Near identity of 3′ RNA secondary structure in bromoviruses and cucumber mosaic virus

The 3′ terminal sequences of RNAs 1, 2, 3 and 4 from each of the three bromoviruses (brome mosaic, cowpea chlorotic mottle and broad bean mottle viruses) and also from cucumber mosaic virus display interviral sequence similarity in addition to strong intraviral homology. Interviral similarity is much more evident when RNA secondary, rather than primary, structures are compared. The last 190 bases of the various RNAs can fold into strikingly similar, extensively base-paired secondary structures whose common features are supported by RNA structure mapping. The extreme 3′ end of each viral RNA can base-pair in two distinct configurations. Bromovirus RNA 3s each contain an unusually accessible internal oligo(A) sequence which, in brome mosaic virus at least, is located in the intercistronic noncoding region. Functional implications of these structural features are discussed.     

Variation of seed α-amylase inhibitors in the common bean

Variation of seed -amylase inhibitors was investigated in 1 154 cultivated and 726 non-cultivated (wild and weedy) accessions of the common bean, Phaseolus vulgaris L. Four -amylase inhibitor types were recognized based on the inhibtion by seed extracts of the activities of porcine pancreatic -amylase and larval -amylase and larval -amylase of the Mexican bean weevil, Zabrotes subfasciatus Boheman. Of the 1 880 accessions examined most (1 734) were able to inhibit porcine pancreatic -amylase activity, but were inactive against the Z. subfasciatus larval -amylase; 41 inhibited only the larval -amylase activity, 52 inhibited the activities of the two -amylases, and 53 did not inhibit the activity of either of the -amylases. The four different inhibitor types were designated as AI-1, AI2, AI-3, and AI-0, respectively. These four inhibitor types were identified by the banding patterns of seed glycoproteins in the range of 14–20 kDa by using SDSpolyacrylamide gel electrophoresis. Additionally, four different banding patterns were recognized in accessions with AI-1, and were designated as AI-1a, 1b, 1c, and 1d. Two different patterns of the accessions lacking an -amylase inhibitory activity were identified and designated as AI-0a and AI-0b. The largest diversity for seed -amylase inhibitors was observed in non-cultivated accessions collected from Mexico where all eight inhibitor types were detected. The possible relationships between the variation of seed -amylase inhibitors and bruchid resistance are discussed.     

Inheritance of seed α-amylase inhibitor in the common bean and genetic relationship to arcelin

The inheritance of seed -amylase inhibitor in the common bean and the genetic relationships among the variants and six arcelin variants in the common bean were investigated by crossing between accessions containing different AI and arcelin variants. All seed proteins in parental, F₁ and F₂ seeds from the crosses were examined by Western-blot analysis. All F₁ seeds gave combined AI banding patterns from parents on the blotting membranes. The segregation of F₂ seeds for AI variants indicated that the polypeptides of AI variants were inherited as single co-dominant units. Moreover, AI and arcelin behaved as a single block in crosses, indicating a close linkage relationship between the genes controlling these proteins.     

Discovery of the first virus, the tobacco mosaic virus: 1892 or 1898?

Two scientists contributed to the discovery of the first virus, Tobacco mosaic virus. Ivanoski reported in 1892 that extracts from infected leaves were still infectious after filtration through a Chamberland filter-candle. Bacteria are retained by such filters, a new world was discovered: filterable pathogens. However, Ivanovski probably did not grasp the full meaning of his discovery. Beijerinck, in 1898, was the first to call 'virus', the incitant of the tobacco mosaic. He showed that the incitant was able to migrate in an agar gel, therefore being an infectious soluble agent, or a 'contagium vivum fluidum' and definitively not a 'contagium fixum' as would be a bacteria. Ivanovski and Beijerinck brought unequal but decisive and complementary contributions to the discovery of viruses. Since then, discoveries made on Tobacco mosaic virus have stood out as milestones of virology history.     

