Full genome sequence of a peste des petits ruminants virus (PPRV) from Ghana.

The full genome of a peste des petits ruminants virus (PPRV) isolated from a sheep lung sample collected in Ghana, Western Africa, in 2010, has been sequenced. Phylogenetic analysis demonstrated that the virus clustered within the lineage II clade while comparison of its full genome with those of other PPRV strains revealed the highest identity (96.6 %) at a nucleotide level with the PPRV strain Nigeria/76/1. This is the first full genome sequence generated for a PPRV lineage II isolated since 1976.

The therapeutic alliance in medical-based interventions impacts outcome in treating alcohol dependence.

This study examined the relationship of the therapeutic alliance and treatment outcomes for alcohol-dependent patients receiving naltrexone or placebo and one of three different types of clinical interventions, including two medical-based (non-specialty) treatments. This is a secondary analysis of a 24-week randomized, placebo-controlled, clinical trial of 100mg/day of naltrexone or placebo for patients with DSM-IV alcohol dependence. Patients were also randomized to one of three interventions: (1) medication clinic only, (2) medication clinic plus BRENDA (an intervention promoting pharmacotherapy), or (3) medication clinic plus cognitive behavioral therapy (CBT). Early in treatment, patients and clinicians completed the working alliance inventory (WAI). Regression analyses were conducted to determine the predictive validity of the WAI on percent days abstinent and percent of sessions attended over the clinical trial. In the medication clinic only condition, the clinicians' WAI total score was marginally correlated to percent of visits attended (p=.057) but not percent days abstinent. In the medication clinic plus BRENDA condition, clinicians' WAI total score was positively correlated with percent days abstinent (p=.013) but not percent visits attended. No significant relationships were found between the WAI scores and either outcome measure in the CBT condition or for any of the patient rated assessments. To our knowledge, this is the first published report providing some support for the importance of the therapeutic alliance in medical interventions for alcohol dependence but only in the context of the clinicians' ratings. The absence of other effects underscores the need for further research.

Detection and Genome Analysis of a Lineage III Peste Des Petits Ruminants Virus in Kenya in 2011.

In May 2011 in Turkana County, north-western Kenya, tissue samples were collected from goats suspected of having died of peste des petits ruminant (PPR) disease, an acute viral disease of small ruminants. The samples were processed and tested by reverse transcriptase PCR for the presence of PPR viral RNA. The positive samples were sequenced and identified as belonging to peste des petits ruminants virus (PPRV) lineage III. Full-genome analysis of one of the positive samples revealed that the virus causing disease in Kenya in 2011 was 95.7% identical to the full genome of a virus isolated in Uganda in 2012 and that a segment of the viral fusion gene was 100% identical to that of a virus circulating in Tanzania in 2013. These data strongly indicate transboundary movement of lineage III viruses between Eastern Africa countries and have significant implications for surveillance and control of this important disease as it moves southwards in Africa.

Peste Des Petits Ruminants in Benin: Persistence of a Single Virus Genotype in the Country for Over 42 Years.

Peste des petits ruminants (PPR) is a contagious and often fatal disease affecting sheep and goats. Currently, it is endemic in Africa, the Middle and Near East, the Indian subcontinent and China. Understanding the molecular epidemiology and evolution of PPR virus (PPRV) can assist in the control of the transboundary spread of this economically important disease. We isolated PPRV from pathological and swab samples collected 42 years apart (1969 and 2011) in Benin, West Africa, and sequenced the full genome of two isolates (Benin/B1/1969 and Benin/10/2011). Phylogenetic analysis showed that all of the characterized isolates clustered within viral lineage II and that the 2011 isolates fell into two distinct subgroups. Comparison of the full genome sequences revealed a 95.3% identity at the nucleotide level, while at the protein level, the matrix protein was the most conserved between the two viruses with an identity of 99.7% and only one amino acid substitution over the 42-year sampling period. An analysis of specific amino acid residues of known or putative function did not identify any significant changes between the two viruses. A molecular clock analysis of complete PPRV genomes revealed that the lineage II viruses sampled here arose in the early 1960s and that these viruses have likely persisted in Benin since this time.

Co-circulation of Peste-des-Petits-Ruminants Virus Asian lineage IV with Lineage II in Nigeria.

Peste-des-petits-ruminants (PPR), a major small ruminant transboundary animal disease, is endemic in Nigeria. Strains of the causal agent, peste-des-petits-ruminants virus (PPRV), have been differentiated into four genetically distinct lineages based on the partial sequence of the virus nucleoprotein (N) or fusion (F) genes. Peste-des-petits-ruminants virus strains that were identified initially in Africa were grouped into lineages I, II and III and viruses from Asia were classified as lineage IV and referred to as the Asian lineage. Many recent reports indicate that the Asian lineage is now also present in Africa. With this in mind, this study was conducted to reassess the epidemiology of PPRV in Nigeria. A total of 140 clinical samples from 16 sheep and 63 goats with symptoms suggestive of PPR were collected from different states of Nigeria during a four-year period (2010-2013). They were analysed by the amplification of fragments of the N gene. Results for 33 (42%) animals were positive. The phylogenetic analysis of the N gene sequences with those available in GenBank showed that viruses that were detected belong to both lineage II and IV. Based on an analysis of the N gene sequences, the lineage IV isolates grouped into two clades, one being predominant in the north-eastern part of the country and the other found primarily in the southern regions of the country. This study reports the presence of PPRV Asian lineage IV in Nigeria for the first time.

