Isolation And Identification Of A New Strain Of Its Pathogenicity

Novel duck reovirus(NDRV)disease is a group of immunosuppressive diseases caused by NDRV and is characterised by haemorrhage or necrosis on the surface of the liver and spleen organs of ducks.Since 2017,outbreaks of the disease have occurred in meat duck breeding areas in Shandong,Henan and Jiangsu.The affected ducks are depressed,with poor herd immunity and large areas of haemorrhage and necrosis on the surface of the liver and spleen visible on autopsy.Due to the reduced level of immune expression,infected ducks are susceptible to secondary bacterial infections or co-infection with other pathogens,resulting in increased mortality,reduced slaughter rate and reduced egg production,which has had a huge economic impact on the meat and breeding duck industry in China.In this study,a strain of NDRV was isolated from naturally infected meat ducks.A SYBR Green Ⅰ fluorescent quantitative RT-PCR assay was developed for the σC gene sequence of this strain,and the method was applied to animal infection tests to investigate the distribution of viral load in various organs at different infection periods.In addition to the determination of virus infection,the cytokine expression trends of liver and spleen organs of infected ducks at different infection periods were determined by relative quantitative q RT-PCR,and the expression patterns of various cytokines in vivo after virus infection were summarized and analyzed to provide a theoretical basis for the immune control of the Novel duck reovirus.(1)In this study,the virus was isolated from meat duck tissues with obvious spleen necrosis in Linyi,Shandong Province,and the pure virus suspension caused the death of the duck embryos after inoculation,RT-PCR results identified the strain as NDRV and the isolate was named strain 2022 LY.The isolates were sequenced and compared with Avian reovirus(ARV)and Muscovy duck reovirus(MDRV).The isolate σC was sequenced and compared with Avian reovirus(ARV)and Muscovy duck reovirus(MDRV)for homology and genetic evolutionary analysis.The homology results showed that the sequence of σC gene of 2022 LY isolate had low homology with the sequence of σC gene of ARV,and the sequence similarity was only 38.2%-40.7%;The sequence homology with the σC gene of MDRV was50.7%-51.5%;the highest homology was with NDRV,with 93.4%-96.5% sequence similarity.The genetic evolutionary results show that the 2022 LY isolate is in the same evolutionary lineage as the NDRV strain and that it is relatively distant from the σC genes of ARV and MDRV.(2)A pair of specific amplification primers was designed based on the NDRV S1 gene with an amplification length of 156 bp.The amplified target fragment was ligated into the p MD18-T vector,sequenced by constructing a recombinant plasmid and extracting a plasmid standard.The plasmid standards were diluted in a 10-fold concentration gradient,and the reaction conditions were optimised by different concentrations of plasmid templates and a fluorescence quantification standard curve was constructed to finally establish the NDRV SYBR Green Ⅰ fluorescence quantification RT-PCR method.Statistical tests and multiple sample analysis of the method showed that it was reproducible,sensitive,specific and met the quantitative requirements for subsequent animal testing.(3)Pathogenicity studies and analysis of NDRV 2022 LY isolates in susceptible animals.The test group was inoculated with 0.5 m L(TCID50 of 10-5.75/0.1 m L)of virus solution by intramuscular injection and the control group was inoculated with 0.5 m L of sterile saline in the same way,and three ducks were randomly selected from both groups for dissection at days 1,2,3,5,7 and 14 after infection.Mortality of infected ducks was counted during the test period,and pathological histological sections were collected from severely lesioned tissues,and the viral load of infected ducks was determined using the established SYBR Green I fluorescent quantitative RT-PCR method.The levels of IL-2,IL-6,IL-8,IFN-α,IFN-β,TNF-α,MX,OAS and PKR in liver and spleen tissues were measured and analysed on days 1,2,3,5,7 and 14 after NDRV infection,using the GAPDH gene as an internal reference and a relative q RT-PCR method.The results of the animal tests showed that the mortality rate in the attack group reached 36.7%,with the dead ducks showing typical lesions of enlarged and haemorrhagic livers,hard and necrotic spleens and widespread haemorrhage in the lungs.Pathological histological findings showed diffuse hepatocellular steatosis,extensive hepatic sinusoidal stasis and multiple vascular stasis in the liver tissue of the test group;Large areas of parenchymal cell necrosis,nuclear fragmentation or lysis are seen in spleen tissue;multiple vascular bruises and dilatations are seen in lung tissue,multiple intra-para-bronchial haemorrhages are seen,and large respiratory capillary bruises.Fluorescence quantification showed that NDRV was mainly concentrated in the liver and spleen organs,with the viral load in each organ peaking on day 3 after infection and gradually decreasing in each organ thereafter.The results of cytokine studies showed that cytokines such as IL,IFN and TNF were overwhelmingly upregulated in the liver and spleen after NDRV infection,and these data suggest that an effective antiviral immune response against NDRV can be established in both the liver and the spleen in a timely manner.In summary,this study successfully isolated a strain of NDRV and determined the distribution pattern of the virus in artificially infected ducks by constructing a fluorescent quantitative assay.By examining the pathogenicity of the virus in infected ducks and the changes in cytokines induced by the virus in liver and spleen tissues after infection,it provides a reliable theoretical basis for subsequent immune control of NDRV.