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Characterization of epitopes on zucchini yellow mosaic potyvirus coat protein permits studies on the interactions between strains.

Monoclonal antibodies (MAbs) raised against the coat protein of zucchini yellow mosaic potyvirus (ZYMV) were characterized by epitope mapping using synthetic oligopeptides. Two mutant viruses with a mutation in the amino acid sequence important for epitope recognition in vitro were obtained by site-directed mutagenesis of a full-length cDNA of ZYMV. Two MAbs, CC11 and DD2, could distinguish specifically between these mutants in mixed infections, or after sequential inoculations of muskmelons. Sequential inoculations of the mutants and analysis with MAbs CC11 and DD2 revealed that cross-protection was established between these quasi-isogenic strains within 48 h.

Amino Acid Sequence↗

Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids.

Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid.

Base Sequence↗

Cloning and sequencing of TT virus genotype 6 and expression of antigenic open reading frame 2 proteins.

The near-full-length genome of a TT virus (TTV) (HEL32), closely related to the previously uncharacterized genotype 6, was cloned and sequenced. The genomic organization of HEL32 was compared to 41 published near-full-length TTV sequences representing 17 genotypes. In the majority of genomes, the open reading frame (ORF) 2 region was divided into two separate ORFs, 2a and 2b. The ORF2a sequence was conserved among all genotypes, while the ORF2b region showed more variability. The two corresponding putative proteins of HEL32 were expressed in prokaryotes and their antigenic potential was studied. IgM and IgG antibodies to the respective ORF2-encoded proteins, fp2a and fp2b, and the presence of TTV DNA were studied in the sera of 89 constitutionally healthy adults. By immunoblot using the small TTV proteins as antigens, strong IgM and IgG reactivities were found in 9 and 10% of subjects, respectively. Follow-up studies for 12-15 years of three subjects showed either a persistent coexistence of IgM and TTV DNA or the appearance of viral DNA regardless of pre-existing antibodies. The low prevalence of IgG could be due to the weak immunogenicity of these probably non-structural proteins or to a genotype-specific antibody response. By nested PCR of the conserved ORF2a region, the prevalence of TTV DNA was 85%. TTV genotype 6 sequences were found by specific PCR in 3 of 35 (8.6%) subjects. The low prevalence of TTV IgG compared to the high TTV DNA prevalence, the coexistence of antibodies and viral DNA and the appearance of TTV DNA regardless of pre-existing antibodies suggest that the B-cell immunity against these minor TTV proteins would not be cross protective.

Adult↗

Multiple stress responses in Streptococcus mutans and the induction of general and stress-specific proteins.

The authors have previously demonstrated that Streptococcus mutans shows an exponential-phase acid-tolerance response following an acid shock from pH 7.5 to 5.5 that enhances survival at pH 3.0. In this study the response of S. mutans H7 to acid shock was compared with the responses generated by salt, heat, oxidation and starvation. Prior induction of the acid-tolerance response did not cross-protect the cells from a subsequent challenge by the other stresses; however, prior adaptation to the other stresses, except heat (42 degrees C), protected the cells during a subsequent acid challenge at pH 3.5. Starvation by fivefold dilution of the basal medium (BM) plus fivefold reduction of its glucose content increased the numbers of survivors 12-fold, whereas elimination of glucose from fivefold-diluted BM led to a sevenfold enhancement compared to the control cells; this indicated a relationship between the acid and starvation responses. The stress responses were further characterized by comparing the 2D electrophoretic protein profiles of exponential-phase cells subjected to the various stress conditions. Cells were grown to exponential phase at pH 7.5 (37 degrees C) and then incubated for 30 min under the various stress conditions in the presence of 14C-labelled amino acids followed by cell extraction, protein separation by 2D gel electrophoresis and image analysis of the resulting autoradiograms. Using consistent twofold or greater changes in IOD % as a measure, oxidative stress resulted in the upregulation of 69 proteins, 15 of which were oxidation-specific, and in the downregulation of 24 proteins, when compared to the control cells. An acid shock from pH 7.5 to 5.5 enhanced synthesis of 64 proteins, 25 of them acid-specific, while 49 proteins exhibited diminished synthesis. The dilution of BM resulted in the increased formation of 58 proteins, with 11 starvation-specific proteins and 20 showing decreased synthesis. Some 52 and 40 proteins were enhanced by salt and heat stress, with 10 and 6 of these proteins, respectively, specific to the stress. The synthesis of a significant number of proteins was increased by more than one, but not all stress conditions; six proteins were enhanced by all five stress conditions and could be classified as general stress proteins. Clearly, the response of S. mutans to adverse environmental conditions results in complex and diverse alterations in protein synthesis to further cell survival.

