Genetic Evolution And Pathogenicity Of H7N9 Subtype Avian Influenza Virus In South China In 2017

In March 2013,the first confirmed case of H7N9 influenza virus was found in Anhui province,China.By January 25,2018,1566 laboratory-confirmed H7N9 cases had been reported,including 613 deaths over twenty provinces of China.The H7N9 not only caused loss of the poultry industry,but also threatened human public health.In 2017,41 H7N9avian influenza viruses?AIVs?were isolated from Guangdong in South China.The molecular characteristics,genetic evolution and pathogenicity of the viruses were analyzed in our study.In this study,the cleavage site in HA protein of the 41 H7N9 AIVs was PKGKRTAR?G or PKRKRTAR?G,which was a characteristic of highly pathogenic avian influenza virus.All viruses had 5 amino acids deleted in the neck of NA protein.In our study,no substitutions in the NA protein were related to drug resistance.All viruses were E at 627 sites of PB2 protein,which was the characteristic of AIVs from birds.All viruses had mutations at the 31 sites of M2 protein?S31N?,which was related to amantadine resistance.Molecular evolution analysis of the 41 H7N9 AIVs found that viruses were divided into three lineages,including Yangtze River Delta Lineage A,Yangtze River Delta Lineage B and Pearl River Delta lineage.The HA and NA genes of 40 viruses were derived from the H7 and N9 AIV,respectively,while the other six internal genes were derived from H9N2AIV.So,the 40 viruses were double?H7 and H9 subtypes?reassortants virus.The M gene of Q1 virus was derived from H5 AIV,while the other seven internal genes were derived from H7 and H9 AIV.So,the Q1 virus was a triple?H5,H7 and H9 subtypes?reassortants virus.Therefore,the 41 H7N9 AIVs were recombined from different subtypes,and belonged to different genotypes,thus showed genetic diversity.In order to understand the pathogenicity and transmission of the H7N9 AIVs,chickens inoculated viruses with the dose of 0.1 mL 10⁸EID₅₀/0.1 mL.The results showed that the chickens inoculated by Q8 and Q9 viruses all died within 4 day post-infection?DPI?,and the viral titer of all organs were 5.5-7.58 log₁₀EID₅₀/0.1 mL at 3DPI.Chickens in the contact group of Q8 and Q9 viruses all died within 7DPI.The experimental results of chickens inoculated viruses with the dose of 0.1 mL 10⁶EID₅₀/0.1 mL showed that the chickens inoculated by the 10 viruses all died within 4DPI,and the titer of all tissues were6.58-9.25 log₁₀EID₅₀/0.1 mL at 2DPI,but the Q28 and Q43 viruses titer of all tissues were3.83-7.42 log₁₀EID₅₀/0.1 mL at 2DPI.Chickens in the contact group of the 9 viruses all died within 7DPI,but 2 chickens in the Q43 virus contact group were survival until 14DPI.Therefore,the H7N9 AIVs had different pathogenicity and transmissibility to chickens in South China in 2017.In order to understand the pathogenicity of the H7N9 AIVs in mice,BALB/c mice were intranasally?i.n.?inoculated viruses with the dose of 0.05mL 10⁶EID₅₀/0.05 mL.The results showed that all viruses could be detected in the lung at 3DPI and 5DPI,and the viral titer at 5DPI were obviously higher than that of at 3DPI(1-6.67 log₁₀EID₅₀/0.1 mL,1-5.92log₁₀EID₅₀/0.1 mL).However,the replication ability of viruses in all tissues was different.The titer of Q25,Q28 and Q52 viruses were highest in all tissues(1.08-6.67 log₁₀EID₅₀/0.1mL),but the Q43 titer virus were lowest in all tissues(1-1.58 log₁₀EID₅₀/0.1 mL).Therefore,the H7N9 AIVs had different pathogenicity to mice in South China in 2017.41 H7N9 AIVs were highly pathogenic avian influenza viruses in our study.All of them occurred internal-genotype recombination and presented genetic diversity.Most viruses had high pathogenicity and transmissibility in chickens,and some viruses had high pathogenicity in mice.Therefore,our study confirmed that the H7N9 AIVs still threatened the poultry health,and had a risk of transmission among mammals.