Molecular Epidemiological Investigation And Cross-protection Study Of Avian Reovirus In East China

Avian reovirus（ARV）can cause various diseases in poultry.In recent years,there is an increasing trend of ARV infection in Chinese chickens.ARV is one of the most important avian viruses causing clinical diseases in poultry and resulting in severe economic loss.Some novel ARV strains were isolated from vaccinated broiler flocks and caused the difficulty of ARV prevention and control.In order to understand the epidemic status of ARV in China in recent years and the molecular genetic evolution,pathogenicity,and vaccine protection of epidemic strains,this study collected suspected ARV samples from East China from January 2019 to September2022 to study the epidemiology,genomic genetic evolution,pathogenicity,and vaccine protection of ARV.The specific content is as follows:1.Epidemiological survey in East China from January 2019 to September 20222076 tissue samples of suspected ARV clinical symptoms and 2631 serum samples from 256 chicken farms in Shandong,Fujian,Jiangsu,Zhejiang,and Anhui provinces from January 2019 to September 2022 were collected for testing.The RT-PCR test results showed that 277 samples from 69 chicken farms were ARV nucleic acid positive（with a positive rate of 13.34%）,with infection mainly concentrated at the age of 2-6 weeks（accounting for 84.8%）.The susceptible species were mainly white feather broilers,followed by broiler breeders.The ELISA antibody test results of serum samples showed that 2160 samples from 59 chicken farms were positive for ARV antibodies（with a positive rate of 82.10%）.According to annual statistics,the positive rates of ARV pathogens in collected samples from 2019 to 2022were 5.4%,11.2%,20.1%,and 12.8%,respectively;The positive rates of antibodies were 84.73%,78.28%,87.5%,and 70.23%,respectively.277 ARV nucleic acid positive samples were further isolated using LMH cell virus,and a total of 90 ARV virus strains were obtained.Combining the genetic evolutionary characteristics ofσC gene and the serological typing pattern of ARV virus,the classical 6 clusters（genotypes）of ARV（1,2,3,4,5 and 6）were further subdivided into 11 smaller branches（genotypes）,including 1.1,1.2,1.3,2.1,2.2,3,4.1,4.2,4.3,5 and 6.According to the new classification criteria,the 90 strains isolated in this study belonged to 8 genotypes,including 1.2,1.3,2.1,2.2,3,4.2,4.3 and 5,the 1.3,2.2,4.3 and 5 genotypes were the main prevalent genotypes in East China.2.Whole-genome sequencing analysis of different genotypes of ARVThe subcluster of 1.3,2.2,4.3 and 5,which are the most prevalent strains in East China,were screened for whole-genome sequencing,including 20191774,201910415,20206919 and 20209560.The results of gene sequence comparison showed that all the 10 genomic fragments of the four strains were from ARV,but they were different from the ARV strains in Gen Bank,and all of them had different degrees of fragment recombination or nucleotide site mutation,among which the strain 201910415 had intra-fragment recombination in the L2 gene.3.Establishment of ARV Taq Man fluorescence quantitative RT-PCR assayBy comparing the ARV sequence information in Gen Bank and the ARV sequence information of different genotypes in this study,we designed primers and probes in the conserved region of M3 gene,prepared standards,and established a Taq Man fluorescence quantitative RT-PCR assay to detect ARV of all genotypes.The results showed that the linearity of the standard curve of the method was good,with the correlation coefficient R² reaching 0.999 and the amplification efficiency 108.196%;the sensitivity was high,and the minimum viral template concentration was 1.0×10¹copies/μL,which was 100 times higher than that of common RT-PCR;the specificity was strong,and no cross-reactivity occurred with other common avian diseases;the reproducibility was good,and the coefficient of variation of intra-group replicates was less than 1%;the detection range was wide.The detection range is wide,and it can detect the S1133 vaccine strain and all genotypes of strains currently prevalent in East China.The method established in this study can be widely applied to the qualitative and quantitative detection of ARV in samples from various provinces in China.4.Establishment of animal infection modelsIn this study,the ELD₅₀ of four popular representative strains,20191774,201910415,20206919 and 20209560,was determined.20206919,which had the lowest ELD₅₀ toxicity,was selected as the challenge strain,and the attacking test was conducted on 1-day-old SPF chickens and 5-day-old white broiler chickens by foot pad inoculation,oral administration and oral cohabitation.The results showed that SPF chickens could cause foot pad swelling and could be used as anchallenge model.SPF chickens infected by oral and oral cohabitation did not show obvious clinical signs,but antibodies turned positive and broiler chickens attacked by different inoculation methods all showed typical clinical signs and could be used as a pathogenicity model.5.Study on the pathogenicity of different genotypes of ARV in broiler chickensIn this study,the pathogenicity of 20191774,201910415,20206919 and20209560 was studied in broiler chickens.The results showed that all four strains caused clinically similar symptoms such as swollen foot pads and joints in white broiler chickens,but no mortality was observed.Four prevalent strains of ARV caused symptoms such as viral arthritis and growth inhibition in broiler chickens,among which 20191774（cluster1.3）had the most severe effect on body weight and caused the most severe damage to foot pad tendons,and had the highest viral load and tissue viral load,and was presumed to be the most pathogenic.The next most pathogenic strain was 201910415（cluster2.2）,while 20209560（cluster5）and 20206919（cluster4.3）were relatively less pathogenic.6.Protective study of two commercial vaccines against different genotypes of ARVTo verify whether the current commercial vaccines can protect against infection with the currently prevalent ARV strains,two ARV vaccines commonly used in the market were selected for immunization of SPF chickens in this study,using the currently prevalent representative strains 20191774（cluster1.3）,201910415（cluster2.2）,20206919（cluster4.3）and 20209560（cluster5）for challenge.Vaccine A was a domestic polyvalent vaccine and vaccine B was an imported vaccine.The results showed that the protection effect of immunization vaccine A was good to 100%for cluster 1.3,4.3 and 5 representatives,while the protection rate was only 20%for 2.2branch representatives;the protection rate of immunization vaccine B was 0%for cluster 1.3,2.2,4.3 and 5 representatives.7.Experimental Study on the Protective Effects of Preparing ARV Candidate Vaccines in the Laboratory on Different Genotypes of Virus StrainsIn this study,the virulent and prevalent 1.3,2.2 and 5 branch representative strains 20191774,201910415 and 20209560 from 90 prevalent strains were selected as vaccine candidates.These three strains were proliferated by LMH cells to prepare an inactivated vaccine in oil emulsion in the ratio of 1:1:1.The prepared inactivated vaccine was immunized against SPF chickens and attacked using the representative strains 20191774,201910415,20206919 and 20209560 from branches 1.3,2.2,4.3and 5.The results showed that SPF chickens immunized with inactivated polyvalent vaccine did not develop disease,and the immune protection rate was 100%,and the candidate vaccine had good protection effect against different genotypic strains.