Detection and monitoring of human bocavirus 1 infection by a new rapid antigen test.

Clinically relevant diagnosis of human bocavirus 1 (HBoV1) is challenging, as the virus is frequently detected in asymptomatic patients, and cofindings with other respiratory viruses are common. The clinical value of current diagnostic methods, such as PCR, is therefore low, and alternative diagnostic strategies are needed. We describe for the first time the use of an antigen detection assay for the rapid identification of HBoV1 in a paediatric patient with respiratory tract infection symptoms. We estimate the duration of active HBoV1 infection to be 6 days.

Status and prospects of plant virus control through interference with vector transmission.

Most plant viruses rely on vector organisms for their plant-to-plant spread. Although there are many different natural vectors, few plant virus-vector systems have been well studied. This review describes our current understanding of virus transmission by aphids, thrips, whiteflies, leafhoppers, planthoppers, treehoppers, mites, nematodes, and zoosporic endoparasites. Strategies for control of vectors by host resistance, chemicals, and integrated pest management are reviewed. Many gaps in the knowledge of the transmission mechanisms and a lack of available host resistance to vectors are evident. Advances in genome sequencing and molecular technologies will help to address these problems and will allow innovative control methods through interference with vector transmission. Improved knowledge of factors affecting pest and disease spread in different ecosystems for predictive modeling is also needed. Innovative control measures are urgently required because of the increased risks from vector-borne infections that arise from environmental change.

The association of recombination events in the founding and emergence of subgenogroup evolutionary lineages of human enterovirus 71.

Enterovirus 71 (EV71) is responsible for frequent large-scale outbreaks of hand, foot, and mouth disease worldwide and represent a major etiological agent of severe, sometimes fatal neurological disease. EV71 variants have been classified into three genogroups (GgA, GgB, and GgC), and the latter two are further subdivided into subgenogroups B1 to B5 and C1 to C5. To investigate the dual roles of recombination and evolution in the epidemiology and transmission of EV71 worldwide, we performed a large-scale genetic analysis of isolates (n = 308) collected from 19 countries worldwide over a 40-year period. A series of recombination events occurred over this period, which have been identified through incongruities in sequence grouping between the VP1 and 3Dpol regions. Eleven 3Dpol clades were identified, each specific to EV71 and associated with specific subgenogroups but interspersed phylogenetically with clades of coxsackievirus A16 and other EV species A serotypes. The likelihood of recombination increased with VP1 sequence divergence; mean half-lives for EV71 recombinant forms (RFs) of 6 and 9 years for GgB and GgC overlapped with those observed for the EV-B serotypes, echovirus 9 (E9), E30, and E11, respectively (1.3 to 9.8 years). Furthermore, within genogroups, sporadic recombination events occurred, such as the linkage of two B4 variants to RF-W instead of RF-A and of two C4 variants to RF-H. Intriguingly, recombination events occurred as a founding event of most subgenogroups immediately preceding their lineage expansion and global emergence. The possibility that recombination contributed to their subsequent spread through improved fitness requires further biological and immunological characterization.

Evolutionary dynamics and temporal/geographical correlates of recombination in the human enterovirus echovirus types 9, 11, and 30.

The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.

Typing of enteroviruses by use of microwell oligonucleotide arrays.

We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

Wide-range antifungal antagonism of Paenibacillus ehimensis IB-X-b and its dependence on chitinase and beta-1,3-glucanase production.

Previously, we isolated a strain of Bacillus that had antifungal activity and produced lytic enzymes with fungicidal potential. In the present study, we identified the bacterium as Paenibacillus ehimensis and further explored its antifungal properties. In liquid co-cultivation assays, P. ehimensis IB-X-b decreased biomass production of several pathogenic fungi by 45%-75%. The inhibition was accompanied by degradation of fungal cell walls and alterations in hyphal morphology. Residual medium from cultures of P. ehimensis IB-X-b inhibited fungal growth, indicating the inhibitors were secreted into the medium. Of the 2 major lytic enzymes, chitinases were only induced by chitin-containing substrates, whereas beta-1,3-glucanase showed steady levels in all carbon sources. Both purified chitinase and beta-1,3-glucanase degraded cell walls of macerated fungal mycelia, whereas only the latter also degraded cell walls of intact mycelia. The results indicate synergism between the antifungal action mechanisms of these enzymes in which beta-1,3-glucanase is the initiator of the cell wall hydrolysis, whereas the degradation process is reinforced by chitinases. Paenibacillus ehimensis IB-X-b has pronounced antifungal activity with a wide range of fungi and has potential as a biological control agent against plant pathogenic fungi.

