Molecular Epidemiology And Phylogenetic Analysis Of H3N2and Hlnl Influenza Among Pet Dogs In Guangxi

Canine influenza (CI) is a highly infectious respiratory disease caused by canine influenza virus(CIV).CIV has been detected among pet dogs in Guangxi, but it has not been that popular so far. However, since pet dogs as a kind of companion animals which have intimate relationship with human, it will has great significance for public health of humanto make a research on CIV among pet dogs.During April to December in2013,326nasal swabs of pet dogs from several cities in Guangxi were collected, and RT-PCR aiming at gene M was used for influenza detection of nasal swabs.44positive samples were detected, which reached to positive rate of13.4%. Four influenza viruses were isolated from positive samples by MDCK and embryonated chicken eggs, which were named A/canine/Guangxi/L1/2013(H3N2)(L1), A/canine/Guangxi/L2/2013(H3N2)(L2), A/canine/Guangxi/LZ317/2013(H1N1)(LZ317), A/canine/Guangxi/WZ11/2013(H1N1)(WZ11), respectively.According to phylogenetic analysis and nucleotide homology comparison of whole genes in H3N2CIVs, all eight genes segments of both of H3N2CIVs were closely related to Moscow/99-like virus sub-lineages which belong to early human influenza viruses H3N2subtype. And the homology of sequences reached to>99%in each of the eight gene segments compared with Moscow/10/99. Whereas, the whole human H3N2influenza viruses have been introduced into pig population and maintained transmission with pigs until2000s in China. Among all gene segments, PB2(99.6%), PA(99.8%), HA(99.9%) had highest homology with A/swine/Henan/l/2010(H3N2), and gene PB1(99.8%), NP(99.8%) shared most similarity with A/swine/Shandong/3/2005(H3N2), while gene NA(99.7%) and NS(100%) had highest homology with A/swine/Zhejiang/01/2008(H1N2), and gene M was most closely to A/swine/Guangdong/L22/2010(H3N2), and also had highest homology with it(99.8%). The analysis results indicated that it was the first time that the whole gene segments of human influenza virus which had been popular in human-being in early time and introduced into pig population were isolated from pet dogs. Result of deduced amino acid of cracking site of gene HA(PEKQTR↓G) showed that both of H3N2CIVs were low pathogenic strains, and importantly, receptor binding sites(190-196aa) of H3N2CIVs were the same as this influenza in pigs(DQISVYA) not that in human being(DQTSLYT). Besides,2261�'�228S in HA1showed that H3N2CIVs still had receptor binding ability with SAa-2,6-Gal.Result of phylogenetic analysis and nucleotide homology comparison of the whole genes in H1N1CIVs showed that gene HA and NA of LZ317and WZ11belonged to European avian-like lineage and had highest homology with A/swine/J iangsu/zg7/2010(H1N1),A/swine/Jiangsu/zg5/2010(H1N1), respectiv-ely. And gene NP, PB2, PB1, PA, M derived from sub-lineage pdm09/H1N1, with highest homology of98.4～99.4%. And gene NS of LZ317and WZ11isolates were most closely associated with classical swine influenza lineage, having highest homology with A/swine/Guangxi/BB1/2013(H1N1) for96.2%,98.1%, respectively. Interestingly, seven gene segments of LZ317and WZ11isolates including HA(98.1-98.2%), NA(98.2%), NP(99.3-99.4%), NS(96.2-98.1%), PB2(98.8-99.6%), PB1(99.0-99.2%), PA(99.0-99.8%) has very high homology with A/swine/Guangxi/BB1/2013(H1N1) which is a triple ressortant and exists in pigs in Guangxi recently, whereas gene M derived from sub-lineage pdm09/H1N1and had most closely relationship with A/Singapore/ON1079/2009(H1N1) with homology of98.8-99.6%, which was different from A/swine/Guangxi/BB1/2013(H1N1). This result indicated that H1N1CIVs were a novel triple ressortant of European avian-like, pdm09-like, classical swine influenza lineages. The analysis of amino acid of gene HA showed that the190aa,225aa,226aa in both of H1N1CIVs were D, E, Q, while there was difference on228aa,228C for LZ317and228G for WZ11, which meant that both of H1N1CIVs could bind to receptors SAa-2,6-Gal and SAa-2,3-Gal; there was only one basic amino acid(R) at HA1and HA2amino acids cracking site in H1N1CIVs; the588aa,627aa,701aa were E,E,D;353aa in gene PA was K; the92aa of NS1was D, without having PDZ ligand function domain sequence at C-terminal of NS1,all of the analysis stated that H1N1CIVs were low pathogenic viruses. Whereas, the12aa in PB1-F2was termination codon showing that the pathogenicity and transmission would become stronger. The finding of119E,274Y,292C,294D in NA and31N in M2declared that both of H1N1CIVs were resistant to oseltamivir and amantadine but still sensitive to zanamivir.To learn the prevalence of H3N2and H1N1CIV in canine population, serological investigation were conducted among pet dogs in several cities in Guangxi. The result showed that14positive serum samples from2013for H1N1were found, with a positive rate of3.2%, and1positive serum samples from2011for H1N1was found at the same time, however, no one was positive for H3N2CIV, which indicated that recently H3N2CIVs has not been popular among pet dogs in Guangxi yet, while a new triple ressortant H1N1CIV has been small-scale epidemic in pet dogs. The exist of human-origin H3N2CIVs and novel triple ressortant H1N1CIVs proved that pet dogs became an indispensable middle host in ecological circle of influenza virus. Due to the close contact of human and pet dogs, the surveillance of influenza in pet dogs would be strengthened, which will do help for a better monitoring prevalence of human influenza and providing supportive theory of prevention and control of human influenza pandemic and advanced warning.