The Isolation And Identification Of Avian Reovirus Variant Strain And Construction Of Infectious Clone For Avian Reovirus

Avian viral arthritis is an infectious disease of poultry caused by avian reovirus(ARV).It can affect chickens at different ages with different varieties,especially commercial broilers.The disease mainly causes swelling or paralysis of tarsal joint,decrease of drinking water and taking food or even death.In recent years,the incidence rate of Avian viral arthritis has been increasing,which seriously endangers the healthy development of poultry industry in China.In this study,an ARV strain named SDYT was isolated from chicken tendons suspected of ARV infection in Yantai,Shandong Province.The whole genome sequence and pathogenicity of the SDYT strain were analyzed to provide theoretical basis for the prevention and control of ARV.At the same time,the infectious clone of ARV was constructed based on LY383 strain isolated in the laboratory,which provided a theoretical basis for the further study of ARV and a technology platform for the pathogenic mechanism,transmission mechanism and immune control of ARV at the molecular level,which provided a theoretical basis for the further study of ARV,the main research contents are as follows:1.In this study,the virus was isolated from chicken tendons suspected of ARV infection in Yantai,Shandong Province.After inoculating chicken embryo hepatocytes with the disease materials,the syncytial cytopathy appeared.RT-PCR detection and ARV S1 gene sequencing results showed that the virus was ARV which artificially named SDYT,and the whole genome sequence was analyzed.The result of homology analysis showed that the nucleotide homology between SDYT strain and the traditional standard strains S1133 and 1733 was low,54.4%?93.2%and 54.7%?93.1%respectively,and the amino acid homology was 49.2%?99.0%and 49.8%?99.0%respectively;the homology analysis of nucleotide and amino acid between the SDYT strain and other reference strains shows that the S1 and S2 genes have the highest homology with the 17203-M-06 strain,68.9%?93.4%and 70.9%?99.0%respectively;the S3,M3,L2 genes have the highest homology with LY383 strain,89.9%?97.8%and 96.5%?99.5%respectively;the S4 gene has the highest homology with878-Bi-05 strain,92.3%and 99.2%respectively;the M1 and L1 genes have the highest homology with the 2408 strain,90.7%?93.2%and 98.6%?99.2%respectively;the M2 gene has the highest homology with the D1104 strain,93.0%and 97.9%respectively;the L3 gene has the highest homology with 138 strains,90.5%and 96.8%,respectively.The results of genetic evolution analysis showed that SDYT strain was closely related to chicken reovirus;according to the genetic evolution analysis of?C gene,the SDYT strain belonged to genotype4 ARV,while traditional standard strains S1133 and 1733 belonged to genotype 1 ARV;according to the genetic evolution analysis of other genes,SDYT strain and traditional standard strains S1133 and 1733 were in different branches except the genes of?B,M1,L1and L2.In conclusion,the SDYT strain is an ARV variant strain derived from different chicken ARV.2.120 1-day-old healthy Ross 308 broilers were randomly divided into four groups,30in each group,group A was intravenous infection group,group B was footpad infection group,group C was oral infection group,and group D was control group.Each chicken of infection group was inoculated with 0.4m L(TCID₅₀was 10^-5.401/0.1 m L)of virus solution,and each chicken in the control group was inoculated with 0.4m L of normal saline,and the clinical symptoms were observed daily.Three animals were randomly selected from each group on 3,6,9,12,15,18 and 21 days post-inoculation.After weighing,the cloacal swabs,anticoagulant and serum were collected.After the section,the tissues and organs were collected and the samples were tested.The clinical symptoms and the results of section showed that the chickens in each infection group had swelling of joints and inability to lie on the ground,the blood stasis was found under tarsal joint,yellow colloidal mucus in tendon sheath,duodenal congestion,spleen enlargement,kidney enlargement and thymus congestion,but the symptoms of group A and group B were the most serious;The weight growth cruve showed that the weight of each group increased slowly,and group B was the most obvious.The results of antibody test showed that the antibody level of each infection group was slightly higher than that of the control group;The results of cloacal detoxification test showed that the amount of virus excretion in each attack group was higher in the early stage,and then decreased;The results of virus test showed that SDYT strain had viremia,and the virus could be detected in blood at 3 dpi,and then reached the peak at 6 dpi?9 dpi,finally began to decline at 15 dpi;The results of virus loads test showed that the virus was detected in every organ of the attack group,the virus loads in tendon were higher,and the viral loads of bursa farnesis and trachea were lower.The above experiments showed that the artificial infection of SDYT strain could cause viral arthritis,growth inhibition and viral disease in broilers.3.Full length c DNA amplification and homologous recombination were used to carry out reverse genetic operation on LY383 strain.Ten fragments of the whole genome of LY383strain were amplified by high fidelity enzyme,and M1 fragment was mutated as molecular tag.The p Blue Script II SK(+)vector(p BSK vector)was modified to carry T7 promoter and T7 terminator.The target fragment was connected to the promoter and terminator of the vector by homologous recombination.Ten p BSK recombinant plasmids were cotransfected into BSR-T7 cells according to different concentrations after extraction.After 72 hours of culture,the cells were collected and frozen for three times.After centrifugation,the supernatant was aseptically inoculated into SPF chicken embryos for 3 generations blind.The allantoic fluid of chicken embryo was collected for identification.The results of RT-PCR showed that the S1 gene of rescue virus could still be detected after 3 generations of blind transmission.The results of molecular tag detection showed that the molecular tag had been successfully introduced.After 3 generations of inoculation,the yolk rupture and embryo congestion were observed in both rescue virus and parent virus embryos.In this study,10recombinant plasmids of the whole genome of chicken reovirus strain LY383 were successfully cloned.The optimal ratio of 10 plasmids for cotransfection was optimized,and the infectious clone of ARV was constructed,which provided a theoretical basis for further research on the pathogenic mechanism and immune control of ARV.