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The Central African Republic (CAR) is characterized by widespread HIV epidemic with notable prevalence and genetic diversity. We herein analysed the genetic diversity of atypical non‐M HIV‐1 strains. In‐house serotyping assays for variants of HIV‐1 (M, N, O, P) and HIV‐2 were used to test a biological collection of 6092 HIV‐seropositive blood samples collected between 2003 and 2014 at the Institut Pasteur de Bangui. Samples indicative of recombinant M/O groups, HIV‐2, or those that yield doubtful/negative results underwent further PCR tests and sequencing. We found six atypical HIV strains: specifically, three (0.05%) HIV‐1 group O strains (subtype H) detected in samples from 2005, 2008 and 2009, alongside three (0.05%) HIV‐2 strains (two group A and one group B) identified in samples from 2007 and 2009. HIV‐1/O strains showed a genetic link to Cameroon and Gabon strains. This study highlights the dominance of HIV‐1/M in the CAR's HIV epidemic over time and underscores the infrequent occurrence of HIV‐1 group O and HIV‐2 strains. These findings validate the efficacy of WHO‐recommended HIV testing protocols and emphasize the need for adaptive surveillance and management strategies to confront the complexities introduced by the genetic diversity of HIV strains. [ABSTRACT FROM AUTHOR]

Background: The genetic divergence of HIV-1 group O is high relative to pandemic group M, which could impact detection and quantification of plasma RNA. Recent commercial kits for RNA quantification seem to show good performances in HIV-1/O, but discrepancies are still observed. Here, we compare the performances of 3 commercial assays for the RNA quantification of HIV-1/O. Methods: We studied the RNA quantification of 117 clinical samples using Abbott RealTime HIV-1, Cepheid Xpert HIV-1 Viral Load, or Roche Cobas TaqMan HIV-1 v2. First, we conducted a qualitative description, and second, we focused on a quantitative analysis of the results above 40 cp/mL. The degree of agreement between methods and the strength of the correlation of viral load determination were estimated using Bland-Altman plot and Passing-Bablok regression with the Spearman coefficient, respectively. Results: Our 2-by-2 analysis showed that the Abbott and Cepheid assays were very close in terms of correlation and dispersion of points, whereas Roche presented higher values in the highest range of quantification (.5 log10). The Cepheid assay combined better correlation with the consensus value and a lower dispersion of values, leading to an overall better performance of quantification. The quantification was still impacted by intragroup genetic diversity with, here, 1 strain (YBF26). Conclusions: Using a new approach to compare the performances of RNA quantification between more than 2 techniques, we demonstrated that Cepheid could be the most suitable assay for HIV-1/O quantification, although the results from all assays remained strain dependent. [ABSTRACT FROM AUTHOR]

Background Little is known about impact of genetic divergence of human immunodeficiency virus type 1 group O (HIV-1/O) relative to HIV-1 group M (HIV-1/M) on therapeutic outcomes. We aimed to determine if responses to standardized combination antiretroviral therapy (cART) were similar between groups despite strain divergence. Methods We performed an open nonrandomized study comparing the immunological, virological, and clinical responses to cART based on 2 nucleoside reverse transcriptase inhibitors plus 1 ritonavir-boosted protease inhibitor, in naive and paired HIV-1/O vs HIV-1/M infected (+) patients (ratio 1:2), matched on several criteria. The primary endpoint was the proportion of patients with undetectable plasma viral load (pVL, threshold 60 copies/mL) at week (W) 48. Secondary endpoints were the proportion of patients with undetectable pVL at W24 and W96 and CD4 evolution between baseline and W24, W48, and W96. Results Forty-seven HIV-1/O+ and 94 HIV-1/M+ patients were included. Mean pVL at baseline was significantly lower by 1 log for HIV-1/O+ vs HIV-1/M+ patients. At W48, no significant difference was observed between populations with undetectable pVL and differences at W24 and W96 were not significant. A difference in CD4 gain was observed in favor of HIV-1/M at W48 and W96, but this was not significant when adjusted on both matched criteria and pVL at baseline. Conclusions Our data demonstrate similar immunovirological and clinical response between HIV-1/O+ and HIV-1/M+ patients. They also reveal significantly lower baseline replication for HIV-1/O variants, suggesting specific virological properties and physiopathology that now need to be addressed. Clinical Trials Registration NCT00658346. [ABSTRACT FROM AUTHOR]

