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Background: In September 2016, South Africa introduced the Universal Test and Treat (UTT) programme to manage HIV infection. However, the development of drug resistance and sustaining viral suppression are challenges to the success of treatment programmes. This prospective observational study describes virologic, immunologic, and drug resistance profiles in a test and treat cohort in north-eastern South Africa., Methods: Five hundred and thirty-four HIV-1 positive antiretroviral naïve adults entering treatment programmes were enrolled between January 2016 and February 2018. Trends in CD4+ cell count, viral load, and drug resistance by examination of deep sequences were assessed at baseline and every three months, for 24 months., Results: Seventy-five percent were late initiators into ART (that is baseline CD4+ cell counts < 500 cells/microliter) and 16% were early initiators into ART and baseline CD4 was not available for 9%. Eleven percent (12/104) achieved immunological response after 6 months, 39.4% (41 /104) after 12 months, and 97.5% (101/104) after 24 months. Seventy-one percent (381/534) had baseline viral loads >1000 RNA copies/ml. Nine percent (22/246) achieved viral suppression after 3 months, 50% (122/246) after 6 months and 73.6% (181/246) after 12 months. A slower viral suppression was observed for males than females (p value = 0.012). A total of 45.6% (52/114) individuals had at least one drug resistance mutation (DRM) detected at >20% threshold in any of the time points, and the number increased to 55% (63/114) when minor variants were accounted for. Forty-eight percent (14/29) had drug resistance mutations at >5% threshold as early as 3 months into treatment., Conclusion: The UNAIDS target of 95% viral suppression in individuals under treatment was not observed after 12 months of treatment, and this was less successful for males. Adherence and drug resistance monitoring could be beneficial for individuals harbouring resistant viruses early into treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ogola et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Simian immunodeficiency viruses (SIV) have been jumping between non-human primates in West/Central Africa for thousands of years and yet, the HIV-1 epidemic only originated from a primate lentivirus over 100 years ago., Methods: This study examined the replicative fitness, transmission, restriction, and cytopathogenicity of 22 primate lentiviruses in primary human lymphoid tissue and both primary human and chimpanzee peripheral blood mononuclear cells., Findings: Pairwise competitions revealed that SIV from chimpanzees (cpz) had the highest replicative fitness in human or chimpanzee peripheral blood mononuclear cells, even higher fitness than HIV-1 group M strains responsible for worldwide epidemic. The SIV strains belonging to the "HIV-2 lineage" (including SIVsmm, SIVmac, SIVagm) had the lowest replicative fitness. SIVcpz strains were less inhibited by human restriction factors than the "HIV-2 lineage" strains. SIVcpz efficiently replicated in human tonsillar tissue but did not deplete CD4+ T-cells, consistent with the slow or nonpathogenic disease observed in most chimpanzees. In contrast, HIV-1 isolates and SIV of the HIV-2 lineage were pathogenic to the human tonsillar tissue, almost independent of the level of virus replication., Interpretation: Of all primate lentiviruses, SIV from chimpanzees appears most capable of infecting and replicating in humans, establishing HIV-1. SIV from other Old World monkeys, e.g. the progenitor of HIV-2, replicate slowly in humans due in part to restriction factors. Nonetheless, many of these SIV strains were more pathogenic than SIVcpz. Either SIVcpz evolved into a more pathogenic virus while in humans or a rare SIVcpz, possibly extinct in chimpanzees, was pathogenic immediately following the jump into human., Funding: Support for this study to E.J.A. was provided by the NIH/NIAID R01 AI49170 and CIHR project grant 385787. Infrastructure support was provided by the NIH CFAR AI36219 and Canadian CFI/Ontario ORF 36287. Efforts of J.A.B. and N.J.H. was provided by NIH AI099473 and for D.H.C., by VA and NIH AI AI080313., Competing Interests: Declaration of interests None of the authors have competing financial interest related to this study., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: Wastewater-based genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provides a comprehensive approach to characterize evolutionary patterns and distribution of viral types in a population. This study documents the molecular epidemiology of SARS-CoV-2, in Northern South Africa, from January 2021 to May 2022., Methodology: A total of 487 wastewater samples were collected from the influent of eight wastewater treatment facilities and tested for SARS-CoV-2 RNA using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). SARS-CoV-2 positive samples with genome copies/mL ≥1,500 were subjected to allele-specific genotyping (ASG) targeting the Spike protein; 75 SARS-CoV-2 positive samples were subjected to whole genome sequencing (WGS) on the ATOPlex platform. Variants of concern (VoC) and lineages were assigned using the Nextclade and PangoLIN Software. Concordance for VoC between ASG and WGS analyses was determined. Sequence relationship was determined by phylogenetic analysis., Results: Seventy-five percent (365/487) of the influent samples were positive for SARS-CoV-2 RNA. Delta and Omicron VoC were more predominant at a prevalence of 45 and 32%, respectively, and they were detected as early as January and February 2021, while Beta VoC was least detected at a prevalence of 5%. A total of 11/60 (18%) sequences were assigned lineages and clades only, but not a specific VoC name. Phylogenetic analysis was used to investigate the relationship of these sequences to other study sequences, and further characterize them. Concordance in variant assignment between ASG and WGS was seen in 51.2% of the study sequences. There was more intra-variant diversity among Beta VoC sequences; mutation E484K was absent. Three previously undescribed mutations (A361S, V327I, D427Y) were seen in Delta VoC., Discussion and Conclusion: The detection of Delta and Omicron VoCs in study sites earlier in the outbreak than has been reported in other regions of South Africa highlights the importance of population-based approaches over individual sample-based approaches in genomic surveillance. Inclusion of non-Spike protein targets could improve the specificity of ASG, since all VoCs share similar Spike protein mutations. Finally, continuous molecular epidemiology with the application of sensitive technologies such as next generation sequencing (NGS) is necessary for the documentation of mutations whose implications when further investigated could enhance diagnostics, and vaccine development efforts., Competing Interests: DT was employed by Global Biomed Laboratories Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Tambe, Mathobo, Matume, Munzhedzi, Edokpayi, Viraragavan, Glanzmann, Tebit, Mavhandu-Ramarumo, Street, Johnson, Kinnear and Bessong.)

