Restriction fragment length polymorphism analysis for rapid gag subtype determination of human immunodeficiency virus type 1 in South Africa

A rapid method for identification of human immunodeficiency virus Type 1 (HIV-1) gag subtypes was developed based on restriction fragment length polymorphism (RFLP) analysis of 400 or 650 bp long polymerase chain reaction (PCR) fragments encompassing the start of the p17 (400 bp) and part of the p24 (650bp) regions. The consensus sequences of subtypes A-D, the only subtypes identified in South Africa, were analyzed to detect restriction endonucleases which generate unique patterns for each subtype. Four restriction endonucleases were identified: AluI, AccI, SwaI and XmnI. Digestion of a 400 bp fragment with AluI allowed identification of subtype C. Samples not identified were then reamplified, and a 650 bp fragment digested with AccI to identify subtype B, followed by SwaI and XmnI to distinguish between subtypes A and D. This strategy was applied to 87 samples previously subtyped by either sequence analysis of the gag p17 region (n = 33); or heteroduplex mobility assay (HMA) based on the env gene (n = 75); or both (n = 21). Out of the 87 samples, RFLP identified two samples as subtype A, 28 as subtype B, 56 as subtype C and one as a subtype D virus. No discrepancies were found between RFLP gag subtypes and gag sequence subtypes demonstrating the reliability of this method. There was also no discordance between gag RFLP subtypes and env HMA subtypes, suggesting that there were no recombinant viruses detected relating to the genomic regions analyzed. RFLP is an effective technique for the rapid screening in an HIV epidemic of limited diversity, such as in South Africa.     

Determination of human immunodeficiency virus type 1 subtypes in Taiwan by vpu gene analysis

The genetic diversity of human immunodeficiency virus (HIV) type 1 (HIV-1) has been characterized mainly by analysis of the env and gag genes. Information on the vpu genes in the HIV sequence database is very limited. In the present study, the nucleotide sequences of the vpu genes were analyzed, and the genetic subtypes determined by analysis of the vpu gene were compared with those previously determined by analysis of the gag and env genes. The vpu genes were amplified by nested PCR of proviral DNA extracted from 363 HIV-1-infected individuals and were sequenced directly by use of the PCR products. HIV-1 subtypes were determined by sequence alignment and phylogenetic analysis with reference strains. The strains in all except one of the samples analyzed could be classified as subtype A, B, C, E, or G. The vpu subtype of one strain could not be determined. Of the strains analyzed, genetic subtypes of 247 (68.0%) were also determined by analysis of the env or gag gene. The genetic subtypes determined by vpu gene analysis were, in general, consistent with those determined by gag and/or env gene analysis except for those for two AG recombinant strains. All the strains that clustered with a Thailand subtype E strain in the vpu phylogenetic analyses were subtype E by env gene analysis and subtype A by gag gene analysis. In summary, our genetic typing revealed that subtype B strains, which constituted 73.8% of all strains analyzed, were most prevalent in Taiwan. While subtype E strains constituted about one-quarter of the viruses, they were prevalent at a higher proportion in the group infected by heterosexual transmission. Genetic analysis of vpu may provide an alternate method for determination of HIV-1 subtypes for most of the strains, excluding those in which intersubtype recombination has occurred.     

HIV-1 infection among injection and ex-injection drug users from Rio de Janeiro, Brazil: prevalence, estimated incidence and genetic diversity.

BACKGROUND AND OBJECTIVES: Due to their behavioral conditions and vulnerability, injection drug users (IDUs) are prone to multiple simultaneous or sequential infections with distinct HIV-1 subtypes and variants, making them a key population for molecular epidemiology surveillance. In the present study, we evaluated HIV-1 infection seroprevalence, genetic diversity and estimated incidence among IDUs and ex-injection drug users (ex-IDUs) from Rio de Janeiro, Brazil. STUDY DESIGN: Six hundred and eight IDUs and ex-IDUs, recruited between 1999 and 2001, were interviewed and agreed to donate 30 ml of blood. The serologic status for HIV infection was determined by two ELISAs and confirmed by IFA. CD4+ T-cell percentages were assessed by flow cytometry. HIV-1 positive samples were submitted to viral load quantification. DNA samples were PCR amplified and HIV-1 subtypes were determined using env and gag HMA. RESULTS AND CONCLUSIONS: Forty-eight (7.89%) individuals were seropositive for HIV-1 infection. The seroincidence of HIV-1 infection was estimated as 0.76%. HIV-1 env and gag subtyping identified 29 (69%) samples as belonging to subtype B, 7 (16.7%) to subtype F, and 6 (14.3%) discordant env/gag genomes infections, indicating the circulation of recombinant viruses in this population.     