Binding between elongation factor 1A and the 3ʹ-UTR of Chinese wheat mosaic virus is crucial for virus infection

The Chinese wheat mosaic virus (CWMV) genome consists of two positive-strand RNAs that are required for CWMV replication and translation. The eukaryotic translation elongation factor (eEF1A) is crucial for the elongation of protein translation in eukaryotes. Here, we show that silencing eEF1A expression in Nicotiana benthamiana plants by performing virus-induced gene silencing can greatly reduce the accumulation of CWMV genomic RNAs, whereas overexpression of eEF1A in plants increases the accumulation of CWMV genomic RNAs. In vivo and in vitro assays showed that eEF1A does not interact with CWMV RNA-dependent RNA polymerase. Electrophoretic mobility shift assays revealed that eEF1A can specifically bind to the 3ʹ-untranslated region (UTR) of CWMV genomic RNAs. By performing mutational analyses, we determined that the conserved region in the 3ʹ-UTR of CWMV genomic RNAs is necessary for CWMV replication and translation, and that the sixth stem-loop (SL-6) in the 3ʹ-UTR of CWMV genomic RNAs plays a key role in CWMV infection. We conclude that eEF1A is an essential host factor for CWMV infection. This finding should help us to develop new strategies for managing CWMV infections in host plants.     

Isolation of recombinant field strains of Marek’s disease virus integrated with reticuloendotheliosis virus genome fragments

Two Marek's disease virus (MDV) field strains were isolated from chickens with tumors independently from Guangdong and Guangxi provinces, and it was confirmed that there were no co-infections with reticuloendotheliosis viruses (REV) in chicken embryo fibroblast cells (CEF) in indirect fluorescence antibody test (IFA) with REV-specific monoclonal antibodies. By dot blot hybridization and PCR of genomic DNA of MDV-infected CEF, it was indicated that LTR fragments of REV genome were integrated into genome of these two MDV field strains. To amplify and clone the integrated REV LTR with MDV sequence at the junction, 4 primers from REV LTR and 7 primers from MDV genome fragment with REV LTR insertion hot points were synthesized and 28 (4x7) pairs of primers (one from REV and another from MDV for each pair) were used in PCR while using the genomic DNA of both strains as the templates. The sequence data demonstrated that both recombinant field strains contained the same REV LTR inserted into MDV at the identical sites in US fragment of the genomes. From the above, it was speculated that both recombinant field MDVs were originated from a same recombinant virus and spread among chicken flocks in two provinces.     

Assessment of the importance of α-amylase inhibitor-2 in bruchid resistance of wild common bean

Both α-amylase inhibitor-2 (αAI-2) and arcelin have been implicated in resistance of wild common bean (Phaseolus vulgaris L.) to the Mexican bean weevil (Zabrotes subfasciatus Boheman). Near isogenic lines (NILs) for arcelin 1–5 were generated by backcrossing wild common bean accessions with a cultivated variety. Whereas seeds of a wild accession (G12953) containing both αAI-2 and arcelin 4 were completely resistant to Z. subfasciatus, those of the corresponding NIL were susceptible to infestation, suggesting that the principal determinant of resistance was lost during backcrossing. Three independent lines of transgenic azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] expressing αAI-2 accumulated high levels of this protein in seeds. The expression of αAI-2 in these lines conferred protection against the azuki bean weevil (Callosobruchus chinensis L.), likely through inhibition of larval digestive α-amylase. However, although the seed content of αAI-2 in these transgenic lines was similar to that in a wild accession of common bean (G12953), it did not confer a level of resistance to Z. subfasciatus similar to that of the wild accession. These results suggest that αAI-2 alone does not provide a high level of resistance to Z. subfasciatus. However, αAI-2 is an effective insecticidal protein with a spectrum of activity distinct from that of αAI-1, and it may prove beneficial in genetic engineering of insect resistance in legumes.     

Epidemics of Tomato torrado virus,Pepino mosaic virus and Tomato chlorosis virus in tomato crops: do mixed infections contribute to torrado disease epidemiology?

