A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background

Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.

Methods and Findings

In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10⁻⁹ IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.

Conclusion

This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.     

The ABC transporter-encoding gene AFR1 affects the resistance of Cryptococcus neoformans to microglia-mediated antifungal activity by delaying phagosomal maturation

The pathogenic yeast Cryptococcus neoformans has evolved several strategies to survive within phagocytes. Recently, it has been demonstrated that upregulation of the ATP binding cassette transporter-encoding gene antifungal resistance 1 ( AFR1 ) is important not only for determining the resistance of C. neoformans to fluconazole but also in influencing fungal virulence. In the present study, we showed that the fluconazole-resistant AFR1- overexpressing mutant strain was not sensitive to microglia-mediated anticryptococcal activity, as compared with the fluconazole-susceptible isogenic strains, the wild type and the afr1 Δ mutant. Interestingly, although the three strains were phagocytosed to a similar extent, reduced acidification and delayed maturation were observed in phagosomes containing the AFR1 -overexpressing strain with respect to the others. These findings provide the first evidence that upregulation of the AFR1 gene affects C. neoformans –microglia interplay, adding insights to the complexity of cryptococcal virulence and to its unexpected link with azole resistance.     

Regulatory T Cell Expansion in HTLV-1 and Strongyloidiasis Co-infection Is Associated with Reduced IL-5 Responses to Strongyloides stercoralis Antigen

Background

Human strongyloidiasis varies from a chronic but limited infection in normal hosts to hyperinfection in patients treated with corticosteroids or with HTLV-1 co-infection. Regulatory T cells dampen immune responses to infections. How human strongyloidiasis is controlled and how HTLV-1 infection affects this control are not clear. We hypothesize that HTLV-1 leads to dissemination of Strongyloides stercoralis infection by augmenting regulatory T cell numbers, which in turn down regulate the immune response to the parasite.

Objective

To measure peripheral blood T regulatory cells and Strongyloides stercoralis larval antigen-specific cytokine responses in strongyloidiasis patients with or without HTLV-1 co-infection.

Methods

Peripheral blood mononuclear cells (PBMCs) were isolated from newly diagnosed strongyloidiasis patients with or without HTLV-1 co-infection. Regulatory T cells were characterized by flow cytometry using intracellular staining for CD4, CD25 and FoxP3. PBMCs were also cultured with and without Strongyloides larval antigens. Supernatants were analyzed for IL-5 production.

Results

Patients with HTLV-1 and Strongyloides co-infection had higher parasite burdens. Eosinophil counts were decreased in the HTLV-1 and Strongyloides co-infected subjects compared to strongyloidiasis-only patients (70.0 vs. 502.5 cells/mm³, p = 0.09, Mann-Whitney test). The proportion of regulatory T cells was increased in HTLV-1 positive subjects co-infected with strongyloidiasis compared to patients with only strongyloidiasis or asymptomatic HTLV-1 carriers (median = 17.9% vs. 4.3% vs. 5.9 p<0.05, One-way ANOVA). Strongyloides antigen-specific IL-5 responses were reduced in strongyloidiasis/HTLV-1 co-infected patients (5.0 vs. 187.5 pg/ml, p = 0.03, Mann-Whitney test). Reduced IL-5 responses and eosinophil counts were inversely correlated to the number of CD4+CD25+FoxP3+ cells.

Conclusions

Regulatory T cell counts are increased in patients with HTLV-1 and Strongyloides stercoralis co-infection and correlate with both low circulating eosinophil counts and reduced antigen-driven IL-5 production. These findings suggest a role for regulatory T cells in susceptibility to Strongyloides hyperinfection.     

Characterisation of structural genes involved in phytic acid biosynthesis in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.)

Phytate (myo-inositol hexakisphosphate), the major form of phosphorous storage in plant seeds, is an inositol phosphate compound poorly digested by humans and monogastric animals. A major goal for grain crop improvement is the reduction of its content in the seed to improve micronutrient bioavailability and phosphorus utilisation by humans and non-ruminant animals, respectively. We are interested in lowering phytic acid in common bean seed and to this goal we have undertaken a two-strategy approach: the isolation of mutants from an EMS mutagenised population (Campion et al. 2009) and the identification of genes coding for candidate enzymes involved in inositol phosphate metabolism for future targeted mutant isolation and/or study. In this paper we report data referred to the second approach and concerning the isolation and genomic organisation of Phaseolus vulgaris genes coding for myo-inositol 1-phosphate synthase (PvMIPSs and PvMIPSv), inositol monophosphatase (PvIMP), myo-inositol kinase (PvMIK), inositol 1,4,5-tris-phosphate kinase (PvIPK2), inositol 1,3,4-triphosphate 5/6-kinase (PvITPKα and PvITPKβ) and inositol 1,3,4,5,6 pentakisphosphate 2-kinase (PvIPK1). All these genes have been mapped on the common bean reference genetic map of McClean (NDSU) 2007 using a virtual mapping strategy. Bean markers, presumably associated to each gene of the phytic acid pathway, have also been identified. In addition, we provide a picture of the expression, during seed development, of the genes involved in phytic acid synthesis, including those such as MIK, IMP and IPK2, for which this information was lacking.     

