Isolation,Identification And Pathogenicity Of A New Subtype Avian Influenza Virus H5N6 In Hubei Province

Avian Influenza Virus is a single-stranded negative RNA virus belonging to the Orthomyxoviridae family,which is a vital pathogen of poultry.Due to the instability of the viral surface antigen,It's prone to genetic mismatch during the replication process to cause structural changes.Different types of AIV can be classified into high pathogenicity and low pathogenicity according to the pathogenic characteristics of infected avians.H5 has been listed by the World Animal Safety Organization and the World Health Organization as an AIV that has a great threat to humans.In 2014,the first cases of H5N6 infection for human was reported in Sichuan province China,which followed reported that human infected by H5N6 in Guangzhou and Yunnan province.It's is a serious threat to human health because the high pathogenicity can cause acute clinical symptoms even death.The distribution of avian influenza varies from region to region.the AIV epidemic in Hubei has been monitored in recent years,indicating that the highly pathogenic H5N6 new subtype has a high prevalence and new gene rearrangements can occur in a short period of time.The virus type is complex and diverse.Especially in the live poultry market,On the one hand,increasing the possibility of viral mutation.On the other hand,it suggests the potential risk of infecting peopleas as a common place for human contact,where a variety of poultry gather.Therefore,we chose to conduct a survey in the live poultry market to further study the avian influenza virus mutation and its impact on pathogenicity.we conducted AIV monitoring of the live poultry market in Jiang Xia and Huang Pi districts from January to April 2018,collecting 445 anal swabs and fresh feaces from major poultry(chicken,duck).RT-PCR test found that 160 were positive for avian influenza virus,the positive rate was 35.96%,of which the mixed infection rate was 37.5%.The positive H5N1 type samples only accounts for 1.25%,while the H5N6 positive rate is 15%.Extracted from HA and NA sequences for molecular genetic evolution analysis in H5N6 samples.The comparison showed that the detected H5N6 and the H5N6detected in 2014-2016 had some base variations,which belonges to Clade 2.3.4.4,a highly pathogenic new subtype.This reflects that this highly pathogenic H5N6 new subtype has been prevalent in the live poultry market in Wuhan even has a genetic mismatch in recent years,circulated as an advantageous type.In order to avoid the occurrence of human infection H5N6 in Hubei.It is necessary to understand the pathogenicity and infective characteristics of this virus type in addition to grasping the epidemic trend of H5N6.Therefore,the laboratory isolated the highly pathogenic H5N6 subtype(J52)to analyze the high pathogenicity in vivo and vitro experiment.To analyze the high pathogenicity of J52 in human A549 cells and non-human MDCK cells and tissue tropism of the virus in BALB/c mice(including heart,liver,spleen,lung,kidney,brain,intestines)and cytokine changes.Combined with Hemagglut-ination Test and CPE experiment,it can be seen that the replication of MDCK cells and A549 cells is different after viral infection.The symptom of the BALB/c mice were observed after infection.Showed weight loss,chilling convulsions and death.The LD₅₀of J52 to mice was 10^-5.27.Viral nucleic acids can be detected in mouse heart,brain and lung tissues.The lung tissue was subjected to HE staining and the pathological sections were observed under the microscope.The results showed that the degree of lung injury was aggravated with the expansion of the virus in vivo.Pathological section of lung tissue showed that avian influenza virus can cause alveolar tissue damage,causing lymphocyte aggregation,erythrocyte leakage and other inflammatory reactions.The cytokines IL-6,IL-10,IL-1?,TNF-?,IFN-?,and IFN-?were increased in different degrees after infection,and the expression of IL-6was much higher than other cytokines.In summary,the H5N6 subtype is still prevalent as a predominant strain of Avian influenza virus in Wuhan.The virus is susceptible to MDCK cells and A549 cells,but the proliferation rate is different.The lethality of the virus on mice shows high pathogenicity.Our results indicate that we can grasp the variation trend of AIV H5N6type in Hubei,and understand the pathogenicity of H5N6,which has important practical significance for the prevention,taking care of and control of avian influenza in Hubei Province.