- Volume 69, Issue 2, 1988

DNA complementary to the 4.7 kb cymbidium ringspot virus (CyRSV) genome was cloned in Escherichia coli. Recombinant plasmids were screened with a cDNA probe synthesized from randomly primed CyRSV genomic RNA, and two clones were chosen for further analysis. Two additional subclones were prepared after deletion of two sequences in the 3′ region and one clone containing 5′ virus genome sequences was constructed by primer extension. Three different RNA species associated with CyRSV were detected in purified virions and virus-infected tissue. The largest RNA was genomic RNA and the other two subgenomic. Northern blot hybridization indicated that the two subgenomic RNAs had in common a region corresponding to the 3′ region of genomic RNA. All three RNAs were found in tissue in both single- and double-stranded forms. In vitro translation experiments with fractionated virus RNA indicated that the coat protein gene is internally located and that it is followed by a 3′-terminal gene coding for a 22K protein.

B component RNA (B-RNA) of red clover mottle comovirus (RCMV) was not transported between cells of inoculated leaf tissue unless it was co-inoculated with the M component. However when host plants were infected with sunn-hemp mosaic tobamovirus (SHMV) or mutant Ni118 of tobacco mosaic virus (TMV) before superinoculation, RCMV B-RNA was transported readily between cells. Plants were infected with SHMV or Ni118 and, 5 days after superinoculation with RCMV B component, protoplasts were isolated from the infected leaves and inoculated with RCMV M component. About 30% of such protoplasts multiplied RCMV (identified by immunofluorescence microscopy) whereas only 3 to 5% of protoplasts from similarly treated plants not initially infected with SHMV or Ni118 multiplied RCMV. Thus B-RNA spread from mixedly infected (SHMV + B-RNA or Ni118 + B-RNA) cells in the absence of M-RNA to the neighbouring cells. RCMV B-RNA spread in leaves infected with the temperature-sensitive coat protein TMV mutant Ni118 and grown at non-permissive temperature. Thus TMV coat protein is not involved in enabling RCMV RNA to be transported.

Analysis of nucleic acids from grapevine tissues by two-dimensional gel electrophoresis demonstrated the presence of two bands of circular RNA. The smaller RNA contained about 300 nucleotide residues and was identified as hop stunt viroid by nucleotide sequencing. The larger RNA band was a mixture of species and contained similar amounts of two components, referred to as RNA 1a and RNA 1b, and in addition a trace amount of citrus exocortis viroid (CEV) which became detectable only after inoculation of the mixture to tomato. The identity of CEV was determined by probe hybridization and nucleotide sequencing. Both RNAs 1a and 1b are distinct from CEV and have estimated sizes larger than those of CEV and other viroids reported so far. RNA 1a preparations were infectious in cucumber and in tomato and the recovered viroid had unique properties. We have provisionally named this viroid Australian grapevine viroid. Evidence for the autonomous replication of RNA 1b was not obtained.

Attempts were made to label the termini of dsRNAs corresponding to the two genomic RNAs of grapevine chrome mosaic nepovirus (GCMV). It was not possible to label the 5′ ends of the dsRNAs with [γ-32P]ATP, which suggests that a genome-linked protein blocks their 5′ ends. Both dsRNA species were labelled at their 3′ ends with pCp. The 3′-terminal sequences were determined by ‘wandering spot’ or by partial enzymic cleavage analysis. One strand (presumably positive) ended in a poly(A) 30 to 50 nucleotides long whereas the other (presumably negative) ended in 3′-ACCUUUUAAAAAG (RNA1) or 3′-ACCUUUUAAUAAAG (RNA2). The sequences resemble closely those complementary to the 5′ ends of the RNAs of tomato black ring virus (strain S), which is distantly related to GCMV.

Synchronous multiplication of rice gall dwarf virus (RGDV) and an assay of its infectivity in cell monolayers of its vector, the green rice leafhopper Nephotettix cincticeps, are described. The number of foci of infected cells was linearly related to the concentration of virus. The method was about 103 times more sensitive than enzymelinked immunosorbent assay. All the vector cells in monolayers were infected when inocula were dilutions of 10-3.5 of sap from infected plants, 10-4.5 of extracts of viruliferous insects, 10-5.5 of infected monolayer cells or purified virus with A 1cm 260nm = 10-5. RGDV was first detected in monolayer cells 10 h after inoculation, and multiplied 105-fold in the subsequent 40 h. Vector cell monolayers are thus an excellent experimental system for the study of RGDV.