Detection of rice grassy stunt tenuivirus nonstructural proteins p2, p5 and p6 from infected rice plants and from viruliferous brown planthoppers.

The genome of Rice grassy stunt virus (RGSV) consists of 6 ambisense RNA segments, among which RNAs 1, 2, 5 and 6 are equivalent to RNAs 1, 2, 3 and 4, respectively, of Rice stripe virus, the type species of the genus Tenuivirus. The RGSV 36-kDa nucleocapsid protein (N) is encoded on the complementary strand of RNA 5. Here, we studied accumulation of three nonstructural proteins, a 23-kDa p2 protein encoded on vRNA 2 (virus genomic strand), a 22-kDa p5 protein encoded on vRNA 5, and a 21-kDa p6 protein encoded on vRNA 6, from RGSV-infected rice leaf tissues and from viruliferous vector insects (brown planthopper, Nilaparvata lugens) by Western blot analyses. p2, p5 and p6 were detected from RGSV-infected rice leaf homogenates; p2 was detected mostly in the cytoplasmic soluble fraction but also a small amount was detected in the cell-wall, organelle-enriched and crude membrane fractions; p5 and p6 were detected from the cytoplasmic soluble fraction in large amounts. Among individual nymphs of N. lugens raised on RGSV-infected rice leaves, only 20% of insects were positive with the N protein. A large amount of p5 was detected from all the N-positive insects. Small amounts of p2 and p6 were detected only from a subset of the N- and p5-positive insects. p5 may have an essential role in virus infection in both plant and insect hosts, whereas p2 may function in plants such as a cell-to-cell movement protein.

Nucleotide sequence of a Dianthovirus RNA1-like RNA found in grassy stunt-diseased rice plants.

A Dianthovirus RNA1-like RNA (DR1L RNA, 4486 nucleotides in length) was found in grassy stunt-diseased rice plants together with Rice grassy stunt virus (RGSV). DR1L RNA has characteristics common to Dianthovirus RNA1 such as (1) presence of a GGAUUUUUAG potential shifty-heptanucleotide at the end of the 5'-proximal ORF, which encodes a 35-kDa protein, followed by a 77-nucleotide sequence capable of forming a stem-loop structure for an efficient--1 frameshift to express the downstream region in a 96-kDa putative replicase protein, (2) presence of nearly identical 17-nucleotide sequences in the 5'-terminal region and in a region upstream of an ORF encoding a 28-kDa, putative capsid protein (CP), and (3) near identity of the 3'-terminal 20 nucleotides to those of Dianthovirus RNAs. Western blot analysis using an antiserum against the C-terminal domain of the putative CP and RT-PCR analysis using primers specific to DR1L RNA of fractions after sucrose density gradient centrifugation of RGSV nucleoproteins indicated that DR1L RNA is associated with the 28-kDa putative CP but not with the 36-kDa RGSV CP. Two additional ORFs for 15-kDa and 33-kDa proteins were present in DR1L RNA although their expression in plants and functions are not known.

Comparison of nucleotide sequences between northern and southern philippine isolates of rice grassy stunt virus indicates occurrence of natural genetic reassortment.

Rice grassy stunt virus is a member of the genus Tenuivirus, is persistently transmitted by a brown planthopper, and has occurred in rice plants in South, Southeast, and East Asia [corrected]. We determined the complete nucleotide (nt) sequences of RNAs 1 (9760 nt), 2 (4069 nt), 3 (3127 nt), 4 (2909 nt), 5 (2704 nt), and 6 (2590 nt) of a southern Philippine isolate from South Cotabato and compared them with those of a northern Philippine isolate from Laguna (Toriyama et al., 1997, 1998). The numbers of nucleotides in the terminal untranslated regions and open reading frames were identical between the two isolates except for the 5' untranslated region of the complementary strand of RNA 4. Overall nucleotide differences between the two isolates were only 0.08% in RNA 1, 0.58% in RNA 4, and 0.26% in RNA 5, whereas they were 2.19% in RNA 2, 8.38% in RNA 3, and 3.63% in RNA 6. In the intergenic regions, the two isolates differed by 9.12% in RNA 2, 11.6% in RNA 3, and 6.86% in RNA 6 with multiple consecutive nucleotide deletion/insertions, whereas they differed by only 0.78% in RNA 4 and 0.34% in RNA 5. The nucleotide variation in the intergenic region of RNA 6 within the South Cotabato isolate was only 0.33%. These differences in accumulation of mutations among individual RNA segments indicate that there was genetic reassortment in the two geographical isolates; RNAs 1, 4, and 5 of the two isolates came from a common ancestor, whereas RNAs 2, 3, and 6 were from two different ancestors.