Celine Montavon

most env and gag subtype A Hiv-1 Viruses Circulating in West and West Central Africa Are Similar to the Prototype Ag Recombinant Virus Ibng

Summary: The genetic subtype was identified in gag and env of 219 HIV‐1‐positive samples collected in different African countries, 44 from Senegal, 55 from Cameroon, 82 from Gabon, and 38 from Djibouti. In total, 20 (9.1%) samples had discordant subtypes between gag and env, 6 of 44 (13.9%) in Senegal, 4 of 55 (7.2%) in Cameroon, 1 of 38 (2.6%) in Djibouti, and 10 of 82 (12.1%) in Gabon. Subtypes A and G were predominantly involved in the recombination events. Phylogenetic tree analysis of gag showed that an important number of the A sequences form a distinct subcluster with the AG‐IBNG prototype strain (a complex A/G mosaic virus): 27 of 32 (84.3%) in Senegal, 12 of 17 (70.6%) in Nigeria, 24 of 39 (61.5%) in Cameroon, and 38 of 70 (54.3%) in Gabon. Full‐length genome analysis of 3 and additional sequences in pol for 10 such strains confirmed that they have a similar complex A/G mosaic genomic structure. These data suggest that in West Africa, most probably between 60% and 84% of the subtype A viruses are recombinant AG‐IBNG viruses. This finding has potential implications on future vaccine, diagnostic, and treatment strategies. The actual and future role of these viruses in the global pandemic must be monitored in all new molecular epidemiologic studies, a discrimination between subtype A and AG‐IBNG‐like viruses is necessary.

Predominance of Subtype A and G HIV Type 1 in Nigeria, with Geographical Differences in Their Distribution

The purpose of this study was to generate data on the relative prevalences of the HIV-1 subtypes circulating in Nigeria. A total of 252 HIV-1-positive samples collected during an epidemiologic survey conducted in April 1996 were genetically characterized by HMA (heteroduplex mobility assay) and/or sequencing. Samples were collected in Lagos, Calabar, Kano, and Maiduguri. Overall, the predominant env subtypes were A (61.3%) and G (37.5%). Subtype A is more prevalent in the south (p < 0.001), about 70% in Lagos and Calabar, whereas a quarter of the samples was classified as subtype G in these states. In contrast, subtype G is predominant in the north ( < 0.001), representing 58% of the samples in Kano. In the northeastern region, Maiduguri, almost similar proportions of subtype A and G were seen, 49 and 47.4%, respectively. A total of 37 samples was also sequenced in the p24 region from the gag gene; 13 (35%) had discordant subtype designations between env and gag. The majority of the gag (12 of 17) and env (14 of 22) subtype A sequences clustered with the A/G-IBNG strain. Within subtype G, three different subclusters were seen among the envelope sequences. These different subclusters are observed among samples obtained from asymptomatic individuals and AIDS patients from the four Nigerian states studied. In conclusion, we observed a limited number of HIV-1 subtypes circulating in Nigeria, with subtypes A and G being the major env subtypes responsible for the HIV-1 epidemic. Nevertheless, the high rate of recombinant viruses (A/G) and the different A/G recombinant structures indicate a complex pattern of HIV-1 viruses circulating in this country.

Evaluation of Different RNA Extraction Methods and Storage Conditions of Dried Plasma or Blood Spots for Human Immunodeficiency Virus Type 1 RNA Quantification and PCR Amplification for Drug Resistance Testing

ABSTRACT The development and validation of dried sample spots as a method of specimen collection are urgently needed in developing countries for monitoring of human immunodeficiency virus (HIV) infection. Our aim was to test some crucial steps in the use of dried spots, i.e., viral recovery and storage over time. Moreover, we investigated whether dried plasma and blood spots (DPS and DBS, respectively) give comparable viral load (VL) results. Four manual RNA extraction methods from commercial HIV type 1 (HIV-1) VL assays—a QIAamp minikit (Qiagen), the Abbott Molecular sample preparation system, the Nuclisens assay (bioMarieux), and High Pure viral nucleic acid kit (Roche Applied Science)—were compared for VL quantification and PCR amplification for genotypic drug resistance testing on dried spots from spiked plasma and residual samples from HIV-1 patients (n = 47; median VL, 4.13 log10 copies/ml). RNA recovery from DPS was efficient using Nuclisens extraction (median difference, 0.03 log10 copies/ml) and slightly underestimated using the Abbott Molecular sample preparation system (median difference, 0.35 log10 copies/ml). PCR amplification results were in concordance. Measurements from DBS overestimated VL for plasma, with VL results showing <3.7 log10 copies/ml. VL was stable for up to 3 months in spiked DPS stored at 20°C but for only 1 month at 37°C. A faster decline was observed in PCR efficiency: DPS could be stored for 1 week at 37°C and for 1 month at 20°C. In conclusion, the RNA extraction method is an important factor in obtaining reliable RNA quantification and PCR amplification of HIV-1 on DPS/DBS. DBS could be used as an alternative for DPS depending on HIV RNA cutoffs for virological failure. VL measurements remain stable over a longer period than do PCR amplification results.

