Phylogenic Analysis Of H3N2and H1N1Swine Influenza Virus

Swine influenza (S1)also called epidemic swine flu is an acute contagious respiratory disease of pig caused by type A influenza virus(Swine influenza virus, SIV) clinically manifested by paroxysmal cough, fever and difficulty breathing. Pigs of all ages breed and gender can be infected. SIV is a widespread and difficult to eradicate disease in nowadays modern piggery industries.In this study, clinical samples of suspected swine influenza (SI) were collected in the laboratory from different counties of the Guangxi region between August,2012and March,2014. Samples were firstly detected for swine influenza virus (SIV) by RT-PCR and positive samples were attempted to the virus isolation by embryonated eggs inoculation and MDCK cell culture. In the end,5viruses were isolated and their8complete gene fragments amplified. The NCBI-BLAST analysis demonstrated that we have isolated2strains of H3N2and3strains of H1N1namely annotated NN01:A/swine/Guangxi/NN01/2013(H3N2),JG04:A/swine/Guangxi/JG04/2013(H3N2),DX24:A/swine/Guangxi/DX24/2013(H1N1),DX31:A/swine/Guangxi/DX31/2013(H1N1) and BB2:A/swine/G uangxi/BB2/2013(H1N1).Phylogenetic and nucleotides based homologies analysis of the NN01showed that its HA gene clustered with the seasonal human influenza H3N2and displayed the homologies of87.3-98.7%, being very close to the A/swine/Guangxi/NS2783/2010(H3N2) and shared99.0%identity with the JG04strain. Its HA gene clustered with the seasonal human influenza H3N2and displayed the homologies of90.4%-98.3%, being very close to the A/swine/Hong Kong/2503/2011(H3N2) sharing98.7%identity with the JG04strain. The internal genes (PB2, PB1, PA, NP, M and NS) were located in the pdm/09subtype and comparison of each of the genes showed homologies of95.2%-99.6%within themselves.The HA and NA external genes of the JG04were located in the human seasonal influenza(H3N2) and had87.3-98.8%and90.2-98.0%homologies respectively, while its internal genes (PB2,PB1,PA,NP,M) contrarily derived from pdm/09with which the homologies were between96.1%to99.1%; and the NS gene was related with the classic swine influenza with homologies of90.9%-95.0%.The eight gene fragments of the DX24and DX31were located in the pdm/09strain sharing the homologies of95.5-99.2%and were closely related with the2012-2013Finland pdm/09strain; and the8gene fragments of the BB2were all derived from the European avian-like H1N1SIV strain with homologies ranging between87.1%and99.7%being highly related to the Jiangsu H1N1SIV.Upon above, the NN01and JG04have the characteristics of the North America triple reassortant internal gene (TRIG). The external genes of the NN01(HA and NA) were originated from the seasonal influenza, while the internal genes (PB2、 PB1、PA、MP、M and NS) were originated from the pdm/09triple reassortant H3N2SIV.The external genes of the JG04(HA and NA) were originated from the seasonal influenza and the internal genes (PB2、PB1、PA、NP、M and NS) were originated from the pdm/09, while the NS gene derived from the classic swine influenza triple reassortant H3N2SIV. The8genes of the DX24and DX31originated from pdm/09strain and the8genes of the BB2derived from the European avian-likeHIN1SIV.Amino acid analysis of the key point of each gene of the5isolates showed that, the conservative sites related to host in the PB2amino acid sequences were E (Glu) and were conserved at amino acid residue (627aa) illustrating that all the5strains were avian origin. Moreover, the PB2of the JG04strain was T588I and this mutation is highly related with the enhancement of the virulence of the virus. In addition, all the5strains contained the truncated PB1-F2protein for which its functional role is still unclear.The HA protein of the5viruses exhibited the E190D and G225/D (H3Numbering) mutations indicating that they all could bind to the SAa-2,6-Gal receptor and their HA proteolytic cleavage site presented the R amino acid residue which is characteristic of the low pathogenic HA strains. Combined together, the92th amino acid residue (92aa) of the NS1protein was D and the PDZ functional domain was absent speculating that the5strains belonged to the lower pathogenic viruses.The amino acid sequences of the NA gene of the NN01and JG03had a conserved amino acid H at position274aa (N2Numbering), and the isolates DX24, DX31and BB2were275H (N1Numbering) at this position indicating that they were still sensitive to Oseltamivir,. However, the M2protein of the5isolates had S3IN mutations implying their resistance to the amantadine drugs. Combined together, the92th amino acid residue (92aa) of the NS1protein was D and the PDZ functional domain was absent speculating that the5strains belonged to the lower pathogenic viruses. The amino acid sequences of the NA gene of the NN01and JG03had a conserved amino acid H at position274aa(N2Numbering), and the isolates DX24, DX31and BB2were275H (N1Numbering) at this position indicating that they were still sensitive to Oseltamivir,. However, the M2protein of the5isolates had S3IN mutations implying their resistance to the amantadine drugs.