Molecular and phylogenetic analysis of bovine coronavirus based on the spike glycoprotein gene

Bovine coronavirus has been associated with diarrhoea in newborn calves, winter dysentery in adult cattle and respiratory tract infections in calves and feedlot cattle. In Cuba, the presence of BCoV was first reported in 2006. Since then, sporadic outbreaks have continued to occur. This study was aimed at deepening the knowledge of the evolution, molecular markers of virulence and epidemiology of BCoV in Cuba. A total of 30 samples collected between 2009 and 2011 were used for PCR amplification and direct sequencing of partial or full S gene. Sequence comparison and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100% bootstrap and 1.00 posterior probability values. The Cuban bovine coronavirus sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique. (C) 2012 Elsevier B.V. All rights reserved.

Phylogenetic analysis of complete genome sequences of hepatitis B virus from an Afro-Colombian community: presence of HBV F3/A1 recombinant strain

Background: Hepatitis B virus (HBV) infection is one of the most prevalent viral infections in humans and represents a serious public health problem. In Colombia, our group reported recently the presence of subgenotypes F3, A2 and genotype G in Bogota. The aim of this study was to characterize the HBV genotypes circulating in Quibdo, the largest Afro-descendant community in Colombia. Sixty HBsAg-positive samples were studied. A fragment of 1306 bp (S/POL) was amplified by nested PCR. Positive samples to S/POL fragment were submitted to PCR amplification of the HBV complete genome. Findings: The distribution of HBV genotypes was: A1 (52.17%), E (39.13%), D3 (4.3%) and F3/A1 (4.3%). An HBV recombinant strain subgenotype F3/A1 was found for the first time. Conclusions: This study is the first analysis of complete HBV genome sequences from Afro-Colombian population. It was found an important presence of HBV/A1 and HBV/E genotypes. A new recombinant strain of HBV genotype F3/A1 was reported in this population. This fact may be correlated with the introduction of these genotypes in the times of slavery.

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of Sao Paulo, Brazil

Background: The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives: This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of Sao Paulo, Brazil. Study design: RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results: From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naive patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion: The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of Sao Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

Clinical, epidemiological and molecular features of the HIV-1 subtype C and recombinant forms that are circulating in the city of São Paulo, Brazil

Abstract Background The city of Sao Paulo has the highest AIDS case rate, with nearly 60% in Brazil. Despite, several studies involving molecular epidemiology, lack of data regarding a large cohort study has not been published from this city. Objectives This study aimed to describe the HIV-1 subtypes, recombinant forms and drug resistance mutations, according to subtype, with emphasis on subtype C and BC recombinants in the city of São Paulo, Brazil. Study design RNA was extracted from the plasma samples of 302 HIV-1-seropositive subjects, of which 211 were drug-naive and 82 were exposed to ART. HIV-1 partial pol region sequences were used in phylogenetic analyses for subtyping and identification of drug resistance mutations. The envelope gene of subtype C and BC samples was also sequenced. Results From partial pol gene analyses, 239 samples (79.1%) were assigned as subtype B, 23 (7.6%) were F1, 16 (5.3%) were subtype C and 24 (8%) were mosaics (3 CRF28/CRF29-like). The subtype C and BC recombinants were mainly identified in drug-naïve patients (72.7%) and the heterosexual risk exposure category (86.3%), whereas for subtype B, these values were 69.9% and 57.3%, respectively (p = 0.97 and p = 0.015, respectively). An increasing trend of subtype C and BC recombinants was observed (p < 0.01). Conclusion The HIV-1 subtype C and CRFs seem to have emerged over the last few years in the city of São Paulo, principally among the heterosexual population. These findings may have an impact on preventive measures and vaccine development in Brazil.

