| The H9N2 subtype avian influenza virus is widespread in my country and has become an endemic disease.Bring huge economic losses to my country's poultry breeding industry.H9N2 AIV has been able to break through the species barrier,without the need for an intermediate host to directly infect people,and can further adapt in the human body,and may even genetically recombine with human influenza viruses,thereby posing hidden dangers to public health safety.H9N2 AIV has been able to break through the species barrier,without the need for an intermediate host to directly infect people,and can further adapt in the human body,and may even genetically recombine with human influenza viruses,thereby posing hidden dangers to public health safety.This article analyzes the genetic evolution and pathogenicity of H9N2 AIVs isolated and identified in Shandong Province from 2017 to2019,and provides a theoretical basis for further understanding of the evolution and variation of H9N2 AIV.1.Isolation and identification of H9N2 subtype avian influenza virusFrom March 2017 to April 2019,a total of 3299 samples were collected from 54 live poultry trading markets,farms and ornamental bird breeding bases in 10 regions of Shandong Province.After PCR experiment identification,it was finally determined that 69H9N2 AIVs were separated and identified.The separation and identification of H9N2 AIV in different locations were different.From 2486 samples in live poultry markets,57 H9N2 positive samples were separated and identified,and 580 samples in farms.10 H9N2 positive samples were isolated and identified in the middle,and 2 H9N2 positive samples were isolated and identified from the 233 samples collected in the ornamental bird breeding base.From the perspective of the isolation of different hosts,62,5 and 2 H9N2 strains were identified and isolated in chickens,pigeons and other ornamental birds,respectively.The isolation and identification rates were 2.77%,1.29%,and 0.86%,respectively.And no H9N2 AIV was isolated and identified on the goose host.The separation and identification results of different sample sources showed that the separation and identification rates in poultry feces,cotton swabs,clinical tissue samples and poultry drinking water were 1.71%,4.27%,5.93% and 7.41%,respectively.Different regions also have different separation rates.Since most of the samples collected in Weifang are clinical samples,the highest separation and identification rate is 13.33%.H9N2 AIV has not been separated and identified in Jinan and Dongying regions.2.Genetic evolution analysis of H9N2 AIVsAccording to different time,location and host,20 strains of H9N2 AIVs were selected and purified for whole genome sequence determination,and the sequencing sequence was compared and analyzed.The results showed that the homology of 20 isolates was PB2 95.31-99.87%,PB1 91.16-99.96%,PA 93.82-99.86%,HA 90.97-99.88%,NP 93.45-100%,NA 93-99.93%,M 89.1-100%,NS 96.66-100%.The 20 isolates formed 18 genotypes,among which the HA genes of all isolates belonged to the Y280 subline,NA,PB1,NP,PA,and NS belonged to the F98 subline,and the PB2,M genes belonged to the G1 subline.Analysis of the internal genetic molecular characteristics of 20 isolates showed that 20 isolates still retain low pathogenicity characteristics,but at the HA 226 locus that affects the virus transmission ability,all isolates have been mutated from Q to L,at the PB2627 position that determines the pathogenicity and transmission ability of the virus to mammals,the 627 amino acid position of 13 isolates is E,which still retains the molecular characteristics of low pathogenicity of avian isolates,while 7 isolates The 627 amino acid position is V.3.Pathogenicity analysis of H9N2 subtype avian influenza virusAfter genetic evolution analysis,five representative strains were screened,and the receptor binding characteristics of four representative H9N2 AIV strains showed that the ability of strains from different sources to bind human receptors was higher than that of avian receptors.ability.The pathogenicity analysis of 5 representative H9N2 AIV strains in mice showed that mice challenged with strain CK/2135/17 and strain CK/98/18 began to lose weight after the challenge.The body weight reached the minimum on the 7th day and the8 th day,decreased by 11.8% and 13.2% respectively.The body weight of the other three groups of mice did not change significantly after challenge,and none of the mice died.The five viruses have different replication abilities in mice,and they can replicate in the lungs and turbinates of mice.In the lungs of mice,the virus strain CK/98/18 has the strongest replication ability,with a virus titer of 106.7 EID50/ml;in the kidneys of mice,only the virus strain CK/98/18 was detected,but the virus replication was poor,the titer is low.Strains GP/1656/19 and CK/98/18 can replicate in the upper respiratory tract of chickens,and the virus can be detected in the trachea and lungs of chickens,and the virus titer in the trachea is higher than that in the lung tissue.Strains GP/1656/19 and CK/932/18 can be transmitted between chickens,and the virus was detected in tracheal and cloaca swabs.However,the transmission efficiency of GP/1656/19 is higher than that of CK/932/18.The H9N2 virus can induce chickens to rapidly produce high-titer specific antibodies.The serum HI test results are all positive for the H9N2 virus,and the HI antibody titer is between 128-2048.In this study,the epidemiological and genetic evolution analysis of the H9N2 subtype avian influenza virus isolated in Shandong Province from 2017 to 2019 revealed some of the biological characteristics of the virus and provided an important basis for the prevention and control of the H9N2 avian influenza virus. |
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