Background: The public health response to the global COVID-19 pandemic has varied widely by region. In Africa, uptake of effective COVID-19 vaccines has been limited by accessibility and vaccine hesitancy. The aim of this study was to compare perceptions of COVID-19 infection and vaccination between pregnant women and non-pregnant adults in four regions of Cameroon, located in Central Africa., Methods: A cross-sectional survey study was conducted at urban and suburban hospital facilities in Cameroon. Participants were randomly selected from a convenience sample of adult pregnant and non-pregnant adults in outpatient clinical settings between June 1st and July 14th, 2021. A confidential survey was administered in person by trained research nurses after obtaining written informed consent. Participants were asked about self-reported sociodemographics, medical comorbidities, perceptions of COVID-19 infection, and vaccination. Descriptive statistics were used for survey responses and univariate and multivariable logistic regression models were created to explore factors associated with COVID-19 vaccine acceptability., Results: Fewer than one-third of participants were interested in receiving the COVID-19 vaccine (31%, 257/835) and rates did not differ by pregnancy status. Overall, 43% of participants doubted vaccine efficacy, and 85% stated that the vaccine available in Africa was less effective than vaccine available in Europe. Factors independently associated with vaccine acceptability included having children (aOR = 1.5; p = 0.04) and higher education (aOR = 1.6 for secondary school vs primary/none; p = 0.03). Perceived risks of vaccination ranged from death (33%) to fetal harm (31%) to genetic changes (1%). Health care professionals were cited as the most trusted source for health information (82%, n = 681)., Conclusion: COVID-19 vaccine hesitancy and misinformation in Cameroon was highly prevalent among pregnant and non-pregnant adults in 2021 while vaccine was available but not recommended for use in pregnancy. Based on study findings, consistent public health messaging from medical professionals about vaccine safety and efficacy and local production of vaccine are likely to improve acceptability., Competing Interests: The authors have declared that no competing interests exist.

South Africa introduced the "diagnose and treat" universal HIV treatment program in September 2016. This program enables all identified HIV-positive patients to immediately start first-line antiretroviral therapy (ART). However, the presence of drug-resistant (DR) viruses in the drug-naive population complicates the choice of ART. We used next-generation sequencing (NGS) to determine the prevalence and diversity of HIV DR mutations in patients entering HIV treatment programs in northern South Africa. RNA was isolated from plasma of drug-naive HIV-1-infected patients. Using reverse transcriptase polymerase chain reaction, the HIV-1- pol gene comprising the complete protease (PR) and the first 900 bp of reverse transcriptase (RT) was amplified and sequenced on an Illumina MiniSeq platform. Consensus sequences were derived at >20% threshold and at >5% threshold using Geneious PRIME ® software version 2020.1.2. HIV-1 surveillance drug resistance mutations (SDRM) were inferred using Calibrated Population Resistance tool in HIV Drug Resistance Database. Viral subtypes were determined using REGA and RIP genotyping tools. The HIV PR/RT region was successfully sequenced from 241 patients. From these, 23 (9.5%) had at least one SDRM detected at >20% threshold, with a prevalence of 9.5% ( n  = 18), 3% ( n  = 7), and 0.4% ( n  = 1) for non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI), and protease inhibitors (PI), respectively. The number of patients with SDRM increased to 31 (12.9%) when minority variants were accounted for at >5% threshold. The most frequent SDRMs based on drug class were; K103N (7.9%-NNRTI), K65R (2.5%-NRTI), and D30N (0.8%-PI). Four cases of dual NRTI/NNRTI mutations were identified. All consensus sequences were subtype C, except three, which were C/A1, C/F1, and C/G recombinants. NGS analysis confirms that individuals entering HIV treatment programs in northern South Africa, habor moderate levels of SDRM, including cases of dual-class drug resistance. Further SDRM studies may be required to better understand resistance in the drug-naive population in the era of "diagnose and treat" in Limpopo Province, South Africa.

Background: South Africa, with one of the highest HIV prevalences in the world, introduced the universal test and treat (UTT) programme in September 2016. Barriers to sustained viral suppression may include drug resistance in the pre-treated population, non-adherence, acquired resistance; pharmacokinetics and pharmacodynamics, and concurrent use of alternative treatments., Objective: The purpose of this review is to highlight potential challenges to achieving sustained viral load suppression in South Africa (SA), a major expectation of the UTT initiative., Methodology: Through the PRISMA approach, published articles from South Africa on transmitted drug resistance; adherence to ARV; host genetic factors in drug pharmacokinetics and pharmacodynamics, and interactions between ARV and herbal medicine were searched and reviewed., Results: The level of drug resistance in the pre-treated population in South Africa has increased over the years, although it is heterogeneous across and within Provinces. At least one study has documented a pre-treated population with moderate (> 5%) or high (> 15%) levels of drug resistance in eight of the nine Provinces. The concurrent use of ARV and medicinal herbal preparation is fairly common in SA, and may be impacting negatively on adherence to ARV. Only few studies have investigated the association between the genetically diverse South African population and pharmacokinetics and pharmacodynamics of ARVs., Conclusion: The increasing levels of drug resistant viruses in the pre-treated population poses a threat to viral load suppression and the sustainability of first line regimens. Drug resistance surveillance systems to track the emergence of resistant viruses, study the burden of prior exposure to ARV and the parallel use of alternative medicines, with the goal of minimizing resistance development and virologic failure are proposed for all the Provinces of South Africa. Optimal management of the different drivers of drug resistance in the pre-treated population, non-adherence, and acquired drug resistance will be beneficial in ensuring sustained viral suppression in at least 90% of those on treatment, a key component of the 90-90-90 strategy.