Production and characterization of poly-(3-hydroxybutyrate) from recombinant Escherichia coli grown on cheap renewable carbon substrates

Although the biopolymer poly-(3-hydroxybutyrate), P[3HB], presents physicochemical properties that make it an alternative material to conventional plastics, its biotechnological production is quite expensive. As carbon substrates contribute greatly to P[3HB] production cost, the utilization of a cheaper carbon substrate and less demanding micro-organisms should decrease its cost. In the present study a 23 factorial experimental design was applied, aiming to evaluate the effects of using hydrolysed corn starch (HCS) and soybean oil (SBO) as carbon substrates, and cheese whey (CW) supplementation in the mineral medium (MM) on the responses, cell dried weigh (DCW), percentage P[3HB] and mass P[3HB] by recombinant Escherichia coli strains JM101 and DH10B, containing the P[3HB] synthase genes from Cupriavidus necator (ex-Ralstonia eutropha). The analysis of effects indicated that the substrates and the supplement and their interactions had positive effect on CDW. Statistically generated equations showed that, at the highest concentrations of HCS, SO and CW, theoretically it should be possible to produce about 2 g L(1) DCW, accumulating 50% P[3HB], in both strains. To complement this study, the strain that presented the best results was cultivated in MM added to HCS, SBO and CW ( in best composition observed) and complex medium (CM) to compare the obtained P[3HB] in terms of physicochemical parameters. The obtained results showed that the P[3HB] production in MM (1.29 g L(-1)) was approximately 20% lower than in CM (1.63 g L(-1)); however, this difference can be compensated by the lower cost of the MM achieved by the use of cheap renewable carbon sources. Moreover, using differential scanning calorimetry and thermogravimetry analyses, it was observed that the polymer produced in MM was the one which presented physicochemical properties (Tg and Tf) that were more similar to those found in the literature for P[3HB].

Characterization of Primary Isolates of HIV Type 1 CRF28_BF, CRF29_BF, and Unique BF Recombinants Circulating in Sao Paulo, Brazil

We report for the first time the genetic and biological characterization of 10 HIV-1 primary isolates representing CRF28_BF and CRF29_BF together with additional unique BF recombinant forms (URFs) obtained by PBMC cocultivation. Recombination is an important factor promoting the increase in the genetic diversity of HIV-1. Notably, more than 20% of HIV-1 sequences worldwide were recombinants. Several recombinant viruses were reported in Brazil, and six circulating recombinant forms (CRFs) have been identified (CRF28_BF, CRF29_BF, CRF31_BC, CRF39_BF, CRF40_BF, and CRF46_BF). CRF28_BF and CRF29_BF were found to infect almost 30% of the patients in Sao Paulo State. The near full-length genomes of these 10 primary isolates were amplified by nested PCR in three overlapping segments, purified, and sequenced. Three samples were related to CRF28_BF, three to CRF29_BF, and four were unique recombinant forms (URFs), as determined by their breakpoint profile determined with the jpHMM program. Additionally, the coreceptor usage of these isolates was investigated in vitro using GHOST assays, which revealed three dual-tropic (X4/R5) viruses, four lymphotropic (X4) viruses, and three macrophage-tropic (R5) viruses with different V3-loop motifs, which challenges the notion that GWGR-carrying viruses are macrophage-tropic only. In sum, we report a much-anticipated well-characterized panel of viruses representing CRF28_BF, CRF29_BF, and URFs from Sao Paulo State, Brazil.

HEV infection in swine from Eastern Brazilian Amazon: Evidence of co-infection by different subtypes

Hepatitis E virus (HEV) is a fecal-orally transmitted member of the genus Hepevirus that causes acute hepatitis in humans and is widely distributed throughout the world. Pigs have been reported as the main source of genotypes 3 and 4 infection to humans in non-endemic areas. To investigate HEV infection in pigs from different regions of Para state (Eastern Brazilian Amazon), we performed serological and molecular analyses of serum, fecal and liver samples from 151 adult pigs slaughtered between April and October 2010 in slaughterhouses in the metropolitan region of Belem, Para. Among the animals tested, 8.6% (13/151) were positive for anti-HEV IgG but not for anti-HEV IgM. HEV RNA was detected in 4.8% (22/453) of the samples analyzed and 9.9% (15/151) of the animals had at least one positive sample. Phylogenetic analysis showed that all sequences belonged to genotype 3 that were related to human isolates from other non-endemic regions, suggesting that the isolates had zoonotic potential. Subtypes 3c and 3f were simultaneously detected in some pigs, suggesting co-infection by more than one strain and/or the presence of a recombinant virus. These results constitute the first molecular and serologic evidence of swine HEV circulation in the Eastern Brazilian Amazon. (C) 2012 Elsevier Ltd. All rights reserved.

