Effects of Grapevine Leafroll associated Virus 3 (GLRaV-3) and duration of infection on fruit composition and wine chemical profile of Vitis vinifera L. cv. Sauvignon blanc.

In order to determine the effects of Grapevine Leafroll associated Virus 3 (GLRaV-3) on fruit composition and chemical profile of juice and wine from Vitis vinifera L. cv. Sauvignon blanc grown in New Zealand, composition variables were measured on fruit from vines either infected with GLRaV-3 (established or recent infections) or uninfected vines. Physiological ripeness (20.4°Brix) was the criterion established to determine the harvest date for each of the three treatments. Date of grape ripeness was strongly affected by virus infection. In juice and wine, GLRaV-3 infection prior to 2008 reduced titratable acidity compared with the uninfected control. Differences observed in amino acids from the three infection status groups did not modify basic wine chemical properties. In conclusion, GLRaV-3 infection slowed grape ripening, but at equivalent ripeness to result in minimal effects on the juice and wine chemistry. Time of infection produced differences in specific plant physiological variables.

Identification and validation of reference genes for normalization of transcripts from virus-infected Arabidopsis thaliana.

Real-time quantitative polymerase chain reaction (qPCR) of complementary DNA is now a standard method for studies of gene expression. However, qPCR can identify genuine variation only when transcript quantities are accurately normalized to an appropriate reference. To identify the most reliable reference genes for transcript quantification by qPCR, we describe a systematic evaluation of candidate reference genes of Arabidopsis thaliana ecotype Columbia-0 (Col-0). Twelve genes were selected for transcript stability studies by qPCR of complementary DNA prepared from Arabidopsis leaf tissue infected with one of five plant viruses (Cauliflower mosaic virus, Tobacco mosaic virus, Tomato spotted wilt virus, Turnip mosaic virus, and Turnip yellow mosaic virus). The F-box family protein, elongation factor 1-α, sand family protein, and protodermal factor 2 gene transcripts showed the most stable accumulation, whereas a traditionally used reference gene, Actin8, showed the least stable accumulation as measured by the geNorm algorithm. The data furnish plant virologists with reference genes for normalization of qPCR-derived gene expression in virus-infected Arabidopsis and will be beneficial to the selection and design of primers targeting orthologous genes in other plant species.

RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double-stranded RNA feeding.

RNA interference (RNAi) or gene silencing is typically induced in insects by the injection of double-stranded RNAs (dsRNAs), short interfering RNAs, or through the use of hairpin constructs in transgenic insects. Here we demonstrate in the horticultural pest, Epiphyas postvittana (Lepidoptera: Tortricidae), that RNAi can be triggered by oral delivery of dsRNA to larvae. Transcript levels of a larval gut carboxylesterase gene (EposCXE1) were reduced to less than half that of controls within 2 days of being fed EposCXE1 dsRNA. Transcript levels of the pheromone binding protein gene (EposPBP1) were reduced in adult antennae by feeding larvae EposPBP1 dsRNA. Knockdown of EposPBP1 transcripts was observed for the first 2 days after adult eclosion but recovered to wild-type levels at 4 days posteclosion. The potential mechanisms involved in the initiation, movement and amplification of the silencing signal are discussed.

An in vitro-selected RNA-binding site for the KH domain protein PSI acts as a splicing inhibitor element.

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

Dark green islands in plant virus infection are the result of posttranscriptional gene silencing.

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.

Three approaches to use of questioning by clinical lecturers: a pilot study.

In the current health care climate nurses require very good problem solving and critical thinking skills. Questioning as a teaching strategy is viewed as one way to promote such student learning. Using a comparative descriptive quantitative and a qualitative approach, this pilot study investigated the types of questions asked of students by lecturers working within the preceptorship model in the clinical setting. A convenience sample of five volunteer nursing lecturers were tape recorded during their interactions with undergraduate students (n = 8). Initially two auditing approaches were used to analyse the interview data: relevant parts of Mogan and Warbinek's (1994) Observation of Nursing Teachers in Clinical Settings instrument (ONTICS Tool) and Craig and Page's (1981) conceptual framework as adapted by Sellappah, Hussey, Blackmore and McMurray (1998). The data were further analysed by qualitative content analysis. This study supported the findings of the ONTICS tool and Sellappah et al's framework that teachers asked predominantly directive style and low level questions. What the two approaches did not adequately capture was the complexity of the lecturers' questioning behaviours or the effects of contextual factors. The content analysis process however, identified three broad categories forming a model that effectively integrated aspects of the context of the lecturer/student interaction. It also represented lecturer questioning behaviours more comprehensively. The preliminary model offered has the potential to highlight the importance of lecturers asking questions that lead students to extend their thinking about practice. It could also contribute to student learning by assisting lecturers to understand the value and critical nature of their questioning and serve as a framework for staff development.

A study of acute respiratory disease in the community of Port Chalmers. III. Efficacy of influenza virus subunit vaccines in 1973, 1974 and 1975.

An evaluation of the efficacy of influenza virus subunit vaccine was undertaken during a study of acute respiratory disease in the semi-isolated community of Port Chalmers, New Zealand. In 1973, the administration of a vaccine containing A/England/42/72(H3N2) and B/Roma/1/67 stains was found to produce HI antibody titres greater than or equal to 1:40 to the A and B components in 50 percent of 32 subjects. There was no significant NI antibody response to the A component. During an epidemic of A/Port Chalmers/I/73(H3N2) occurring three to four months after vaccination, vaccinees were not protected from clinical infection. Sixty and ninety-nine subjects received vaccine containing A/Port Chalmers/1/73 and B/Hong Kong/8/73 in 1974 and 1975 respectively. In 1974 all 60 subjects received a second dose of vaccine which was shown to have little effect on the distribution of HI antibody titres. The benefits of annual vaccination in this general practice are unclear.

A study of acute respiratory disease in the community of Port Chalmers. I. Illnesses within a group of selected families and the relative incidence of respiratory pathogens in the whole community.

A study of respiratory diseases in the semi-isolated community of Port Chamlers, New Zealand, began in April 1973. The intensive surveillance of a selected group fo 26 families involved the weekly reporting of illness, the collection of specimens for virus, Group A streptococci and Mycoplasma pneumoniae isolation and the collection of sera at 6-month intervals. A total of 956 illnesses were reported during 32 months. The median number of illnesses per year were: infants 4.4, children 2.5, female adults 2.4 and male adults 2.0. Of all these illnesses, 57% were upper respiratory, 31% were lower respiratory and 9% were enteric. The severity of these illnesses was not greater than would be expected in open communities. Surveillance by pathogen isolation only of the whole community through the patients in the general practice was carried out concurrently. A total of 640 nasopharyngeal swab specimens were collected from which 161 viruses, 47 Group A streptococci and 2 M. pneumoniae were isolated. The overall isolation rate was 33%. The similarities between the epidemiological patterns of respiratory disease in the open community and the isolated community are discussed.