Torrado disease was first observed in protected tomato crops in the Murcia province of Spain in spring 2001, causing serious concern to regional tomato producers. The disease-causing agent was initially identified as a picorna-like bipartite plant RNA virus, now known as Tomato torrado virus (ToTV), but several additional torradoviruses inducing similar disease symptoms have been described more recently. We studied the incidence of torradoviruses between 2005 and 2008 in two parts of Murcia (Spain) where tomato crops are grown commercially. We also analysed the potential association among ToTV, Pepino mosaic virus (PepMV) and Tomato chlorosis virus (ToCV) in samples showing torrado symptoms of varying severity. ToTV was the only torradovirus found in the samples (predominantly as single infections), but double and triple infections comprising ToTV, PepMV and/or ToCV were also detected. There was no evidence of a specific association among the viruses as the frequencies of mixed infections did not deviate from those expected to occur by chance. Statistical analysis of the potential association between torrado symptoms and the type of infection (single or multiple) was inconclusive. To determine whether co-infections with ToTV and PepMV have any marked influence on the torrado disease, we analysed torrado symptom severity and virus accumulation in tomato plants experimentally infected with ToTV-CE, PepMV-Sp13 and PepMV-PS5 in single and mixed infections. The severity of the torrado symptoms was not affected by the presence of PepMV. In single infections, the ToTV titre remained very low, reaching its maximum in the early stages of infection and declining rapidly thereafter, whereas the disease symptoms became more severe over the same timescale. In mixed infections, the accumulation of both ToTV and PepMV was altered with respect to single infections, and the magnitude of this alteration appeared to be virus and strain specific. Therefore, ToTV and PepMV mixed infections may modulate the epidemiology of both viruses in a complex way by altering virus fitness. The impact of our studies on efforts to track and prevent the spread of torrado disease is discussed.     

Presence of double‐stranded RNAs and virus‐like particles in the entomopathogenic fungus Metarhizium anisopliae

Nucleic acids from 41 strains of Metarhizium anisopliae, obtained from different parts of the world were extracted and examined by electrophoresis. Strong bands of double‐stranded RNA (dsRNA) were detected in two isolates from Brazil, V215 and V291, which had, respectively, 13 and 9 distinct bands ranging in size from ca. 0.75 to 3.5 kb. Icosahedral virus‐like particles (VLPs) (ca. 33 nm in diameter) were observed by transmission electron microscopy in extracts of these isolates. The VLPs and dsRNA were both absent from a clone of the isolate V291 which had been subcultured successively on solid medium. Bioassays against the aphid Myzus persicae showed no detectable difference in virulence between the clone of V291 which contained dsRNA and the clone that did not.     

Evolutionary relationships among proteins in the phytohemagglutinin-arcelin-α-amylase inhibitor family of the common bean and its relatives

The common bean, Phaseolus vulgaris, contains a family of defense proteins that comprises phytohemagglutinin (PHA), arcelin, and -amylase inhibitor (AI). Here we report eight new derived amino acid sequences of genes in this family obtained with either the polymerase chain reaction using genomic DNA, or by screening cDNA libraries made with RNA from developing beans. These new sequences are: two AI sequences and arcelin-4 obtained from a wild accession of P. vulgaris that is resistant to the Mexican bean weevil (Zabrotes subfasciatus) and the bean weevil (Acanthoscelides obtectus); an AI sequence from the related species P. acutifolius (tepary bean); a PHA and an arcelin-like sequence from P. acutifolius; an AI-like sequence from P. maculatus; and a PHA sequence from an arcelin-5 type P. vulgaris. A dendrogram of 16 sequences shows that they fall into the three identified groups: phytohemagglutinins, arcelins and AIs. A comparison of these derived amino acid sequences indicates that one of the four amino acid residues that is conserved in all legume lectins and is required for carbohydrate binding is absent from all the arcelins; two of the four conserved residues needed for carbohydrate binding are missing from all the AIs. Proteolytic processing at an Asn-Ser site is required for the activation of AI, and this site is present in all AI-like sequences; this processing site is also found at the same position in certain arcelins, which are not proteolytically processed. The presence of this site is therefore not sufficient for processing to occur.     

A review of MHC‐based mate preferences and fostering experiments in two congenic strains of mice

We review our studies of mate choice with two MHC‐congenic strains of mice. This work was stimulated by findings from Yamazaki and colleagues showing that male mice exhibited mate preferences for females whose MHC‐haplotype was different from their own, while female mice exhibited either no preference or a weak preference for males of a particular MHC‐haplotype (see Beauchamp et al., 1988). Since these findings were unexpected (mate choice theory predicts that females will be more selective than males), we studied the preferences of mice from two additional MHC‐congenic strains to assess the generality of the previous findings. Specifically, the goals of our research were: (1) to determine the mate preferences of congenic mice with MHC‐haplotypes derived from wild populations, (2) to compare the mate preferences of male and female mice in a test situation where each sex has a clear opportunity to make a choice, and (3) to estimate effects of cross‐fostering on each sex. This revised version was published online in July 2006 with corrections to the Cover Date.     