Autoradiography↗

Starvation survival in Listeria monocytogenes: characterization of the response and the role of known and novel components.

The starvation survival response (SSR) of Listeria monocytogenes EGD is induced under glucose- or multiple-nutrient-, but not amino-acid limitation. 0.01-0.2% of the population remain viable even after 20 d and the survivors show a reduced cell size and increased cross-protection to several environmental stresses. The development of the SSR may therefore be important in L. monocytogenes survival in the food environment. The initiation, but not the maintenance, of the SSR involves both protein and cell wall biosynthesis. It is also likely that nutrients released from dead cells are recycled to allow survival of the remaining population. To define the molecular mechanisms involved in the initiation, maintenance and release from the SSR the role of known, and novel, components was examined. The well-characterized regulators SigB and PrfA are both required for the full SSR and effect stress resistance during growth and starvation. A transposon mutagenesis screen identified two novel loci with roles in the SSR and stress resistance. Characterization of the transposon insertion sites revealed a putative homologue of the gene yulB from Bacillus subtilis and a gene of unknown function. The potential individual and combined roles of the SSR components are discussed.

Bacterial Proteins↗

Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves.

Small round viruses (SRV) were isolated from the faeces of diarrhoeic calves from three farms. All three SRV preparations caused diarrhoea experimentally in gnotobiotic calves. Each preparation contained viral particles of two morphological types, "astrovirus-like" and "calicivirus-like", and from one preparation the two particle types were separated from each other. The calicivirus-like agent ("Newbury agent") was 33 nm in diameter, and caused diarrhoea in gnotobiotic calves with villous atrophy and D-xylose malabsorption. This virus did not infect cell cultures. The astrovirus-like agent did not cause diarrhoea in two gnotobiotic calves; however, it infected cell cultures (primary calf kidney) and the infected cells immunofluoresced with convalescent gnotobiotic-calf antiserum. The astrovirus-like agents in the three preparations were antigenically related. Experiments in calves showed that there was a degree of cross-protection between the three SRV preparations, as judged by the presence or absence of diarrhoea, but that at least three unrelated pathogens were present.

Acute Disease↗

Screening of TnphoA mutants of Vibrio cholerae O139 for identification of antigens involved in colonisation.

A new serogroup of Vibrio cholerae non-O1, designated as O139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between O1 and O139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by O1 strains and colonising antigens are known to be protective, investigation of the colonising antigens of O139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising mutants did not express a 40-kDa outer-membrane protein.

Animals↗

Molecular resolution of a complex of potyviruses infecting solanaceous crops at the centre of origin in Peru.

Peru is a centre of origin and domestication of the potato, pepper and tomato (family Solanaceae). Many potyviruses (genus Potyvirus) that infect these crops were described 20-30 years ago. However, definitive classification of these viruses as distinct species remains unresolved for several reasons, including their close serological relationships, similar symptomatology in test plants and lack of genomic sequence data. Using samples collected from Peru, we have determined the complete genomic sequence of two strains of Peru tomato virus (PTV) as well as near-complete sequences for two additional PTV strains. We also obtained partial sequences of four strains of Potato virus V (PVV). Comparisons with genomic sequences of Wild potato mosaic virus (WPMV), Potato virus Y (PVY), Pepper mottle virus (PepMoV), Potato virus A (PVA) and other potyviruses established that all these viruses constitute different taxa (species). Phylogenetic comparisons indicated that PTV, PVV and WPMV are the most closely related species which, together with PepMoV, PVY, Pepper yellow mosaic virus and Pepper severe mosaic virus, constitute a group that is distinguishable from other potyviruses. Therefore, the members of this group may share a common ancestor. PVA does not belong to this group. PVV and PTV were also closely related serologically. However, PTV did not cross-protect against PVV and WPMV in tobacco plants or complement systemic infection of PVV and WPMV in pepper plants. Two biologically and phylogenetically distinguishable strain groups were identified within PTV and PVV. In future studies, the sequence data and virus-specific primers and probes for PTV, PVV and WPMV described in this study will enable accurate indexing of plants with respect to either single or mixed infection with these viruses.