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences

Intra-and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton-and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F-S parameters. In addition, high values of haplotype diversity ((H) over cap) and low values of nucleotide diversity (pi), combined with a high number of low frequency haplotypes and values of theta(pi)<theta(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.

Phylogeny of Lundia (Bignoniaceae) based on ndhF and PepC sequences

The circumscription of genera belonging to tribe Bignonieae (Bignoniaceae) has traditionally been complex, with only a few genera having stable circumscriptions in the various classification systems proposed for the tribe. The genus Lundia, for instance, is well characterized by a series of morphological synapomorphies and its circumscription has remained quite stable throughout its history. Despite the stable circumscription of Lundia, the circumscription of species within the genus has remained problematic. This study aims to reconstruct the phylogeny of Lundia in order to refine species circumscriptions, gain a better understanding of relationships between taxa, and identify potential morphological synapomorphies for species and major clades. We sampled 26 accessions representing 13 species of Lundia, and 5 outgroups, and reconstructed the phylogeny of the genus using a chloroplast (ndhF) and a nuclear marker (PepC). Data derived from sequences of the individual loci were analyzed using parsimony and Bayesian inference, and the combined molecular dataset was analyzed with Bayesian methods. The monophyly of Lundia nitidula, a species with a particularly complex circumscription, was tested using Shimodaira-Hasegawa (SH) test and the approximately unbiased test for phylogenetic tree selection (AU test). In addition, 40 morphological characters were mapped onto the tree that resulted from the analysis of the combined molecular dataset in order to identify morphological synapomorphies of individual species and major clades. Lundia and most species currently recognized within the genus were strongly supported as monophyletic in all analyses. One species, Lundia nitidula, was not resolved as monophyletic, but the monophyly of this species was not rejected by the AU and SH tests. Lundia sect. Eriolundia is resolved as paraphyletic in all analyses, while Lundia sect. Eulundia is monophyletic and supported by the same morphological characters traditionally used to circumscribe this section. The phylogeny of Lundia contributed important information for a better circumscription of species and served as basis the taxonomic revision of the genus.

In silico identification of conserved intercoding sequences in Leishmania genomes: Unraveling putative cis-regulatory elements

In silico analyses of Leishmania spp. genome data are a powerful resource to improve the understanding of these pathogens' biology. Trypanosomatids such as Leishmania spp. have their protein-coding genes grouped in long polycistronic units of functionally unrelated genes. The control of gene expression happens by a variety of posttranscriptional mechanisms. The high degree of synteny among Leishmania species is accompanied by highly conserved coding sequences (CDS) and poorly conserved intercoding untranslated sequences. To identify the elements involved in the control of gene expression, we conducted an in silico investigation to find conserved intercoding sequences (CICS) in the genomes of L major, L infantum, and L braziliensis. We used a combination of computational tools, such as Linux-Shell, PERL and R languages, BLAST, MSPcrunch, SSAKE, and Pred-A-Term algorithms to construct a pipeline which was able to: (i) search for conservation in target-regions, (ii) eliminate CICS redundancy and mask repeat elements, (iii) predict the mRNA's extremities, (iv) analyze the distribution of orthologous genes within the generated LeishCICS-clusters, (v) assign GO terms to the LeishCICS-clusters. and (vi) provide statistical support for the gene-enrichment annotation. We associated the LeishCICS-cluster data, generated at the end of the pipeline, with the expression profile oft. donovani genes during promastigote-amastigote differentiation, as previously evaluated by others (GEO accession: GSE21936). A Pearson's correlation coefficient greater than 0.5 was observed for 730 LeishCICS-clusters containing from 2 to 17 genes. The designed computational pipeline is a useful tool and its application identified potential regulatory cis elements and putative regulons in Leishmania. (C) 2012 Elsevier B.V. All rights reserved.

