Physical and Chemical Barriers in the Larval Midgut Confer Developmental Resistance to Virus Infection in Drosophila.

Insects can become lethally infected by the oral intake of a number of insect-specific viruses. Virus infection commonly occurs in larvae, given their active feeding behaviour; however, older larvae often become resistant to oral viral infections. To investigate mechanisms that contribute to resistance throughout the larval development, we orally challenged Drosophila larvae at different stages of their development with Drosophila C virus (DCV, Dicistroviridae). Here, we showed that DCV-induced mortality is highest when infection initiates early in larval development and decreases the later in development the infection occurs. We then evaluated the peritrophic matrix as an antiviral barrier within the gut using a Crystallin-deficient fly line (Crys-/-), whose PM is weakened and becomes more permeable to DCV-sized particles as the larva ages. This phenotype correlated with increasing mortality the later in development oral challenge occurred. Lastly, we tested in vitro the infectivity of DCV after incubation at pH conditions that may occur in the midgut. DCV virions were stable in a pH range between 3.0 and 10.5, but their infectivity decreased at least 100-fold below (1.0) and above (12.0) this range. We did not observe such acidic conditions in recently hatched larvae. We hypothesise that, in Drosophila larvae, the PM is essential for containing ingested virions separated from the gut epithelium, while highly acidic conditions inactivate the majority of the virions as they transit.

Contrasting Patterns of Virus Protection and Functional Incompatibility Genes in Two Conspecific Wolbachia Strains from Drosophila pandora.

Wolbachia infections can present different phenotypes in hosts, including different forms of reproductive manipulation and antiviral protection, which may influence infection dynamics within host populations. In populations of Drosophila pandora two distinct Wolbachia strains coexist, each manipulating host reproduction: strain wPanCI causes cytoplasmic incompatibility (CI), whereas strain wPanMK causes male killing (MK). CI occurs when a Wolbachia-infected male mates with a female not infected with a compatible type of Wolbachia, leading to nonviable offspring. wPanMK can rescue wPanCI-induced CI but is unable to induce CI. The antiviral protection phenotypes provided by the wPanCI and wPanMK infections were characterized; the strains showed differential protection phenotypes, whereby cricket paralysis virus (CrPV)-induced mortality was delayed in flies infected with wPanMK but enhanced in flies infected with wPanCI compared to their respective Wolbachia-cured counterparts. Homologs of the cifA and cifB genes involved in CI identified in wPanMK and wPanCI showed a high degree of conservation; however, the CifB protein in wPanMK is truncated and is likely nonfunctional. The presence of a likely functional CifA in wPanMK and wPanMK's ability to rescue wPanCI-induced CI are consistent with the recent confirmation of CifA's involvement in CI rescue, and the absence of a functional CifB protein further supports its involvement as a CI modification factor. Taken together, these findings indicate that wPanCI and wPanMK have different relationships with their hosts in terms of their protective and CI phenotypes. It is therefore likely that different factors influence the prevalence and dynamics of these coinfections in natural Drosophila pandora hosts.IMPORTANCEWolbachia strains are common endosymbionts in insects, with multiple strains often coexisting in the same species. The coexistence of multiple strains is poorly understood but may rely on Wolbachia organisms having diverse phenotypic effects on their hosts. As Wolbachia is increasingly being developed as a tool to control disease transmission and suppress pest populations, it is important to understand the ways in which multiple Wolbachia strains persist in natural populations and how these might then be manipulated. We have therefore investigated viral protection and the molecular basis of cytoplasmic incompatibility in two coexisting Wolbachia strains with contrasting effects on host reproduction.

Haplotypes in CCR5-CCR2 , CCL3 and CCL5 are associated with natural resistance to HIV-1 infection in a Colombian cohort / Los haplotipos en CCR5-CCR2 , CCL3 y CCL5 se asocian con resistencia natural a la infección por el HIV-1 en una cohorte colombiana

