The characterization of a subgenomic RNA and in vitro translation products of oat chlorotic stunt virus.

Oat chlorotic stunt virus (OCSV) is a 35 nm icosahedral plant virus comprising of a single capsid protein with a Mr of 48.2 kDa and a 4.1 kb single stranded, positive sense genomic RNA. Northern blot analysis detected a single 3' terminal subgenomic RNA in extracts from infected plants, which was also found to be encapsidated. Virion RNA directs the synthesis of a 23 kDa polypeptide in a rabbit reticulocyte in vitro translation system. Primer extension analysis has been used to map the end of both the genomic and subgenomic RNA's, and has shown the genomic size to be 4115 nucleotides in length. The results have enabled a model for the genome expression to be proposed.

An improved method for the detection of Tospoviruses using the polymerase chain reaction.

A reverse transcription-polymerase chain reaction (RT-PCR)-based assay for the detection of tomato spotted wilt virus (TSWV) has been improved and extended to enable the detection of additional Tospoviruses. In addition to TSWV-specific primers, two further pairs of primers have been designed, one pair which specifically detects impatiens necrotic spot virus (INSV) and another which detects all Tospoviruses tested, including TSWV, INSV, tomato chlorotic spot virus and groundnut ringspot virus. An improved, rapid RNA extraction method is also described.

The nucleotide sequence and proposed genome organization of oat chlorotic stunt virus, a new soil-borne virus of cereals.

The complete genomic sequence of a new virus, first found infecting oats in Wales, UK, has been determined. The genome is a positive-sense ssRNA molecule, 4114 nucleotides in length, examination of which indicates the presence of four ORFs. The first ORF initiating at the 5' terminus (ORF1) encodes a protein with a predicted M(r) of 23476 (p23). ORF2 extends through the amber termination codon of ORF1 to give a protein with a predicted M(r) of 84355 (p84). The readthrough domain of p84 contains amino acid sequence similarities with a number of putative RNA-dependent RNA polymerases. ORF3 is in a different reading frame from ORF1/2 and encodes a protein with an M(r) of 48231 (p48), identified as the coat protein by direct peptide sequencing. ORF4 nests within ORF3 but is in a different frame from it and codes for a protein with a predicted M(r) of 8220 (p8). Comparisons of peptide sequence, particularly within the putative polymerase region and within the S domain of the coat protein, highlight similarities with members of both the tombusvirus and carmovirus groups. The coat protein region shows most similarity with members of the tombusvirus group, whilst the size and predicted strategy of the genome seem to be intermediate between that of the carmovirus and tombusvirus groups. These features highlight possible evolutionary links with each group whilst being distinct from both. We propose the name of oat chlorotic stunt for this new virus.

The detection of tomato spotted wilt virus using the polymerase chain reaction.

A polymerase chain reaction (PCR) based assay was used to detected a number of UK tomato spotted wilt virus (TSWV) isolates in total RNA extractions made from infected plant material. Extracts were reverse transcribed and the resultant cDNA amplified by PCR, using oligonucleotide primers specific for a 276 base pair fragment of the L RNA segment. Assay products were electrophoresed on agarose gels and visualised by ethidium bromide staining. The viral origin of the product produced was confirmed by sequencing, with the data obtained having very high homology with previously published L RNA sequence data. The specificity and sensitivity of the RT-PCR assay, in comparison with existing tests, is discussed.

Sweet potato feathery mottle potyvirus (C1 isolate) virion and RNA purification.

A procedure for the purification of a Peruvian isolate (C1) of sweet potato feathery mottle potyvirus (SPFMV) and infective RNA has been developed. The use of Hepes [N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid] buffer containing urea and sodium EDTA as a base for tissue extraction and virus suspension enabled good yields of virus (35-50 mg/100 g) to be obtained from Nicotiana benthamiana L. Domin. A short RNA isolation procedure yielded infectious RNA, from which ds cDNA of nearly genome size could be obtained. Sweet potato feathery mottle potyvirus, Purification, RNA isolation, cDNA synthesis.

Structure of tobraviral particles: a model suggested from sequence conservation in tobraviral and tobamoviral coat proteins.

Comparisons of the coat protein sequences of four tobraviruses with those of seven tobamoviruses indicate that these proteins share a common evolutionary origin. Numerous amino acids for which specific functions have been identified in the molecular structure of the tobacco mosaic virus vulgare protein have identical or closely similar counterparts among the tobraviral proteins. These include those with roles in the hydrophobic core of the protein, those that contribute to the RNA binding site and those involved in the control of virus assembly. We suggest a model for the structure of the tobraviral particle that not only offers an explanation for the greater diameter of the tobraviral particle but also confirms an early suggestion for RNA placement within this particle.

An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi type B and Clostridium perfringens type D epsilon components of multivalent sheep vaccines.

Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins. The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test.

A model for the generation of tobacco rattle virus (TRV) anomalous isolates: pea early browning virus RNA-2 acquires TRV sequences from both RNA-1 and RNA-2.

Comparison of the 5'-terminal sequences of several tobraviruses suggests that the RNA-2 molecule of the tobacco rattle virus (TRV) anomalous isolate TCM arose from pea early browning virus (PEBV) RNA-2 by acquisition of 3' and 5' sequences from TRV RNA-1 and RNA-2 molecules, respectively. We have identified a region of homology in the RNA-2 molecules of PEBV, TRV and pepper ringspot virus which could have facilitated this recombination.

Construction and analysis of infectious transcripts synthesized from full-length cDNA clones of both genomic RNAs of pea early browning virus.

