The application of single-strand conformation polymorphism (SSCP) technique for the analysis of molecular heterogeneity of grapevine virus A

The results of the analysis of grapevine virus A (GVA) isolates by single-strand conformation polymorphism (SSCP) confirm that this technique is very helpful in rapid and relatively low cost preliminary analysis of molecular heterogeneity of viruses. The results clearly show that the reliability of SSCP analysis of GVA depends on oligonucleotide primers for successful RT-PCR amplification of the highest possible number of molecular variants of the virus. Among 7 pairs of GVA-specific primers designed in different laboratories only two, those from Canada (117038 and C7273) and Switzerland (MP and CPdt), allowed positive RT-PCR amplification of all our isolates of the virus mechanically transmitted from various grapevines to Nicotiana benthamiana. With SSCP analysis of 238 bp DNA fragments complementary to part of ORF5 of GVA, produced by RT-PCR using the first pair of primers, we were able to detect 1-35 nt differences between GVA isolates. The DNA fragments, about 986 bp, complementary to part of ORF3 and ORF4, ORF5 and 3'UTR of GVA, produced by RT-PCR using the second pair of primers, were useful for SSCP analysis only after their digestion with the restriction enzyme DdeI. The results strongly suggest that SSCP analysis of 238 nt fragment of ORF5 of GVA along with DdeI/SSCP analysis of about 986 nt 3'terminal fragment of the virus allow rapid and reliable determination of the number of dominant nt sequence variants of GVA present in a single N. benthamiana or grapevine plant.

Single-strand conformation polymorphism (SSCP), cloning and sequencing reveals two major groups of divergent molecular variants of grapevine leafroll-associated virus 3 (GLRaV-3)

The SSCP and RE/SSCP analysis of ORF5, ORF6 and ORF7 of 25 local and overseas isolates of GLRaV-3 showed only two kinds of distinct SSCP profiles for each of these genomic regions. It suggested low molecular variability of the virus. Fragments of the 5’UTR + ORF1a, 3’ terminal part of ORF1a, ORF4, ORF5, ORF6 and ORF7 of three isolates, representing distinct SSCP profiles, were cloned and sequenced. Results revealed that although the 3’terminal half of the genome (ORF4-7) and the sequence located in the 3’ terminal part of ORF1a were relatively similar among isolates (91.3-96.2 % nt identity), their 5’terminal parts (88 nt of 5’UTR and adjacent first 329 nt of ORF1a) were clearly divergent (81.6-81.8 % nt identity). Analysis of this divergent part of GLRaV-3 for an additional 11 isolates showed that they cluster in two distinct molecular groups, sharing 94.7-99.7 % and 80.8-85.1 % nt identity within and between groups respectively. The correlation between the molecular groups and SSCP profiles of the 209 nt fragment of ORF5 of GLRaV-3 strongly suggests that SSCP analysis of this easily RT-PCR amplified region can be used for rapid identification of divergent molecular variants of the virus in field-collected grapevine samples.

Grapevine leafroll-associated virus 3.

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management

An HLA-I signature favouring KIR-educated Natural Killer cells mediates immune control of HIV in children and contrasts with the HLA-B-restricted CD8+T-cell-mediated immune control in adults

Natural Killer (NK) cells contribute to HIV control in adults, but HLA-B-mediated T-cell activity has a more substantial impact on disease outcome. However, the HLA-B molecules influencing immune control in adults have less impact on paediatric infection. To investigate the contribution NK cells make to immune control, we studied >300 children living with HIV followed over two decades in South Africa. In children, HLA-B alleles associated with adult protection or disease-susceptibility did not have significant effects, whereas Bw4 (p = 0.003) and low HLA-A expression (p = 0.002) alleles were strongly associated with immunological and viral control. In a comparator adult cohort, Bw4 and HLA-A expression contributions to HIV disease outcome were dwarfed by those of protective and disease-susceptible HLA-B molecules. We next investigated the immunophenotype and effector functions of NK cells in a subset of these children using flow cytometry. Slow progression and better plasma viraemic control were also associated with high frequencies of less terminally differentiated NKG2A+NKp46+CD56dim NK cells strongly responsive to cytokine stimulation and linked with the immunogenetic signature identified. Future studies are indicated to determine whether this signature associated with immune control in early life directly facilitates functional cure in children