Ethnopharmacological Relevance: Medicinal plants are used in the management of Human Immunodeficiency Virus and Acquired Immunodeficiency Syndrome (HIV/AIDS) in many developing country settings where HIV-1 subtype C drives the epidemic. Efforts to identify plant derived molecules with anti-HIV properties require reproducible assay systems for routine screening of selected plant compounds. Although a number of standardized HIV-1 pseudoviruses have been generated to assess infectivity, replicability or reproducibility, HIV-1 subtype C (HIV-1-C) pseudoviruses have not been comprehensively characterized to identify inhibitory plant substances., Aim of the Study: The current study aimed at developing an HIV-1-C pseudovirus assay, and evaluate plant substances targeting reverse transcriptase (RT) activity., Materials and Methods: HIV-1 subtype C pseudoviruses containing a luciferase reporter gene were generated by transfection of human 293T cells. HIV-1 subtype B (HIV-1-B) wild type pseudoviruses and mutants resistant to nucleoside and non-nucleoside RT inhibitors were also generated and used as controls. Selected plant substances and the RT inhibitors Zidovudine (AZT) and Nevirapine (NVP), were used to evaluate inhibition. Pseudovirus infectivity was determined by luciferase measurement in CF2/CD4 + /CCR5 cells, and cytotoxicity was determined using the MTT assay. AZT and NVP inhibited wild type pseudoviruses in a dose dependent manner, with IC 50 values in the nanomolar range., Results: Pseudoviruses harbouring RT drug resistance mutations were poorly suppressed by AZT and NVP. Catechin, obtained from Peltophorum africanum inhibited HIV-1-C and HIV-1-B pseudoviruses with selective indices of 6304 μM (IC 50 : 0.49 μM, CC 50 : 3089 μM) and 1343 μM (IC 50 : 2.3 μM, CC 50 : 3089 μM), respectively; while the methanol root crude extract of Elaeodendron transvaalense gave IC 50 values of 11.11 μg/ml and 16.86 μg/ml, respectively., Conclusion: The developed HIV-1-C pseudovirus assay can be used to screen plant substances for RT inhibition, and may have utility in settings with limited access to high level biosafety facilities., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Entry inhibitors, such as Maraviroc, hold promise as components of HIV treatment and/or pre-exposure prophylaxis in Africa. Maraviroc inhibits the interaction between HIV Envelope gp120 V3-loop and CCR5 coreceptor. HIV-1 subtype C (HIV-1-C) is predominant in Southern Africa and preferably uses CCR5 co-receptor. Therefore, a significant proportion of HIV-1-C CXCR4 utilizing viruses (X4) may compromise the effectiveness of Maraviroc. This analysis examined coreceptor preferences in early and chronic HIV-1-C infections across Africa., Methods: African HIV-1-C Envelope gp120 V3-loop sequences sampled from 1988 to 2014 were retrieved from Los Alamos HIV Sequence Database. Sequences from early infections (< 186 days post infection) and chronic infections (> 186 days post infection) were analysed for predicted co-receptor preferences using Geno2Pheno [Coreceptor] 10% FPR, Phenoseq-C, and PSSMsinsi web tools. V3-loop diversity was determined, and viral subtype was confirmed by phylogenetic analysis. National treatment guidelines across Africa were reviewed for Maraviroc recommendation., Results: Sequences from early (n = 6316) and chronic (n = 7338) HIV-1-C infected individuals from 10 and 15 African countries respectively were available for analyses. Overall, 518/6316 (8.2%; 95% CI 0.7-9.3) of early sequences were X4, with Ethiopia and Malawi having more than 10% each. For chronic infections, 8.3% (95% CI 2.4-16.2) sequences were X4 viruses, with Ethiopia, Tanzania, and Zimbabwe having more than 10% each. For sequences from early chronic infections (< 1 year post infection), the prevalence of X4 viruses was 8.5% (95% CI 2.6-11.2). In late chronic infections (≥ 5 years post infection), X4 viruses were observed in 36% (95% CI - 16.3 to 49.9), with two countries having relatively high X4 viruses: South Africa (43%) and Malawi (24%). The V3-loop amino acid sequence were more variable in X4 viruses in chronic infections compared to acute infections, with South Africa, Ethiopia and Zimbabwe showing the highest levels of V3-loop diversity. All sequences were phylogenetically confirmed as HIV-1-C and clustered according to their co-receptor tropism. In Africa, Maraviroc is registered only in South Africa and Uganda., Conclusions: Our analyses illustrate that X4 viruses are present in significantly similar proportions in early and early chronic HIV-1 subtype C infected individuals across Africa. In contrast, in late chronic infections, X4 viruses increase 3-5 folds. We can draw two inferences from our observations: (1) to enhance the utility of Maraviroc in chronic HIV subtype C infections in Africa, prior virus co-receptor determination is needed; (2) on the flip side, research on the efficacy of CXCR4 antagonists for HIV-1-C infections is encouraged. Currently, the use of Maraviroc is very limited in Africa.

Background: The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) genes A3D, A3F, A3G and A3H have all been implicated in the restriction of human immunodeficiency virus type 1 (HIV-1) replication. Polymorphisms in these genes are likely to impact viral replication and fitness, contributing to viral diversity. Currently, only a few studies indicate that polymorphisms in the A3 genes may be correlated with infection risk and disease progression., Methods: To characterize polymorphisms in the coding regions of these APOBEC3 genes in an HIV-1 infected population from the Limpopo Province of South Africa, APOBEC3 gene fragments were amplified from genomic DNA of 192 HIV-1 infected subjects and sequenced on an Illumina MiSeq platform. SNPs were confirmed and compared to SNPs in other populations reported in the 1000 Genome Phase III and HapMap databases, as well as in the ExAC exome database. Hardy-Weinberg Equilibrium was calculated and haplotypes were inferred using the LDlink 3.0 web tool. Linkage Disequilibrium (LD) for these SNPS were calculated in the total 1000 genome and AFR populations using the same tool., Results: Known variants compared to the GRCh37 consensus genome sequence were detected at relatively high frequencies (> 5%) in all of the APOBEC3 genes. A3H showed the most variation, with several of the variants present in both alleles in almost all of the patients. Several minor allele variants (< 5%) were also detected in A3D, A3F and A3G. In addition, novel R6K, L221R and T238I variants in A3D and I117I in A3F were observed. Four, five, four, and three haplotypes were identified for A3D, A3F, A3G, and A3H respectively., Conclusions: The study showed significant polymorphisms in the APOBEC3D, 3F, 3G and 3H genes in our South African HIV1-infected cohort. In the case of all of these genes, the polymorphisms were generally present at higher frequencies than reported in other 1000 genome populations and in the ExAC exome consortium database .

Background: Entry inhibitors, such as Maraviroc, bind to CCR5 inhibiting entry of CCR5 utilizing viruses (R5 viruses). In the course of HIV infection, CXCR4 utilizing viruses (X4 viruses) may emerge and outgrow R5 viruses, and potentially limit the effectiveness of Maraviroc. The use of Maraviroc is reserved for salvage therapy in South Africa., Objective: In this study, we examined the frequency of R5 and X4 viruses, using next generation sequencing, in patients under treatment to draw inferences on the utility of Maraviroc in a South African population., Study Design: Proviral DNA was isolated from peripheral blood mononuclear cells (PBMC) of 72 chronically HIV infected patients on antiretroviral treatment. HIV V3 loop gene was amplified and sequenced on an Illumina MiniSeq platform. Viral subtypes were determined by the jumping profile Hidden Markov Model (jpHMM) and REGA genotyping tools. De Novo consensus sequences were derived for the majority and minority populations for each patient using Geneious ® software version 8.1.5. HIV-1 tropism was inferred using PSSMsinsi, Geno2pheno and Phenoseq-C web-based tools., Results: Quality V3 loop sequences were obtained from 72 patients, with 5 years (range: 0-16) median duration on treatment. Subtypes A1, B and C viruses were identified at frequencies of 4% (3/72), 4% (3/72) and 92% (66/72) respectively. Fifty four percent (39/72) of patients exclusively harboured R5 viral quasispecies; and 21% (15/72) exclusively harbored X4 viral quasispecies. Twenty five percent of patients (18/72) harbored dual/mixture of R5X4 quasispecies. Of these 18 patients, about 28% (5/18) harbored the R5+X4, a mixture with a majority R5 and minority X4 viruses, while about 72% (13/18) harbored the R5X4+ mixture with a majority X4 and minority R5 viruses. The proportion of all patients who harbored X4 viruses either exclusively or dual/mixture was 46% (33/72). Thirty-five percent (23/66) of the patients who were of HIV-1 subtype C harboured X4 viruses (χ 2  = 3.58; p = .058), and 57% of these (13/23) harbored X4 viruses exclusively. CD4 + cell count less than 350 cell/μl was associated with the presence of X4 viruses (χ 2  = 4.99; p = .008)., Conclusion: The effectiveness of Maraviroc as a component in salvage therapy may be compromised for a significant number of chronically infected patients harboring CXCR4 utilizing viruses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Recombinant Newcastle Disease virus (rNDV) vectored vaccines are safe mucosal applicable vaccines with intrinsic immune-modulatory properties for the induction of efficient immunity. Like all viral vectored vaccines repeated inoculation via mucosal routes invariably results to immunity against viral vaccine vectors. To obviate immunity against viral vaccine vectors and improve the ability of rNDV vectored vaccines in inducing T cell immunity in murine air way we have directed dendritic cell targeted HIV-1 gag protein (DEC-Gag) vaccine; for the induction of helper CD4 + T cells to a Recombinant Newcastle disease virus expressing codon optimized HIV-1 Gag P55 (rNDV-L-Gag) vaccine., Methods: We do so through successive administration of anti-DEC205-gagP24 protein plus polyICLC (DEC-Gag) vaccine and rNDV-L-Gag. First strong gag specific helper CD4 + T cells are induced in mice by selected targeting of anti-DEC205-gagP24 protein vaccine to dendritic cells (DC) in situ together with polyICLC as adjuvant. This targeting helped T cell immunity develop to a subsequent rNDV-L-Gag vaccine and improved both systemic and mucosal gag specific immunity., Results: This sequential DEC-Gag vaccine prime followed by an rNDV-L-gag boost results to improved viral vectored immunization in murine airway, including mobilization of protective CD8 + T cells to a pathogenic virus infection site., Conclusion: Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves viral vectored immunization, including mobilization of protective CD8 + T cells to a pathogenic virus infection site such as the murine airway., (© 2017 The Authors. Immunity, Inflammation and DiseasePublished by John Wiley & Sons Ltd.)

