Isolation And Identification Of Novel Duck Reovirus And Selection Of Attenuated Strains

Novel duck reovirus（NDRV）infection can cause spotted hemorrhage or necrosis in the liver of ducks,and white necrosis and hemorrhage in the spleen to varying degrees.It can cause the death of many breeds of ducklings and cause serious economic losses to the duck industry.It is urgent to study an effective new reovirus vaccine for prevention and control.In this study,liver and spleen of dead ducks were collected from 5 suspected NDRV infection cases in Guangdong,Fujian,Jiangxi and other places,and the virus was isolated and identified by cell inoculation,RT-PCR and other methods.The 5disease material samples can proliferate on Vero cells after successive passages,showing cell giant fusion lesions;RT-PCR detects NDRV positive,duck plague virus（DPV）,Muscovy duck reovirus（MDRV）,Muscovy duck small Virus（MPV）and Tembusu virus（TMUV）tested negative.Sequencing and analysis of the S gene clones of the 5 isolated virus strains showed that the 5 cell-derived virus strains had high homology,with S gene nucleotide homology of 87.5%-99.3%and amino acid homology of 90.6%-100%,the nucleotide homology with the currently popular NDRV strain is 87.3%-100%,and the amino acid homology is 85%-100%.Phylogenetic tree analysis shows that the 5 strains are in the same waterfowl reovirus gene.Type 2 branch.In summary,this study isolated five NDRV strains and named them NDRV-CJH-GD20,NDRV-FJ19,NDRV-ZSS-FJ20,NDRV-LRS-GD20,NDRV-CS-JX20.The representative strain NDRV-CJH-GD20 was selected as the NDRV-induced attenuated parental virus,and a proliferation system was constructed on BHK-21 cells.The mutation was induced by 5-fluorouracil（5-FU）for 40 generations to obtain NDRV-CJH-F10,F15,F20,F25,F30,F35,F40 and other generations of GD20 induce virus.The S gene of the above 7 generations of induced virus strains was sequenced and subjected to mutation analysis.Compared with the original strain,the S gene of F40had 30 base mutation sites,which caused 13 amino acid site mutations,includingδC protein.6 amino acid mutations（T55M,A57T,S148G,N161D,M223L,D234G）,1amino acid mutation inδA protein（L130p）,3 amino acid mutations inδB protein（P131S,H132L,A245T）,3 amino acids inδNS protein Mutations（L136P,R284,L389Q）.The above 7 generations of virus strains were induced to challenge the 1-day-old Muscovy ducklings at a dose of 2×10⁵ TCID₅₀.After 7 days of challenge,the weight change of the parent strain group was significantly reduced,and the amount of toxin discharged from anal and throat swabs was significantly higher.In the blank group,there was no significant difference between the induced strains;the necropsy observation showed that there was no obvious lesion in the F25～F40 group,the RT-PCR test result was negative,and the pathological section showed normal tissue structure.After 7 days of immunization with 7 strains of each generation,artificially infected with the parental strain NDRV-CJH-GD20 virus solution 2×105 TCID50,the body weight of each group did not change significantly after 7 days,and the F0,F35,and F40 groups showed massive hemorrhage in the liver.Necrosis,severe spleen enlargement,white necrosis and hardening,liver pathological observation showed inflammatory cell hyperplasia,extensive necrosis,loss of splenic lymphocytes,naked endothelial cells,degeneration and necrosis,the positive rate of RT-PCR detection was70%-100%.F10,F15,F20,F30 showed needle-shaped necrosis of the liver and moderate necrosis of the spleen.Some ducks showed hepatic inflammatory cell proliferation and necrosis,lack of lymphocytes in the spleen,and degeneration and necrosis.The positive rate of RT-PCR detection was negative.Both are 30%.There were no obvious lesions in the necropsy and pathological sections of the F25 group,and the RT-PCR test was negative.The results showed that compared with other generation virus strains induced by NDRV-CJH-GD20,the F25 generation virus strain has been weakened and retains good immunogenicity.The F25 virus strain was passaged 20 times in BHK-21 cells（F25-1～F25-20）,and it was passed 5 times in Muscovy ducks（P1～P5）.The determination of the S gene of the F25-20 virus found that compared with F25,F25-20 has 3 amino acid site mutations,of whichδA has 1mutation site（S353P）,δB 1 mutation site（N347D）,andδNS 1 mutation site（H375R）.RT-PCR detection of P1～P5 showed that the results of P1 and P2 were positive,and the results of P3～P5 were negative.Sequencing of the P2 S gene clone showed that compared with the F25 generation,the S gene of P2 has 1 onδB Amino acid mutation site.The results show that F25 is relatively stable in BHK-21,and the animal regression experiment has not found a strong virulence.It can be used as a candidate strain for NDRV attenuated vaccine.Three strains isolated from different regions,NDRV-ZSS-FJ20,NDRV-LRS-GD20,and NDRV-CS-JX20,were artificially infected with young Muscovy ducks immunized for 7 days at a dose of 2×105 TCID50.The results showed that the three strains were positive for the liver of the control group.Different sizes of bleeding spots,necrotic spots,splenomegaly and severe necrosis,the positive rate of RT-PCR detection was 100%,the spleen/liver of the three immunized muscovy ducks had no obvious pathological changes,and the pathological structure was normal and no obvious pathological changes.,The results of RT-PCR for each group were negative.