Characteristics of RNA silencing in plants: similarities and differences across kingdoms.

RNA silencing is a collective term that encompasses the sequence of events that leads to the targeted degradation of cellular mRNA and thus to the silencing of corresponding gene expression. RNA silencing is initiated after introduction into the host genome of a gene that is homologous to an endogenous gene. Transcription of the introduced gene results in the formation of double-stranded RNA (dsRNA) that is cut into smaller dsRNA species termed small interfering RNAs (siRNAs) by an RNaseIII-like enzyme called 'Dicer'. siRNAs associate with a protein complex termed the 'RNA-induced silencing complex' (RISC), which mediates the binding of one strand of siRNAs with mRNAs transcribed from the native 'target' gene. The binding of siRNAs with native gene mRNAs earmarks native gene mRNAs for destruction, resulting in gene silencing. In plants, RNA silencing appears to serve as a defence mechanism against viral pathogens and also to suppress the activity of virus-like mobile genetic elements. In an apparent response to RNA silencing, some plant viruses express suppressors of RNA silencing. RNA silencing also is directly implicated in the regulation of the function(s) of microRNAs, which are the key determinants in an additional cellular mechanism related to the translational repression of genes, the effect of which ultimately impinges on development. The high degree of sequence similarity that exists between genes involved in RNA silencing in widely different organisms underscores the conserved nature of many aspects of the RNA silencing mechanism. However, depending (for example) on the precise nature of the target gene involved, there also are significant differences in the silencing pathways that are engaged by various organisms.

Dysfunctionality of a tobacco mosaic virus movement protein mutant mimicking threonine 104 phosphorylation.

Replication of tobacco mosaic virus (TMV) is connected with endoplasmic reticulum (ER)-associated membranes at early stages of infection. This study reports that TMV movement protein (MP)-specific protein kinases (PKs) associated with the ER of tobacco were capable of phosphorylating Thr(104) in TMV MP. The MP-specific PKs with apparent molecular masses of about 45-50 kDa and 38 kDa were revealed by gel PK assays. Two types of mutations were introduced in TMV MP gene of wild-type TMV U1 genome to substitute Thr(104) by neutral Ala or by negatively charged Asp. Mutation of Thr(104) to Ala did not affect the size of necrotic lesions induced by the mutant virus in Nicotiana tabacum Xanthi nc. plants. Conversely, mutation of Thr to Asp mimicking Thr(104) phosphorylation strongly inhibited cell-to-cell movement. The possible role of Thr(104) phosphorylation in TMV MP function is discussed.

Simultaneous assessment of occupational exposures from multiple worker groups.

The methods developed by Rappaport et al. [Ann. Occup. Hyg. 39 (1995) 469] and Lyles et al. [J. Agri. Bio. Environ. Stat. 2 (1997a) 64; Ann. Occup. Hyg. 41 (1997b) 63]) for assessing workplace exposures on a group-by-group basis are extended to allow for the simultaneous assessment of data from multiple worker groups within the same industry. These extended methods allow models to be fit simultaneously to data on all groups in a study, even when some of the groups might not contribute adequate information to be modeled separately. We assume that the exposures are log-normally distributed, and that they can be adequately modeled by a mixed effects regression model with parameters for exposure levels and for between- and within-worker variance components. Simultaneously analyzing data from multiple groups is only advantageous when at least one of these variance components can be assumed to be homogeneous across the groups. Here, we advocate testing an assumption of homogeneous within-worker variance components, sigma(2)(w,h), using a likelihood ratio test to choose between a full model (distinct sigma(2)(w,h) for each group) and a reduced model (common sigma(2)(w) across groups). We then develop a procedure, which is conditional on the results of the likelihood ratio test, for testing whether or not each group of workers is overexposed to the contaminant of interest. This modeling and testing procedure was applied to 39 different data sets, each containing data for multiple groups, from a wide variety of industries. For these data, the testing procedure generally resulted in the same conclusion regarding overexposure under both models, even in those data sets where the within-worker variance components appeared to be quite heterogeneous. We also conducted a small simulation study to estimate the significance level of the proposed testing procedure, and found that the significance levels tended to be adequately close to the specified nominal level when a likelihood ratio test with significance level of at least 0.01 was used as a preliminary test. Additionally, we make specific recommendations for designing studies and suggest a method for determining whether engineering and administrative controls or individual-level interventions would be of most benefit to an overexposed group of workers.