Background: Combination antiretroviral therapy (cART) has significantly reduced HIV morbidity and mortality in both developed and developing countries. However, the sustainability of cART may be compromised by the emergence of viral drug resistance mutations (DRM) and the cellular persistence of proviruses carrying these DRM. This is potentially a more serious problem in resource limited settings., Methods: DRM were evaluated in individuals with unsuppressed viral loads after first or multiple lines of cART at two sites in rural Limpopo, South Africa. Seventy-two patients with viral loads of >1000 copies/ml were recruited between March 2014 and December 2015. Complete protease (PR) and partial Reverse Transcriptase (RT) sequences were amplified from both plasma RNA and paired proviral DNA from 35 of these subjects. Amplicons were directly sequenced to determine subtype and DRM using the Stanford HIV Drug Resistance Interpretation algorithm., Results: Among the 72 samples, 69 could be PCR amplified from RNA and 35 from both RNA and DNA. Sixty-five (94.2%) viruses were subtype C, while one was subtype B (1.4%), one recombinant K/C, one recombinant C/B and one unclassified. Fifty-eight (84%) sequences carried at least one DRM, while 11 (15.9%) displayed no DRM. DRM prevalence according to drug class was: NRTI 60.8% NNRTI 65.2%, and PI 5.8%. The most common DRMs were; M184V (51.7%), K103N (50%), V106M (20.6%), D67N (13.3%), K65R (12%). The frequency of the DRM tracked well with the frequency of use of medications to which the mutations were predicted to confer resistance. Interestingly, a significant number of subjects showed predicted resistance to the newer NNRTIs, etravirine (33%) and rilpivirine (42%), both of which are not yet available in this setting. The proportion of DRM in RNA and DNA were mostly similar with the exception of the thymidine analogue mutations (TAMs) D67N, K70R, K219QE; and K103N which were slightly more prevalent in DNA than RNA. Subjects who had received cART for at least 5 years were more likely to harbour >2 DRM (p < 0.05) compared to those treated for a shorter period. DRM were more prevalent in this rural setting compared to a neighbouring urban setting., Conclusion: We found a very high prevalence of NRTI and NNRTI DRM in patients from rural Limpopo settings with different durations of treatment. The prevalence was significantly higher than those reported in urban settings in South Africa. The dominance of NNRTI based mutations late in treatment supports the use of PI based regimens for second line treatment in this setting. The slight dominance of TAMs in DNA from infected PBMCs compared to plasma virus requires further studies that should include cART subjects with suppressed virus. Such studies will improve our understanding of the pattern of drug resistance and dynamics of viral persistence in these rural settings.

The HIV-1 replication cycle requires the nucleocytoplasmic export of intron-containing viral RNAs, a process that is ordinarily restricted. HIV overcomes this by means of the viral Rev protein, which binds to an RNA secondary structure called the Rev response element (RRE) present in all unspliced or incompletely spliced viral RNA transcripts. The resulting mRNP complex is exported through interaction with cellular factors. The Rev-RRE binding interaction is increasingly understood to display remarkable structural plasticity, but little is known about how Rev-RRE sequence differences affect functional activity. To study this issue, we utilized a lentiviral vector assay in which vector titer is dependent on the activity of selected Rev-RRE pairs. We found that Rev-RRE functional activity varies significantly (up to 24-fold) between naturally occurring viral isolates. The activity differences of the Rev-RRE cognate pairs track closely with Rev, but not with RRE activity. This variation in Rev activity is not correlated with differences in Rev steady state protein levels. These data suggest that Rev sequence differences drive substantial variation in Rev-RRE functional activity between patients. Such variation may play a role in viral adaptation to different immune milieus within and between patients and may be significant in the establishment of latency. The identification of differences in Rev-RRE functional activity in naturally occurring isolates may also permit more efficient production of lentiviral vectors., Competing Interests: Author Disclosure Statement No competing financial interests exist.

A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

High throughput screening of a 4096 compound library of boronic acid and acridine containing branched peptides revealed compounds that have dissociation constants in the low nanomolar regime for HIV-1 RRE IIB RNA. We demonstrate that branched peptide boronic acids A5 , A6 , and A7 inhibit the production of p24, an HIV-1 capsid protein, in a dose-dependent manner.

Despite only 30,000 group O HIV-1 infections, a similar genetic diversity is observed among the O subgroups H (head) and T (tail) (previously described as subtypes A, B) as in the 9 group M subtypes (A-K). Group O isolates bearing a cysteine at reverse transcriptase (RT) position 181, predominantly the H strains are intrinsically resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, their susceptibility to newer antiretroviral drugs such as etravirine, maraviroc, raltegravir (RAL), and elvitegravir (EVG) remains relatively unknown. We tested a large collection of HIV-1 group O strains for their susceptibility to four classes of antiretroviral drugs namely nucleoside RT, non-nucleoside RT, integrase, and entry inhibitors knowing in advance the intrinsic resistance to NNRTIs. Drug target regions were sequenced to determine various polymorphisms and were phylogenetically analyzed. Replication kinetics and fitness assays were performed in U87-CD4(+)CCR5 and CXCR4 cells and peripheral blood mononuclear cells. With all antiretroviral drugs, group O HIV-1 showed higher variability in IC50 values than group M HIV-1. The mean IC50 values for entry and nucleoside reverse transcriptase inhibitor (NRTI) were similar for group O and M HIV-1 isolates. Despite similar susceptibility to maraviroc, the various phenotypic algorithms failed to predict CXCR4 usage based on the V3 Env sequences of group O HIV-1 isolates. Decreased sensitivity of group O HIV-1 to integrase or NNRTIs had no relation to replicative fitness. Group O HIV-1 isolates were 10-fold less sensitive to EVG inhibition than group M HIV-1. These findings suggest that in regions where HIV-1 group O is endemic, first line treatment regimens combining two NRTIs with RAL may provide more sustained virologic responses than the standard regimens involving an NNRTI or protease inhibitors.