Development, standardization and refinement of procedures for evaluating effects of endocrine active compounds on development and sexual differentiation of Xenopus laevis

Xenopus laevis has been introduced as a model to study effects of endocrine-active compounds (EAC) on development and sexual differentiation. However, variable and inconsistent data have raised questions about the reliability of the test methods applied. The current study was conducted in two laboratories to develop, refine, and standardize procedures and protocols. Larvae were exposed in flow-through systems to 17β-estradiol (E2), at concentrations from 0.2 to 6.0 μg E2 L−1 in Experiment 1A, and 0.015 to 2.0 μg E2 L−1 in Experiment 1B. In both studies survival (92%, 99%) and percentage of animals that completed metamorphosis (97%, 99%) indicated reproducible biological performance. Furthermore, minor variations in husbandry led to significant differences in snout-to-vent length, weight, and gonad size. In Experiment 1A, almost complete feminization occurred in all E2 treatment groups whereas a concentration response was observed in Experiment 1B resulting in an EC50 of 0.12 μg E2 L−1. The final verified protocol is suitable for determining effects of EAC on development and sexual differentiation in X. laevis

PARV4 prevalence, phylogeny, immunology and coinfection with HIV, HBV and HCV in a multicentre African cohort

Background The seroprevalence of human parvovirus-4 (PARV4) varies considerably by region. In sub-Saharan Africa, seroprevalence is high in the general population, but little is known about the transmission routes or the prevalence of coinfection with blood-borne viruses, HBV, HCV and HIV. Methods To further explore the characteristics of PARV4 in this setting, with a particular focus on the prevalence and significance of coinfection, we screened a cohort of 695 individuals recruited from Durban and Kimberley (South Africa) and Gaborone (Botswana) for PARV4 IgG and DNA, as well as documenting HIV, HBV and HCV status. Results Within these cohorts, 69% of subjects were HIV-positive. We identified no cases of HCV by PCR, but 7.4% were positive for HBsAg. PARV4 IgG was positive in 42%; seroprevalence was higher in adults (69%) compared to children (21%) (p Conclusions This characterisation of PARV4 infection provides enhanced insights into the epidemiology of infection and co-infection in African cohorts, and provides the foundations for planning further focused studies to elucidate transmission pathways, immune responses, and the clinical significance of this organism.</p

Grapevine leafroll-associated virus 3

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by the Frontiers Research Foundation. The published article can be found at: http://www.frontiersin.org/Microbiology.Keywords: Ampelovirus, GLRaV-3, Grapevine leafroll disease, Closteroviridae, Genetic variantsKeywords: Ampelovirus, GLRaV-3, Grapevine leafroll disease, Closteroviridae, Genetic variant

Are men well served by family planning programs?

Although the range of contraceptives includes methods for men, namely condoms, vasectomy and withdrawal that men use directly, and the Standard Days Method (SDM) that requires their participation, family planning programming has primarily focused on women. What is known about reaching men as contraceptive users? This paper draws from a review of 47 interventions that reached men and proposes 10 key considerations for strengthening programming for men as contraceptive users. A review of programming shows that men and boys are not particularly well served by programs. Most programs operate from the perspective that women are contraceptive users and that men should support their partners, with insufficient attention to reaching men as contraceptive users in their own right. The notion that family planning is women’s business only is outdated. There is sufficient evidence demonstrating men’s desire for information and services, as well as men’s positive response to existing programming to warrant further programming for men as FP users. The key considerations focus on getting information and services where men and boys need it; addressing gender norms that affect men’s attitudes and use while respecting women’s autonomy; reaching adolescent boys; including men as users in policies and guidelines; scaling up successful programming; filling gaps with implementation research and monitoring & evaluation; and creating more contraceptive options for men