Isolation And Characteristics Of A Reovirus From Short-nosed Fruit Bats (Cynopterus Sphinx)

Wild short-nosed fruit bats samples from Shaoguan City Guangdong Province were scrunched and inoculated in Vero-E6 cells. Two virus strains were isolated. One virus isolated from short-nosed fruit bats didn't caused cytopathic effects (CPE) until fourth-passaged on Vero-E6 cells. Infected cells emerged granulating, shrinking, rounding and falling off. After three times freeze-thaw, cells and culture medium were harvested for electron microscopy. Virus particles were nonenveloped, double capsid and icosahedral symmetry. This virus was designated Bat/China/2003(B/03). Hemagglutination test indicated that the virus could agglutinate healthy human type O red cells, but could not agglutinate the red cells of SPF chicken, experimental common bovine, rat and guinea pig red cells. This virus is tolerant to chloroform treatment and pH3.0. Virus loses infectivity in water bath 50*C 1 h. 1M Mgcl₂ can enhance resistance of virus to heat treatment and increase infectivity. Nucleic acid electrophoresis on agarose gels suggested that the genome of this virus was divided into large, medium and small parts. Specific primers according to mammalian reovirus were used for Reverse Transcription Polymerase Chain Reaction (RT-PCR). Appropriate specific products were amplified by RT-PCR. NCBI BLAST analysis indicated that this segment shared the highest identity with Ndelle virus who was a member of the genus Orthoreovirus, and the homology was 91.2%. DNAMAN Multiple Alignment analysis of nucleotide sequences with mammalian reovirus serotype 1(T1L), 2(T2J) and 3(T3D) found that homologies were 89.9%, 76.9% and 89.9%, respectively. So we can deduce this virus is a member of the virus family Reoviridae.We determinated the sequences of reovirus B/03 strain isolated from short-nosed fruit bats. Integrated sequences of 10 ORFs were determinated by RT-PCR. Molecular biological softwares were used to analyse homologies of nucleotide sequences and amino acid sequences, drawing phylogenetic tree, analyzing fuctional and structural domains of each protein encoding by virus genes, and then predicting function of each protein. Comparison results of proteins of B/03 strain with those of other members in the family Reoviridae suggested that LI, L2, L3, Ml, M2, S1, S2 and S4 encoded viral structural protein λ3, λ2, λ1, μ2, μ1, σ1,σ2 and σ3, respectively. M3, S1 and S3 encoded viral nonstructural protein μNS, σlS and σNS, respectively(S1 encoded two proteins, structural protein σ1 and nonstructural protein σ1S).Analysis of the functional and structural domains suggested that λ3 protein contained conserved domain of RNA-dependent RNA polymerase (RdRp), which confirmed that λ3 was RdRp. λ2 protein included the activity domains of guanylytransferase and methyltransferase, deducing λ2 was important for capping of mRNA during virus replication. We found Zinc finger domain and activity domain of RNA helicase in amino-terminal of λ1 protein, which predicted that λ1 protein could bind dsRNA and possessed ATPase activity. μ2 protein included two NTP-binding motifs that maybe possessed nucleotide triphosphatase function. There were four hydrophilicity domains in carboxy-terminal of μ1 protein predicted to occur in loops or turns at the surface of the protein. These regions may importantfor the host immune response to this protein, a\ protein had a hepta peptide repeat pattern (a-b-c-d-e-f-g)n, which can form a-helical coiled-coil structure, it maybe important to heamagglutinin. There were four potential glycosylation sites in ol protein, which suggested that ol maybe a glycoprotein. Carboxy-terminal of o2 protein had an amphipathic a-helix similar to the E. coli DNA-dependent RNA polymerase p subunit, predicting o2 protein maybe can bind to dsRNA. a3 protein had Zinc finger domain at amino-terminal and dsRNA binding domain at carboxy-terminal. We found a hepta peptide repeat pattern (a-b-c-d-e-f-g\ that can form a-helical coiled-coil structure at carboxy-terminal of uNS protein, which maybe important for binding cell skeletons. There was also a conserved domain of ATPase at amino-terminal of uNS protein, which suggested that this protein may have ATPase activity. olS protein had many basic amino acids, a-helix and a glycosylation site, but its function is unclear. oNS protein was abundant to Cys and a-helix, it was predicted to bind ssRNA.The X3 protein (RdRp) of B/03 strain shared the highest identity with the virus of family Reoviridae, genus Orthoreovirus (91.9%⁹8.2%). On the basis of this result, we concluded that B/03 strain should belong to Reoviridae, genus Orthoreovirus. Comparing the proteins of B/03 strain with those of other members in the genus Orthoreovirus, we found that B/03 strain shared the highest identity with mammalian orthoreovirus (MRV). Phylogenetic tree suggested that genes of B/03 strain belonged to the MRV species, not belonging to orthoreovirus isolated from bats. We suggested that the reovirus B/03 strain isolated from short-noseed fruit bats should be classified into family Reoviridae, genus Orthoreovirus and mammalian orthoreovirus species. SI gene encoded type sepecific antigen protein. Phylogenetic tree suggested that SI gene of B/03 strain may belong to MRV serotype 1. While we still need more evidence to classify B/03 strain to serotype 1.Sequences comparison of B/03 strain suggested that different segments shared the higest identify with different reovirus strains. It is suggested that this virus may emerge from reassortment during long term nature infection.