Background: CCR5-using (r5) HIV-1 predominates during asymptomatic disease followed by occasional emergence of CXCR4-using (x4) or dual tropic (r5x4) virus. We examined the contribution of the x4 and r5 components to replicative fitness of HIV-1 isolates., Methods: Dual tropic r5x4 viruses were predicted from average HIV-1 env sequences of two primary subtype C HIV-1 isolates (C19 and C27) and from two patient plasma samples (B12 and B19). Chimeric Env viruses with an NL4-3 backbone were constructed from the B12 and B19 env sequences. To determine replicative fitness, these primary and chimeric dual tropic HIV-1 were then competed against HIV-1 reference isolates in U87.CD4 cells expressing CXCR4 or CCR5 or in PBMCs ± entry inhibitors. Contribution of the x4 and r5 clones within the quasispecies of these chimeric or primary HIV-1 isolates were then compared to the frequency of x4, r5, and dual tropic clones within the quasispecies as predicted by phenotypic assays, clonal sequencing, and 454 deep sequencing., Results: In the primary HIV-1 isolates (C19 and C27), subtype C dual tropic clones dominated over x4 clones while pure r5 clones were absent. In two subtype B chimeric viruses (B12 and B19), r5 clones were >100-fold more abundant than x4 or r5/x4 clones. The dual tropic C19 and C27 HIV-1 isolates outcompeted r5 primary HIV-1 isolates, B2 and C3 in PBMCs. When AMD3100 was added or when only U87.CD4.CCR5 cells were used, the B2 and C3 reference viruses now out-competed the r5 component of the dual tropic C19 and C27. In contrast, the same replicative fitness was observed with dualtropic B12 and B19 HIV-1 isolates relative to x4 HIV-1 A8 and E6 or the r5 B2 and C3 viruses, even when the r5 or x4 component was inhibited by maraviroc (or AMD3100) or in U87.CD4.CXCR4 (or CCR5) cells., Conclusions: In the dual tropic HIV-1 isolates, the x4 replicative fitness is higher than r5 clones but the x4 or x4/r5 clones are typically at low frequency in the intrapatient virus population. Ex vivo HIV propagation promotes outgrowth of the x4 clones and provides an over-estimate of x4 dominance in replicative fitness within dual tropic viruses.

Twenty-five years ago, an aberrant HIV-1 (now classified as HIV-1 group O) was described from a Cameroonian HIV patient living in Belgium. The epicenter of group O was later found to be in Central Africa, overlapping with the geographical location of the central chimpanzees (Pan troglodytes troglodytes) and western gorillas (Gorilla gorilla), the likely original hosts of group O. Although the prevalence of group O has remained low at 1-2% in Cameroon, some European countries (France, Spain, Belgium) with strong colonial ties to Central Africa have reported the highest prevalence out of Africa. The sequence diversity between HIV-1 group O and M strains is huge, reaching 50 and 30% in the envelope and pol, respectively. This diversity has hindered diagnosis, monitoring, and treatment of group O-infected patients. Due to the intrinsic presence of the C181 mutation in group O, more than 60% of the approximately 30,000 individuals that live with this virus are faced with the challenge of drug resistance to some currently used antiretroviral therapies, notably the non-nucleoside reverse transcriptase inhibitors. Despite its susceptibility to most antiretroviral therapies, some group Os show a high variable baseline susceptibility to enfuvirtide (T20) and maraviroc. Group O strains are the least fit among all HIV-1 and -2 and restrict tetherin using their Nef but not Vpu as reported for group M. Although limited follow-up studies indicate that the natural course of group O is similar to that of M, these viruses are dominantly CCR5 tropic even late in infection, suggesting slow disease progression. This review summarizes important findings that marked the discovery, origin, spread, evolution, pathogenesis, and treatment of group O within the last 25 years.

The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous "CRF" B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events.

It has been hypothesized that a single mutation at a highly conserved amino acid site (HCS) can be severely deleterious to HIV in most if not all isolate-specific genetic backgrounds. Consequently, potentially universal HIV-1 vaccines exclusively targeting highly conserved regions of the viral proteome have been proposed. To test this hypothesis, we examined the impact of 10 Gag-p24 and 9 Env-gp120 HCS single mutations on viral fitness. In the original founder sequence of the subject in whom these mutations were identified, all Gag-p24 HCS mutations significantly reduced viral replication fitness, including 7 that were lethal. Similar results were obtained at 9/10 sites when the same mutations were introduced into the founder sequences of two epidemiologically unlinked subjects. In contrast, none of the 9 Env-gp120 HCS mutations were lethal in the original founder sequence, and four had no fitness cost. Hence, HCS mutations in Gag-p24 are likely to be severely deleterious in different HIV-1 subtype B backgrounds; however, some HCS mutations in both Gag-p24 and Env-gp120 fragments can be well tolerated. Therefore, when designing HIV-1 immunogens that are intended to force the virus to nonviable escape pathways, the fitness constraints on the HIV segments included should be considered beyond their conservation level.

Objectives: To determine the impact of HIV-1 subtype on treatment outcomes and the emergence of drug resistance in the resource limited setting of Kampala, Uganda., Design: The Joint Clinical Research Centre (JCRC) in Kampala, Uganda has provided over 2000 drug-resistant genotypes (DRGs) over the past 10 years as standard of care for patients failing therapy and 1403 from treatment-naive and experienced patients over the past 10 years have been analyzed for this study., Method: Viral loads, CD4 cell count, treatment histories and other relevant clinical data was compared with the infecting HIV-1 subtype and DRGs of Ugandan patients failing treatment., Results: Patients failing HAART with DRGs (n = 937) were more frequently infected with subtype D than expected on the basis of the subtype distribution in the treatment-naive population (n = 655) in Kampala (P < 0.001). Higher proportions of treatment failures among subtype D-infected patients were driven by resistance to nucleoside reverse transcriptase inhibitors (NRTI) (P < 0.0002) more than to non-NRTIs (P > 0.04) or protease inhibitors., Conclusion: Higher rates of treatment failure among subtype D as compared with subtype A-infected Ugandans was analogous to the faster disease progression in subtype D-infected patients. The mechanism(s) by which drug resistance may emerge faster in subtype D HIV-1 may relate to higher replicative fitness and increased propensity for a CXCR4 tropism.