Selection,development and testing of phage‐resistant strains of Serratia entomophila for grass grub control

Use of the bacterium Serratia entomophila as an inundative biological control agent for the New Zealand grass grub (Costelytra zealandica) depends on the consistent production of high yields of the bacterium in liquid fermentation. Following the phage related failure of several S. entomophila fermentations, a programme was initiated to isolate phage‐resistant strains. No naturally occurring strains were found to be resistant to eight stocks of phage isolated from either grass grub larvae or the fermenter. Therefore, ethylmethane sulphonate was used to generate phage‐resistant mutants. Strains which showed cross resistance to all eight phage test stocks were tested for pathogenicity towards grass grub. Several strains showing unimpaired pathogenicity were selected for further tests. Four of these phage‐resistant strains produced high cell yields, even when grown in the presence of high numbers of fermenter‐derived phage. Phage‐resistant strains have subsequently been produced in bulk in industrial fermenters, for use in large‐scale field trials throughout New Zealand.     

Use of ELISA and GUS‐transformed strains to study competition between pathogenic and non‐pathogenic Fusarium oxysporum for root colonization

To characterize the ability of different strains of Fusarium oxysporum to colonize roots, and to analyze competition for root colonization between pathogenic and non‐pathogenic strains of F. oxysporum, it was necessary to develop specific labelling techniques for quantification of root colonization. Two methods were selected: the production of polyclonal antibodies, and the use of GUS‐transformed strains of F. oxysporum. The polyclonal antibodies recognized infected plants, and gave a minimum reaction with healthy plants, but were not specific for individual strains of F. oxysporum. These antibodies enabled total density of F. oxysporum to be assessed on roots, by ELISA. Metabolic activity of the root population of GUS‐marked strains was assessed by measuring the glucuronidase activity. Strains showed a diversity in their ability to colonize roots: patterns of root colonization were similar, but the intensity and the speed of colonization differed according to the plant—fungus combination used. Results demonstrated competition between the pathogenic and the non‐pathogenic strains for root colonization. In the presence of the non‐pathogenic strain Fo 47, the competition seems to be reciprocal, affecting both the pathogen and non‐pathogenic strain. Other non‐pathogenic strains reduced root colonization by the pathogenic strain, but some strains did not reduce the metabolic activity of the pathogen, suggesting that different mechanisms are involved in the interaction between pathogenic and non‐pathogenic F. oxysporum.     

Effect of cyclopentene ��,�¡�-triketone disodium salt on the activity of hydrolases and the state of tobacco mosaic virus particles in tobacco leaves

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.     

Why are dengue virus serotypes so distantly related? Enhancement and limiting serotype similarity between dengue virus strains

Dengue virus, the causative agent of dengue fever, has four major serotypes characterized by large genetic and immunological distances. We propose that the unusually large distances between the serotypes can be explained in the light of a process of antibody-dependent enhancement (ADE) leading to increased mortality. Antibody-dependent enhancement results from a new infection with a particular serotype in an individual with acquired immunity to a different serotype. Classical dengue fever causes negligible mortality, but ADE leads to the risk of developing the significantly more dangerous dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). A mathematical model is presented that describes the epidemiological dynamics of two serotypes of a pathogen where there is the possibility of co-infection and reinfection by a different serotype, along with increased mortality as a result of enhancement. We show that if there is no or slightly increased mortality after reinfection (enhancement), serotypes with a small immunological distance can stably coexist. This suggests that a cloud of serotypes with minor serological differences will constitute the viral population. By contrast, if enhancement is sufficiently great, a substantial immunological distance is necessary for two serotypes to stably coexist in the population. Therefore, high mortality owing to enhancement leads to an evolutionarily stable viral community comprising a set of distantly separated serotypes.     

Non‐verbal interaction of Anglo‐Canadian,Jewish‐Canadian,and French‐Canadian physicians with their young,middle‐aged,and elderly patients

This study investigates the role that non‐verbal communication plays in establishing and maintaining rapport among physicians and their young, middle‐aged, and elderly patients.

Eleven physicians with a variety of patients were studied in the project in Montreal. Five physicians were Anglo‐Canadian, three were respectively Jewish‐Canadian and French‐Canadian. Three additional physicians were part of a pilot project.

Patient‐doctor interviews were videotaped by means of a stationary camera fitted with a wide‐angle lens placed in the doctors’ offices. Physical examinations were not videotaped. After each session, patients filled out a questionnaire elucidating the success of the interview.