Lethal and sublethal effects of preimaginal treatments with two pyrethroids on the life history of the egg parasitoid <Emphasis Type="Italic">Telenomus busseolae</Emphasis>

The egg parasitoid Telenomus busseolae Gahan (Hymenoptera: Scelionidae) is one of the most important natural enemies of the Mediterranean corn stem borer, Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae). The effects of lethal and sublethal concentrations of two commonly used pyrethroids, deltamethrin and cyfluthrin, on emergence, fecundity, longevity, and offspring sex ratio of T. busseolae exposed during different preimaginal stages were studied. The insecticide, the tested concentrations of the insecticide (field-recommended concentration and three dilutions with twofold concentration decreases), and the stage of parasitoid preimaginal development (first and second larval stages and pupae) all significantly influenced the percentage of emergence. Life history parameters of parasitoids exposed to the highest and lowest tested concentrations of the two insecticides (3.0 and 0.375 ml l⁻¹ for cyfluthrin and 0.5 and 0.0625 ml l⁻¹ for deltamethrin) at eight days post-oviposition (pupal stage) were calculated. The intrinsic rate of increase, the total progeny produced, and the net reproductive rate of the parasitoid were all adversely affected by the field-recommended concentrations of at least one insecticide. However, the longevity, the length of the oviposition period, and the offspring sex ratio were unaltered by the two insecticides. The effects of sublethal concentrations of the insecticides, applied at preimaginal stages, on the responses of T. busseolae females to host abdominal scales, were studied in open-arena bioassays. Cyfluthrin altered both the residence time and linear speed of the parasitoid in the host scale-containing areas, whereas deltamethrin had no effect. Our results indicate that, in addition to lethal effects, sublethal effects should also be considered when evaluating insecticide consequences on non-targets.     

Comparing the influences of ecological and evolutionary factors on the successful invasion of a fungal forest pathogen

The fungal forest pathogen Heterobasidion annosum has been introduced from North America into Italy and is now associated with high mortality of Italian stone pines. Due to the presence of a closely related native H. annosum taxon, this pathosystem presents an unusual opportunity to test specific ecological and evolutionary factors influencing fungal invasions. Comparative inoculation experiments on Scots pine cuttings and on seedlings of European and North American pines failed to identify significant increased pathogenicity of North American genotypes on European hosts congruent with lack of host-pathogen co-evolution. However, spore trappings indicate that while reproductive potential of native H. annosum was significantly reduced in the dry season, that of the invasive taxon was consistently high regardless of season. Ecological differences between the native and exotic taxon may therefore facilitate this invasion. Understanding which factors enhance this emerging forest disease is important both for biotic invasion theory and for disease control.     

The oligodeoxynucleotide sequences corresponding to never-expressed peptide motifs are mainly located in the non-coding strand

Background  

We study the usage of specific peptide platforms in protein composition. Using the pentapeptide as a unit of length, we find that in the universal proteome many pentapeptides are heavily repeated (even thousands of times), whereas some are quite rare, and a small number do not appear at all. To understand the physico-chemical-biological basis underlying peptide usage at the proteomic level, in this study we analyse the energetic costs for the synthesis of rare and never-expressed versus frequent pentapeptides. In addition, we explore residue bulkiness, hydrophobicity, and codon number as factors able to modulate specific peptide frequencies. Then, the possible influence of amino acid composition is investigated in zero- and high-frequency pentapeptide sets by analysing the frequencies of the corresponding inverse-sequence pentapeptides. As a final step, we analyse the pentadecamer oligodeoxynucleotide sequences corresponding to the never-expressed pentapeptides.     

Aquaporins in the wild: natural genetic diversity and selective pressure in the PIP gene family in five Neotropical tree species

Background  

Tropical trees undergo severe stress through seasonal drought and flooding, and the ability of these species to respond may be a major factor in their survival in tropical ecosystems, particularly in relation to global climate change. Aquaporins are involved in the regulation of water flow and have been shown to be involved in drought response; they may therefore play a major adaptive role in these species. We describe genetic diversity in the PIP sub-family of the widespread gene family of Aquaporins in five Neotropical tree species covering four botanical families.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.