Hypersusceptibility (HS) to inhibition by different antiretroviral drugs (ARVs) among diverse HIV-infected individuals may be a misnomer because clinical response to treatment is evaluated in relation to subtype B infections while drug susceptibility of the infecting virus, regardless of subtype, is compared to a subtype B HIV-1 laboratory strain (NL4-3 or IIIB). Mounting evidence suggests that HS to different ARVs may result in better treatment outcome just as drug resistance leads to treatment failure. We have identified key amino acid polymorphisms in the protease coding region of a non-B HIV-1 subtype linked to protease inhibitor HS, namely, 17E and 64M in CRF02&#95;AG. These HS-linked polymorphisms were introduced in the BD6-15 CRF02&#95;AG molecular clone and tested for inhibition using a panel of protease inhibitors. In general, suspected HS-linked polymorphisms did increase susceptibility to specific protease inhibitors such as amprenavir and atazanavir, but the combination of the 17E/64M polymorphisms showed greater HS. These two mutations were found at low frequencies but linked in a sequence database of over 700 protease sequences of CRF02&#95;AG. In direct head-to-head virus competitions, CRF02&#95;AG harboring the 17E/64M polymorphisms also had higher replicative fitness than did the 17E or the 64M polymorphism in the CFR02&#95;AG clone. These findings suggest that subtype-specific, linked polymorphisms can result in hypersusceptibility to ARVs. Considering the potential benefit of HS to treatment outcome, screening for potential HS-linked polymorphisms as well as preexisting drug resistance mutations in treatment-naïve patients may guide the choice of ARVs for the best treatment outcome.

Since the beginning of the AIDS pandemic, and following the discovery of the human immunodeficiency virus (HIV) as the etiological agent of the disease, it was clear that the virus gains access to the human host predominantly through the mucosal tissue after sexual exposure. As a consequence, the female genital tract (vaginal and cervical), as well as the rectal, penile, and oral mucosae have been extensively studied over the last thirty years towards a better understanding of--and to develop strategies to prevent--sexual HIV transmission. This review seeks to describe the biology of the events leading to HIV infection through the human mucosa and introduce some of the approaches attempted to prevent the sexual transmission of HIV.

Since the isolation of HIV, multiple transmissions are thought to have occurred between man and other old-world primates. Assessment of samples from apes and human beings with African equatorial forest ancestry has traced the origin of HIV-1 to chimpanzees, and dated its most recent common ancestor to 1908. The evolution of HIV-1 has been rapid, which has resulted in a complex classification, worldwide spread, and intermixing of strains; at least 48 circulating recombinant forms are currently identified. In addition to posing a nearly insurmountable challenge for diagnosis, treatment, vaccine development, and prevention, this extreme and divergent evolution has led to differences in virulence between HIV-1 groups, subtypes, or both. Coincidental changes in human migration in the Congo river basin also affected spread of disease. Research over the past 25 years and advances in genomic sequencing methods, such as deep DNA sequencing, have greatly improved understanding and analysis of the thousands to millions of full infectious HIV-1 genomes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Natural evolution in primate lentiviral reverse transcriptase (RT) appears to have been constrained by the necessity to maintain function within an asymmetric protein composed of two identical primary amino acid sequences (66 kDa), of which one is cleaved (51 kDa). In this study, a detailed phylogenetic analysis now segregates groups O and M into clusters based on a cysteine or tyrosine residue located at position 181 of RT and linked to other signature residues. Divergent evolution of two group O (C181 or Y181) and the main (Y181 only) HIV-1 lineages did not appreciably impact RT activity or function. Group O RT structural models, based on group M subtype B RT crystal structures, revealed that most evolutionarily linked amino acids appear on a surface-exposed region of one subunit while in a noncatalytic RT pocket of the other subunit. This pocket binds nonnucleoside RT inhibitors (NNRTI); therefore, NNRTI sensitivity was used to probe enzyme differences in these group O and M lineages. In contrast to observations showing acquired drug resistance associated with fitness loss, the C181Y mutation in the C181 group O lineage resulted in a loss of intrinsic NNRTI resistance and was accompanied by fitness loss. Other mutations linked to the NNRTI-resistant C181 lineage also resulted in altered NNRTI sensitivity and a net fitness cost. Based on RT asymmetry and conservation of the intricate reverse transcription process, millions of years of divergent primate lentivirus evolution may be constrained to discrete mutations that appear primarily in the nonfunctional, solvent-accessible NNRTI binding pocket.

Objectives: Several drug resistance and secondary mutations have been described in HIV-1 viruses from patients undergoing antiretroviral therapy. In this study, we assessed the impact of the protease substitution T74S on the phenotype and on the replicative fitness in HIV-1 subtypes B and C., Methods: HIV-1 molecular clones carrying subtype B or C proteases had these coding regions subjected to site-directed mutagenesis to include T74S alone or in combination with four known protease inhibitor (PI) primary drug resistance mutations. All clones were used in a phenotypic assay to evaluate their susceptibility to most commercially available PIs. The impact of T74S on virus fitness was also assessed for all viruses through head-to-head competitions and oligonucleotide ligation assays to measure the proportion of each virus in culture., Results: Viruses of both subtypes carrying T74S did not have their susceptibility altered to any tested PI. Viruses with the four resistance mutations showed strong resistance to most PIs with fold changes ranging from 5 to 300 times compared with their wild-type counterparts. Surprisingly, the addition of T74S to the multiresistant clones restored their susceptibilities to indinavir and ritonavir and partially to lopinavir, close to those of wild-type viruses. Most 74S-containing viruses were more fit than their 74T counterparts., Conclusions: Our results suggest that T74S is not a major drug resistance mutation, but it resensitizes multiresistant viruses to certain PIs. T74S is a bona fide accessory mutation, restoring fitness of multidrug-resistant viruses in both subtypes B and C. T74S should be further studied in clinical settings and considered in drug resistance interpretation algorithms.