The use of a task-based exposure assessment model (T-BEAM) for assessment of metal fume exposures during welding and thermal cutting.

Elevated disease rates have been documented among construction workers for cancer, pneumonoconiosis, asbestosis, and silicosis. However, methodologies for exposure assessment in construction are not well described in the U.S. literature. Working through a cooperative agreement with the National Institute for Occupational Safety and Health (NIOSH), the Center to Protect Workers' Rights--a research arm of the Building and Construction Trades Department, AFL-CIO--has developed and used a "Task-Based Exposure Assessment Model (T-BEAM)" for construction. The characteristic elements of T-BEAM are: (1) an emphasis on the identification, implementation, and evaluation of engineering and work practice controls; and (2) use of experienced, specially trained construction workers (construction safety and health specialists) in the exposure assessment process. A task-based approach was used because tasks, or specialized skills, form the single greatest thread of continuity in the dynamic environment of construction. Workers in the construction industry come from several crafts and are typically employed by a large number of contractors throughout their career. Project types (e.g., residential or industrial rehabilitation) are also highly variable and present unique health risks. Finally, because construction involves building, renovating, or dismantling physical surroundings, the work site is constantly changing. Between 1995 and 1996, T-BEAM was applied to the collection of approximately 200 personal exposure measurements associated with "hot work tasks"--welding and thermal cutting. Data were collected with the assistance of specially trained, journeyman ironworkers, pipe fitters, and boilermakers on nine construction sites located throughout the United States. Portable local exhaust ventilation was provided to participating contractors with the intent of measuring its impact on exposure. Results indicate that data collected in a standardized, systematic fashion from multiple work sites can be used to characterize exposures among sampled trades. Comparison of results to American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit values (TLVs) demonstrate a significant health hazard among sampled trades posed by welding and thermal cutting fume, manganese, nickel, and chromium VI. Direct estimates of the probability of exceeding the ACGIH TLV for respirable particulate suggests that boilermakers (100%) and ironworkers (71%) are at greatest risk. Other task variables evaluated with respect to exposure include task, whether work was performed indoors or outdoors, intermittency of work, and use of ventilation. Use of local or mechanical ventilation reduced mean exposures to fumes significantly.

Application of mixed models to assess exposures monitored by construction workers during hot processes.

Particulate exposures were assessed among construction workers engaged in hot processes in four jobs (boilermakers, ironworkers, pipefitters and welder-fitters) at nine sites in the U.S. After being trained by occupational hygienists, the workers obtained shift-long personal samples at each site for total particulates (TP). Selected samples were also assayed for manganese (Mn), nickel (Ni), and chromium (Cr). Workers provided information about process- and task-related covariates that were present on the days of monitoring. Data were investigated with mixed-model regression analyses that designated the jobs and covariates as fixed effects and the worker and error terms as random effects. Results indicated that the within-worker variance components, but not the between-worker variance components, could be pooled among jobs. Mean air levels for a given agent varied by roughly six to 100 fold among the jobs, with boilermakers and ironworkers experiencing much higher levels of TP and Mn than pipefitters and welder-fitters. Limited data also suggested that welder-fitters were exposed to greater levels of Ni and Cr than pipefitters. Sufficient sample sizes were available to evaluate the effects of covariates upon exposures to TP and Mn. As expected, processes involving more than 50% hot work led to substantially higher levels of TP and Mn than those involving shorter durations of hot work. Local-exhaust or mechanical ventilation reduced exposure to TP (but not Mn) by as much as 44%, and shielded or manual arc welding increased exposure to Mn (but not TP) by about 80%. Parameters estimated with these mixed models were used to calculate probabilities that workers were exposed at levels above U.S. occupational exposure limits (OELs). Regarding TP and Mn, these calculations suggested that 26-95% of exposures to boilermakers and pipefitters and 2-13% of exposures to pipefitters and welder-fitters exceeded the current Threshold Limit Values. Among welder-fitters, limited data also pointed to probabilities of 2-50% for exceeding particular OELs for Ni and Cr. Using the significance of the estimated random-worker effects as a gauge for the uniformity of exposure within a job, administrative or engineering changes appear appropriate for reducing exposures to boilermakers and ironworkers, while individual personal environments should be investigated for pipefitters and welder-fitters.

Replication in the phloem is not necessary for efficient vascular transport of tobacco mosaic tobamovirus.