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.

The complete nucleotide sequence of PEBV RNA2 reveals the presence of a novel open reading frame and provides insights into the structure of tobraviral subgenomic promoters.

The 3374 nucleotide sequence of RNA2 from the British PEBV strain SP5 has been determined. The RNA includes three open reading frames flanked by 5' and 3' noncoding regions of 509 and 480 nucleotides. The open reading frames specify coat protein, a 29.6K product homologous to the 29.1K product of TRV(TCM) RNA2 and a 23K product not homologous to any previously described protein. The homology demonstrated between the coat proteins of PRV, TRV and PEBV indicates a common evolutionary origin for these proteins. Upstream of each ORF are located sequences homologous to those with which subgenomic RNAs of other tobraviruses start. Subgenomic RNAs for the expression of the three ORFs may start at these points. On all five tobraviral RNA2 molecules sequenced to date, these sequences were found upstream of the coat protein ORF in association with a strongly-conserved potential secondary structural element. Similar potential structures were identified upstream of other tobraviral ORFs. These structures may contribute to the activity of the tobraviral subgenomic promoter.

Protein synthesis in cucumber mosaic virus-infected tobacco leaves, pre-infected cells and protoplasts.

Polypeptide synthesis has been investigated in cucumber mosaic virus-infected tobacco by radiolabelling and polyacrylamide gel electrophoresis. In addition to the coat protein polypeptide of molecular weight 27,000, others of higher molecular weight ca. 120,000 to greater than or equal to 160,000 (probably two) and possibly one of ca. 32,000--34,000 were detected in the particulate fractions only. A polypeptide of ca. 53,000--57,000 was particularly evident in leaves systemically infected using a differential temperature treatment. Two polypeptides of low molecular weight, ca. 23,000--24,000 and 21,000, were only observed in pre-infected cells and protoplasts and probably represent coat protein degradation products.

Protein and RNA composition of a mild (W) and a severe (P6) strain of cucumber mosaic virus.

The protein and RNA composition of mild (W) and severe (P6) chlorosis-inducing strain of cucumber mosaic virus (CMV) have been compared. The coat polypeptide of both strains migrated with an apparent molecular weight of 26.9 +/- 0.4 (95%) x 10(3) on SDS polyacrylamide gels. They also co-migrated, and showed no anomaly in SDS binding, on SDS agarose gels. Their amino acid compositions were similar except for small differences in the number of alanine, glycine and proline residues. Analysis by double immuno-diffusion revealed a fast migrating antigen, which showed a reaction of identity between strains, and also a slow migrating antigen which showed a reaction of partial identity. Both CMV-P6 and CMV-W possessed a normal genome complement of RNAs 1 to 4. In addition, an isolate of both strains was found that contained large quantities of satellite RNA 5. However, its presence did not modify symptom severity in tobacco or tomato.

The infection of cucumber mesophyll protoplasts with tobacco mosaic virus.

Protoplasts from the first leaf mesophyll of cucumber plants were isolated by an 18 hours combined petinase/cellulase treatment. Conditions favouring the infection of these protoplasts with tobacco mosaic virus (TMV), and the accumulation of infective virus up to 96 hours after inoculation have been studied. Infection of approximately 5--10 per cent of the protoplats, revealed by indirect fluorescent antibody staining, was achieved by pre-treatment of the cells in 0.01 M citrate-buffered mannitol (CBM), pH 5.2 with 2 mug/ml poly-L-ornithine followed by centrifugation and direct resuspension of the cells in the same mixture together with 2 to 4 mug/ml TMV. Higher concentrations of the polycation and buffer were toxic to the protoplasts. Under the best conditions, virus yields of approximately 10-20 mug TMV/10(6) protoplasts were attained, while after 72 hours incubation, significant amounts of virus could often be recovered from the incubation medium. Addition of actinomycin D to cultures of protoplasts 2 hours post-inoculation partially inhibited development of infectivity.

Investigation on the infection of cucumber mesophyll protoplasts with cucumber mosaic virus.

Isolated protoplasts from the first leaf mesophyll of cucumber plants have been successfully infected in vitro with cucumber mosaic virus (CMV). Virus instability before, during and after inoculation of the protoplasts resulted in low infectivities when extracts were assayed on cowpea; however, viral RNA extraction improved the bioassay technique. Attempts to optimize inoculation and incubation of protoplasts are outlined, incorporating the improved assay.

Ribosomal RNA metabolism in cucumber leaf mesophyll protoplasts.

Aspects of the metabolism of RNA have been studied in enzymatically isolated protoplasts from cotyledon and first leaf mesophyll tissue of two cultivars of cucumber. The first leaf mesophyll protoplasts incorporated (3H)-uridine into ribosomal RNA at a constant rate for up to 25 hr in a simple salts medium and for up to 45 hr in a growth medium. Pulse-chase labelling experiments on such preparations showed a rapid dilution of the intracellular (3H)-uridine pool(s) and a high metabolic rate in the cells in one cultivar but not in another. Gel electrophoretic analysis of the RNA from both cotyledon and first leaf protoplasts showed that both protoplast types incorporated either (14C)- or (3H)-uridine into ribosomal RNA species. Incorporation of (3H)-uridine into chloroplasts RNA was minimal in cotyledon protoplasts, but significant in leaf protoplasts. Greater incorporation into the chloroplast RNA species could be achieved by longer pulses. Synthesis of all of the ribosomal RNA species was sensitive to actinomycin D at 10 and 25 mug/ml concentrations in all protoplasts tested.