Use of Degenerate Primers for Partial Sequencing and RT-PCR-Based Assays of Grapevine Leafroll-Associated Viruses 4 and 5.

ABSTRACT Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.

Replication of wild-type and mutant clones of satellite tobacco mosaic virus in Nicotiana benthamiana protoplasts.

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.

Characterization of deletion and frameshift mutants of satellite tobacco mosaic virus.

A series of frameshift and deletion mutations was created in the genome of satellite tobacco mosaic virus (STMV) by modifying full-length cDNA clones of the type strain, from which biologically active transcripts could be synthesized in vitro. Deletions and frameshift mutations in the 5' open reading frame had no effect, compared to wild-type STMV, on RNA accumulation, systemic movement, or the symptoms induced by STMV in Nicotiana tabacum co-inoculated with tobacco mild green mosaic tobamovirus (TMGMV). This implies that the protein encoded by this reading frame is not necessary for biological activity. Deletions and frameshift mutations in the coat protein open reading frame resulted in decreased accumulation of STMV RNA in N. tabacum, although these mutants were still capable of systemic movement, presumably in a nonencapsidated or free RNA form. Furthermore, the mild symptoms induced in tobacco by co-inoculations of wild-type STMV/TMGMV or infection with TMGMV alone were altered to severe systemic necrosis when plants were co-inoculated with these STMV coat protein mutants and TMGMV. Mutants within the 3' untranslated region were much less able to accumulate in TMGMV-infected plants than was wild-type STMV, and under some growth conditions did not accumulate to detectable levels.

Point mutations in cauliflower mosaic virus gene VI confer host-specific symptom changes.

Mutants of cauliflower mosaic virus (CaMV) strain D4 have been characterized with regard to host-specific phenotypes that resulted from specific changes in the viral DNA sequence. Both the mutant and the wild-type viruses infect a brassicaceous host, Brassica campestris, systemically, giving indistinguishable symptoms. However, in the solanaceous host Datura stramonium, which was systemically infectible by the wild-type virus, mutants induced necrotic local lesions at 21 degrees C and above, and a veinal necrosis at lower temperatures. The mutants differed from the parental D4 strain by having single base changes in gene VI. The necrotic phenotype could be selected during serial passage of D4 in B. campestris or created by site-directed mutagenesis within the gene VI coding region. Full-length 62-kDa gene VI gene products were detected in extracts of plants infected with the mutant strains, as were two smaller proteins derived from the same coding region. The relationship of the host-specific phenotypes of the mutants to the detected gene VI-encoded proteins is discussed in the context of the natural variation found in this gene.

Survial from acute renal failure with and without multiple organ dysfunction.

A 10-year retrospective analysis has been carried out of 114 patients dialysed for acute renal failure. Fifty-eight patients, predominantly suffering from multiple organ failure, required treatment in an Intensive Therapy Unit (ITU); 56 less severely ill patients were treated in a Renal Unit. Overall survival in the former group was 36% and in the latter group 63%. In the first 5 years of the study, survival in the ITU patients was 31% and in the second 5 years, was 38% in spite of a trend towards increased severity of illness. These results challenge the view that haemodialysis is rarely worth-while in patients with multiple organ failure, and suggest that current management techniques have improved prognosis. The most important adverse factors continue to be old age, sepsis and gastrointestinal disease.

Day care surgery under general anaesthesia in a purpose built unit.

A survey of 200 patients undergoing surgery under general anaesthesia in a purpose-built day case unit is described. The response rate was 80%. The results demonstrate a high incidence of minor post-operative morbidity, but, despite this, there was a very high degree of patient satisfaction with the service provided in the unit. Suggestions are made with regard to day care surgery under general anaesthesia.