RESUMEN Introduction: Variants in genes encoding for HIV-1 co-receptors and their natural ligands have been individually associated to natural resistance to HIV-1 infection. However, the simultaneous presence of these variants has been poorly studied. Objective: To evaluate the association of single and multilocus haplotypes in genes coding for the viral co-receptors CCR5 and CCR2, and their ligands CCL3 and CCL5, with resistance or susceptibility to HIV-1 infection. Materials and methods: Nine variants in CCR5-CCR2, two SNPs in CCL3 and two in CCL5 were genotyped by PCR-RFLP in 35 seropositive (cases) and 49 HIV-1-exposed seronegative Colombian individuals (controls). Haplotypes were inferred using the Arlequin software, and their frequency in individual or combined loci was compared between cases and controls by the chi-square test. A p' value <0.05 after Bonferroni correction was considered significant. Results: Homozygosis of the human haplogroup (HH) E was absent in controls and frequent in cases, showing a tendency to susceptibility. The haplotypes C-C and T-T in CCL3 were associated with susceptibility (p'=0.016) and resistance (p'<0.0001) to HIV-1 infection, respectively. Finally, in multilocus analysis, the haplotype combinations formed by HHC in CCR5-CCR2, T-T in CCL3 and G-C in CCL5 were associated with resistance (p'=0.006). Conclusion: Our results suggest that specific combinations of variants in genes from the same signaling pathway can define an HIV-1 resistant phenotype. Despite our small sample size, our statistically significant associations suggest strong effects; however, these results should be further validated in larger cohorts.

ABSTRACT Introducción. Algunas variantes en genes que codifican los correceptores del HIV-1 y sus ligandos se han asociado individualmente a la resistencia natural frente a dicha infección. Sin embargo, su presencia simultánea ha sido poco estudiada. Objetivo. Evaluar la asociación de haplotipos individuales y multilocus en genes que codifican los correceptores virales CCR5 y CCR2 y sus ligandos CCL3 y CCL5 con la resistencia o la propensión a la infección por el HIV-1. Materiales y métodos. Nueve variantes en CCR5-CCR2, dos en CCL3 y dos en CCL5 fueron genotipificadas mediante reacción en cadena de la polimerasa de polimorfismos de longitud de fragmentos de restricción (Restriction Fragment Length Polymorphism-PCR-RFLP) en 35 individuos seropositivos (casos) y 49 seronegativos expuestos (controles) de Colombia. Los haplotipos se infirieron utilizando el programa Arlequín, y su frecuencia individual o combinada se comparó en los casos y los controles mediante la prueba de ji al cuadrado. Se consideró significativo un valor de p'<0,05 después de la corrección de Bonferroni. Resultados. La homocigosis del haplogrupo humano (HH) E estaba ausente en los controles y era frecuente en los casos, es decir, con tendencia hacia la propensión. Los haplotipos C-C y T-T en CCL3 se asociaron con la propensión (p'=0,016) y la resistencia (p'<0,0001), respectivamente. Por último, en el análisis multilocus, el haplotipo combinado formado por HHC en CCR5-CCR2, T-T en CCL3 y G-C en CCL5 se asoció con la resistencia (p'=0,006). Conclusión. Los resultados de este estudio sugieren que ciertas combinaciones específicas de variantes en los genes de una misma vía de señalización pueden definir un fenotipo resistente al HIV-1. Aunque el tamaño de la muestra era pequeño, las asociaciones estadísticamente significativas sugieren un efecto considerable; sin embargo, estos resultados deben validarse en cohortes de mayor tamaño.

Haplotypes in CCR5-CCR2, CCL3 and CCL5 are associated with natural resistance to HIV-1 infection in a Colombian cohort.

INTRODUCTION: Variants in genes encoding for HIV-1 co-receptors and their natural ligands have been individually associated to natural resistance to HIV-1 infection. However, the simultaneous presence of these variants has been poorly studied. OBJECTIVE: To evaluate the association of single and multilocus haplotypes in genes coding for the viral co-receptors CCR5 and CCR2, and their ligands CCL3 and CCL5, with resistance or susceptibility to HIV-1 infection. MATERIALS AND METHODS: Nine variants in CCR5-CCR2, two SNPs in CCL3 and two in CCL5 were genotyped by PCR-RFLP in 35 seropositive (cases) and 49 HIV-1-exposed seronegative Colombian individuals (controls). Haplotypes were inferred using the Arlequin software, and their frequency in individual or combined loci was compared between cases and controls by the chi-square test. A p' value ;0.05 after Bonferroni correction was considered significant. RESULTS: Homozygosis of the human haplogroup (HH) E was absent in controls and frequent in cases, showing a tendency to susceptibility. The haplotypes C-C and T-T in CCL3 were associated with susceptibility (p'=0.016) and resistance (p';0.0001) to HIV-1 infection, respectively. Finally, in multilocus analysis, the haplotype combinations formed by HHC in CCR5-CCR2, T-T in CCL3 and G-C in CCL5 were associated with resistance (p'=0.006). CONCLUSION: Our results suggest that specific combinations of variants in genes from the same signaling pathway can define an HIV-1 resistant phenotype. Despite our small sample size, our statistically significant associations suggest strong effects; however, these results should be further validated in larger cohorts.

