Whole Genome Sequence Analysis And Experimental Transmission In Guinea Pigs Of H9N2Avian Influenza Viruses Isolated From Shandong

Avian influenza (AI) is one of the most serious infectious diseases of poultry caused byAvian influenza virus (AIV). The disease was first discovered in Italy in1878and thenprevailed in many countries and regions. Notably, the low pathogenic AIV H9N2has beenspreading widely, leading to enormous economic losses to the poultry industry. Although thepathogenicity of H9N2AIV was trivial, it could cause high morbidity and lethality to poultrywhen co-infected with other pathogens. In1994, Chen Bolun first isolated a H9N2AIV strainfrom diseased chickens and verified the evidence of H9N2AIV in our country. Throughinvestigation, we discovered that H9N2AIV had caused serious epidemics in Shandongprovince, China, from winter2009to spring2010.In order to facilitate the molecular epidemiology surveillance for movements of all genesof H9N2AIVs in in Shandong province, China, a H9N2AIV isolated from Shandongprovince in2008was used. The eight full-length cDNAs of the virus genes were amplified byRT-PCR, and inserted into pMD18-T vector, and the recombinant plasmid was transformed incompetent Top10E.Coli, then sequenced after identification through enzyme cutting. Thenucleotide sequences were compared and analyzed for the homology and phylogeneticrelationships between the isolate A/Chicken/Shandong/01/2008and some other isolates ofH9N2subtype using professional software. The results showed that isolateA/Chicken/Shandong/01/2008can be classified into Eurasian lineage, and shared the highesthomology to the corresponding nucleotide sequences of the representative strains of Eurasianlineage, implying relevant genes evolving in homothetic route. Amino acid analysis showedthat the sequences at the cleavage site between HA1and HA2was P-A-R-S-S-R, which is acharacter of low pathogenic avian influenza virus. At the receptor-binding site, the viruscarried Leu-234which was specific to the human influenza virus-like receptor.The transmission ability among mammals was investigated using guinea pigs divided into inoculate group, direct contact group and aerosol contact group.Virus shedding wasdetected by collecting nasal washes at two days intervals. Air samples in isolator werecollected with AGI-30for the detection of the concentration of AIV. The seroconversion wasassessed by the hemagglutination inhibition (HI) test. AIV aerosols were detected between6and10dpi, reaching its peak concentration of1.19×105copies/m3air at8dpi. Viruses wereisolated from the nasal washes of the inoculated and direct contact guinea pigs at day2and4post inoculation (dpi), respectively. Seroconversion occurred in all inoculated and directcontact guinea pigs at7and14dpi, respectively. No viral shedding or seroconversion wasdetected in any aerosol contact guinea pigs. The results showed that after being infected withAIV, the guinea pigs could excrete viruses via nasal cavity, thus leading to contamination offeedstuff and drinking water, as well as viral transmission; infected guinea pigs may excreteviruses to form aerosols which may be transmitted by air flow.In this study, infection dose of H9N2subtype avian influenza virus through aerosol routefor guinea pigs was quantified. The viral aerosols were nebulized using a microbial aerosolgenerator and then the female guinea pigs were exposed for60min for infections. Infectionwas determined by detecting the virus shedding. The aerosol median infective dose (aID50)was calculated using the Bliss method (weighted regression method). The results showed thataID50of aerosol infection was1.32×105copies/L air, demonstrating that the aerosols couldinfect guinea pigs at certain concentrations in experimental condition, which was a greatthread to humans.