A novel protein refolding protocol for the solubilization and purification of recombinant peptidoglycan-associated lipoprotein from Xylella fastidiosa overexpressed in Escherichia coil

Xylella fastidiosa is a Gram-negative xylem-limited plant pathogenic bacterium responsible for several economically important crop diseases. Here, we present a novel and efficient protein refolding protocol for the solubilization and purification of recombinant X. fastidiosa peptidoglycan-associated lipoprotein (XfPal). Pal is an outer membrane protein that plays important roles in maintaining the integrity of the cell envelope and in bacterial pathogenicity. Because Pal has a highly hydrophobic N-terminal domain, the heterologous expression studies necessary for structural and functional protein characterization are laborious once the recombinant protein is present in inclusion bodies. Our protocol based on the denaturation of the XfPal-enriched inclusion bodies with 8 M urea followed by buffer-exchange steps via dialysis proved effective for the solubilization and subsequent purification of XfPal, allowing us to obtain a large amount of relatively pure and folded protein. In addition, XfPal was biochemically and functionally characterized. The method for purification reported herein is valuable for further research on the three-dimensional structure and function of Pal and other outer membrane proteins and can contribute to a better understanding of the role of these proteins in bacterial pathogenicity, especially with regard to the plant pathogen X. fastidiosa. (C) 2012 Elsevier Inc. All rights reserved.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five (TM) cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A 'site-specific' proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification. Copyright (C) 2011 S. Karger AG, Basel

A Recombinant Protein Based on Trypanosoma cruzi P21 Enhances Phagocytosis

Background: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Findings: Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent on the PI3-kinase signaling pathway. Conclusions: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

MRKAd5 HIV-1 Gag/Pol/Nef Vaccine-Induced T-Cell Responses Inadequately Predict Distance of Breakthrough HIV-1 Sequences to the Vaccine or Viral Load

Background: The sieve analysis for the Step trial found evidence that breakthrough HIV-1 sequences for MRKAd5/HIV-1 Gag/Pol/Nef vaccine recipients were more divergent from the vaccine insert than placebo sequences in regions with predicted epitopes. We linked the viral sequence data with immune response and acute viral load data to explore mechanisms for and consequences of the observed sieve effect. Methods: Ninety-one male participants (37 placebo and 54 vaccine recipients) were included; viral sequences were obtained at the time of HIV-1 diagnosis. T-cell responses were measured 4 weeks post-second vaccination and at the first or second week post-diagnosis. Acute viral load was obtained at RNA-positive and antibody-negative visits. Findings: Vaccine recipients had a greater magnitude of post-infection CD8+ T cell response than placebo recipients (median 1.68% vs 1.18%; p = 0.04) and greater breadth of post-infection response (median 4.5 vs 2; p = 0.06). Viral sequences for vaccine recipients were marginally more divergent from the insert than placebo sequences in regions of Nef targeted by pre-infection immune responses (p = 0.04; Pol p = 0.13; Gag p = 0.89). Magnitude and breadth of pre-infection responses did not correlate with distance of the viral sequence to the insert (p. 0.50). Acute log viral load trended lower in vaccine versus placebo recipients (estimated mean 4.7 vs 5.1) but the difference was not significant (p = 0.27). Neither was acute viral load associated with distance of the viral sequence to the insert (p>0.30). Interpretation: Despite evidence of anamnestic responses, the sieve effect was not well explained by available measures of T-cell immunogenicity. Sequence divergence from the vaccine was not significantly associated with acute viral load. While point estimates suggested weak vaccine suppression of viral load, the result was not significant and more viral load data would be needed to detect suppression.

Synthetic Phytochelatin Surface Display in Cupriavidus metallidurans CH34 for Enhanced Metals Bioremediation

This work describes the effects of the cell surface display of a synthetic phytochelatin in the highly metal tolerant bacterium Cupriavidus metallidurans CH34. The EC20sp synthetic phytochelatin gene was fused between the coding sequences of the signal peptide (SS) and of the autotransporter beta-domain of the Neisseria gonorrhoeae IgA protease precursor (IgA beta), which successfully targeted the hybrid protein toward the C. metallidurans outer membrane. The expression of the SS-EC20sp-IgA beta gene fusion was driven by a modified version of the Bacillus subtilis mrgA promoter showing high level basal gene expression that is further enhanced by metal presence in C. metallidurans. The recombinant strain showed increased ability to immobilize Pb2+, Zn2+, Cu2+, Cd2+, Mn2+, and Ni2+ ions from the external medium when compared to the control strain. To ensure plasmid stability and biological containment, the MOB region of the plasmid was replaced by the E. coli hok/sok coding sequence.