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Objective: The HIV epidemic in Cameroon is characterized by a high level of strain diversity despite a relatively low prevalence of infection. In this study, HIV strains infecting blood donors in Cameroon were characterized to determine the prevalence of subtypes and intersubtype recombinants and if strain prevalence was changing over time. Methods: From 1996 through 2004, 676 HIV-infected blood donations were collected at blood banks in Douala and Yaounde, Cameroon. A subset of the HIV-1 group M strains (n = 574) were classified based on phylogenetic analysis of viral sequences from the gag p24, pol integrase, and env gp41 regions. Results: HIV-1 group M accounted for 97.3% (n = 658) of infections, whereas group O was present in 2.2% (n = 15) and HIV-2 in 0.4% (n = 3). Within the group M infections, 14 subtypes and circulating recombinant forms (CRFs) and unique recombinant forms (URFs) were identified. Overall, CRF02_AG accounted for 58.2% of infections, URFs 14.8%, and levels of subtypes, A, B, C, D, F2, and G, and CRFs, 01, 06, 09, 11, 13, 22, and 37, varied from 0.2% to 6.1%. Evaluation of HIV strains present in the donor population over this 9-year period showed no substantial changes in the proportion of infections caused by each subtype and CRF, the percentage of intersubtype recombinants, or the strain composition of the URFs. Conclusions: HIV-1 strain diversity in Cameroon did not significantly change, suggesting a mature and relatively stable epidemic.

In the Brazilian HIV-1 epidemic subtypes B, C, and F1 are cocirculating in the high risk population groups, and there is a high prevalence of intersubtype recombinant forms. The dynamic nature of the HIV epidemic in Brazil led us to study HIV-1 subtypes present in HIV-infected blood donations collected from 2001 to 2003. Donations from 91 seropositive donors were evaluated. Genetic subtype was obtained for 88 specimens based on sequence analysis of gag p24, pol IN, and env gp41 IDR. HIV-1 subtype B was the predominant strain present in the donor population (73.9%). A significant prevalence of intersubtype recombinants of subtypes B and F1 was found (22.7%). Subtype C (1.1%) and F1 (2.3%) were rare. None of the B/F1 recombinants is CRF28_BF or CRF29_BF. The high level of unique B/F1 recombinant strains in this population demonstrates the dynamic and complex nature of the HIV epidemic in Brazil.

Texto completo: acesso restrito. p. 237–243 Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-08-23T11:03:09Z No. of bitstreams: 1 1-s2.0-S0166093406000395-main.pdf: 212674 bytes, checksum: 411c7cb3b1da7567c1b1dc64cd693538 (MD5) Approved for entry into archive by Rodrigo Meirelles (rodrigomei@ufba.br) on 2013-11-16T13:11:50Z (GMT) No. of bitstreams: 1 1-s2.0-S0166093406000395-main.pdf: 212674 bytes, checksum: 411c7cb3b1da7567c1b1dc64cd693538 (MD5) Made available in DSpace on 2013-11-16T13:11:51Z (GMT). No. of bitstreams: 1 1-s2.0-S0166093406000395-main.pdf: 212674 bytes, checksum: 411c7cb3b1da7567c1b1dc64cd693538 (MD5) Previous issue date: 2006 The combination of automated sample preparation and real-time RT-PCR for measurement of HIV-1 viral load has the potential to significantly enhance throughput, reduce operator-associated error, and increase assay sensitivity and dynamic range. In this study, RNA was extracted from the plasma of 91 HIV-1 seropositive Brazilian blood donors using the Abbott m2000sp™ automated sample preparation system. Viral loads measured using the RealTime™ HIV-1 (RealTime HIV-1) assay and the Abbott m2000rt™ instrument were compared to values obtained in the LCx® HIV RNA quantitative assay. Subtype was determined for 89 of 91 specimens by sequence/phylogenetic analysis of three genomic regions: gag p24, pol integrase, and env gp41. The panel included 69 subtype B, 1 C, 2 F, and 17 recombinant strains. Eighty-seven specimens were quantified by both assays. Two specimens were quantified only in RealTime HIV-1. Two additional specimens below the detection limit of both assays were also negative on PCR amplification. Viral load results were highly correlated, and good agreement was observed between assays with 90% of values within 0.5 log10 copies/ml. The RealTime HIV-1 assay and m2000 system offer the advantages of automation while providing reliable quantification of diverse HIV strains.

