Isolation and characterization of H7N9 avian influenza A virus from humans with respiratory diseases in Zhejiang, China

•Two novel H7N9 avian influenza A virus were firstly isolated from the positive infection cases identified in Zhejiang Province, China last year.•Significant amino acid mutations were found in both two isolates.•Mean rate of evolution of HA gene was estimated to be 5.74E−3 (95%: 3.82E−3 to 7.79E−3) while the NA gene showed 2.243E−3 (4.38E−4 to 3.79E−3) s/s/y.•Phylogenetic results indicated that the novel H7N9 originated from the virus that infected birds.•Live poultry markets played an important role in whole H7N9 circulation. In 2013, the novel reassortant avian-origin influenza A (H7N9) virus was reported in China. Through enhanced surveillance, infection by the H7N9 virus in humans was first identified in Zhejiang Province. Real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) was used to confirm the infection. Embryonated chicken eggs were used for virus isolation from pharyngeal swabs taken from infected human patients. The H7N9 isolates were first identified by the hemagglutination test and electron microscopy, then used for whole genome sequencing. Bioinformatics software was used to construct the phylogenetic tree and for computing the mean rate of evolution of the HA gene in H7Nx and NA in HxN9. Two novel H7N9 avian influenza A viruses (A/Zhejiang/1/2013 and A/Zhejiang/2/2013) were isolated from the positive infection cases. Substitutions were found in both Zhejiang isolates and were identified as human-type viruses. All phylogenetic results indicated that the novel reassortant in H7N9 originated in viruses that infected birds. The sequencing and phylogenetic analysis of the whole genome revealed the mean rate of evolution of the HA gene in H7NX to be 5.74E−3 (95% Highest posterior density: 3.8218E−3 to 7.7873E−3) while the NA gene showed 2.243E−3 (4.378E−4 to 3.79E−3) substitutions per nucleotide site per year. The novel reassortant H7N9 virus was confirmed by molecular methods to have originated in poultry, with the mutations occurring during the spread of the H7N9 virus infection. Live poultry markets played an important role in whole H7N9 circulation.