Analysis Of Genetic Evolution Of H9N2 Subtype Avian Influenza Virus Strain And Preparation Of HA And NA Gene Nucleic Acid Standard

Avian influenza(AI)is an infectious disease caused by avian influenza virus(AIV),which results in a variety of symptoms from mild respiratory symptoms to severe systemic septicemia.Because of the difference of pathogenicity,avian influenza can be divided into high pathogenicity(HPAI)and low pathogenicity(LPAI).The pathogenicity lead to different symptoms of poultry death.According to the difference of hemagglutinin(HA)and neuraminidase(NA)on the surface of virus,avian influenza virus can be classified into various subtypes,including 18 HA subtypes and 11 NA subtypes.Avian influenza virus infects not only birds such as chicken,quail,duck and wild fowl,but also mammals such as pigs,horses and humans.Because of the low morbidity rate of H9N2 subtype avian influenza virus,the symptoms of avian influenza virus are not obvious or no symptom was observed after infection,which results in H9N2 epidemic in a large area of poultry and brings great economic losses to the breeding industry.Therefore,the prevention and control of H9N2 subtype avian influenza virus is very important.In this study,H9N2 subtype avian influenza virus gene sequences were obtained from GenBank,and HA and NA gene phylogenetic trees were drawn to understand the epidemic situation of H9N2 subtype avian influenza virus.The HA and NA genes of 18 strains of H9N2subtype avian influenza virus isolated strains and 2 strains of reference strains in Jilin area were amplified and sequenced.The evolutionary analysis of HA and NA gene was carried out to grasp the epidemic trend of H9N2 subtype avian influenza virus isolates in Jilin area in China,and to analyze the relationship between kinship and evolution of isolated strains.Specific primers and probes were designed for 5 typical strains of H9N2 subtype virus HA gene isolated in China,and fluorescent PCR detection methods were established respectively.Then,the HA and NA genes of H9N2 subtype influenza virus were obtained by in vitro transcription to obtain HA-cRNA and NA-cRNA,and cRNA was used as a template to prepare standard for detection of HA gene and NA gene of H9N2 subtype avian influenza virus,providing a reference material for the detection of H9N2 subtype avian influenza virus.1.HA and NA gene sequences of H9N2 subtype avian influenza virus were obtained from GenBank,including 1619 HA gene and 1104 NA gene.According to the information of time,region and host,the evolutionary trees were drawn.The results showed that influenza viruses in China were divided into north American and Eurasian lineages,HA gene into five branches,and NA gene into four branches.Each branch of HA and NA gene covered most regions of China,and there was no obvious difference in geographical distribution among the branches.The virus strains were mostly from Guangdong,Shandong and some coastal areas,and the host was mainly chicken.The properties of HA and NA gene were amplified and sequenced in 18 strains of H9N2 subtype influenza virus and 2 reference strains in Jilin area,the evolution analysis of HA and NA gene was carried out,and the results show that all isolates are distributed from the first to third branch of HA gene and the first to second branch of NA gene.The 5-branch of HA gene belongs to the North American lineage,which represents the strain with TY66 and has fewer strains of 11 strains,and the isolates mostly come from wild birds and waterfowl.All the other strains belong to Eurasia lineage,among which only 6 strains belong to the fourth branch.The representative strain is HKG1,most of which are from coastal areas and waterfowls.Eighty seven strains of BJ94 were found earlier in the third branch.The isolates were closely related to the representative strain BJ94.There were 1515 strains in the first and second branches,and most of the isolates were concentrated in these branches.The fourth branch of NA gene is the north American lineage represented by TY66,there are eight strains in this lineage and the other three are Eurasian lineages.HKG1 is the representative strain of the third branch.There are only6 strains of this branch virus,and the majority of the strains are waterfowls.There are 213strains in the second branch,and the time of isolation is earlier.There are 887 strains in the first branch,and most of the isolates in recent years are concentrated in this branch.2.In order to distinguish the strains isolated from different branches,a specific fluor escent PCR method was established.In this study,a pair of specific primers and probes were designed according to the HA gene sequences of representative strains of H9N2 su btype influenza viruses from 5 branches,the standard curve of fluorescent PCR was con structed by using standard positive plasmid as template,and the detection method of flu orescent PCR was established.The results showed that the diluted plasmids could be amp lified to 10~2 copies/?L and 10~1 copies/?L by fluorescent PCR,but the other strains were not amplified,which proved that the method had good sensitivity and specificity.The s tability experiment indicated that the recombinant plasmid 10~8 copies/?L~10~6 copies/?L c ould beamplifiedthree times and showed a good gradient curve with stability.3.In order to improve the accuracy of detection of H9N2 subtype influenza virus,the HA and NA genes of the two tested strains were amplified and the recombinant plasmids containing T7 promoter were constructed.After sequencing,a large number of HA-cRNA and NA-cRNA fragments could be rapidly obtained by in vitro transcription.The measured product concentrations were 346 ng/?L and 301 ng/?L,respectively.After successive dilution,the products were used as templates for Real-time RT-PCR detection,and the standard curves were drawn.The results showed that the measured values were basically in a straight line,and the amplification curves were good,which could be used as reference materials for nucleic acid detection.Then the homogeneity,stability and uncertainty of the certified reference material were analyzed.The results show that the uniformity of the certified reference material meets the requirements of the certified reference material,and the test group was stable during the stability monitoring period.In order to reduce the possibility of H9N2 subtype avian influenza outbreak in the world,we should grasp the epidemic trend of H9N2 subtype avian influenza in our cou ntry in time,reform monitoring and preventive measures,and guarantee the healthy bree ding and food safety.