Isolation And Identification Of Novel Duck Orthoreovirus

In recent years,Many meat duck farms in China have been infected with an infectious disease characterized by swollen tarsal joints,unstable standing and blocked growth and development.The main clinical features are swelling of tarsal joints,unstable standing of affected ducks and obstruction of growth and development.Autopsy revealed suppurative bleeding at the swollen joint and tendon rupture;Liver swelling,bleeding,necrosis and irregular necrosis of spleen.It is initially suspected that it is caused by the novel duck orthoorthoreovirus(N-DRV),but the clinical manifestations of the disease are not exactly the same as the "spleen necrosis" disease of Peking duck.In this study,pathogens were isolated from the joints,liver and spleen of diseased ducks sent for examination in a meat duck farm in Linyi,Shandong Province.In this study,the pathogen was isolated from the diseased duck joints,liver and spleen of a meat duck farm in Linyi,Shandong Province.After virus proliferation,sequencing and whole genome sequencing,a strain of N-DRV was isolated and named LY20,The mortality,clinical manifestation and detoxification law of the strain were understood through regression animal experiment,which provided a theoretical basis for the prevention and control of N-DRV.In this study,the virus was isolated from the tendon and viscera of duck suspected of NDRV infection by chicken embryo hepatocytes.After virus infection,the cells showed lesions characterized by cell fusion,which was consistent with the pathological characteristics of NDRV.The cytotoxicity was detected by RT-PCR ?C sequencing showed that the nucleotide homology between the virus and N-DRV reference strain XT18 strain was 98.9%,named LY20 strain,and analyzed its whole genome sequence.The results showed that the whole genome sequence of LY20 strain was 23419 bp,including 10 segments.Homology analysis showed that each segment of LY20 strain had the highest homology with N-DRV.The results of genetic evolution analysis showed that each genomic segment of LY20 strain was in the NDRV branch.In the genetic evolution analysis tree of L1,L2,L3,M1,M3,S2 and S4 genes,NDRV was closer to MDRV,and ARV was in a single large branch.The homology of L2,L3,M2,M3,S1 and S3 genes with N-DRV reference strain XT18 strain was the highest(nt:98.3%?99.6%;aa: 97?99.7%);M1 gene has the lowest homology among all segments and the highest homology with N-DRV reference strain HN5 d strain(nt: 97.5%;aa: 94.2%).The homology comparison of S1 gene showed that LY20 strain had low homology with MDRV and ARV,with ARV reference strain S1133(nt: 45.2%;aa: 11.3%),and lower homology with MDRV reference strain 815-12(nt: 33.4%;aa: 14.5%).The homology comparison results of?C gene showed that the homology with MDRV reference strain 815-12 was(nt: 49.6%;aa:42.2%),which was higher than that with ARV reference strain S1133(nt: 39.6%;aa:28.3%).The results of genetic evolution analysis of S1 gene showed that N-DRV,MDRV and ARV were in different branches,which were close to the genetic evolution of ARV and far from MDRV.In ?C gene genetic evolution tree,N-DRV is closely related to MDRV,but far from ARV.120 1-day-old healthy Cherry Valley ducks were randomly divided into four groups with30 ducks in each group.Group A was the foot pad Infection group,group B was the muscle Infection group,group C was the oral Infection group and group D was the control group.Each animal in the infection group was inoculated with 0.2 ml virus solution,and each animal in the control group was inoculated with 0.2 ml normal saline.The clinical symptoms were observed every day.Three ducks were randomly selected at 1 dpi,3 dpi,5 dpi,7 dpi,10 dpi,14 dpi,18 dpi and 22 dpi after infection.The cloacal cotton swabs were collected,blood was collected and serum was separated.Then,the tissues and organs such as liver,spleen,lung,kidney,bursa fabricius,thymus and tendon were dissected for virus load detection.The main clinical symptoms of meat ducks in the infection group were depression,slow growth and development,swelling of joints and severe paralysis.The symptoms in the foot pad infection group were the most obvious.The symptoms of autopsy were bleeding and necrosis of liver,severe necrosis of spleen,swelling of bursa of fabricius and bleeding of thymus,A few meat ducks in the virus Infection group had lung bleeding,necrosis,kidney swelling and bleeding.Deformation of the femoral head,subcutaneous bleeding,suppuration and congestion in the joint cavity,and brownish red mucus exudation.The symptoms in foot pad Infection group were the most serious,followed by group muscle Infection group and group oral Infection group.The weight test data showed that the weight growth of each drug Infection group was slow,and foot pad Infection group was the most significant.The detection results of tissue viral load showed that the virus could be detected in all collected tissues,and the viral load in spleen and bursa of fabricius was higher.The rule of blood detoxification showed that the virus could be detected in the blood one day after infection,with 3-5dpi reaching the peak and14 dpi reaching the second peak.The detoxification rule of cotton swabs showed that the detoxification amount of the virus Infection group was higher in the early stage of infection.The antibody level test results showed that the antibody levels of the virus Infection group were higher than those of the control group.The above experimental results showed that artificial infection with LY20 strain could cause viral arthritis,liver and spleen necrosis and growth inhibition in meat ducks.