Identification of subtype C human immunodeficiency virus type 1 by subtype-specific PCR and its use in the characterization of viruses circulating in the southern parts of India

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-kappaB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.     

High genetic diversity of HIV-1 strains in Chad,West Central Africa

The genetic diversity of HIV-1 strains in Chad was documented with a total of 107 samples from patients attending the general hospital in N'Djamena, the capital city of Chad. The genetic subtypes were identified in the V3-V5 env and p24 gag regions by sequence and phylogenetic tree analyses. Of the 107 strains, 78 had the same subtype/CRF designation between env and gag. Four subtypes and three CRFs were found to cocirculate: subtype A, 20.5%; subtype D, 18.7%; CRF02_AG, 13.1%; CRF11_cpx, 13.1%; subtype G, 3.7%; CRF01_AE, 2.8%; and subtype F1, 0.9%. The remaining 29 strains (27%) had discordant subtypes or CRF designations between env and gag; in 15 of these 29 strains, a CRF was involved in the recombination event, and 10 were subtype G in gag and subtype A in env, forming a separate subcluster within subtypes G and A. Subtype D strains represent almost 20% of the HIV-1 strains circulating in Chad and form a separate subcluster in gag and env. Nearly full-length genome sequencing for two such strains (99TCD-MN011 and 99TCD-MN012) revealed that they represent nonrecombinant subtype D variants. Compared with neighboring countries, the genetic subtype distribution of HIV-1 strains in Chad is unique for several reasons: lower prevalence of CRF02, high prevalence of CRF11 and subtype D, and absence of CRF06. These data clearly show that subtype distribution is very heterogeneous in Africa, probably the result of different founder effects.     

Distribution of HIV-1 subtypes among HIV-seropositive patients in the interior of Côte d'Ivoire

Limited data exist on the distribution of HIV-1 subtypes in C?te d'Ivoire. The aim of this study is to describe the distribution of genetic subtypes of HIV-1 strains in six regions of C?te d'Ivoire. In 1997, we consecutively collected blood from 172 HIV-1-infected patients from six regional tuberculosis treatment centers. Peripheral blood mononuclear cells (PBMCs) from these people were analyzed by a restriction fragment-length polymorphism (RFLP) assay that involves a sequential endonuclease digestion of a 297-base pair polymerase chain reaction (PCR) fragment; plasma samples were tested by a V3-loop peptide enzyme immunoassay (PEIA). DNA sequencing of the protease or env genes was performed on all samples discordant in the two assays as well as a random sample of the concordant subtyped samples. Of 172 specimens, 3 were PCR-negative, and 169 were putatively classified as subtype A by RFLP. The 3 PCR-negative samples were unequivocally subtyped A by PEIA. Of the 169 RFLP subtype A samples, 159 (94%) were subtyped A by PEIA. Of the 10 discordant samples, PEIA testing classified 3 as subtype C, 2 as D, and 5 as F. Sequencing of the env gene classified these samples as 1 subtype A, 4 Ds, and 5 Gs. Thus, 163 (95%) of the specimens were subtype A, 3 subtype D, 4 subtype G, 1 A/D, and 1 A/G (IbNG) circulating recombinant forms (CRF). In conclusion, most HIV-1-infected tuberculosis patients throughout the interior of C?te d'Ivoire are infected with HIV-1 subtype A, which are very likely the A/G (IbNG) CRF. The uniform distribution of this subtype makes C?te d'Ivoire a potential site for vaccine trials.     

Human immunodeficiency viruses type 1 subtypes circulating in Sspain.

Genetic subtypes of Human immunodeficiency viruses type 1 (HIV-1) were investigated in 101 HIV-1-infected individuals living in Spain from 1993 to 1998. Samples selected randomly from the HIV clinic population included 29 Spanish native born subjects (28.7%) and 72 foreigners (71.3%). Proviral DNA extracted from peripheral blood mononuclear cells (PBMCs) or viral RNA isolated from plasma was amplified, and endonuclease restriction analysis was carried out on polymerase chain reaction (PCR) products. Restriction fragment length polymorphism (RFLP) analysis on the HIV-1 protease region enabled the characterisation of the different HIV genotypes infecting these individuals. Overall, 38 subjects (37.6%) carried non-B subtypes (A in 26, C in 2, D in 1, E in 2, and F in 7), 31 (81. 6%) of them being immigrants. Direct sequence analysis of PCR products and/or a specific serological assay confirmed the data obtained by RFLP in most individuals tested. In conclusion, different HIV-1 subtypes are circulating currently in Spain, with non-B HIV-1 subtypes being confined mostly to immigrants.     