Capsicum↗

Characterization of pathogenic and non-pathogenic African swine fever virus isolates from Ornithodoros erraticus inhabiting pig premises in Portugal.

Ten African swine fever virus isolates from the soft tick Ornithodoros erraticus collected on three farms in the province of Alentejo in Portugal were characterized by their ability to cause haemadsorption (HAD) of red blood cells to infected pig macrophages, using restriction enzyme site mapping of the virus genomes and by experimental infection of pigs. Six virus isolates induced haemadsorption and four were non-haemadsorbing (non-HAD) in pig macrophage cell cultures. The restriction enzyme site maps of two non-HAD viruses, when compared with a virulent HAD isolate, showed a deletion of 9.6 kbp in the fragment adjacent to the left terminal fragment and of 1.6 kbp in the right terminal fragment and an insertion of 0.2 kbp in the central region. The six HAD viruses isolated were pathogenic and produced typical acute African swine fever in pigs and the four non-HAD isolates were non-pathogenic. Pigs that were infected with non-HAD viruses were fully resistant or had a delay of up to 14 days in the onset of disease, after challenge with pathogenic Portuguese viruses. Non-HAD viruses could be transmitted by contact but with a lower efficiency (42-50 %) compared with HAD viruses (100 %). The clinical differences found between the virus isolates from the ticks could have implications for the long-term persistence of virus in the field because of the cross-protection produced by the non-pathogenic isolates. This may also explain the presence of seropositive pigs in herds in Alentejo where no clinical disease had been reported.

African Swine Fever Virus↗

Vector competence of Culicoides species and the seroprevalence of homologous neutralizing antibody in horses for six serotypes of equine encephalosis virus (EEV) in South Africa.

Field-collected Culicoides species (Diptera: Ceratopogonidae) were fed on horse blood-virus mixtures containing one of the six serotypes of equine encephalosis virus (EEV1 to EEV6). The virus mean titres in the bloodmeals varied between 6.1 and 7.0 log10TCID50/mL. Of 19 Culicoides species assayed after 10 days extrinsic incubation at 23.5 degrees C, five yielded the challenge virus, namely Culicoides (Avaritia) imicola Kieffer (EEV1-6), C. (A.) bolitinos Meiswinkel (EEV1, 2, 4, 6), C. (Meijerehelea) leucostictus Kiefer (EEV1, 2), C. (Culicoides) magnus Colaço (EEV1) and C. (Hoffmania) zuluensis de Meillon (EEV2). Virus recovery rates ranged from 0.5 to 13%. The mean levels of viral replication differed between serotypes and Culicoides species and ranged from 1.0 to 2.3 log10TCID50/midge. Culicoides midges shown in this study to be susceptible to oral infection with EEV are widely distributed in South Africa but differ considerably in their abundance, host preference and breeding sites. Of 1456 horses tested, 1144 (77%) had antibody to EEV. Homologous virus-neutralizing antibodies to all six serotypes were detected in individual horses from all eight geographical provinces of South Africa. The distribution, prevalence, and the rate of exposure to individual serotypes varied significantly between regions. The potential for vectoring of EEV in the field by several Culicoides species with unique ecologies and lack of cross-protection to re-infection with multiple serotypes highlights some of the mechanisms that are likely to play a role in the virus' natural maintenance cycle and the highly efficient level of countrywide transmission amongst South African horses.

Animals↗

T cell clones from a non-leprosy exposed subject recognize the Mycobacterium leprae 18-kD protein.