Background. To obtain reliable clinical data of human immunodeficiency virus type 1 group O (HIV-1/O) infection, and immunovirological responses to combination antiretroviral therapy (cART), in a large series of 101 patients. Methods. Piecewise linear models were used to estimate CD4 count before and after cART initiation. Kaplan-Meier survival curves were used to estimate time to reach clinical stage C before antiretroviral therapy (ART) and to analyze time to achieve a plasma viral load (pVL) <40 copies/mL following cART initiation. Immunovirological response was assessed at the most recent visit in patients on active follow-up. Results. Data showed a 16.6% cumulative probability of reaching stage C within 5 years following diagnosis, and a mean CD4 decrease of -30.5 cells/µL/year. cART initiation in ART-naive patients led to a mean CD4 gain of 147 cells/µL after 12 months, and to a median pVL of <40 copies/mL after 3.8 months for 89.3%. Initiation with a nonrecommended nonnucleoside reverse transcriptase inhibitor-based vs a ritonavir-boosted protease inhibitor-based regimen resulted in a much smaller gain of around 100 CD4 cells/µL after 1 year. Patients on follow-up since 2007 had a median CD4 count of 498 cells/µL, and 87% had a pVL <40 copies/mL at the most recent follow-up visit. Conclusions. This work provides unique data on HIV-1/O infection, in favor of a milder natural evolution than HIV-1 group M (HIV-1/M) and of a highly efficient current management, based on HIV-1/M guidelines, despite genetic divergence. Studies of comparable HIV-1/M and HIV-1/O populations are needed to confirm these results. [ABSTRACT FROM AUTHOR]

The human immunodeficiency virus, HIV, is characterized by a tremendously high genetic diversity, leading to the currently known circulating HIV types, groups, subtypes, and recombinant forms. HIV-1 group O is one of the most diverse forms of HIV-1 and has been so far related to Cameroon or individuals originating from Cameroon. In this study, we investigated in Cameroon, the evolution of this viral group from 2006 to 2013, in terms of prevalence, genetic diversity and public health implications. Our results confirmed the predominance of HIV-1 group M (98.5%), a very low prevalence (< 0.02%) for HIV-1 group N and P, and HIV-2 in this country. HIV-1 group O was found at around 0.6% (95% confidence interval: 0.4–0.8%), indicating that the frequency of this virus in Cameroon has remained stable over the last decades. However, we found an extensive high genetic diversity within this HIV-1 group, that resulted from previous steady increase on the effective number of HIV-1 group O infections through time, and the current distribution of the circulating viral strains still does not allow classification as subtypes. The frequency of dual infections with HIV-1 group M and group O was 0.8% (95% confidence interval: 0.6–1.0%), but we found no recombinant forms in co-infected patients. Natural resistance to integrase inhibitors was not identified, although we found several mutations considered as natural polymorphisms. Our study shows that infections with HIV-1 group O can be adequately managed in countries where the virus circulates, but this complex virus still represents a challenge for diagnostics and monitoring strategies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antiretroviral drug resistance is mostly linked to a complex interaction of several amino acids with variable importance or a single amino acid. To facilitate the interpretation of observed mutation patterns, hospital university centers have developed several interpretation systems. All the currently available interpretation algorithms evolved, are being continuously updated and have been improved during the last decade. Some discrepancies are still evident that are partially smoothened by link of the individual programs with other systems. After the interpretation of HIV-1 group M subtype B mutations, a refined algorithm for the other group M subtypes was developed followed by the interpretation of HIV-1 group O and HIV-2 mutations. The process of improvement is ongoing, due to the better understanding and interpretation of single and cluster mutations and the availability of new antiretroviral substances. The knowledge gained from the experience of HIV drug resistance testing has been used to establish the interpretation of HBV polymerase mutations and will be extended for the treatment of HCV infected with protease inhibitors. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Introduction: HIV-1 group O (HIV-O), mainly found in Cameroon, has a very high genetic diversity with consequences on the diagnosis and treatment of patients, requiring the development of specific tools. Objective: We present the currently available tools for the specific detection of HIV-O and its therapeutic monitoring, and the first RES-O data, a French network for the identification and monitoring of patients infected by HIV-O. Method: The diagnosis of infection was confirmed by group-specific envelope serotyping. The viral load was assessed by a specific technique, LTR-O, developed in the laboratory and compared to the nonspecific kit RealTime HIV-1 (Abbott). The sequencing of antiretroviral target regions (Protease, Reverse Transcriptase (RT), Integrase and Gp41), was performed by specific primers. The analysis of resistance mutations was performed with the ANRS algorithm used for HIV-M. Results: HIV-O infection was confirmed for 117 patients. Measuring viral load showed the two techniques were equivalent, but with a tendency to under-quantification for the Abbott technique greater than 1 log for 5% of samples. 70 to 100% of the studied strains had at least 10 mutations in the Protease, four4 in the RT, and one in Gp41, resulting in a natural genotypic resistance to some anti-retroviral molecules. Discussion: The diagnosis and monitoring of HIV-O infection is now possible. However, the impact of this variant''s natural polymorphism on response to treatment remains undocumented. [Copyright &y& Elsevier]

