Isolation And Identification Of Tupaia Belangeri Orthoreovirus And The S1Gene Recombinant Plasmid Construction And The S1Genomic Characteristics Analysis

The taxonomic status of tree shrew is between the primitive insectivorous and the original primates, known as the Scandentia.Pathogenic viruses can harm acutely the life and health of laboratory tree shrews;however,few papers exist regarding natural pathogenic virus infection in this species.Six feces of the tree shrews died from diarrhea were collected and infected Vero cell line and resulted in CPE after72h. The CPE was shown granulating, shrinking, rounding, seining and falling off. The isolation was shown spherical, double-layered capsid, about75nm in diameter by electron microscopy. The genome of the purified isolation was shown10segments in a3:3:4typical arrangement by polyacrylamide gels (PAGE) electrophoresis. The isolation was confirmed by RT-PCR assays targeting the conserved region of L1gene and sequence analysis and reconstruction of a phylogenetic tree. It was concluded that the isolation was a Tupaia Orthoreovirus(TRV) belonging to Mammalian Orthoreovirus (MRV).At present there was few research reports about Tupaia Orthoreovirus(TRV). As for the genome information of TRV had nothing to be reported,it was necessary to construct the S1recombinant plasmid,for revealing the molecular characteristics of TRV SI genome. The S1genome of the isolation was sequenced using4pairs of specific primers derived from the published sequences of MRV. The cDNA fragments of TRV S1gene were amplified by RT-PCR, and ligated into pMD19-T Vector, then transformed into E. coli DH5a, and the S1recombinant plasmid was constructed successfully. The sequencing results indicated TRV S1genome was1463bp, included two ORF, encoded two protiens δ1and δ1s. The difference of the S1genome segments of TRV with typical strains, which of TIL,T2J, T3D and NEDV were very obviously, and the deduced amino acid sequence similarity of TRV withTIL, T2J, T3D and NEDV were86%,44%,23.7%,23.2%. The phylogenetic tree based on deduced amino acid of S1genome segment supported the same serotype viral strains located in the same evolution branch. According to the phylogenetic tree, the serotype of TRV was identified as serotype I1. The predicted results of deduced protein indicated δ1and δ1s proteins were hydrophilicity proteins, without signal peptide sequence.2. The δ1Protein contained3N-glycosylation sites,5T phosphorylation sites,5S phosphorylation sites,1D phosphorylation site,2P phosphorylation sites,1Y phosphorylation site. The δ1S Protein contained3N-glycosylation sites,1T phosphorylation site,4S phosphorylation sites.3. The beta sheet were capital ingredient in the secondary structure of δ1S Protein with the Antigen index significantly in20-80aa,95-135aa,180-195aa,210-225aa,230-245,265-295aa,310-340aa,390-440aa..The alpha helix structure and beta sheet were capital ingredient in the secondary structure of δ1S Protein with the Antigen index significantly in1～18aa,58-67aa.As a zoonotic virus, the MRV isolation which was first isolated from wild tree shrews is very important to develop the viral quality control standards of the tree shrews and provides scientific data for the prevention of the zoonotic tree shrew-to-human transmission. The S1recombinant plasmid was constructed successfully.It is very important to revealing the molecular characteristics of TRV, the the pathogenic mechanism and the relation between TRV and diseases.