A cross-sectional study was undertaken among drug-naïve HIV patients at the University Hospital in Ouagadougou shortly before and after the introduction of large-scale antiretroviral therapy (ART) in Burkina Faso. Baseline clinical and virological data as well as protease (PR) and 5' reverse transcriptase (RT) sequences from 104 HIV infected patients were analyzed. Genotypic classification revealed the following subtypes and recombinant forms: CRF06&#95;cpx, n = 46 (44.2%); CRF02&#95;AG, n = 39 (37.5%); subtype A, n = 4 (3.8%); CRF09&#95;cpx, n = 2 (1.9%); and unclassified, n = 13 (12.5%). Bootstrap analysis of CRF02&#95;AG and CRF06&#95;cpx viruses showed that >80% had a similar structure to their respective prototypes. The prevalence of primary drug resistance mutations was 12.5%, all mutations arising in the RT sequences in accordance with the dominance of this drug class in Burkina Faso. The mutations were distributed as follows: NRTI (10.6%): M41L (n = 2), D67N (n = 2), K70K/E (n = 2), L210W (n = 1), T215S/Y (n = 2), and K219K/Q (n = 2); NNRTI (6.1%): K103K/N (n = 2), Y181C (n = 2), G190G/A (n = 1), and P236P/L (n = 1). Subtype specific secondary polymorphisms such as K20I and M36I in the PR were observed in almost all patients. Drug resistance mutations occurred at similar frequencies (12.8% and 10.8%, respectively) among patients infected with CRF02&#95;AG and CRF06&#95;cpx. Some subtype specific polymorphisms were observed within important HLA epitopes, including B35, B7, and A2 in the RT, and A*6802 in the PR sequences. The observed resistance mutations are most likely to have been transmitted based on the timing of the study but prior undocumented use of ART cannot be excluded.

Human immunodeficiency virus type 1 (HIV-1) subtype C is the dominant subtype globally, due largely to the incidence of subtype C infections in sub-Saharan Africa and east Asia. We compared the relative replicative fitness (ex vivo) of the major (M) group of HIV-1 subtypes A, B, C, D, and CRF01&#95;AE and group O isolates. To estimate pathogenic fitness, pairwise competitions were performed between CCR5-tropic (R5) or CXCR4-tropic (X4) virus isolates in peripheral blood mononuclear cells (PBMC). A general fitness order was observed among 33 HIV-1 isolates; subtype B and D HIV-1 isolates were slightly more fit than the subtype A and dramatically more fit than the 12 subtype C isolates. All group M isolates were more fit (ex vivo) than the group O isolates. To estimate ex vivo transmission fitness, a subset of primary HIV-1 isolates were examined in primary human explants from penile, cervical, and rectal tissues. Only R5 isolates and no X4 HIV-1 isolates could replicate in these tissues, whereas the spread to PM1 cells was dependent on active replication and passive virus transfer. In tissue competition experiments, subtype C isolates could compete with and, in some cases, even win over subtype A and D isolates. However, when the migratory cells from infected tissues were mixed with a susceptible cell line, the subtype C isolates were outcompeted by other subtypes, as observed in experiments with PBMC. These findings suggest that subtype C HIV-1 isolates might have equal transmission fitness but reduced pathogenic fitness relative to other group M HIV-1 isolates.

Human lymphocyte antigen (HLA)-restricted CD8(+) cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.

The presence of antigen-specific cellular immune responses may be an indicator of long-term asymptomatic HIV-1-disease. The detection of cellular immune responses to infection with different subtypes of HIV-1 may be hampered by genetic differences of immunodominant antigens such as the capsid protein CAp24. In Nouna, Burkina Faso, HIV-1 circulating recombinant forms CRF02&#95;AG and CRF06&#95;cpx are the 2 major strains detectable in HIV-1-infected individuals, while subtype B strains prevail in Europe and North America. Amino acid sequences of CAp24 were assessed in blood samples from 10 HIV-1-infected patients in Nouna, Burkina Faso. Production of interferon-gamma (IFN-gamma) in peripheral blood CD4(+) lymphocytes in response to recombinant HIV-1 proteins derived from clade B (including CAp24(NL4-3)) was measured using a modified flow-cytometry-based whole blood short term activation assay (FASTimmune, BDBiosciences). IFN-gamma production following stimulation with a whole length CAp24 protein derived from clade B (CAp24(NL4-3)) was additionally quantified in comparison to a CAp24 protein derived from CRF02&#95;AG (CAp24(BD6-15)) in 16 HIV-1-infected patients in Heidelberg, Germany. Amino acid sequence identity of CAp24 obtained from patients in Nouna ranged between 86 and 89% when compared to the clade B CAp24(NL4-3) consensus sequence, between 90 and 95% when compared to the circulating recombinant form CRF06&#95;CPX consensus sequence, and between 92 and 96% when compared to the CAp24(BD6-15) consensus sequence. Significant numbers of HIV-1-specific CD4(+) lymphocytes producing IFN-gamma were detected in 4 of 10 HIV-1-infected patients. In 7 of 16 patients in Heidelberg, recombinant CAp24(BD6-15) stimulated IFN-gamma-production in CD4(+) lymphocytes to a similar extent as the clade B-derived CAp24(NL4-3). Thus, antigen-specific CD4(+) lymphocytes from both West African and European patients infected with different strains of HIV-1 show relevant cross-clade recognition of HIV-1 CAp24 in a flow-cytometry-based whole blood short term activation assay.

Objective: Determine the pattern of drug resistance among HIV infected drug exposed patients failing therapy in Ouagadougou, Burkina Faso., Methods: The protease (PR) and reverse transcriptase (RT) of 87 samples from 75 treatment exposed HIV infected patients failing therapy were PCR amplified, sequenced, subtyped and analyzed for the presence of drug resistance mutations., Results: The most common drugs used were 3TC, AZT (or d4T) and EFV. The dominant subtypes were CRF06&#95;cpx (48%) and CRF02&#95;AG (40%). The prevalence of resistance mutations among patients failing therapy was: PR inhibitors (PI), 40%; non-nucleoside RT-inhibitors (NNRTI), 76% and nucleoside RT-inhibitors (NRTI), 85%. Dominant mutations included M46I (37%), 154V (26%), V82A/T/F (30%) in PR; K103N (44%), G190A/S (16%) and T215F/Y (48%) (NRTIs) in RT. Some resistance mutations, notably D67N/G, K70R and L210W (thymidine analogue mutations-TAMs); K101E, V179E in RT, 154V, V82A/T/F and L90M in PR were significantly higher among CRF06&#95;cpx than CRF02&#95;AG strains (P < 0.05). Although not significant, other TAMs (M41L, T215F/Y, K219Q/E) also occurred more frequently among CRF06&#95;cpx strains as well., Conclusion: There is a high prevalence of drug resistance mutations among ARV exposed patients in Burkina Faso with an unexpected subtype-specific difference. Validation of this result will require larger sample sizes and in vitro drug susceptibility studies with CRF06&#95;cpx strains.

CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.

Reference ranges for peripheral blood lymphocyte subsets were generated for 186 healthy adults in Burkina Faso using single-platform flow cytometry. CD4(+) T-cell counts ranged from 631 to 1,696 cells microl(-1); they were lower in males (n = 97) than in females (n = 89), whereas natural killer cell counts were higher.