An HIV-1 group N infection, 02CM-DJO0135, was identified among specimens collected in 2002 at the D'Joungolo Hospital, Yaoundé, Cameroon. Sequences were obtained from viral RNA extracted from plasma for regions of LTR-gag, pol-vif, and env. The virus amplified from the specimen is closely related to a previously reported group N virus, 02CM-DJO0131, that was also collected at this hospital in 2002. Although the viral sequences for the two isolates differ, their close relationship suggests that the two specimens are linked. No patient histories are available for 02CM-DJO0131 and 02CM-DJO0135; the specimens could have been drawn from a husband/wife, mother/child, or a single individual. However, differences in seroreactivity indicate that it is unlikely that the specimens were drawn from the same patient. This report documents the second case that suggests linkage between group N-infected individuals and indicates that there is ongoing transmission of HIV-1 group N in Cameroon.

HIV-1 is classified into three groups, M (major), N (non-M non-O), and O (outlier); each group arose from a separate transmission of SIVcpz into humans. HIV-1 group N was recently discovered and infections with this virus are rare with only eight documented cases. All group N infections have been found in Cameroon and there is no evidence of direct linkage between the infected patients. We report here the identification of HIV-1 group N infections in a husband and wife. The group N infection in the husband, 1131-03, was identified first based on seroreactivity in peptide EIAs and confirmed by PCR amplification of group N viral sequences. Subsequently the wife, 1015-04, was evaluated and confirmed to also be infected with a group N virus. Near full-length viral genomes were amplified and sequenced from each patient's specimen. The low level of diversity between the two viral sequences provides evidence of horizontal transmission of group N from one spouse to the other. Patient 1131-03 was receiving antiviral therapy consisting of reverse transcriptase inhibitors; the treatment appears effective for suppression of group N viral replication based on apparently low viral load in plasma specimens collected from the patient and the absence of drug resistance mutations in RT sequences amplified from 1131-03. This report brings to 10 the number of group N infections identified and to 5 the number of group N genomes sequenced. Although group N infections continue to be rare, group N is a pathogenic virus and its prevalence needs to be monitored.

HIV-1 group O strains have a level of genetic diversity similar to that of strains in group M; however, group O has not been readily classified into genetic subtypes. Phylogenetic classification of group O has been hindered by the limited sequence information available. To facilitate phylogenetic analysis, we sequenced the gag p24 (693 nt), pol p32 (864 nt), and env gp160 (approximately 2700 nt) genes from 39 group O-infected specimens. These specimens include 32 plasma samples collected in Cameroon between 1996 and 1999, 2 specimens collected in the United States, and 5 infections previously isolated in Equatorial Guinea. Phylogenetic analysis of HIV-1 group O sequences resulted in the identification of five clusters that are maintained across gag, pol, and env, generally supported by high bootstrap values, and approximately equidistant from each other. In addition to the group O clusters, several isolates branch independently and are equidistant from the other group O isolates. Cluster I comprises greater than 50% of the group O isolates and is a diverse set of isolates that is subdivided into subclusters. The average intra-, sub-, and intercluster distances for group O are similar to the corresponding distances for group M subtypes. The five group O clusters have characteristics similar to those of group M subtypes. Thus the data presented may form the basis for classification of group O into subtypes. However, full-length genomes representing each group O cluster will be required to formalize a group O subtype classification.

