Isolation And Identification Of Novel Duck Reovirus From Dead Embryos And Weak Ducklings

The novel duck reovirus disease is a common disease in the duck industry of China,which is characterized by irregular hemorrhagic-necrotic lesions in the liver and spleen.Ducklings infected with NDRV showed symptoms of mental depression,rough coat,anorexia,and death.As an immunosuppressive and vertically transmitted disease,the infection of NDRV leads to a decline in immunity,which causes mixed infections and secondary infections.Therefore,the NDRV disease cause serious economic losses to the duck industry in our country.In this study,fresh disease materials were collected regularly from the Liuhe hatchery of Tai’an and PCR technology was used to detect the infection rate of NDRV.Based on this,theσC gene sequence of ten NDRV isolates were sequenced and analyzed.One of the ten NDRV isolates,which caused obvious duck embryo lesions,was finally selected for pathogenicity analysis.This study provided a reliable theoretical basis for the scientific NDRV prevention and the vaccine development.To understand the prevalence of the disease in Shandong Province,497 fresh disease materials were collected in 9 batches,including 332 dead embryos,110 weak duckling and 55unfertilized eggs.PCR detection results showed that the total infection rate of NDRV was52.7%and the detection rates of NDRV were 53.9%,26.3%and 98.2%in dead embryos,weak ducklings and unfertilized eggs,respectively.Among them,the detection rate of NDRV in unfertilized eggs is the highest.To determine the genetic relationship between the newly isolated strain and the existing strain of reovirus,theσC gene sequence of 10 NDRV isolates was amplified in this study,and Megline 7.0 software was used to construct theσC gene phylogenetic tree and homology analysis.The analysis showed that NDRV,MDRV and ARV are separately in different evolutionary branches,both NDRV and MDRV are in the branch of waterfowl-origin reovirus.The SD-22,SD-11 and SD-62 NDRV isolates are close to each other and have the closest relationship with the TH11 strain.The other seven isolates are close to each other.TheσC gene sequence homology of 10 isolated strains was 94.1%-99.9%.TheσC gene sequence homology between the NDRV isolated strain and reference strain was 93.5%-97.4%.The homology of theσC gene sequence with ARV was the lowest,38.8%-43.2%.In this study,the SD-32 strain,which caused obvious duck embryo lesions and no other pathogenic infections,was finally selected from 10 NDRV isolates to do further analysis.The SD-32 isolate could produce obvious cytopathic effects on DF1 cells.The TCID₅₀of the isolate is 10^-6.052/0.1m L.Seventy healthy 1d cherry valley ducklings were randomly divided into two groups,35 ducks/group.The infected group was injected with 0.2 m L of virus fluid into the leg muscles,and the control group was injected with the same amount of stroke-physiological saline solution.The clinical symptoms,necropsy lesions and body weight changes of challenged ducks were observed.At 1DPI,3DPI,5DPI,7DPI,14DPI,and21DPI,4 ducks were randomly selected from each group to be killed.The immune organ index,the viral load of each tissue and organ,and the changes in the serum levels of IL-4,IL-6,IFN-ɑand IFN-γwere observed.The results showed that 1d ducklings infected with NDRV had symptoms of mental depression and white loose feces but no natural death.Necropsy showed varying degrees of hemorrhage and necrosis of the heart,liver and spleen,enlarged thymus,needle-shaped hemorrhage,and restrained weight gain.The immune organ index results showed that the thymus index was significantly lower than the control group at5DPI,the spleen index was significantly lower than the control group at 7DPI,and the thymus and bursa index at 14DPI were significantly lower than the control group.The results of the serum detoxification law showed that the virus could be detected on the first day after infection.The first peak of detoxification appeared at 3DPI and the second small peak at21DPI.The results of the viral load of each tissue showed that it could detect the presence of virus at 1DPI in all tissues,the virus content in the spleen was the highest,and the trend of viral load changes in each tissue and organ basically coincided with the trend of detoxification in the blood.The results of serum cytokine changes showed that the expression level of IL-4and IL-6 were very significantly up-regulated at 5DPI,the expression level of IFN-ɑand IFN-γwere significantly up-regulated at 5DPI,and the expression of cytokines was diverse.