Nucleotide sequence of the gene coding for SEC14p in Candida (torulopsis) glabrata.

A gene coding for SEC14p from Candida glabrata has been cloned and characterized. Nucleotide (nt) sequence analysis reveals an open reading frame of 909 bp and predicts the synthesis of a polypeptide of 302 amino acid (aa) residues. Comparison of nt and aa sequences shows that the gene exhibits a much higher homology to the Saccharomyces cerevisiae (72% and 87%, respectively) than to the Candida albicans (55% and 65%, respectively) SEC14 gene.

Isolation and characterization of a gene encoding alpha-tubulin from Candida albicans.

A gene encoding the alpha-tubulin of Candida albicans has been cloned and characterized. Nucleotide sequence analysis reveals the presence of an intron within the structural gene and predicts the synthesis of a polypeptide of 448 amino acid residues. Comparison of nucleotide and amino acid sequences with the Saccharomyces cerevisiae alpha-tubulin encoding genes shows a 75% homology and about 92% similarity respectively. In contrast to S. cerevisiae, C. albicans appears to possess only one gene for alpha-tubulin which is able to functionally complement a S. cerevisiae cold-sensitive tub1 mutant.

Neutrophil-activating protein (HP-NAP) versus ferritin (Pfr): comparison of synthesis in Helicobacter pylori.

We recently reported that the neutrophil-activating protein (HP-NAP) of Helicobacter pylori is capable of binding iron in vitro. To more fully understand the relationship between iron and HP-NAP the synthesis of HP-NAP was compared to that of Pfr, another iron-binding protein of H. pylori. Synthesis of HP-NAP and Pfr in growing cultures of H. pylori was analysed under iron depletion and iron, copper, nickel and zinc overload. The synthesis of HP-NAP and Pfr in H. pylori was also analysed under conditions of varying pH and oxidative stress. In addition, recombinant HP-NAP and Pfr were produced in Escherichia coli to assess the contribution of the two proteins to increased survival of E. coli under heavy metal overload. Our data reveal that both HP-NAP and Pfr accumulate in the stationary phase of growth. HP-NAP synthesis is not regulated by iron depletion or overload or by the presence of copper, nickel or zinc in liquid medium and it does not confer resistance to these metals when produced in E. coli. Except for an increase in the synthesis of Pfr at pH 5.7 neither the pH or oxidative stress conditions investigated had an affect on the synthesis of either protein. An increase in Pfr synthesis was observed under iron overload and a decrease was observed under conditions of copper, nickel and zinc overload confirming previous reports. Recombinant Pfr, as well as conferring resistance to iron and copper as previously reported, also conferred resistance to zinc overload when produced in E. coli.

Human IL-1 receptor antagonist from Escherichia coli: large-scale microbial growth and protein purification.

Interleukin-1 receptor antagonist (IL-1ra) is a recently discovered cytokine which specifically inhibits IL-1 pro-inflammatory activities in various experimental conditions. In this work, the growth conditions of a recombinant E. coli strain which in laboratory studies expressed human IL-1ra mostly in insoluble form, have been optimized at the level of 6-1 bioreactors and then scaled up to a 50-1 process. As a result, a high amount (0.43 g l-1 of microbial culture) of soluble, active IL-1ra has been directly obtained in the large-scale cell lysate with no need for protein solubilization. Also, an efficient purification procedure has been developed for the soluble protein, based on cation exchange expanded bed adsorption directly followed by anion exchange chromatography. This process, which does not include any intermediate dialysis step or gradient elutions, can be easily scaled up to larger production volumes and is therefore well-suited for manufacturing. As a result of the overall optimization study, more than 12 g of pure IL-1ra have been obtained from a single 50-1 fermentation run, without any denaturation/renaturation process. The final product, whose identity and purity have been checked also by MALDI-TOF and ESI-MS, shows full biological activity both in cellular assays and in in vivo experiments with Cynomolgus monkeys.

Population characteristics of Irish Helicobacter pylori isolates: a tRNA-associated locus.

