Establishment Of Duplex RT-PCR Method On Muscovy Duck Reovirus Strains With Two Different Pathogenicity And Induction Of Apoptosis

Muscovy duck reovirus?MDRV?is a strongly infectious virus in muscovy ducks,infected ducks are characterized mainly by white necrotic foci in liver and other clinical symptoms such as weak feet,reluctance to move,swelling of toe joints and tarsal joints.Several MDRVs strains have been isolated from diseased ducks by our laboratory since 2001,among which two strains of different pathogenicity are named as YB and YJL strain respectively.Our previous sequence analysis had showed that S4 gene homology between YB strain and MDRV France strain 89026 or 89330 was up to more than 94%,while S1 gene of YJL strain was found to be more than 96%identical to the avian reovirus?ARV?S1133 strain or 176 strain counterpart,S class-encoded genes of YB and YJL strain just shared less than 30%sequence identity.YB strain showed high pathogenicity clinically and caused a high mortality,while YJL strain just showed low pathogenicity to muscovy ducks.As the similarity of clinical symptoms and pathogenesis induced by these two strains brought great difficulty to differential diagnosis merely through clinical diagnosis,so the establishment of a fast,efficient and specific method for distinguishing different virulence strains clinically was of great significance.Firstly,based on the genomic differences of YB and YJL strain,two pairs of specific primers were designed respectively according to P10-coding gene and P17-coding gene.Through optimization of PCR conditions,a detection method of duplex RT-PCR was established,which could be used for differential diagnosis of these two strains.The results showed that:the expect fragment of 288bp of YB strain and 441 bp of YJL strain could be simultaneously amplified by duplex RT-PCR,while amplification results of other muscovy duck pathogens were negative.The optimum reaction conditions for duplex RT-PCR were as follows:95? 5min,94 C 1min,55? 45 s,72?lmin,30 cycles,72 ? 10min.The RNA template amount of YB or YJL strain used in this detection could be as low as 10pg.Detection of 60 copies of clinical samples by single RT-PCR and duplex RT-PCR showed a coincidence rate of 100%.With a good specificity,sensitivity and repeatability,this method could be used for differential diagnosis of these two different virulence strains fast and accurately.Considering the existence of mixed infection of different virulence strains in muscovy ducks,which increased difficulties of disease prevention,it appeared to be of great importance to explore pathogenesis of MDRV infection.During our study of MDRV infection,apoptosis performance of the host immune organs and target organs induced by MDRV was found.In this study,virus-induced apoptosis was investigated through interactions between MDRVs strains?YB and YJL strain?and host,primary muscovy duck embryo fibroblasts?MDEF?.Firstly,TCID₅₀ of this two strains were determined on MDEF,secondly,followed by exposure of MDEF to two different doses of YB and YJL strain respectively,cell viability of MDEF at 12h,24h,36h,48h,72h,96h postexposure were measured by MTS assay to determine suitable inoculation dose and optimal time point.Then qualitative and quantitative analyses of virus-induced apoptosis were carried by using fluorescence microscopy,DNA Ladder assay and flow cytometry;finally,activation levels of caspase-3 of 12 copied of cell samples were measured by Western bloting.The results showed that:TCID₅₀ of YB strain on MDEF was much higher than YJL strain,about three times;MTS assay indicated that inflection point of cell viability curve in challenge group was at 48h,and there was a significant decrease in cell viability,thereinto,cell viability of YB group was lower than YJL group,moreover,effect of high dose YB group?YB-H?on cell viability were consistent with low dose YB group?YB-L?,while variation of low dose YJL group?YJL-L?is not obvious,so the doses of YB-L and YJL-H groups were chosen as test groups;results of fluorescence staining indicated that both of YB and YJL groups could induced early apoptosis,and apoptosis of YB group was more obvious than that of YJL group in the late stage;DNA ladder assay of YB and YJL groups both showed typical DNA ladder,in which YB group was more obvious than YJL group,while control group showed negative;flow cytometry demonstrated that apoptotic levels of YB group were higher than YJL group,and apoptotic level of liver of YB group?75%?was much higher than that of spleen?only 23%?,apoptosis of liver tissue in YB group during the apparent period converged on middle-late stage,whereas that of spleen distributed in each period,as for YJL group,there was no difference in apoptotic levels between liver and spleen?around 10%?;detection of apoptosis on MDEF revealed that apoptotic levels of test group at 48h postinoculation significantly increased than the control group,apoptosis of YB group at 72h postinoculation converged on middle-late stage,while that of YJL group distributed in every period;activation levels of caspase-3 were measured by Western bloting,results showed that a mass of pre-caspase-3?32KD?were detected in either liver or spleen of the control group,activated caspase-3 large subunit?17KD?were rarely detected;on the contrary,expression of pre-caspase-3 in both liver and spleen of YB group was significantly reduced compared with the control group,meanwhile expression of activated Caspase-3 was obviously increased;as for YJL group,there was just a slight increased amount of activated caspase-3 expression in liver and spleen compared with the control group.As regards MDEF group,expression of pre-/caspase-3 of each groups at 48h postinoculation did not change significantly,while expression of activated caspase-3 of YB group at 72h postinoculation had a visible promotion compared with the control group;though activation levels of caspase-3 of YJL group at 72h postinoculation was significantly lower than that of YB group,there was still an apparent increase over 48h.In present study,a kind of fast and efficient duplex RT-PCR was established with regard to two different virulence strains,which could provide reference for laboratory diagnosis and production practice,then from the point of apoptosis,pathogenesis of the two strains were studied,and infection ability,inflection point of cell viability curve and apoptosis-inducing ability of the two strains were determined,thereby providing basic data for further research of viral protein function and related signal pathways.