in swine and turkey populations in the United States. In 2004, we isolated two H3N2 triple reassortant viruses from two turkey breeder flocks in Ohio and Illinois. Infected hens showed no clinical signs, but experienced a complete cessation of egg production. In this study, we evaluated three triple reassortant H3N2 isolates of turkey origin and one isolate of swine origin for their transmission between swine and turkeys. Although all 4 viruses tested share high genetic similarity in all 8 genes, only the Ohio strain (A/turkey/Ohio/313053/04) was shown to transmit efficiently both ways between swine and turkeys. One isolate, A/turkey/North Carolina/03, was able to transmit from pigs to turkeys but not vice versa. Neither of the other two viruses transmitted either way. Sequence analysis of the HA1 gene of the Ohio strain showed one amino acid change (D to A) at residue 190 of the receptor binding domain upon transmission from turkeys to pigs. The Ohio virus was then tested for intraspecies transmission in three different avian species. The virus was shown to replicate and transmit among turkeys, replicate but does not transmit among chickens, and did not replicate in ducks. Identifying viruses with varying inter- and intra-species transmission potential should be useful for further studies on the molecular basis of interspecies transmission.

The emergence of novel influenza viruses among pigs in North America due to interspecies transmission and reassortment

In 1997–1998, H3N2 influenza viruses of different genotypes emerged among North American pigs. Triple reassortant H3N2 viruses containing human, classical swine and avian virus genes spread widely within the swine population and gave rise subsequently to reassortant H1N2 and H1N1 viruses. Together with wholly avian viruses recovered from North American pigs since 1999, these viruses emphasize that influenza virus interspecies transmission and reassortment in pigs can occur throughout world. They provide important research tools with which the genetic factors that control interspecies transmission can be investigated.

Phylogenetic analysis of H1N2 isolates of influenza A virus from pigs in the United States

Twenty-four H1N2 influenza A viruses were newly isolated from pigs in the United States. These isolates originated from 19 farms in 9 different swine producing states between 1999 and 2001. All farms had clinical histories of respiratory problem and/or abortion. The viral isolates were characterized genetically to determine the origin of all eight gene segments. The results showed that all H1N2 isolates were reassortants of classical swine H1N1 and triple reassortant H3N2 viruses. The neuraminidase (NA) and PB1 genes of the H1N2 isolates were of human origin, while the hemagglutinin (HA), nucleoprotein (NP), matrix (M), non-structural (NS), PA and PB2 polymerase genes were of avian or swine origin. Fifteen of the 24 H1N2 isolates were shown to have a close phylogenic relationship and high amino acid homology with the first US isolate of H1N2 (A/SW/IN/9K035/99). The remaining nine isolates had a close phylogenic relationship with classical swine influenza H1N1 in the HA gene. All other genes including NA, M, NP, NS, PA, PB1 and PB2 showed a close phylogenic relationship with the H1N2 (A/SW/IN/9K035/99) strain and triple reassortant H3N2 viruses. However, PB1 genes of two isolates (A/SW/KS/13481-S/00, A/SW/KS/13481-T/00) were originated from avian influenza A virus lineage. These results suggest that although there are some variations in the HA genes, the H1N2 viruses prevalent in the US swine population are of a similar genetic lineage.

The emergence of novel swine influenza viruses in North America

Since 1997, novel viruses of three different subtypes and five different genotypes have emerged as agents of influenza among pigs in North America. The appearance of these viruses is remarkable because there were no substantial changes in the overall epidemiology of swine influenza in the United States and Canada for over 60 years prior to this time. Viruses of the classical H1N1 lineage were virtually the exclusive cause of swine influenza from the time of their initial isolation in 1930 through 1998. Antigenic drift variants of these H1N1 viruses were isolated in 1991–1998, but a much more dramatic antigenic shift occurred with the emergence of H3N2 viruses in 1997–1998. In particular, H3N2 viruses with genes derived from human, swine and avian viruses have become a major cause of swine influenza in North America. In addition, H1N2 viruses that resulted from reassortment between the triple reassortant H3N2 viruses and classical H1N1 swine viruses have been isolated subsequently from pigs in at least six states. Finally, avian H4N6 viruses crossed the species barrier to infect pigs in Canada in 1999. Fortunately, these H4N6 viruses have not been isolated beyond their initial farm of origin. If these viruses spread more widely, they will represent another antigenic shift for our swine population, and could pose a threat to the world's human population. Research on these novel viruses may offer important clues to the genetic basis for interspecies transmission of influenza viruses.