Isolation And Identification And Biological Characteristics Of Two Low Pathogenic Avian Influenza Viruses

Avian influenza(AI)is a highly contagious disease caused by Avian influenza Virus(AIV).At present,there are 18 subtypes(H1-H18)of HA,11 subtypes of NA(N1-N11),and the combinations of HA and NA subtypes form different subtypes of avian influenza viruses.According to the ability to cause disease,AI is divided into three kinds,the Highly Pathogenic Avian Influenza(Highly Pathogenic Avian state-run,HP-AI),Mildly Pathogenic Avian Influenza(Mildly Pathogenic Avian state-run,MP-AI)and lowly Pathogenic Avian Influenza(Lowly Pathogenic Avian state-run,LP-AI).Since the first case of H9N2 avian influenza has been reported in 1994,it occurs in all parts of China.The H9N2 strain does not show obvious clinical symptoms,and it can expel of toxin constantly after infecting the host,it is difficult to prevent and control of avian influenza.This experiment provides a theoretical basis for the prevention and control of bird flu virus.1.Isolation and identification of Avian influenza virus.This experiment collected the pathological tissue from the large chicken farms in Jingmen and Daye,Hubei province.Currently,it is used to proliferate the virus by injecting chicken embryo.After infecting to the allantoic cavities of SPF chicken embryo which are 9-10 days age,collecting allantoic fluid contain virus and carrying out a series of experiment,such as blood clotting test,blood clotting inhibition test,and screening to the two strains of avian influenza virus subsquently.According to subtype identification,two H9N2 subtypes of Avian influenza virus were obtained and named A/chicken/Daye DY0602/2017(for short DY0602)and A/chicken/Jinmen/JM0305/2017(for short JM0305)by means of influenza virus named standard system respectively.The two strains H9 subtype of Avian influenza virus were conducted to blood clot characteristics text,EID50(chicken embryos half infection),MDT(chicken embryos minimal lethal dose of the average death time),ICPI(brain disease index)and so on,which are correlation tests to study the biological characteristics.The biological characteristics of two H9 subtype avian influenza viruses,DY0602 and JM0305,correspond to the basic characteristics of avian influenza.The EID50 is 10-8.42 EID50/0.1 ml,10-7.42 eid50/0.1 ml respectively.The MDT value is 72 h and 91 h respectively.The ICPI values are all 0.2.Whole-genome sequencing and analysis of avian influenza virus.In order to further understand the molecular epidemic characteristics of H9N2 subtype of two avian influenza virus strains isolated from diseased chickens in a poultry farms,the whole genome sequencing was carried on firstly.After obtaining sequences,they were analyzed,and uploaded to NCBI,obtained registration number.Followed by comparison and analysis the homologous relationships of two strains of subtype H9N2 Avian influenza virus strains with representatives in all branches,which have been uploaded in NCBI,two kinds of amino acid protein loci of cracking of HA and NA,receptor binding sites and the system evolution.The gene open reading frames of two strains of PB2,PB1,PA,HA,NP,NA,M,NS are composed of 2280,2274,2151,1683,1497,1401,759,366 bases,which are encoding 759,757,716,560,498,466,252,97,121 amino acid residues respectively.The typical features of these two viral HA lysis sites with low pathogenic avian influenza viruses are RSSR/GLF.These two H9 subtypes of avian influenza viruses are all in accordance with the CK-BJ-like sub-branch of China's Eurasian branch,which is in line with the strains isolated from China.3.The study of pathogenicity of avian influenza virus in quail.To further study about low pathogenicity avian influeTo further study about low pathogenicity avian influenza virus' s the biological characteristics of H9N2 subtype,we infected two strains of H9N2 subtype avian influenza virus to 4 weeks quails.After 24 h,sending the cohabitation quail,testing its clinical symptoms,detoxification,serum antibody and Yang,and viscera histologic changes.A nd then observing the influences of the H9N2 subtype avian influenza virus to pathogenicity of quail and the ability to contact transmission.Results show that the two strains of H9 subtype of avian influenza virus can through the larynx and cloacal detoxification after hosting.Although they can not cause obvious clinical symptoms and death,internal organs have different degree of pathological changes in pathological analysis.Serum antibodies can be detected at the 7d and 14 d after the infection.It shows that two H9N2 subtype avian influenza viruses have good horizontal propagation ability in quail flocks.In recent years,due to changes in the human environment,the virus in the natural world has also been adapted to changes in the environment through its own variation.The continuous mutation of the bird flu virus has also brought great difficulties to the prevention and control ofbird flu in China.In this study,the isolation and identification of avian influenza virus epidemicstrains and the study of some biological characteristics were conducted in order to understandthe prevalence of AIV and the characteristics of strains in Hubei and its neighboring provincesand cities.At the same time,the whole genome sequencing and analysis of chicken strainsJM0305 strain and DY0602 strain were completed in this study.The pathogenic characteristicsof chicken-derived AIV were initially understood and the effects of the strains on other specieswere explored.In addition,the pathogenicity test of the two strains on earthworms also provideda certain theoretical basis for the prevention and control of the disease.