Sequence Note: Identification of All HIV Type 1 Group M Subtypes in Senegal, a Country with Low and Stable Seroprevalence

A total of 343 HIV-1-positive samples obtained between June 1996 and March 1999 was genetically characterized in the envelope region by HMA and/or sequencing. The env subtype distribution was as follows: 290 (84.6%) A, 22 (6.5%) B, 16 (4.7%) C, 8 (2.5%) D, 1 (0.03%) E, 1 (0.03%) F1, 4 (1.2%) G, and 1 (0.03%) H. For 77 samples the p24 region from the gag gene was also sequenced, and for 9 (11.6%) the subtypes between env and gag were different. Phylogenetic tree analysis showed the predominance of AG-IBNG-like viruses among gag and env subtype A sequences. HMA is relatively simple and requires less sophisticated technical facilities compared with sequencing, and in Senegal 323 (94.2%) of the 343 samples could be identified by this technique. However, in the actual configuration of the assay, discrimination between the recombinant AG-IBNG-like recombinant viruses, which are predominant in Senegal, and the nonrecombinant subtype A viruses is not possible.

Scale-up of antiretroviral treatment in sub-Saharan Africa is accompanied by increasing HIV-1 drug resistance mutations in drug-naive patients.

Objectives:To evaluate the frequency and progression over time of the WHO-defined transmitted HIV-1 drug resistance mutations (DRMs) among antiretroviral treatment (ART)-naive HIV-1-infected patients in Cameroon. Design:We analyzed HIV-1 DRM data generated from 369 ART-naive individuals consecutively recruited between 1996 and 2007 in urban and rural areas in Cameroon. Methods:HIV-1 drug resistance genotyping was performed in the pol gene using plasma samples and surveillance DRMs were identified using the 2009 WHO-DRM list. Results:We observed in Yaounde, the capital city, an increasing prevalence of DRMs over time: 0.0% (none of 61 participants) in 1996–1999; 1.9% (one of 53 participants) in 2001; 4.1% (two of 49 participants) in 2002; and 12.3% (10 of 81 participants) in 2007. In the rural areas with more recently implemented ART programs, we found DRMs in six of 125 (4.8%) ART-naive individuals recruited in 2006–2007. DRMs identified in both areas included resistance mutations to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and non-NRTIs (NNRTIs) that might impair the efficacy of available first-line and second-line treatments. Conclusion:This report showed an increase in transmitted DRMs in areas where antiretroviral drugs were introduced earlier, although other factors such as natural viral polymorphisms and acquired DRMs through exposure to antiretroviral cannot be totally excluded. Further surveillances are needed to confirm this evolution and inform public health policies on adequate actions to help limit the selection and transmission of drug-resistant HIV, while scaling up access to ART in developing countries.

CRF06-cpx: a new circulating recombinant form of HIV-1 in West Africa involving subtypes A, G, K, and J.