Analysis of 3555 HIV-seropositive specimens, collected in Cameroon from 2002 to 2006, led to the identification of four HIV-1 group N infections based on differential seroreactivity to HIV env-derived peptides and proteins and confirmation by nucleic acid amplification. Group N prevalence continues to be low accounting for only 0.1% of HIV infections in Cameroon. Near full-length genomic sequences were obtained from viral RNA or proviral DNA by PCR amplification of overlapping fragments for three isolates, 06CM-U14296, 06CM-U14842, and 02CM-SJGddd. Two genome segments, partial pol and env-nef, were obtained from viral RNA for the fourth isolate, 02CM-TIM0217. With the four group N isolates identified in this study and group N sequences previously reported, eight near full-length and five partial genome sequences are now available. Despite genetic divergence from HIV-1 group M and O, all of the group N infections evaluated by five commercial HIV immunoassays were detected.

Recently, we reported a high level of HIV-1 strain diversity in patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Based on phylogenetic analysis of gag p24, pol integrase, and env gp41 sequences, subtypes A, B, C, D, and G, and CRF02_AG, as well as unique recombinant forms were identified. Subtype G accounted for 25% of the infections in the Saudi population and this high prevalence was unexpected. Although subtype G is found in west central Africa, pure subtype G strains are uncommon. To further characterize the subtype G infections in Saudi Arabia, six strains that appeared to be pure subtype G were selected for full genome sequencing. Near full-length genomes were obtained using RT-PCR amplification to generate overlapping fragments from viral RNA extracted from plasma. The six strains are not subtype G throughout their entire genome. Four isolates have a recombinant structure composed of CRF02_AG and subtype G and share three identical breakpoints. This recombinant form defines a new CRF designated CRF43_02G. The remaining two isolates are CRF25_cpx, a circulating recombinant form identified in Cameroon composed of subtypes A and G and unclassified segments. Reanalysis of the previously reported Saudi HIV-1 partial genome sequences revealed additional isolates classified as CRF43_02G and CRF25_cpx and one isolate was reclassified to CRF22_01A. Identification of CRF43_02G in Saudi Arabia could indicate a transmission network within the country. Alternatively, the new CRF could have been introduced from an external source where this CRF is not yet recognized.

Saudi Arabia has a very low prevalence of HIV infections and nothing is known about HIV strains present in the population. Here specimens were collected from 62 HIV-1-infected patients at the King Faisal Specialist Hospital and Research Center, Jeddah, Saudi Arabia. Viral sequences were PCR amplified using primers for HIV-1 group M in gag p24, pol integrase, and env gp41 and genetic subtype was determined by phylogenetic analysis. HIV-1 viral sequences were amplified from 56 of the 62 specimens. Based on phylogenetic analysis of viral sequences, subtype C was the most common subtype present and accounted for 39.3% of the infections followed by subtype G (25%), subtype B (17.9%), subtype D (3.6%), and subtypes A and CRF02_AG (1.8% each). In addition, for six specimens subtype classifications were discordant between gag, pol, and/or env; these intersubtype recombinant viruses account for 10.7% of the infections and consisted of recombinants of subtypes A/CRF01, A/CRF02, A/G, B/G, and D/CRF02. The high HIV-1 strain diversity suggests that there have been multiple introductions of HIV-1 into Saudi Arabia from several sources. Within the study population, there were five husband/wife pairs. For each pair, the viral sequences obtained were closely related to each other showing that heterosexual transmission occurred.

The high level of HIV genetic diversity has important implications for screening, diagnostic testing and patient monitoring. Continued diversification and global redistribution of HIV groups, subtypes and recombinants make it imperative that serological and molecular assays be designed and evaluated to ensure reliable performance on all HIV infections. Recognizing the importance of this issue, we initiated a comprehensive program to monitor global diversification of HIV, search for newly emerging variants, assemble large-volume panels of genetically and geographically diverse strains, and develop strategies to determine the impact of HIV diversity on assays used for detecting and monitoring HIV infection. Efforts to identify and characterize rare and emerging HIV strains have lead to the identification of HIV-1 group O, group N, and dual infections of groups M and O. A panel of plasma specimens was established that includes specimens collected from 12 countries in Africa, Asia, Europe, and South America; the panel comprises infections due to HIV-1 group M subtypes A, B, C, D, F, and G, as well as CRF01, CRF02, and unique recombinant forms, group N, and group O. Serological and molecular characterization of this unique panel has provided vital sequence data to support assay development and an invaluable source of well-defined specimens to evaluate and compare assay performance. The ability to address the challenge posed by ongoing evolution of HIV and the emergence of new variants requires continued surveillance of global HIV strain diversity, a sound scientific foundation for assay development, and suitable panels to evaluate and validate assay performance. J. Med. Virol. 78:S24–S29, 2006. © 2006 Wiley-Liss, Inc.