Plant viruses move systemically from one leaf to another via phloem. However, the viral functions needed for systemic movement are not fully elucidated. An experimental system was designed to study the effects of low temperature on the vascular transport of the tobacco mosaic tobamovirus (TMV). Vascular transport of TMV from lower inoculated leaves to upper non-inoculated leaves via a stem segment kept at low temperature (4 degrees C) was not affected. On the other hand, several experiments were performed on tobacco leaves to demonstrate that virus replication did not occur at the same temperature. The data suggest that replication of TMV in the phloem of wild-type tobacco plants is not necessary for the vascular transport of TMV, and that the virus moves with photoassimilates as suggested previously.

Characterization of the coat protein gene of mite-transmitted blackcurrant reversion associated nepovirus.

The nucleotide sequence of the 3' terminal 3105 nucleotides (nt) of RNA2 of blackcurrant reversion associated virus (BRAV), the first mite-transmitted member of the nepovirus group, has been determined. The sequence contains an open reading frame of 1744 nt in the virus-sense strand, a 3' untranslated region of 1360 nt and a 3' poly(A) tail. Analysis of the amino-terminal residues of purified coat protein (CP) suggests that the CP gene is located between nts 1361 and 2959 (from the 3' terminus) in the RNA2, and that Asp/Ser is the proteolytic cleavage site of CP in the RNA2 encoded polyprotein. The predicted translation product from the CP gene is a polypeptide of 533 amino acids with a calculated Mr of 57 561. The amino acid sequence of BRAV CP showed highest similarity to blueberry leaf mottle virus (BLMV), and tomato ringspot virus (ToRSV), two members of the proposed sub-group three of nepoviruses possessing large RNA2 components. Nucleic and amino acid sequence comparisons between BRAV CP and the CPs of other nepoviruses indicate that specific conserved nepovirus CP domains occur in the BRAV CP thus confirming that BRAV is a member of the subgroup three of nepoviruses. reserved.

Selection of single-chain variable fragment antibodies to black currant reversion associated virus from a synthetic phage display library.

ABSTRACT Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

Purification and properties of a new virus from black currant, its affinities with nepoviruses, and its close association with black currant reversion disease.

ABSTRACT Black currant reversion is a virus-like disease whose causal agent has not been identified. In rooted cuttings of a black currant plant affected with the severe form of the disease, pronounced chlorotic line patterns and ringspots developed in newly emerging leaves. From such symptom-bearing leaves, a virus was mechanically transmitted with difficulty to Chenopodium quinoa and, from this host, to other herbaceous test plants. The virus was purified and partially characterized, and the purified viri-ons were used for antiserum production. Virus particles were isometric, approximately 27 nm in diameter, and sedimented as two nucleoprotein components. They contained a protein species with a molecular mass of 55 kDa, which was readily degraded into a 54-kDa protein and two major RNA components of about 6,700 and 7,700 nucleotides (nt), each with a poly(A) tail. Most of these properties are shared by nepoviruses, but the virus was serologically unrelated to 14 nepoviruses or putative nepovi-ruses tested. However, the deduced sequence of 1,260 nt at the 3' end of one of the viral RNA species was distinct from any known viral sequence, except that it contained short regions of homology to the 3' terminal sequences of RNAs of seven other nepoviruses and two comovi-ruses. To detect this virus in Ribes plants, primers were designed from the known sequence to amplify a 210-nt region of the cDNA of the virus RNA using an immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) protocol. Using this assay for the virus, we associated its presence with two recognized forms of black currant reversion disease occurring in Finland, Scotland, or New Zealand. We also detected the virus in vector gall mites from reverted plants and in black currant plants on which such vector mites had fed. However, the virus was not detected by IC-RT-PCR in known healthy Ribes plants; in Ribes plants free from reversion, but affected by three other distinct virus-like diseases of Ribes; or in plants infected with arabis mosaic, strawberry latent ringspot, or raspberry ringspot nepoviruses. These data suggest that this virus may be the causal agent of reversion disease, and it is tentatively called black currant reversion associated virus.

Ergonomics and construction: a review of potential hazards in new construction.

This article reviews potential ergonomic hazards in new construction work, summarizing findings from published literature and from a 15-month investigation of health hazards on a new construction site in suburban Washington, D.C. The review is structured to follow the sequence of events in the construction process. Ergonomic solutions are included where they have been developed. Highway work and renovation and demolition of existing structures, the segments of construction that are growing, are not discussed here. However, many of the same problems and principles apply to other sectors of construction.