Vitamin D modulates the expression of HLA-DR and CD38 after in vitro activation of T-cells.

OBJECTIVE: Vitamin D (VitD) is an anti-inflammatory hormone; however, some evidence shows that VitD may induce the expression of activation markers, such as CD38 and HLA-DR. We explored its effect on the expression of these markers on CD4+ and CD8+ T-cells in vitro, and their potential correlations in vivo. MATERIALS AND METHODS: CD38 and HLA-DR expression was measured by flow cytometry in PHA/IL-2-activated mononuclear cells cultured under VitD precursors: three cholecalciferol (10-11M, 10-9M, 10-7M; n=11) and two calcidiol (40 ng/mL, 80 ng/mL; n=9) concentrations. The correlation between the expression of these markers in freshly isolated blood cells and serum levels of calcidiol was also explored (n=10). RESULTS: Cholecalciferol at 10-7M increased the proportion of CD4+ CD38+ and CD8+ CD38+ cells, and decreased CD8+HLA-DR+ cells. As co-expression, it increased the CD38+HLA-DR- and decreased CD38-HLA-DR+ subpopulations in both CD4+ and CD8+ T-cells, and decreased CD4+CD38-HLA-DR- and CD8+ CD38+HLA-DR+; whereas both calcidiol concentrations decreased the proliferation of CD38-HLA-DR- and CD38-HLA-DR+ subpopulations. Both forms of VitD increased the number of CD38 molecules per cell. In contrast, there was a positive but non-significant correlation between serum calcidiol levels and the expression of CD38 and HLA-DR in CD4+ and CD8+ T-cells. CONCLUSION: Although no significant correlations were observed in vivo in healthy subjects, VitD treatment in vitro modulated immune activation by increasing the expression of CD38 and decreasing the proliferation of HLA-DR+ and resting cells, which may correlate with improved effector and decreased proliferative capabilities. These results highlight the potential use of VitD as therapeutic strategy in immune disorders.

Precursor Forms of Vitamin D Reduce HIV-1 Infection In Vitro.

BACKGROUND: Although the anti-HIV-1 effects of vitamin D (VitD) have been reported, mechanisms behind such protection remain largely unexplored. METHODS: The effects of two precursor forms (cholecalciferol/calciol at 0.01, 1 and 100 nM and calcidiol at 100 and 250 nM) on HIV-1 infection, immune activation, and gene expression were analyzed in vitro in cells of Colombian and Italian healthy donors. We quantified levels of released p24 by enzyme-linked immunosorbent assay, of intracellular p24 and cell-surface expression of CD38 and HLA-DR by flow cytometry, and mRNA expression of antiviral and immunoregulatory genes by real-time reverse transcription-polymerase chain reaction. RESULTS: Cholecalciferol decreased the frequency of HIV-1-infected p24CD4 T cells and levels of p24 in supernatants in a dose-dependent manner. Moreover, the CD4CD38HLA-DR and CD4CD38HLA-DR subpopulations were more susceptible to infection but displayed the greatest cholecalciferol-induced decreases in infection rate by an X4-tropic strain. Likewise, cholecalciferol at its highest concentration decreased the frequency of CD38HLA-DR but not of CD38HLA-DR T-cell subsets. Analyzing the effects of calcidiol, the main VitD source for immune cells and an R5-tropic strain as the most frequently transmitted virus, a reduction in HIV-1 productive infection was also observed. In addition, an increase in mRNA expression of APOBEC3G and PI3 and a reduction of TRIM22 and CCR5 expression, this latter positively correlated with p24 levels, was noted. CONCLUSIONS: VitD reduces HIV-1 infection in T cells possibly by inducing antiviral gene expression, reducing the viral co-receptor CCR5 and, at least at the highest cholecalciferol concentration, by promoting an HIV-1-restrictive CD38HLA-DR immunophenotype.