HIV appears to have diverged into several lineages upon multiple zoonotic introductions from the nonhuman primates. The HIV-2 and HIV-1 groups M, N, and O likely represent different cross-species transmission events. The radial evolution of group M in multiple clades or subtypes is likely due to adaptation and expansions in the human hosts. It is not well understood why HIV strains such as HIV-1 subtype C in particular or group M in general have spread disproportionately as compared to other subtypes, groups, or types, which often remained geographically constrained to local epidemics. Host genetic effects, transmission bottlenecks, social/behavioral and environmental limitations, founder effect and other viral factors could have contributed to variable spread through the human population. Even after transmission, viruses evolve at different rates during disease progression. Recent studies have explored phenotypic differences between HIV types, groups, and subtypes in attempts to explain or understand this radial evolution and expansion. This review explores some of the important aspects relating to fitness during disease progression, during global distribution of different HIV subtypes, and related to circulation of recombinant forms in the epidemic.

Summary: : On introduction of a program for prevention of mother-to-child transmission (PMTCT) of HIV in Nouna, rural Burkina Faso, we determined HIV prevalence in this region to be 3.6%, which is significantly lower than the 7% reported for 2 major cities of Burkina Faso. Forty-three samples from drug-naive pregnant women and patients before introduction of antiretroviral therapy (ART) were genotypically characterized in gag, pol, and env regions. One individual each was infected with HIV-2 or dually infected with HIV-1 and HIV-2. The most dominant HIV-1 subtypes were CRF02&#95;AG and CRF06&#95;cpx, similar to what has been observed in other West African countries. A discordant genotype was observed in almost half of the analyzed samples, with most putative recombinants deriving from CRF02&#95;AG and CRF06&#95;cpx. Recently reported strains like the CRF09&#95;cpx and the sub-subtype A3 as well as some unique recombinant forms of HIV like D/D/CRF02&#95;AG and CRF02&#95;AG/CRF02.AG/CRF&#95;09cpx were also detected. Analysis of drug resistance-associated polymorphisms detected the nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations K103N/E and V118I in 1 individual each, suggesting transmission of drug-resistant viruses or prior use of antiretroviral drugs. Resistance-associated polymorphisms (K20I and M36I) were prevalent in the complete protease (PR) region, but no primary drug resistance mutations were detected. Analysis of the HR1 and HR2 regions of gp41, important for T-20 sensitivity, revealed no known resistance mutations but several polymorphisms of unknown importance. Monitoring for drug resistance mutations among naive subjects is important in this area on introduction of antiretroviral drugs.

A seroprevalence study was carried out of six different human pathogenic viruses, namely human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus (HTLV), human herpesvirus type 8 (HHV-8), and dengue virus among pregnant women and blood donors from rural (Nouna) and urban (Ouagadougou) Burkina Faso, West Africa. A total of 683 samples from blood donors (n = 191) and pregnant women (n = 492) were collected from both sites and screened for the different virus infection markers resulting in the following prevalence values for Nouna or Ouagadougou, respectively: HIV 3.6/4.6, anti-HBV core (anti-HBc) 69.6/76.4, HBV surface antigen (HBsAg)14.3/17.3, HCV 2.2/1.5, HTLV 1.4/0.5, HHV-8 11.5/13.5, dengue virus 26.3/36.5. Individuals aged > or =25 years were more likely to be infected with HIV than those below 24 years (P < 0.05). Infection with HIV increased the likelihood of co-infection with other viruses, such as HHV-8, HBV and HTLV. Co-infection studies involving five viruses (HBV-HBsAg, HHV-8, HIV, HCV, and HTLV) showed that 4.8% (33/683) of the studied population were dually infected, with HBsAg+ HHV-8 (13/33), HBsAg+HIV (8/33) and HIV+HHV-8 (8/33) being the most common co-infections. Of the population studied 0.6% (4/683) was triply infected, the most common infection being with HBV+HIV+HHV-8 (3/4). There was no difference in the prevalence of HIV, anti-HBc, HBsAg, HCV, HTLV, and HHV-8 either among blood donors or pregnant women in urban or rural setting, while dengue virus prevalence was relatively lower in rural (26.3%) than in urban (36.5%) Burkina Faso., (Copyright 2006 Wiley-Liss, Inc.)

In this report, we describe the construction and characterization of the first full-length infectious molecular clone from the Cameroonian HIV-1 group O primary isolate MVP8913. Virus obtained after transfection of the proviral clone pCMO2.3 replicated to levels comparable to its parental isolate in the human T-cell line PM-1, although replication was reduced by fivefold in peripheral blood mononuclear cells (PBMC) and was barely detectable in primary monocyte-derived macrophages (MDM). Phylogenetic analysis of the complete proviral sequence revealed a closer relationship to ANT70 than to MVP5180, the two prototypic group O primary isolates. All reading frames for structural and accessory genes were open except for vpr that contained an in-frame stop codon. In the nef gene, a mutation disrupting the functionally important myristoylation signal was observed. Repairing the defect in nef enhanced replication in PBMC and MDM, although repairing the vpr defect only affected replication in MDM, consistent with the known phenotypes of vpr and nef mutants in HIV-1 group M viruses. Repairing both vpr and nef showed an additive effect, but the resulting virus was still impaired compared to the parental isolate. This defect was overcome when the gag-pol coding region was exchanged for that from another O-type isolate giving rise to the proviral clone pCMO2.5. Virus obtained from pCMO2.5 replicated with similar kinetics as the parental O-type isolate in both PBMC and MDM, making this proviral clone a valuable tool for further studies on functional characteristics of HIV-1 group O viruses.

Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells.

In this study we report the molecular and biological characteristics of 19 HIV-1 primary isolates obtained in April 1999 from 47 HIV-1-infected individuals living mainly in western Cameroon. Discontinuous portions of gag, pol, and env were amplified by polymerase chain reaction and directly sequenced. Phylogenetic analysis of these sequences showed that all were of HIV-1 group M with the following genotypes: A(gag)/A(pol)/A(env) (n = 4), A(gag)/AG(pol)/AG(env) (n = 2), AG(gag)/A(pol)/AG(env) (n = 1), AG(gag)/U(pol)/AG(env) (n = 1), AG(gag)/AG(pol)/AG(env) (n = 6), G(gag)/G(pol)/G(env) (n = 3), F2(gag)/F2(pol)/F2(env) (n = 1), and a novel A(gag)/J(pro/rt)/A(int)/U(env) complex recombinant (n = 1). This A/J/U recombinant shared the same gag-pol cross-over point with known CRF02.AG viruses and 99CMBD6, an AG recombinant from our panel of isolates. The biological phenotype of most of the isolates correlated with the clinical status of the patient. Six isolates were syncytium inducing (SI) on MT-2 cells whereas 13 isolates were of the non-syncytium-inducing phenotype (NSI). Coreceptor usage by these isolates determined on GHOST cells correlated with their biological phenotype, as all SI isolates used CXCR4 and all NSI isolates used CCR5. Our results show a high predominance of subtype A (mainly CRF02.AG-like viruses) in western Cameroon and fewer HIV-1 subtypes compared with other parts of Cameroon. Genetic variability was, however, not reflected in the biological characteristics of the isolates. The presence of a novel A/J/U complex recombinant from this region further emphasizes the role of recombination in the global evolution of HIV.