Abstract
The pandemic H1N1 (pH1N1) influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV), were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST) cells with swine-derived endemic H1N2 (MN745) and pH1N1 (MN432) yielded two reassortant H1N2 viruses (R1 and R2), both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log10 TCID50/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism.
Highlights
The pandemic H1N1 influenza A virus, first isolated from humans in 2009, quickly spread to numerous other species, including swine, cats and ferrets [1,2,3,4]
The first reassortant swine influenza virus (SIV) was identified in Hong Kong in 2010 and it contained a pH1N1derived neuraminidase (NA) gene, a Eurasian-lineage hemagglutinin (HA) gene, and six internal genes derived from a conserved combination of avian, human and swine genes, which has been known as the triple reassortant internal gene cassette (TRIG) [6]
Generation of reassortant viruses in vitro Routine characterization of SIV isolated from diagnostic samples identified MN745 as a H1N2 virus with homology to the d-subcluster and Minnesota/0432/2010 H1N1 (MN432) as H1N1 with homology to pandemic H1N1 influenza A virus (pH1N1) based upon HA, M and NA gene sequencing
Summary
The pandemic H1N1 influenza A virus (pH1N1), first isolated from humans in 2009, quickly spread to numerous other species, including swine, cats and ferrets [1,2,3,4]. Coinfection of endemic SIV and pH1N1 in swine generated numerous reassortant viruses. A H1N2 reassortant was isolated in the United Kingdom that contained HA and NA genes derived from endemic SIV and the six internal genes from pH1N1 [7]. In Italy, a H1N2 reassortant virus was isolated with all gene segments derived from pH1N1 except NA [8]. A H1N1 reassortant virus was isolated in Germany with the same pattern of recombination [9]. A swine H3N2 and pandemic H1N1 reassortant has been reported in Canadian mink and pigs [10]
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