Human Virology

We attempted to define optimal conditions for amplification of low copy number HIV-1 RNA sequences in plasma samples, applying improved conditions for nucleic acid extraction and amplification. Several methodologic parameters were evaluated, including methods of RNA extraction, volumes of plasma samples, proportion of extracted RNA used as a template for amplification, and reverse transcriptase-DNA polymerase enzyme combination employed in cDNA synthesis and polymerase chain reaction amplification. With this improved assay, we were able to obtain sufficient amounts of amplified material for direct sequencing in 97% of all plasma samples in our study, including 88% of samples with viral loads <80 copies/mL, 78% of samples with viral loads <50 copies/mL, and even 2 (67%) of 3 samples with <20 copies/mL. This procedure could be useful for testing resistance mutations in patients undergoing highly active antiretroviral therapy, in which the viral load is commonly <400 copies...

Human parvovirus B19 is a member of the family Parvoviridae. It can be transmitted congenitally from an infected mother to the foetus. The virus has been implicated in hydrops foetalis, spontaneous abortion, aplastic crisis and acute symmetric polyarthropathy. The present study was carried out to determine the seroprevalence of Human Parvovirus B19 IgG and IgM antibodies and to determine the risk factors associated with acquisition of the virus. A total of 326 blood samples were collected from consenting pregnant women who were attending antenatal clinic in Abuja, Nigeria. Structured questionnaire was used to obtain data on socio-demography and risk factors. The sera were obtained from samples were analyzed for IgG and IgM antibodies of human parvovirus B19 using SERION ELISA Classic Parvovirus B19 IgG/IgM kits. A total of 171 (52.5%) of women had IgG antibody to Parvovirus B19 while 31 (9.5%) were positive for IgM antibody. Demographics including age group, educational status, occupation, type of marriage, number of children, history of blood transfusion and trimester of pregnancy were statistically associated with prevalence of IgG antibody (p ≤ 0.05). There was a significant association (χ 2 = 12.571; p = 0.028) between age group and IgG antibody. The age group > 40 years old had the highest prevalence of IgG antibodies while the age group 21-25 years (p ≤ 0.05) had the least (39.3%). Similarly, there was significant association (χ 2 = 39.602; p < 0.001) between trimester and IgG antibody with the highest IgG antibody prevalence recorded in second trimester. Human Parvovirus B19 is prevalent in Abuja, Nigeria. Ten out of 100 pregnant women are likely to have current human parvovirus B19 infection. So, there is an urgent need to embark on mass education and enlightenment campaign to increase awareness of the general populace on the virus and its acquisition.

The introduction of highly active antiretroviral therapy (HAART) has significantly improved life expectancy of HIV-infected patients; nevertheless, it does not eliminate the virus from hosts, so a cure for this infection is crucial. Some strategies have employed the induction of anti-HIV CD8+ T cells. However, the high genetic variability of HIV-1 represents the biggest obstacle for these strategies, since immune escape mutations within epitopes restricted by Human Leukocyte Antigen class I molecules (HLA-I) abrogate the antiviral activity of these
cells. We used a bioinformatics pipeline for the determination of such mutations, based on selection pressure and docking/refinement analyses. Fifty HIV-1 infected patients were recruited; HLA-A and HLA-B alleles were typified using sequence-specific oligonucleotide approach, and viral RNA was extracted for the amplification of HIV-1 gag, which was bulk sequenced and aligned to perform selection pressure analysis, using Single Likelihood
Ancestor Counting (SLAC) and Fast Unconstrained Bayesian Approximation (FUBAR) algorithms. Positively selected sites were mapped into HLA-I-specific epitopes, and both mutated and wild type epitopes were modelled using PEP-FOLD. Molecular docking and refinement assays were carried out using AutoDock Vina 4 and FlexPepDock. Five positively selected sites were found: S54 at HLA-A*02 GC9, T84 at HLA-A*02 SL9, S125 at HLA-B*35 HY9, S173 at HLA-A*02/B*57 KS12 and I223 at HLA-B*35 HA9. Although some mutations have been previously described as immune escape mutations, the majority of them have not been reported. Molecular docking/refinement analysis showed that one combination of mutations at GC9, one at SL9, and eight at HY9 epitopes could act as immune escape mutations. Moreover, HLA-A*02-positive patients harbouring mutations at KS12, and HLA-B*35-positive patients with mutations at HY9 have significantly higher plasma viral loads than patients lacking such mutations. Thus, HLA-A and -B alleles could be shaping the genetic diversity of HIV-1 through the selection of potential immune escape mutations.