Sequence Analysis Of Muscovy Duck Reovirus S-Class Genome And S C-ELISA Assay For The Detection Of Mdrv

The S-class genome s A/s B/s NS/s C-encoding genes of S12 and S14 were cloned andsequenced. The identity of s A/s NS-encoding genes showed 76.0%-77.1% and 78.4%-79.6% atnucleotide level and 89.5%-91.2% and 91.6%-92.7% at amino acid level between DRV and ARV,respectively. While the identity of group-specific and type-specific neutralizing antibodies inducers B/s C-encoding genes showed only 52.5%-55.1% and 2.7%-9.9% at nucleotide level and61.4-62.0%, and 22.6%-26.7% at amino acid level between DRV and ARV, respectively. The lowlevel identity of s B/s C proteins demonstrated that DRV and ARV are antigenic differently. TheS12/S14 and French Muscovy duck reovirus 89026 isolate are highly related. The homology of sA/s B/s NS/s C-encoding genes were 90.0%, 93.6%, 87.9%-88.0%, and 93.1% at nucleotide level,and 97.1%, 94.3%, 95.7%-95.9%, and 93.7% at amino acid level between DRV S12/$14 and 89026,respectively. Phylogenetic analyses demonstrate that DRV are quite different from ARV and shouldgive a precise classification for DRV in Orthoreovirus genus.The minor core protein s C-encoding gene of Muscovy duck reovirus was cloned into theprokaryotic expression vector pET32a. The recombinant plasmid pET32a-s C was amplified andextracted after being transformed into E.coli DH5a competent cells. Restriction analysis withEcoRⅠand SacⅠand sequences analysis indicated that the recombinant plasmid was inserted withcorrect open reading frame. The fusion protein about 50 ku was produced after induction with 0.15mmol/L IPTG of E.coli competent cells transformed with pET32a-sC. The SDS-PAGE andWestern-bloting test indicated that the fusion protein reacted with the convalescents sera of duckinfected with Muscovy duck reovirus. The indirect ELISA method was developed by using thepurified fusion s C protein as coating antigen. The optimal concentration of s C was 5μg/ml, thedilution of serum sample was 1:40; The results showed that preparation of an ELISA by using sCas coating antigen in detecting 50 field duck sera in comparison with the AGIP were more sensitiveand specific than agar gel immuno-diffusion AGIP test. The results suggest that presence ofantibody against viral protein sC in duck may be a good indicator by the sC-ELISA for detectionof duck infection with reovirus.