Although Mycobacterium leprae shares many protein antigens with other mycobacterial species, there is a degree of specificity in the T cell response to the organism. This is evident in the failure of cross-protection between mycobacterial species and the specific unresponsiveness to M. leprae in lepromatous leprosy patients. The antigenic basis of this specificity is unresolved, but the M. leprae 18-kD protein is one candidate because of its restricted distribution and the isolation of M. leprae-specific T cell clones reactive with the protein from M. leprae-vaccinated subjects. In the course of analysing the human T cell repertoire to mycobacteria we have isolated further CD4+ T cell clones reactive with this protein from a subject who had never been exposed to M. leprae. These clones did not respond to other mycobacteria, including M. tuberculosis and M. bovis (BCG). In addition, they were unreactive with the M. tuberculosis 16-kD protein which has recently been shown to have limited amino acid identity with the M. leprae 18-kD protein. Both clones reacted with peptide 38-50 from the M. leprae 18-kD protein, the T cell response to which is restricted by HLA-DR4. Although homologues for the gene encoding the M. leprae 18-kD antigen have been identified in M. avium and M. intracellulare, the clones failed to respond to preparations of M. avium. Both clones secreted interferon-gamma (IFN-gamma) and tumour necrosis factor-beta (TNF-beta) and were cytolytic against autologous targets pulsed with peptide 38-50 or the 18-kD protein. The nature of the antigen which stimulates this apparently 'M. leprae-specific' response is unknown. Nevertheless the recognition of the 18-kD protein by individuals not exposed to leprosy indicates that this protein may not be suitable as a reagent to distinguish between infection with M. leprae and other pathogenic mycobacteria.

Adult↗

Vibrio vulnificus serovar A: an emerging pathogen in European anguilliculture.

The spread of the emerging pathogen Vibrio vulnificus biotype 2 serovar A in Danish anguilliculture is reported. Serovar A was originally isolated in a Spanish eel farm in 2000 and occurred in Denmark in the summer of 2004, affecting eels of 5-10 g body weight cultured in fresh water. The Danish eels showed clinical signs different from those reported for Spanish eels, such as severe haemorrhages in the head and gill region with necrosis of the soft tissues. Danish isolates were biochemically and serologically identical to Spanish serovar A strains and also highly virulent for eels by both intraperitoneal injection and immersion challenges. Vaccination with Vulnivaccine, a vaccine against V. vulnificus serovar E, cross-protected eels against serovar A. The LD(50) for experimentally infected vaccinated animals was significantly higher than for non-vaccinated animals.

Animals↗

Adaptive larval thermotolerance and induced cross-tolerance to propoxur insecticide in mosquitoes Anopheles stephensi and Aedes aegypti.

Fourth-instar larvae of mosquitoes Anopheles stephensi and Aedes aegypti normally died within 90 min at 43 degrees C. Pre-exposure to high but sublethal temperatures conferred adaptive thermotolerance, dependent on the temperature and the duration of pre-exposure. Adaptive cross-tolerance to propoxur (a carbamate insecticide) was also induced in larvae by pre-exposing them to sublethal temperatures. Pre-exposure to sublethal concentrations of propoxur was found to confer cross-thermotolerance to a lower extent. These results suggest that the shock proteins (e.g. heat shock proteins) induced by unrelated stress factors play an important role in the development of adaptive cross-protection (stress response) to other stress conditions.

Aedes↗

Adaptive evolution of the Bordetella autotransporter pertactin.

The virulence factor pertactin is expressed by the closely related pathogens Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. Pertactin is an autotransporter involved in adherence of Bordetella species to the lung epithelium of mammalian hosts, and it is an important component of most current acellular pertussis vaccines. These three species produce immunologically distinct pertactin molecules, resulting in a lack of cross-protection against B. parapertussis and probably also against B. bronchiseptica. Variation in pertactin is not only inter-specific, but also occurs between isolates from the same species. Knowledge about codons that are under positive selection could facilitate the development of more broadly protective vaccines. Using different nucleotide substitution models, pertactin genes from B. bronchiseptica, B. parapertussis and B. pertussis were compared, and positively selected codons were identified using an empirical Bayesian approach. This approach yielded 15 codons predicted to be under diversifying selection pressure. These results were interpreted in an immunological context and may help in improving future pertussis vaccines.

Bacterial Outer Membrane Proteins↗

Chemical structure of the lipid A of Escherichia coli J-5.

