Study On Characteristics Of The Phylogenesis And Infection Of Swine H3N2 Influenza Viruses

Swine influenza is an acute respiratory disease caused by influenza A virus. The primary clinical manifestations of viral infection are fever and acute respiratory distress. It is difficult to diagnose and treat the disease for frequent co-infecting with other respiratory bacteria or viruses. Various subtypes of influenza virus had been isolated from pig populations, but only three subtypes, H1N1 H1N2 and H3N2, were endemic in pig populations worldwide. As pigs can serve as intermediate hosts of interspecies transmission or as mixing vessels, new strains of influenza virus could be generated by reassortment between viruses of avian and human origin, some of which may transmit to other species including humans. Therefore, it is important to monitor the epidemiology of swine influenza, which can not only prevent the epidemic of influenza in animal and human populations, but also provide theoretical basis for developing a candidate vaccine against influenza virus.To investigate the antigenicity of three swine H3N2 isolates in this study, antisera were prepared for the hemagglutination inhibition (HI) test. The results showed that A/swine/Jilin/5/2007 was similar to A/swine/Jilin/19/2007, and both of them are far to A/swine/Jilin/37/2008, which suggested all of those isolates from Jilin province have being evolving.At the same time, we compared the deduced amino acid sequences of the hemagglutinin 1 (HA1) gene from the three swine H3N2 isolates against the representatives of five lineages available in GenBank. Concerning the three swine H3N2 isolates, two or four amino acid substitutions were observed at the major antigenic sites (A and B) of the HA1 molecule compared with Moscow/10/99, the representative for contemporary human lineage. The sites of substitutions in 2007 isolations are almost the same, while completely different from the 2008 isolate. Analysis of the receptor-binding sites revealed that all of the H3N2 isolates may infect human, for the residues of 226 V and 228 S identical to the New York/99-like sublineage of the contemporary human lineage. Except eight common sites in the HAs, A/swine/Jilin/5/2007 and A/swine/Jilin/19/2007 possessed an additional glycosylation site at position 126, and A/swine/Jilin/5/2007 acquired another one at position 246. The addition of new carbohydrate side chains to HA may have provided the virus with the ability to evade antibody pressure. Phylogenies of the whole genome of the three swine H3N2 influenza viruses from Jilin province revealed that two distinguishable groups of H3N2 influenza viruses were present in pigs: the wholly contemporary human-like H3N2 viruses (represented by the Moscow/10/99-like sublineage) and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses, both co-circulating in pig populations. This is the first report on reassortment between mammal H3N2 and avian H5 viruses.Influenza virus infection is initiated by the specific attachment of the virus to cellular, mediated by the interaction of the viral hemagglutinin with the receptor, either SAα2,3Gal or SAα2,6Gal. A modified solid-phase enzyme-linked assay was used for the investigation of the three isolates. The results showed that three H3N2 influenza viruses bound to both types of receptors, while the binding activity of A/swine/Jilin/5/2007 is higher than the other two isolates. Moreover, all the isolates bound to SAα2,3Gal with a higher affinity than to SAα2,6Gal, implying they have the potential to infect human.To evaluate the pathogenic potential between the reassortant virus and the unreassortant virus, the pathogenicity was tested in mice. Experimental inflection suggested that enhanced pathogenicity strains will be generated by reassortment between H5 and H3N2 viruses. The inoculums were administered intranasally to mice, followed by monitor daily for clinical signs of disease and body weight until day 14 postinoculation. The lungs were collected for histopathologic evaluation. The results showed that the body weight and the lungs histopathology induced by A/swine/Jilin/37/2008, the reassortant virus containing M and NS genes of H5, were more serious than A/swine/Jilin/5/2007, all the gene segments of which were human origin, suggested that A/swine/Jilin/37/2008 may represent a potential threat in the emergence of new human viruses.In conclusion, two distinguishable groups of H3N2 influenza viruses were co-circulating in pig populations of Jilin province: the wholly human contemporary human-like H3N2 viruses and double-reassortant viruses containing genes from contemporary human H3N2 viruses and avian H5 viruses. This is the first description of an instance between human origin H3N2 and avian H5 influenza viruses in nature. Importantly, all viruses have the ability of interspecies transmission for possessing two receptor-binding properties, and animal experiment suggested that this reassortant virus is more pathogenic to BALB/c mice than other isolates. So it emphasizes the importance of reinforcing swine influenza virus surveillance.