Abstract: Clinical specificity, analytical and clinical sensitivities, reproducibility, and subtype/genotype coverage of the cobas TaqScreen MPX Test, a multiplex nucleic acid test with expanded coverage of HIV variants, were determined. A total of 72,281 blood donations were evaluated. The 95% limit of detection (LOD) for the MPX Test inclusive viruses was determined by testing six dilutions of WHO or Roche standards. Over 3000 high-risk and confirmed seropositive specimens were tested with the MPX and COBAS AmpliScreen Tests. Ten subtypes of HIV-1 Group M, HIV-1 Group O, HIV-2 A and B, HBV genotypes A-H, and HCV genotypes 1–6 were tested with the MPX Test. Reproducibility panels were evaluated at three testing sites across three lots. Clinical specificity in pools was 99.99%. There was one HBV yield case. The LODs for HIV-1 Group M, HCV, and HBV were 49, 11, and 3.8IU/mL, respectively, and 89 and 59.3copies/mL for HIV-1 Group O and HIV-2, respectively. Concordance between the MPX and the AmpliScreen Tests was 94.9%. Clinical sensitivity based on AmpliScreen comparison was 97.8–99.5%. All genotype/subtype replicates were detected at three times the LOD. Reproducibility was 98.3–100%. In conclusion, the MPX Test is robust and covers HIV-1 Group O and HIV-2. [Copyright &y& Elsevier]

To define a routine algorithm for the specific diagnosis and complete follow-up of HIV-1 group O (HIV-O) infections in Cameroun.During 18 months, samples referred to Centre Pasteur du Cameroun for HIV testing or viral monitoring were screened for HIV-O infection with an in-house serotyping assay. HIV-O viral load was quantified by real-time polymerase chain reaction in the LTR gene and resistance genotyping was performed on pol and env sequences.Of the 7030 samples tested, 78 HIV-O infections (1.1%) were identified, including 7 M and O dually seroreactive samples (9%). All treatment-naive patients and 59% of the patients receiving HAART had detectable viral loads. Analysis of pol sequences from 15 treatment-naive patients revealed a high number of polymorphisms in the protease region, with natural residues implicated in genotypic resistance to tipranavir and saquinavir for HIV-1 group M according to the Agence Nationale de Recherches sur le Sida et les Hépatites virales algorithm. Six patients (40%) harbored the 181C mutation conferring natural resistance to nonnucleoside reverse transcriptase inhibitors. Among antiretroviral-treated patients, major resistance mutations described for HIV-1 group M were found.HIV-O prevalence remains relatively low in Cameroun. The cocirculation of groups M and O in this country leads to replicative dual infections. HIV-O-infected patients in this region can now benefit from effective and specific tools for a complete monitoring of infection. However, further studies are needed to understand long-term response to antiretrovirals of these complex variants. [ABSTRACT FROM AUTHOR]

HIV-1 group O infections remains essentially restricted to central Africa, and particularly Cameroon, although isolated cases have been reported in Western countries. Genomic differences explain why commercial tests used to quantify HIV-1 group M plasma load are unsuitable for HIV-1 group O. This lack of a quantitative tool hinders the clinical management of HIV-O-infected patients. We have therefore developed a real-time PCR assay, based on LightCycler technology, to quantify HIV-1 group O RNA in plasma. The primers were selected in the LTR 3′ region. Forty-eight plasma samples containing strains belonging to the different HIV-1 type O clades (O:A, O:B and O:C) were tested. RNA was quantifiable in 40 of these samples. RNA was always detected in samples from untreated patients, except for one patient infected by a highly divergent strain. The kinetics of plasma viral load were also examined in seven patients for whom clinical and immunologic follow-up data were available. HIV-1 group O plasma load was high in the absence of treatment and correlated negatively with the CD4 cell count. Serial samples obtained during treatment allowed us to compare viral load changes with immunologic outcome. Despite the high initial cost of acquiring the required cycling device, the per-sample cost of this real-time quantitative PCR assay for HIV-1 group O is low, making it suitable for use in endemic zones. [Copyright &y& Elsevier]

Human immunodeficiency virus type 1 (HIV-1) requires a programmed −1 ribosomal frameshift to produce Gag–Pol, the precursor of its enzymatic activities. This frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2 nt (loop 1) and 20 nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the frameshift efficiency to near wild-type level. The difference between the frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission. [Copyright &y& Elsevier]