Summary: Phylogenetic analysis of numerous strains of HIV‐1 isolated from diverse geographic origins has revealed three distinct groups of HIV‐1: groups M, N, and O. Within group M, subtypes, sub‐subtypes and circulating recombinant forms (CRFs) exist. Recently, two near‐full‐length genomes of similar complex mosaic viruses containing fragments of subtypes A, G, I, and J were described in patients from Burkina Faso (BFP‐90) and Mali (95ML‐84). Here, we report on the characterization of two additional full‐length genome sequences with similar mosaic structure in epidemiologically unlinked individuals from Senegal (97SE‐1078) and Mali (95ML‐127). Phylogenetic and recombinant analysis confirmed that the previously described strains, BFP‐90 and 95ML‐84, were indeed a new CRF of HIV‐1, which we can now designate as CRF06‐cpx. This new CRF fits the complex (cpx) designation, because four different subtypes (A, G, K, and J) were involved in the mosaic genome structure. The fragment in the pol gene, which was initially characterized as unknown in the BFP‐90 strain and subsequently as subtype I in the 95ML‐84 strain, is now, with the recent description of the new K subtype, clearly identified as subtype K. CRF06‐cpx circulates in Senegal, Mali, Burkina Faso, Ivory Coast, and Nigeria, although the exact prevalence remains to be determined. Importantly, this new variant has also been documented on other continents (Europe [France] and Australia), showing that these viruses are spreading not only locally but globally.

Identification of a New Circulating Recombinant Form of HIV Type 1, CRF11-cpx, Involving Subtypes A, G, J, and CRF01-AE, in Central Africa

In this study, we characterized three full-length genome sequences with a similar mosaic structure from epidemiologically unlinked individuals from Cameroon (97CM-MP818) and the Central African Republic (99CF-MP1298 and 99CF-MP1307). Phylogenetic and recombinant analysis confirmed that the three strains had a similar complex recombinant genome, which we can designate now as CRF11-cpx. This new CRF was composed of successive fragments of subtype A, G, J, and CRF01-AE. The previously reported GR17 virus from a Greek patient infected in the Democratic Republic of Congo (DRC) has a similar structure and should be considered as the prototype strain of CRF11-cpx. This new CRF circulates in Cameroon, Central African Republic, Gabon, and DRC, although the exact prevalences remain to be determined.

Sequence Note The Identification of a Complex A/G/I/J Recombinant HIV Type 1 Virus in Various West African Countries

In this sequence note we describe the full-length genome sequence of an HIV-1 isolate originating from the west African country of Mali. The phylogenetic tree analysis from the near full-length genome shows that the 95ML84 strain forms a separate cluster, supported by 100% of the bootstrap values, with the previously described A/G/J/? mosaic virus BFP90 from Burkina Faso. Additional analysis showed that throughout the genome the lowest diversity was seen between the 95ML84 and the BFP90 viruses, and bootscan analysis showed a similar complex genomic structure. In addition to the initial report describing the BFP90 virus as an A/G/J/? recombinant, our data show that for the BFP90 and 95ML84 strains the unclassified region corresponds to subtype I. The A/G/I/J BFP90 and 95ML84 strains represent the fifth and most complex circulating recombinant form of HIV-1 detected so far, and our data show its presence in various West African countries. Subtype I and J sequences, initially considered rare, seem to have broadened their geographical spread by way of these recombinant forms.

Emergence of complex and diverse CRF02-AG/CRF06-cpx recombinant HIV type 1 strains in Niger, West Africa.

On the basis of partial env and gag subtyping, we documented that the majority of HIV-1 strains circulating in Niger were CRF02-AG (54.3%) or CRF06-cpx (18.1%) and that 9% of the samples were possible recombinants between CRF02 and CRF06. To determine in more detail the precise structure of these viruses we sequenced the full-length genomes for three such strains (97NE-003, 00NE-036, and 00NE-095). From the bootscan and phylogenetic tree analysis it is evident that the new viruses are the result of recombination events between CRF02-AG and CRF06-cpx strains. Importantly, each virus had a different complex recombinant structure with multiple breakpoints, leading to viruses with complex mosaic patterns.

Predominance of CRF02-AG and CRF06-cpx in Niger, West Africa

A total of 110 HIV-1-positive samples obtained in 1997 (n = 44) and 2000 (n = 66) were genetically characterized in the V3-V5 envelope region and the p24 gag region. The majority of the strains were CRF02-AG (54.3%) or CRF06-cpx (18.1%) in env and gag. More than 9% of the samples were recombinants between CRF02 and CRF06; 9 were CRF06 in env but CRF02 in gag, and for one sample the opposite was seen. Overall for 23 (20.9%) samples, the subtype designation was different between env and gag, and in 20 of these 23 samples a CRF was involved in the recombination event. No significant differences were seen between subtype distributions in 1997 and 2000, except that the proportion of recombinants increased from 13.6% in 1997 to 27.2% in 2000.