The Molecular Characteristics And Infectious Cloning Vector Of S Gene-group Derived From Muscovy Duck Reovirus Virulent Strain And Attenuated Strain

Muscovy duck reovirus(MDRV)is the etiological agent of a contagious disease with the main feature of soft feet,a lot of white needle-like necrosis point shown on liver and spleen surface causing huge economic losses in the Muscovy duck farming in China.The CA vaccine strain can effectively prevent this disease,while the molecular mechanism of the pathogenesis and attenuation of MDRV are not clear.In this research,we selected MDRV-MW9710 virulent strain and MDRV-CA attenuated strain as the material and respectively constructed infectious cloning plasmid vectors of MDRV S gene-group,results are as follows:1.A gene-molecular cloning method constructed to obtain the S gene-group of virulent strain and attenuated strain:Firstly we extracted viral RNA,designed gene-specific primers for the whole S gene-group,by RT-PCR and DNA sequencing,and obtained the S gene-group for both MDRV strains.The results showed that both strains had the same size of S1,S2,S3,S4,which were 1324 bp,1201 bp,1191 bp,1124 bp,respectively.The nucleotide sequence of two strains compared to the MDRV reference strain shared more than 98%nucleotide identity,which further confirmed the obtainment of S gene-group for both MDRV strains.2.The discrepancy and physicochemical properties analysis of deduced amino acid sequences encoded by MDRV S gene-group:Results based on DNAstar software showed that the amino acid substitution occurred at 2 positions within ?A(A/V at 301 and 409),12 positions within ?B(S/P at 40 and 218,V/A at 5,W/R at 21,M/T at 65,P/S at 85,I/T at 96,Y/H at 113,S/F at 120,D/G at 244,G/S at 265 and A/T at 347 respectively),1 position within ?NS(S/T at 273),3 positions within p10(H/Y at 70 and 73,A/V at 46),8 positions within ?C(H/Y at 162,F/I at 167,T/I at 168,S/F at 172,P/S at 197,T/M at 198,P/F at 206,L/F at 264 respectively).Compared to the virulent strain,the molecular weight of ?B goes up and PI decrease,but the variation of ?C in those two aspects is in an opposite way.3.The infectious cloning vector's construction of MDRV S gene-group:Referring to the idea of successfully constructed MRV infectious clone,we constructed a viral vector of p-MDRV,with Sap I restriction site.S gene-group of these two strains was amplified with primers including Sap I site,then by digested,purified,connected to the p-MDRV vector.Sequencing results showed that the infectious cloning vectors were constructed successfully with the right orientation.The conclusions:This study identified the differences of S gene-group and its'encoding proteins of virulent strain and attenuated strain and successfully constructed the infectious cloning vectors of S gene-group.This study provides methods and experience for the further clone of L and M gene-groups of MDRV,builds the foundation of infectious clone in Muscovy duck reovirus and lays a good foundation for the further development of MDRV in molecular mechanism of the pathogenesis and attenuation.