Isolation,characterization and genome sequence of a birnavirus strain from flounder <Emphasis Type="Italic">Paralichthys </Emphasis><Emphasis Type="Italic">olivaceus</Emphasis> in China

A birnavirus strain, Paralichthys olivaceus birnavirus (POBV), was isolated and characterized from cultured flounder in China, and its complete genomic sequence was subsequently determined. The virus could induce cytopathic effects (CPE) in four of seven fish cell lines and was resistant to chloroform, 5-iodo-2′-deoxyuridine, acid and alkaline pH, and heat treatment. Purified virus particles had a typical icosahedral shape, with a diameter of approximately 55–60 nm. The genomic segments A and B of POBV were 3,091 and 2,780 bp in length and shared many of the features of the members of the family Birnaviridae. Segment A contained two partially overlapping ORFs encoding a polyprotein, pVP2-VP4-VP3, and a nonstructural protein, VP5, while segment B had only one ORF encoding for the VP1, a viral RNA-dependent RNA polymerase (RdRp). This is the first report about a birnavirus strain from a new non-salmonid host in China and its complete genome sequence. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The complete genome sequences of POBV reported here have been deposited in the GenBank database under accession no EU161285 and EU161286, respectively.     

Identification and sequence analysis of the <Emphasis Type="Italic">Condylorrhiza vestigialis</Emphasis> MNPV <Emphasis Type="Italic">p74</Emphasis> gene

The baculovirus Condylorrhiza vestigialis multiple nucleopolyhedrovirus (CoveMNPV), isolated from C. vestigialis infected larvae in Paraná (Brazil), was identified in our laboratory. A full-length clone was obtained from the CoveMNPV genome, of the gene that encodes the homolog to baculoviral p74, essential for oral infectivity which was then sequenced and characterized. The CoveMNPV p74 gene (GenBank accession number EU919397) contains an ORF of 1935 bp that encodes a deduced protein of 73.61 kDa. The phylogenetic affiliations of the CoveMNPV gene were determined by a heuristic search of 40 aligned baculovirus p74 nucleotide sequences using maximum parsimony (PAUP 4.0b4a). The phylogenetic analysis placed CoveMNPV within lepidopteran nucleopolyhedrovirus (NPV) Group I, Clade A, as being the closest to Choristoneura fumiferana defective NPV.     

The complete genome sequence of a third distinct baculovirus isolated from the true armyworm, <Emphasis Type="Italic">Mythimna unipuncta</Emphasis>, contains two copies of the <Emphasis Type="Italic">lef</Emphasis>-<Emphasis Type="Italic">7</Emphasis> gene

A baculovirus isolate from a USDA Forest Service collection was characterized by electron microscopy and analysis of its genome sequence. The isolate, formerly referred to as Pseudoletia (Mythimna) sp. nucleopolyhedrovirus #7 (MyspNPV#7), was determined by barcoding PCR to derive from the host species Mythimna unipuncta (true armyworm) and was renamed Mythimna unipuncta nucleopolyhedrovirus #7 (MyunNPV#7). The occlusion bodies (OBs) and virions exhibited a size and morphology typical for OBs produced by the species of genus Alphabaculovirus, with occlusion-derived virions consisting of 2–5 nucleocapsids within a single envelope. The MyunNPV#7 genome was determined to be 148,482 bp with a 48.58% G+C nucleotide distribution. A total of 159 ORFs of 150 bp or larger were annotated in the genome sequence, including the 38 core genes of family Baculoviridae. The genome contained six homologous repeat regions (hrs) consisting of multiple copies of a 34-bp imperfect palindrome. Phylogenetic inference from concatenated baculovirus core gene amino acid sequence alignments placed MyunNPV#7 with group II alphabaculoviruses isolated from other armyworm and cutworm host species of lepidopteran family Noctuidae. MyunNPV#7 could be distinguished from other viruses in this group on the basis of differences in gene content and order. Pairwise nucleotide distances suggested that MyunNPV#7 represents a distinct species in Alphabaculovirus. The MyunNPV#7 genome was found to contain two copies of the late expression factor-7 (lef-7) gene, a feature not reported for any other baculovirus genome to date. Both copies of lef-7 encoded an F-box domain, which is required for the function of LEF-7 in baculovirus DNA replication.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Biochemical and molecular characterization of a putative endoglucanase in <Emphasis Type="Italic">Magnaporthe</Emphasis><Emphasis Type="Italic">grisea</Emphasis>

Microbial pathogens secrete an array of cell wall-degrading enzymes to break down the structure of the host cell wall to facilitate colonization of the host tissue. To better understand their role in the pathogenesis, a putative endoglucanase from Magnaporthe grisea was characterized in this paper. SignalP-3.0 analysis indicates that the protein encoded by gene MGG_02532.5 in M. grisea (named MgEGL1 for M . g risea endoglucanase 1 ) contains a secretory signal peptide. Multiple alignment shows that MgEGL1 has high level of homology to endoglucanases (EC 3.1.1.4) from Aspergillus nidulans and Trichoderma reesei. The three proteins share a conserved catalytic domain, but only the one from T. reesei contains a cellulose binding module. MgEGL1 was constitutively expressed with the highest level in mycelia and the lowest in conidia. Interestingly, the MgEGL1 RNA could be alternatively processed when cultured in vitro and after infection of rice. Expression analysis confirmed that the MgEGL1 is a secreted protein. Its endoglucanase activity was assayed by Congo red plates, and further confirmed by the dinitrosalicylic acid method. The finding in this paper will provide the basis for further determination of the biochemical properties of the endoglucanase protein and its relevant function in fungal pathogenesis.     