HIV-1 strain diversity was examined in a study population that consisted of hospital and clinic patients from seven cities and villages located in the northwestern regions of Cameroon. Specimens were screened using a serological algorithm designed to identify HIV-1 group M, N, and O, and SIVcpz-like infections followed by RT-PCR amplification to characterize the infecting virus. The results show that the HIV epidemic in northwest Cameroon is dominated by HIV-1 group M CRF02_AG infections (57%). Additional group M subtypes present include A, D, F2, G, and CRF01_AE. Based on discordant subtype classification between gag and env sequences, a high percentage (23%) of viral strains appear to be unique intersubtype recombinants with the majority (88%) involving recombination with CRF02_AG. Group O prevalence is low accounting for only 0.4% of HIV infections. However, group O strain diversity is high; isolates from clades I, IV, and V, as well as unclassified and recombinant strains, were found. Three dual infections by HIV-1 group M and group O were identified and characterized. In two specimens, both group M and O sequences were amplified in gag, pol, and env suggesting the presence of both viruses. Analysis of the third specimen shows the presence of a group O virus and an intergroup M/O recombinant virus. Finally, no infections due to HIV-1 group N or SIVcpz-like strains were found in the study population.

HIV-infected plasma specimens, collected in Cameroon between 1999 and 2002, were screened for HIV-1 group N and SIVcpz infections using a serological screening algorithm based on immunoassays with antigens derived from HIV-1 group M, N, and O, and SIVcpz strains. Specimens with reactivity to group N and SIVcpz antigens were characterized by RT-PCR and sequence analysis to identify the infecting virus. Although several specimens were serotyped as potential group N or SIVcpz infections, only one group N infection was confirmed. The specimen, 02CM-DJO0131, was collected in 2002 from a hospital patient at the D'Joungolo Hospital, Yaoundé. The virus genome was amplified as seven overlapping fragments comprising 8938 nucleotides. Phylogenetic analysis shows that 02CM-DJO0131 branches with group N sequences. With this study, three near full-length sequences are now available for group N. While we confirm the presence of group N in the Cameroonian population, group N infections continue to be rare and difficult to identify.

Human immunodeficiency virus type 1 (HIV-1) is classified into three distinct groups; M (major), N (non-M/non-O), and O (outlier). Group M strains are further subclassified into subtypes, subsubtypes, and circulating recombinant forms (CRF). While the level of genetic diversity within group O is similar to that between group M subtypes, group O has not been classified into subtypes. A previous study, based on the phylogenetic analyses of the gag p24, pol p32, and env gp160 sequences from 39 group O isolates, laid the foundation for the classification of group O subtypes. Five phylogenetic clusters, I-V, were identified that have characteristics analogous to group M subtypes. However, a complete phylogenetic analysis and classification of group O requires the availability of at least two full-length and one partial genomes for each group O phylogenetic cluster. In this study, 15 group O isolates were selected for full genome sequencing. Phylogenetic analysis of the 15 sequences with eight additional group O genomes supports the classification of three group O subtypes (I-III) and the potential existence of one CRF (IV) and at least one additional subtype (V). The group O subtypes are equidistant to each other and lack subsegments of other subtypes. The intra- and intersubtype genetic distances for group O are similar in magnitude to the corresponding distances for group M subtypes. Intersubtype recombination was identified in three of the 23 (13%) group O genomes. Formal classification of group O subtypes should be forthcoming pending the analysis of additional group O genomes and agreement of the HIV nomenclature committee.