Isolation And Identification Of A Novel Duck Reovirus SL Strain And Establishment Of A Quantitative PCR Detection Method

Novel duck reovirus（NDRV）disease is a disease characterized by spleen enlargement,hemorrhage and necrosis in ducks.The disease has no obvious onset cycle,and ducks of all strains can be infected,and can be transmitted in two ways,horizontal and vertical.Under natural conditions,the morbidity and mortality of the disease are relatively low,but infection in ducks can lead to developmental disorders.And immunosuppression caused a variety of complications,resulting in high mortality,and brought great harm to my country’s waterfowl breeding industry.In this study,a reovirus was isolated from the spleen of sick ducks in Longchang,Sichuan,named SL strain,and its pathogenicity was studied;the whole genome of SL strain was determined by next-generation sequencing technology,Its genetic evolution relationship was analyzed;TaqMan and SYBR Green Ⅰ real-time quantitative PCR detection methods were established according to sequence characteristics,and a total of 176 clinical samples from different farms in 10 provinces including Shandong,Chongqing,and Sichuan during 2019-2021 were analyzed.At the same time,it was compared with the traditional RT-PCR detection method,and the detection rate of the three methods was compared,which laid the foundation for the clinical diagnosis and in-depth research of NDRV.The main research content is divided into the following four parts:1.Isolation,identification and pathogenicity test of a novel duck reovirusA virus was isolated from ducks suffering from spleen necrosis in Longchang,Sichuan,named SL strain.The duck embryo passaging test showed that when it was passed to the F9generation,90%of the duck embryos died in 60-120 hours,and the embryos had spotty hemorrhages.The spleen and liver have foci of necrosis.It was confirmed to be duck reovirus by RT-PCR method combined with sequence determination.In the animal regression test,both the infected ducks and the dead ducks showed developmental retardation and opisthotonus symptoms.The autopsy found that the liver had necrotic foci,splenomegaly,infarction,and scattered bleeding points,which were consistent with the clinical symptoms.Histopathological observation found that the liver and spleen tissues of the diseased ducks were infiltrated by heterophilic granulocytes and lymphocytes,and there were mixed thrombus and red pulp congestion in the splenic vein.Use F9 generation allantoic fluid10-fold gradient dilution,take 4 dilutions to inoculate healthy duck embryos,5 in each group,and continue to observe for 120h,using the Reed-Muench method to calculate the median lethal dose of the virus to duck embryos is 10^-5.32 ELD50/0.2ml.Five dilutions of F9generation allantoic fluid were used to inoculate 5-day-old healthy ducks,10 in each group,and were observed for 7 consecutive days.The median lethal dose of the strain to ducklings was 10^-4.2ELD50/ml.2.Sequencing and analysis of the whole genome of the novel duck reovirus SL strainWhole-genome sequencing of SL strains using next-generation sequencing technology and prediction and functional annotation of gene-encoded proteins,the results showed that the whole genome of this strain consists of 10 segments with a total length of 23580bp and a GC content of 49.62%,encodingλA（L1）andλB（L2）respectively,λC（L3）,μA（M1）,μB（M2）,μC（M3）,σA（S2）,σB（S3）,σNS（S4）,σC,P10,P17（S1）,The sequencing results were uploaded to the NCBI database to obtain 10 accession numbers:S4-S1:MW196679-MW196682,M3-M1:MW196683-MW196685,L3-L1:MW196686-MW196688.Among the proteins for functional annotation,exceptμNS,which is a non-structural protein,the others are structural proteins,which are involved in different biological processes of virus replication.for 10genome segments By comparative analysis,it was found that the SL strain had a high similarity（87.02%-99.48%）with the novel duck reovirus（NDRV）,followed by the similarity with the Muscovy duck reovirus（MDRV）（80.09%）.-98.77%),while the similarity with chicken-derived reovirus（ARV）was the lowest（70.04%-91.15%）.The phylogenetic tree showed that the 10 gene segments of the SL strain were all located in the main branch formed by NDRV and MDRV,except that the M1 segment was located in the same branch as MDRV（SH12）,the L1 segment was located in the same branch as MDRV and NDRV,and was in the same branch as XT18 and XT18.Except for the closest distance between GX-Y7,all other gene segments are located in the same branch as NDRV（GX-Y7,XT18）and have the closest distance.It is inferred that the SL strain may be the result of gene recombination between MDRV and NDRV,indicating that the separation in this experiment the SL virus strain is a novel duck reovirus.3.Establishment of fluorescent quantitative PCR detection method for novel duck reovirusA pair of primers were designed and synthesized according to the S1 gene of the novel duck reovirus SL strain to construct a positive plasmid standard.A pair of specific primers and an MGB probe were designed and synthesized according to the gene sequence of the positive standard,and TaqMan and SYBR Green Ⅰ were established respectively.Two fluorescent quantitative PCR detection methods were used,and the specificity,sensitivity and repeatability of the two methods were compared.The results showed that the correlation coefficients of the standard curves of the two detection methods were both 0.999,and the detection of duck parvovirus（DPV）,duck hepatitis virus type Ⅰ（DHV-1）,duck hepatitis virus type Ⅲ（DHV-3）,duck infectious bronchial.The detection results of inflammatory bowel disease（IBV）,duck tembusu virus（DTMUV）,and goose astrovirus（GAst V）were all negative with good specificity.The minimum detection limit of SYBR Green Ⅰ method is100copies/μL,and the minimum detection limit of TaqMan method is 101copies/μL,and the sensitivity of the former is higher.The intra-and inter-group coefficients of variation of the SYBR Green Ⅰ method and TaqMan method were less than 2.1%and 1.8%,respectively.In conclusion,both fluorescence quantitative methods were successfully established,and the SYBR Green Ⅰ method was especially sensitive.4.The clinical application of the new fluorescent quantitative detection method of duck reovirus and its comparison with the traditional RT-PCR methodTaqMan and SYBR Green Ⅰ were used to detect 176 samples,and compared with RT-PCR detection method.The results showed that SYBR Green Ⅰ detected 146 positive samples,and the detection rate was 83%.Acupuncture detected 152 positives,with a detection rate of 86%,and the traditional RT-PCR detection method had 107 positives,with a detection rate of 61%.The chi-square test was used to analyze the detection rate of the two fluorescence quantitative detection methods.The results showed that P value=0.37>0.05,indicating that the detection rate of the two detection methods was not significantly different,and both were higher than ordinary RT-PCR method.