Human immunodeficiency virus type 1 group O strains have been described as highly divergent, compared with the majority of the viruses classified in group M. To study the diversity and genetic characteristics of group O, we have sequenced the accessory genes of 7 isolates. Analysis of the deduced amino acid sequences for Vif, Vpr, Tat, Vpu, and Rev indicate that most of the functional domains of these proteins, as described for group M viruses, are highly conserved and retained among all the group O strains we have characterized. The only difference concerns the Vif phosphorylation sites, which are absent in all of the group O isolated we have sequence with the exception of two isolates in which only one phosphorylation site was conserved. These sites, present in nearly all of the group M isolates, play a critical role in the regulation of viral replication and infectivity. As described for group M isolates, the vpu gene is the one with the highest diversity among group O viruses. Phylogenetic analysis of these sequences suggests that group O viruses could be differentiated into at least four different clusters. [Copyright &y& Elsevier]

We report a survey of HIV-1 group O infection in Cameroon during 1986 to 1998. The prevalence of HIV-1/O decreased from 0.6% to 0.4%, while HIV-1/M increased from 19.2% to 31.5% from 1994 to 1998. We concluded that HIV-1/O infection is stable in Cameroon and may be declining slightly.

HIV-1 diversity poses major challenges to viral load assays because genetic polymorphisms can impede nucleic acid detection. In addition to the on-going viral diversification within the HIV-1 group M pandemic, HIV-1 genetic diversity is further increased by non-group M infections, such as HIV-1 groups O (HIV-1-O), N and P. We here conducted a systematic evaluation of commercially available PCR assays to detect HIV-1-O isolates. We collected 25 primary HIV-1-O isolates covering all genetic clusters within HIV-1-O. Subsequently, this panel of isolates was tested on eight commercially available quantitative and five qualitative HIV-1 PCR-based assays in serial dilutions. Sequence analyses were performed for severe cases of underquantification or lack of detection. We observed differences between the assays in quantification that depended on the HIV-1-O isolate's subgroup. All three tested HIV-1-O subgroup IV isolates were underquantified by the Roche CAP/CTM >800-fold compared to the Abbott RealTime assay. In contrast, the latter assay underquantified several subgroup I isolates >200-fold. Notably, the Xpert HIV-1 Viral Load test from Cepheid failed to detect two of the HIV-1-O isolates, whereas the Roche Cobas 8800 assay readily detected all isolates. Comparative sequence analyses identified polymorphisms in the HIV-1-O long-terminal repeat and integrase genes that likely underlie inadequate nucleic acid amplification. Potential viral load underquantification should be considered in therapeutic monitoring of HIV-1-O-infected patients. Pre-clinical assessments of HIV-1 diagnostic assays could be harmonized by establishing improved and internationally standardized panels of HIV-1 isolates that cover the dynamic diversity of circulating HIV-1 strains. [ABSTRACT FROM AUTHOR]

Simian immunodeficiency viruses (SIVs) use their Nef proteins to counteract the restriction factor tetherin. However, a deletion in human tetherin prevents antagonism by the Nef proteins of SIVcpz and SIVgor, which represent the ape precursors of human immunodeficiency virus type 1 (HIV-1). To promote virus release from infected cells, pandemic HIV-1 group M strains evolved Vpu as a tetherin antagonist, while the Nef protein of less widespread HIV-1 group O strains acquired the ability to target a region adjacent to this deletion. In this study, we identified an unusual HIV-1 group O strain (RBF206) that evolved Vpu as an effective antagonist of human tetherin. While both RBF206 Vpu and Nef exert anti-tetherin activity in transient-transfection assays, mainly Vpu promotes RBF206 release in infected CD4 + T cells. Although mutations distinct from the adaptive changes observed in group M Vpus (M-Vpus) were critical for the acquisition of its anti-tetherin activity, RBF206 O-Vpu potently suppresses NF-κB activation and reduces CD4 cell surface expression. Interestingly, RBF206 Vpu counteracts tetherin in a largely species-independent manner, degrading both the long and short isoforms of human tetherin. Downmodulation of CD4, but not counteraction of tetherin, by RBF206 Vpu was dependent on the cellular ubiquitin ligase machinery. Our data present the first example of an HIV-1 group O Vpu that efficiently antagonizes human tetherin and suggest that counteraction by O-Nefs may be suboptimal. IMPORTANCE Previous studies showed that HIV-1 groups M and O evolved two alternative strategies to counteract the human ortholog of the restriction factor tetherin. While HIV-1 group M switched from Nef to Vpu due to a deletion in the cytoplasmic domain of human tetherin, HIV-1 group O, which lacks Vpu-mediated anti-tetherin activity, acquired a Nef protein that is able to target a region adjacent to the deletion. Here we report an unusual exception, identifying a strain of HIV-1 group O (RBF206) whose Vpu protein evolved an effective antagonism of human tetherin. Interestingly, the adaptive changes in RBF206 Vpu are distinct from those found in M-Vpus and mediate efficient counteraction of both the long and short isoforms of this restriction factor. Our results further illustrate the enormous flexibility of HIV-1 in counteracting human defense mechanisms., (Copyright © 2017 American Society for Microbiology.)