Isolation And Identification Of A Strain Of NDRV And Establishment Of A High-Resolution Melting (HRM) Method To Differentiate NDRV And MDRV

Since 2005,a new infectious disease,characterized by irregular necrosis and hemorrhage in the liver and plaque necrosis and hemorrhage in the spleen,has emerged in Fujian,Jiangsu,Guangdong and other duck breeding areas in China,which seriously endanger the duck industry.The pathogen has been confirmed as Novel duck reovirus（NDRV）.NDRV and Muscovy Duck Reovirus（MDRV）constitute two genotypes of duck reovirus（DRV）.Both NDRV and MDRV can cause immunosuppression,which not only causes a large number of morbidity and mortality in ducklings,but also makes the infected ducks become stiff ducks,which seriously affects the production performance.It also increases the possibility of secondary infection and co-infection of other pathogens and increases the mortality rate,thus causes great harm to the duck industry in China.In order to study the biological characteristics of the epidemic strains of NDRV in Guangxi,and to establish a new detection method that can simultaneously detect the single or co-infection of NDRV and MDRV,the isolation and identification of NDRV,and establishment of a real-time fluorescent quantitative PCR method for the differential detection of NDRV and MDRV based on high resolution melting（HRM）analysis technology were carried out in this study.The specific research contents are as follows:In the present study,clinical samples of spleen were collected from a suspected case of duckling infection caused by NDRV in a duck farm in Guilin,Guangxi.RT-PCR detection of partial gene fragment of NDRVσB was conducted according to the reference method,and gene cloning,sequencing and analysis of the amplified target fragment of NDRV were performed.Then,the treated samples were inoculated into 9-day-old SPF chicken embryos through the urinary sac for virus isolation and culture.The BHK-21 cells were infected virus isolate for studing the culture characteristics,chloroform sensitivity,thermal sensitivity and other biological characteristics test.Finally,100 ELD₅₀（0.2 m L）virus solution was used to artificially infect 1-day-old Cherry Valley ducks by eye-dropping and nose-dropping for animal regression test.The results showed that the specific band of 586 bp was amplified from the clinical sample;sequence analysis showed that the isolate had high nucleotide sequence similarity with NDRV reference strain,and had the highest nucleotide sequence similarity of 99.49%with DRV/GX-Y7,which belonged to the evolutionary branch of NDRV.The isolated virus was proved to belong to NDRV in duck reovirus and was named as DRV/GX-Y5.After inoculation with NDRV,the embryo died,the whole body and multiple organs of the embryo showed hemorrhage and stunted.After viral infection of BHK-21 cells,the cells were disintegrated and exfoliated to form giant cell clusters,which which is consistent with the culture characteristics of NDRV.The isolated virus is not sensitive to chloroform and is sensitive to heat.The results of artificial infection test showed that ducklings in the challenge group showed varying degrees of clinical manifestations during the test.The diseased ducks showed mental retardation,loss of appetite,occasional lameness,and the mortality rate was 40%（6/15）.Liver and spleen hemorrhage and necrosis could be observed in the autopsy of dead ducks.Pathological section showed a large number of liver cells degeneration and necrosis,no normal liver cell structure,obvious blood stasis.The spleen corpuscle structure is blurred,lymphocyte infiltration,congestion and bleeding are serious.The spleen indexes at 4,7 and 14 days after challenge were measured,and the spleen indexes in the challenge group were significantly lower than those in the control group.The clinical symptoms and pathological manifestations of artificially infected ducklings were consistent with NDRV natural infection.To establish an HRM differential diagnostic method for NDRV and MDRV,referring to the relatively conservative S4 gene sequence shared by NDRV and MDRV of the two genotypes of duck reovirus published in NCBI,a pair of primers was designed and synthe-sized to simultaneously amplify the target fragment containing the genotype specific SNP of two genotypes duck reovirus,NDRV and MDRV,with genotype specific TM values.In this study,the extracted nucleic acid templates of NDRV DRV/GX-Y5 strain and MDRV commercial vaccine（CA strain）were used for gene amplification,cloning and sequencing,and the positive plasmids were extracted to make standard products.The obtained plasmid standard products were subjected to fluorescence amplification,and the HRM differential diagnosis method of NDRV and MDRV was established.The standard curve was plotted,and the test of specificity,sensitivity and repeatability were carried out.The possible co-infection was simulated.The NDRV and MDRV plasmid standard products were mixed with templates at the ratios of 4:0,3:1,2:2,1:3 and 0:4.A total of 43 suspected duck reovirus infection samples from Guangxi were also detected.The results showed that the standard curve equations established by NDRV and MDRV plasmid standard were y=-3.2770x+41.42 and y=-3.3433x+42.53,respectively,and the correlation coefficients R²were 0.9992 and 0.9963,respectively,with good linear relationship.The results of NDRV and MDRV typing showed that the specific Tm values were about 83.1°C and 84.8°C,respectively.The genotyping results were clear,accurate and reproducible.The established method was applied to detect common avian viruses without specific amplification and melting peaks.The established method for the detection of NDRV and MDRV diluted by 10³-10¹¹was selected.The results showed that the minimum detection for NDRV was 11.23 copies/μL,and the minimum detection for MDRV was 9.4 copies/μL.The coefficient of variation（CV%）of intra and inter batch repeatability tests for NDRV and MDRV was≤1.6%,indicating good repeatability.The results of mixed plasmid template detection showed that different proportions of co-infection could present special melting peaks containing NDRV and MDRV genotype-specific melting peaks.The established method was used to detect 43 clinical samples of suspected duck reovirus infection.The positive detection rates of NDRV and MDRV were higher than those of conventional PCR,which confirmed that the established method was highly sensitive and could be used as an effective detection method for the differential diagnosis of NDRV and MDRV in clinic.In conclusion,a strain of NDRV DRV/GX-Y5 was successfully isolated from clinically diseased ducks in Guangxi,and some biological characteristics and genetic evolution of the strain were studied and analyzed.At the same time,a rapid,specific and sensitive HRM method for the differential diagnosis of NDRV and MDRV was established,which provides materials and theoretical basis for the etiology of NDRV and the epidemic situation and disease prevention and control in Guangxi,and provides a new technical means for the differential diagnosis of NDRV and MDRV clinical cases.