Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus （GCRV）, a double stranded RNA virus that infects aquatic animals, often with disastrous effects, belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses, genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies, also called viral factories. Sequences analysis revealed the nonstmctural protein NS80, encoded by GCRV segment 4, has a high similarity with μNS in MRV（Mammalian orthoreovimses）, which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication, the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region （335-742） was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28℃. In addition, serum specific rabbit antibody was obtained by using super purified recombinant NS80（335-742） protein as antigen. Moreover, the expressed protein was able to bind to anti-his-tag monoclonal antibody （mouse） and NS80〈335.742） specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Molecular characterization of a novel reovirus isolated from SARS patients with distinct S1 segment

We reported a novel mammalian reovirus, designed BYD1, isolated from throat swabs of patients with severe acute respiratory syndrome （SARS）, in 2003. In the present study, we firstly compared the genome electrophoretic migration patterns of reovirus BYD1 with 3 prototype reovirus strains by polyacrylamide gel electrophoresis （PAGE） and determined the complete nucleotide sequence of the S1 gene segment of BYD1 by single primer amplification technique. The electropherogram of BYD1 was different from those of the 3 prototype strains and any other reovirus isolates reported before. The entire S1 segment sequence of BYD1 is 1437 bp long with two meaningful open reading frames （ORFs）. The longest ORF encodes σ1, the cell attachment protein, and the second longest ORF supposedly encodes σ1s, an important nonstructural virulence factor. The terminal sequences of SI segment are 5＇GCUA and 3＇UCAUC, which are consistent with those of other mammalian reoviruses. The highest homology of deduced σ1 amino acid sequence is 64% identity with known mammalian reoviruses. Phylogenetic analysis of both S1 nucleotide sequence and σ1 amino acid sequence indicated the BYD1 isolate belonged to a new clade of serotype 2 group. The results of this study showed that the BYD1 S1 segment was markedly different from those of isolates reported before and BYD1 was a novel human reovirus isolate.