The lipopolysaccharide, and particularly its lipid A moiety, of the J-5 mutant of Escherichia coli O111 plays a central role in studies on potential induction of cross-reactive and cross-protective antibodies, however, its chemical and antigenic structure was hitherto unknown. Here, the chemical structure of the J-5 lipid A is reported. It is composed of the bisphosphorylated disaccharide beta-D-GlcpN-4-P-(1-6)-alpha-D-GlcpN-1-P which carries four residues of 3-hydroxytetradecanoic acid, one each at positions 2, 3, 2', and 3'. The hydroxyl groups of the acyl residues at 2' and 3' are esterified with dodecanoic and tetradecanoic acid, respectively. The hydroxyl group at C-6' functions in the lipopolysaccharide as the attachment site of the core oligosaccharide. Furthermore, a new method to isolate the hydrophilic backbone, i.e. the 1,4'-bisphosphorylated glucosamine disaccharide, and its structural analysis by 1H-, 13C-, and 31P-NMR spectroscopy, are described, leading to a new and easier strategy in structural analysis of lipid A from bacterial lipopolysaccharides.

Carbohydrate Sequence↗

Characterization of toxin from different strains of Pasteurella multocida serotype A and D.

Chromosomal DNA from 13 different selected Pasteurella multocida spp. multocida strains of serotypes A and D were isolated and compared. All 10 toxigenic strains were recognized by a DNA probe which included the toxA gene coding for the Pasteurella multocida toxin (PMT). None of the three nontoxigenic strains reacted with the DNA probe. Toxin from the 10 toxigenic strains were isolated and compared. All were found to possess the biological characteristics previously described for the PMT isolated from P. multocida ssp. multocida NCTC 12178, including molecular mass of approx. 143 kDa and reactivity with a series of monoclonal antibodies. Toxin prepared from different toxigenic strains could not be differentiated immunologically by tandem crossed immunoelectrophoresis, Toxin, which was affinity purified from four of the strains and subsequently inactivated by formaldehyde, was cross-protective when used for vaccination of mice before challenge with PMT. It is concluded that the toxin from toxigenic strains of P. multocida ssp. multocida must be very similar, if not identical.

Animals↗

Thermal tolerance of acid-adapted and unadapted Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice.

AIMS: A study was performed to determine D values of acid-adapted and unadapted cells of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice. METHODS AND RESULTS: Salmonella enterica serotype Poona, S. enterica serotype Saphra, two strains of E. coli O157:H7, and two strains of L. monocytogenes were grown in tryptic soy broth (TSB) and TSB supplemented with 1% glucose for 24 h at 37 degrees C. Decimal reduction times (D values) of cells suspended in unpasteurized cantaloupe juice and watermelon juice were determined. Acid-adapted cells of Salmonella and E. coli O157:H7, but not L. monocytogenes, had increased thermal tolerance compared with cells that were not acid-adapted. There was no correlation between soluble solids content of the two types of juice and thermal resistance. CONCLUSIONS: Growth of Salmonella and E. coli O157:H7 in cantaloupe juice, watermelon juice, or other acidic milieu, either in preharvest or postharvest environments, may result in cross protection to heat. The pasteurization conditions necessary to achieve elimination of pathogens from these juices would consequently have to be more severe if cells are habituated to acidic environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Insights from this study provide guidance to developing pasteurization processes to eliminate Salmonella, E. coli O157:H7, and L. monocytogenes in cantaloupe juice and watermelon juice.

Adaptation, Physiological↗

Excitatory action of sympathomimetic amines on 5-hydroxytryptamine receptors of gut.

1. Twenty-two sympathomimetic amines were tested for excitatory activity on isolated guinea-pig ileum, rabbit jejunum, and rat stomach.2. Eight amines contracted all or almost all guinea-pig ileum preparations; five amines contracted half the preparations and relaxed the others. Nine amines were consistently relaxant. Structural requirements for excitatory activity were not clear.3. Six amines contracted rat stomach and only four contracted rabbit jejunum. Only beta-phenylethylamine and (-)-amphetamine contracted all three preparations.4. The excitatory effects of the sympathomimetic amines on guinea-pig ileum seem to be due to an action on 5-hydroxytryptamine receptors. This conclusion is based on evidence that their excitatory action is antagonized by 2-bromolysergic acid diethylamide, by morphine and by desensitization with 5-hydroxytryptamine, and that there is cross-protection between 5-hydroxytryptamine and the excitatory sympathomimetic amines against block by phenoxybenzamine.

Amphetamine↗