BACKGROUND: A strain-variable transfer RNA-associated-locus (trl) was present in 50% of Irish Helicobacter pylori (H. pylori), isolates and did not correlate with the origin of the isolates. AIM: To associate a particular genotype or phenotype to trl status in H. pylori by further screening the isolates from the original study for the presence of known genotypic and phenotypic characteristics. METHODS: Forty two clinical isolates were screened for the presence of the cagA, vacA, iceA1 and vapD genes by Southern or DNA dot blot analysis. Western blot analysis was performed using antibodies to CagA, VacA, Lewis X (Le(x)) and Lewis Y (Le(y)). Plasmids were identified by the alkaline lysis method. RESULTS: The cagA gene was present in 29 (69%) of isolates screened and 21 (50%) produced the CagA protein. The vacA gene was detected in all of the isolates while VacA was expressed in 71.4%. The iceA1 and vapD loci were detected in 73.8% and 71.4% respectively. Le(x) was expressed in 42.9% and Le(y) in 38.1% of the isolates. Expression of both Lewis antigens was detected in 7.1% while in 30.9% neither antigen was detected. Plasmids were present in 47.6%. There was no association between the trl status of isolates and any of the above. There were no significant associations between the phenotypic and genotypic characteristics studied and peptic ulcer disease or non-ulcer dyspepsia. CONCLUSION: The strain-variable tRNA-associated locus is independent of the vacA/VacA, cagA/CagA, Lewis X, Lewis Y, iceA1, vapD and plasmid status in the population of Irish H. pylori isolates studied.

Phylogenetic analysis of rabies viruses from Burkina Faso, 2007.

Genetic characterization of 32 canine rabies viruses circulating in Burkina Faso in 2007 identified two clades both belonging to the Africa 2 lineage. Sequence homology data suggest that transboundary spread is the most likely means of introduction, highlighting an evolving epidemiological situation.

Virulence factors of Helicobacter pylori.

To date a number of virulence factors have been identified and characterised from the gastric pathogen Helicobacter pylori. The vacuolating toxin (VacA) is a major determinant of H. pylori-associated gastric disease. In non-polarised cells, VacA alters the endocytic pathway, resulting in the release of acid hydrolases and the reduction of both extracellular ligand degradation and antigen processing. The toxin forms trans-membrane anion-specific channels and reduces the transepithelial electrical resistance of polarized monolayers. Localization of the VacA channels in acidic intracellular compartments causes osmotic swelling which, together with membrane fusion, leads to vacuole formation. The neutrophil-activating protein of H. pylori (HP-NAP) induces the production of oxygen radicals in human neutrophils via a cascade of intracellular activation events which may contribute to the damage of the stomach mucosa. This protein has recently been shown to be an important antigen in the human immune response to H. pylori infection. In addition, mice vaccinated with recombinant HP-NAP were protected against H. pylori challenge. H. pylori strains that are associated with severe tissue damage and inflammation possess the cag pathogenicity island that contains several genes encoding factors involved in the induction of proinflammatory cytokines/chemokines and of a type IV secretion system involved in the delivery of a highly immunogenic protein, CagA, into eukaryotic cells. Recent advances in our understanding of the involvement of VacA, HP-NAP and the CagA/Type IV secretion system in the H. pylori-associated disease process are discussed in this review.

The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a major 17 kDa antigen of the immune response of infected individuals. Amino acid sequence comparison indicated a high similarity between HP-NAP and both bacterial DNA-protecting proteins (Dps) and ferritins. The structure prediction and spectroscopic analysis presented here indicate a close similarity between HP-NAP and Dps. Electron microscopy revealed that HP-NAP forms hexagonal rings of 9-10 nm diameter with a hollow central core as seen in Dps proteins, clearly different from the 12 nm icositetrameric (24 subunits) ferritins. However, HP-NAP is resistant to thermal and chemical denaturation similar to the ferritin family of proteins. In addition, HP-NAP binds up to 40 atoms of iron per monomer and does not bind DNA. We therefore conclude that HP-NAP is an unusual, small, ferritin that folds into a four-helix bundle that oligomerizes into dodecamers with a central hole capable of binding up to 500 iron atoms per oligomer.

Helicobacter pylori neutrophil-activating protein stimulates tissue factor and plasminogen activator inhibitor-2 production by human blood mononuclear cells.

Helicobacter pylori neutrophil-activating protein (HP-NAP) is a virulence factor that activates phagocytic NADPH-oxidase. The effect of HP-NAP on the production of tissue factor (TF), plasminogen activator inhibitor-2 (PAI-2), and urokinase-type plasminogen activator (u-PA) by human blood mononuclear cells (MNC) was evaluated by using functional and immunological assays and mRNA analysis. HP-NAP induced time- and dose-dependent increases in TF and PAI-2, with a maximal effect at 300 nmol/L (>15-fold increase in antigens). No changes in u-PA were observed. When whole bacteria were used, an H. pylori mutant lacking HP-NAP was significantly less active than the wild-type strain. MNC from a patient with chronic granulomatous disease behaved as do normal cells, which indicates that HP-NAP effects can occur independently of NADPH-oxidase. HP-NAP, by inducing the coordinate expression of cell procoagulant and antifibrinolytic activities, might favor fibrin deposition and contribute to the inflammatory reaction of gastric mucosa elicited by H. pylori.