Molecular Characterization And Infection System Of Rice Black Streaked Dwarf Virus Isolated From Maize

Rice black streaked dwarf virus (RBSDV) and Maize rough dwarf virus (MRDV) are two closely related Fijiviruses. RBSDV was first reported from black streaked dwarf diseased rice in Japan and MRDV from rough dwarf diseased maize in Europe. Differences have been reported in S1, S4, S5 and S6 dsRNA segments by hybridization and in the host ranges of the two viruses. In order to elucidate variation at the complete genome sequence level of RBSDV which in China can causes both maize rough dwarf and rice black streaked dwarf diseases, we extracted viral dsRNA directly from an isolateof Rice black streaked dwarf virus causing rough dwarf disease on maize in Hubei, China. The__cDNAs of viral segments S1, S4, S5 and S6 were sequenced. S1 is 4501 bp in length and codes for a 168 kDa protein, which is predicted to be a RNA-dependent RNA polymerase (RdRp), because it contains the characteristic motif of GDD (Glycine-Aspartate-Aspartate) and other conserved domains. The evolutionary relationship was analyzed by comparisons of amino acids sequences of the RdRp from related reoviruses. S4 is 3617 bp long and contains one ORF which encodes a 135 kDa protein and homology analysis revealed that this protein may be the B-spike structural protein. S5 is 3164 bp in size and contains a large ORF1 and a small overlapping ORF2, whose predicted initiation coden differs from another RBSDV isolate from diseased rice in Zhejiang (RBSDV-Zjr). S6 is 2645 bp in length and encodes a 90 kDa protein, whose amino acid aligns significantly with MAD and SMC in the domain adjacent to C terminus.A prokaryotic expression vector was constructed by inserting the P10 ORF into pGEX-KG for expression in E. coli. The expressed P10 was purified through Glutathione Sepharose 4B and used to immunize a rabbit. The antiserum against PI0 can detect RBSDV from diseased wheat by ELISA and western blotting. For ELISA, as little as 160 ng of P10 was detectable when it was diluted in healthy wheat sap, and also it is possible to discriminate infected from healthy wheat up to a dilution of 1/1280 in buffer.In order to analyze the infectivity of RBSDV, plasmolyzed wheat suspension cells and rice protoplasts were infected with RBSDV crude extracts, and sampled after 48 hours. Positive results were obtained for rice protoplasts, wheat suspension cells and calli by immunodetection with P10 and /or P6. Viral genomic RNA was coated with liposomes to transfect rice protoplasts. Western blotting by P8 antisera provided positive results, but only samples infected with dsRNA and dsRNA/ssRNA mixtures were positive by northern blotting.