Phylogenetic analysis reveals five independent transfers of the chloroplast gene<Emphasis Type="Italic"> rbcL</Emphasis> to the mitochondrial genome in angiosperms

We used the chloroplast gene rbcL as a model to study the frequency and relative timing of transfer of chloroplast sequences to the mitochondrial genome. Southern blot survey of 20 mitochondrial DNAs confirmed three previously reported groups of plants containing rbcL in their mitochondrion, while PCR studies identified a new mitochondrial rbcL. Published and newly determined mitochondrial and chloroplast rbcL sequences were used to reconstruct rbcL phylogeny. The results imply five or six separate interorganellar transfers of rbcL among the angiosperms examined, and hundreds of successful transfers across all flowering plants. By taxonomic criteria, the crucifer transfer is the most ancient, two separate transfers within the grass family are of intermediate ancestry, and the morning-glory transfer is most recent. All five mitochondrial copies of rbcL examined exhibit insertion and/or deletion events that disrupt the reading frame (three are grossly truncated); and all are elevated in the proportion of nonsynonymous substitutions, providing clear evidence that these sequences are pseudogenes.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> and <Emphasis Type="Italic">cagA</Emphasis> and <Emphasis Type="Italic">vacA</Emphasis> gene status in children from Brazil with chronic gastritis

Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1-16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).     

Prevalence of the <Emphasis Type="Italic">ica</Emphasis> operon and insertion sequence IS<Emphasis Type="Italic">256</Emphasis> among <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis> prosthetic joint infection isolates

Joint replacement surgery has improved the quality of life for hundreds of thousands of patients. However, the infection of a joint implant is an important and serious complication, though the prevalence is low. Staphylococcus epidermidis is the most important pathogen involved in foreign-body infections. S. epidermidis is also a commensal that comprises a substantial part of the normal skin flora of humans. The possibility to demonstrate potential specific virulence markers may facilitate the interpretation of the bacteriological findings, as well as the clinical decision. The prevalence of the ica locus and insertion sequence IS256 by using polymerase chain reaction (PCR) among 32 clinical S. epidermidis isolates from prosthetic joint infections (PJIs) and 24 commensal isolates from nares and skin was investigated. Sixteen (50%) of the 32 PJI isolates harbored the ica operon compared with one-third of the commensal isolates obtained from the samples of the skin and nares of healthy individuals. The IS256 was demonstrated in 26 (81%) out of 32 PJI isolates. By contrast, IS256 was found in one of 24 commensal isolates. In conclusion, IS256 may be superior to the ica operon as a marker of the invasive capacity of S. epidermidis, since it was found in most of the PJI isolates, but rarely among commensals.     

Identification of the <Emphasis Type="Italic">het-r</Emphasis> vegetative incompatibility gene of <Emphasis Type="Italic">Podospora anserina</Emphasis> as a member of the fast evolving <Emphasis Type="Italic">HNWD</Emphasis> gene family

In fungi, vegetative incompatibility is a conspecific non-self recognition mechanism that restricts formation of viable heterokaryons when incompatible alleles of specific het loci interact. In Podospora anserina, three non-allelic incompatibility systems have been genetically defined involving interactions between het-c and het-d, het-c and het-e, het-r and het-v. het-d and het-e are paralogues belonging to the HNWD gene family that encode proteins of the STAND class. HET-D and HET-E proteins comprise an N-terminal HET effector domain, a central GTP binding site and a C-terminal WD repeat domain constituted of tandem repeats of highly conserved WD40 repeat units that define the specificity of alleles during incompatibility. The WD40 repeat units of the members of this HNWD family are undergoing concerted evolution. By combining genetic analysis and gain of function experiments, we demonstrate that an additional member of this family, HNWD2, corresponds to the het-r non-allelic incompatibility gene. As for het-d and het-e, allele specificity at the het-r locus is determined by the WD repeat domain. Natural isolates show allelic variation for het-r. Sequence data reported here are available in the Genbank database under accession numbers FJ269240 and FJ269239 for het-r and het-R, respectively.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

<Emphasis Type="Italic">Pepo aphid</Emphasis>-<Emphasis Type="Italic">borne yellows virus</Emphasis>: a new species in the genus <Emphasis Type="Italic">Polerovirus</Emphasis>

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV’s genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

Selective activity of extracts of <Emphasis Type="Italic">Margaritaria discoidea</Emphasis> and <Emphasis Type="Italic">Homalium africanum</Emphasis> on <Emphasis Type="Italic">Onchocerca ochengi</Emphasis>

Background  

The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon.     

<Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">aeruginosa</Emphasis> Biofilms in CF Infection

Pseudomonas aeruginosa is an opportunistic pathogen of immunocompromised hosts. In cystic fibrosis (CF), P. aeruginosa causes acute and chronic lung infections that result in significant morbidity and mortality. P. aeruginosa possesses several traits that contribute to its ability to colonize and persist in acute and chronic infections. These include high resistance to antimicrobials, ability to form biofilms, plethora of virulence products, and metabolic versatility. In P. aeruginosa, a cell-to-cell communication process termed quorum sensing (QS) regulates many of these factors that contribute to its pathogenesis. Recent evidence suggests that the CF lung environment presents a specialized niche for P. aeruginosa. The relationship of P. aeruginosa QS, biofilm formation, and the CF lung environment is discussed.