| H3 subtype avian influenza viruses have such a wide host range from birds to various mammalian species including humans, pigs, dogs, and horses. H3 influenza virus is prone to antigenic drift and shift, which make it a major threat to public health. In China, live poultry markets(LPM) are the highly productive sources of avian influenza viruses(AIVs), where numerous hosts(e.g., chickens, ducks, quails, guineafowl, pigeons etc.) are brought together and kept for days resulted to AIV dissemination, reassortment and interspecies transmission. Coexistence of multiple subtypes of influenza viruses enhances the risk of viral reassortment and mutation of H3 subtype influenza viruses. However, so far, little was known about the evolution and pathogenicity of H3 AIVs circulating in domestic birds in China.In this study, oropharyngeal swabs were collected from fowls at live poultry markets in Shanghai, Zhejiang, Jiangsu, Guangdong and Hebei during 2009 to 2011. Hereinto, seven H3 AIVs were isolated from those swabs, and named A/duck/Shanghai/120-1/2009(H3N8)(120-1/H3N8), A/duck/Nanjing/A1591-1/2010(H3N8)(A1591-1/H3N8), A/chicken/Nanjing/B854-2/2011(H3N8)(B854-2/H3N8), A/duck/Hebei/B1645-2/2011(H3N2)(B1645-2/H3N2), A/duck/Hebei/B1646-2/2011(H3N2)(B1646-2/H3N2), A/duck/Hebei/B1647-1/2011(H3N2)(B1647-1/H3N2) and A/duck/Shanghai/74-1/2009(H3N2)(74-1/H3N2). For the further phylogenetic and molecular characteristic analysis of the virus isolates, the total RNAs of each virus isolate were extracted, with which Two-step RT-PCR was employed to amplify the viral gene segments. The eight segments of each virus were amplified respectively using the universal primers for influenza A virus and the purified PCR products were sequenced. Phylogenetic trees of all eight segments were generated by applying the neighbor-joining method using MEGA6.0. All the NA genes were clustered into Eurasian lineage, except for two N8 genes of B854-2/H3N8 and A1591-1/H3N8 fell into the North American lineage. Intriguingly, the N8 gene of 120-1/H3N8 and the PB2, PB1, NP and NS of 1646-2/H3N2 and B1645-2/H3N2 were closely related with that of highly pathogenic H5N8 viruses circulating in Korea and United State. Seven H3 isolates shared the same amino acid sequence(PEKQTR/GLF) at the cleavage site between HA1 and HA2, indicating they are low pathogenic AIVs. None of these viruses held any amino acid residues reported to be involved in the recognition of human-type receptors, suggesting that all the isolates prefer avian like receptor(α-2, 3- sialic acid). E/D at position 627/701 of the polymerase PB2 protein of the seven H3 isolates suggested that these isolates were short of efficient adaptation in mammals. According to sequences of NA protein, all isolates presented susceptibility to neuraminidase inhibitors. Two isolates, B1645-2/H3N2 and B1646-2/H3N2 possessed an S31 N mutation in the M2(viral ion channel) that confers resistance to the ion channel blockers.To analyze virus growth kinetics on Madin-Darby Canine Kidney Cells(MDCK), Human lung adenocarcinoma epithelial cells(A549) and DF-1 cells, confluent MDCK, A549 and DF-1 cells were infected with each virus at a multiplicity of infection(MOI) of 0.01. The supernatants from infected cells were collected at different time points(12, 24, 36 and 48 hours post infection) and viral titers were determined in embryonated chicken eggs. Growth kinetic results showed that all H3 viruses replicated efficiently in MDCK cells, and replicated to different titers in A549 and DF-1 cell. Notably, B1646-2/H3N2 and B1647-1/H3N2 replicated more efficiently than other H3N2 viruses at 48 and 72 hours post infection in A549 cells. In DF-1 cell, all viruses replicated to lower titers compared with those in MDCK cells.To determine the pathogenicity of the H3 isolates in mice and chicken,groups of 6-week-old female BALB/c mice were anesthetized and inoculated intranasally(i.n.) with 106 EID50 of each virus. Body weights from 0 to 14 days post inoculation(dpi.) were monitored; and viruses in different tissue samples were tested and titrated. No clinical sign was detected in both group, and only slight weight losses in mice were caused by those H3 isolates. The virus titration results showed that all the H3 isolates were replicable in mouse lungs and nasal turbinates without prior adaptation. Based on histopathological examination, all H3 viruses caused mild bronchointerstitial pneumonia, characterized by infiltration of the alveolar lumen with neutrophils and by damage of the alveolar epithelium. Besides, B1646-2/H3N2, 854-2/H3N8 and 120-1/H3N8 were selected for further determination of the pathogenicity according to the phylogenetic analysis. Groups of 6-week-old SPF chickens were inoculated intranasally(i.n.) with 106 EID50 of each virus. Oropharynx and cloaca swabs on 3, 5 and 7 dpi were collected; and viruses in different tissue samples 4 dpi were tested and titrated. No clinical sign was detected in both group, and the virus titration results showed that only B1646-2/H3N2 could replicable in chicken trachea, lung, pancreas, brain and duodenum and may transmit through oropharynx and cloaca.In conclusion, gene reassortment of H3 AIVs circulating in China occurred and resulted in the substitution of N8 genes from North American lineage for NA genes from Eurasian lineage genes in some isolates. All H3 isolates showed the low pathogenic molecular characteristics in birds or mammals. However, it should not be ignored their risk in human health for all of those H3 isolates were replicable in mouse lungs and nasal turbinates without prior adaptation. |
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