Independent introduction of transmissible F/D recombinant HIV-1 from Africa into Belgium and The Netherlands

Most HIV-1 subtype F viruses described so far have been isolated from individuals originating in South America, Romania, or Central Africa. Previous studies have shown that subtype F viruses from these three areas can be distinguished by phylogenetic tree analysis of various parts of the HIV genome. Subtype F strains circulating in Central Africa and classified as subgroup F2 and F3 have relatively large nucleotide distances from strains of subgroup F1, which includes some African strains, along with strains from Romania and South America. Subtype F strains have now appeared in Europe. In this study, we analyzed the complete gag gene and a large fragment of the pol gene of seven strains of African origin that represent the three F subgroups. At least five of the seven strains appear to be intersubtype recombinants. Of four strains circulating in Belgium and the Netherlands, three were F/D mosaics and the fourth harboured a G(gag)/GH(pol)/F3(env) recombinant structure. Two of the three F/D mosaics showed identical breakpoints and were independently introduced in Belgium and the Netherlands. At least two of the mosaics were further transmitted. The remaining three strains of the seven we studied were isolated from individuals in Cameroon. Two included large or smaller F1 fragments in gag and pol. The third strain was subtype D along the entire gag and pol fragment. A parental African subtype F that showed no evidence for recombination was not found.     

Genetic characteristics of variants of human immunodeficiency virus type 1, causing an epidemic among substance abusers in Commonwealth of Independent States countries]

Gag/env nucleotide sequences of human immunodeficiency virus type 1 (HIV-1) variants detected in drug users in Russia, Ukraine, and Belarus are analyzed. Two HIV-1 subtypes A and B circulate in this risk group. Genetic variability within one subtype is no higher than 3.1 and 3.9% for gag and env genes, respectively, suggesting the same source of infection in populations of drug users infected with the same subtype. Recombinant viruses with gagAenvB genotype, genetically related to parental strains of subtypes A and B, circulate in this risk group. This is the first report about HIV-1 recombinant of the two subtypes, for which both parental strains are known, directly confirming the in vivo recombination between different subtypes.     

Nucleotide sequence of gag and env genes of human immunodeficiency virus type 1, found in Russia: detection of new recombinant variants

Phylogenetic analysis of nucleotide sequences of gag and env genes of type 1 human immunodeficiency virus (HIV-1) variants isolated from individuals infected through sexual intercourse or nosocomially (by injections with nonsterile syringes) showed that 5 of 27 (18.5%) isolated strains were recombinants. Two viruses found in the Russian Far East had gagAenvE genotype, three other recombinants had envG genotype; gag gene of one isolate belonged to subtype A and gag genes of two others belonged to subtype D. Detection of new recombinant variants in addition to the A/B recombinant described previously shows that these viruses can contribute to the HIV-1 genetic variability in Russia.     

Risk of HIV-1 transmission by breastfeeding among mothers infected with recombinant and non-recombinant HIV-1 genotypes

BACKGROUND: Viral genotype and intersubtype recombination may influence the rate and/or timing of mother-to-child HIV-1 transmission. METHODS: We determined the HIV-1 subtype of the C2-C5 env and 5'LTR regions from milk and blood samples of 61 Tanzanian mothers who transmitted the virus through breastfeeding and their HIV-1 positive non-transmitting controls. Cases and controls were matched on infant's age at sample collection. All mothers resided in Dar es Salaam, Tanzania. RESULTS: Most infections among cases were due to recombinant viruses (41.0%), followed by HIV-1 subtype A (26.2%), subtype D (19.7%), and subtype C (13.1%). In multivariate analysis including maternal CD4+ cell counts, HIV disease stage, and proviral load in breast milk, the odds of breast milk transmission were 7.2 times higher if the mother carried an intersubtype recombinant genome in comparison to a subtype C virus (p=0.02). Viruses with recombinant LTRs were 4.9 times more likely to be transmitted through breastfeeding than viruses with non-recombinant LTRs of subtype A, C or D combined (p=0.01). CONCLUSIONS: This suggested that intersubtype recombinant genomes, and especially recombination within the LTR, might render HIV-1 more fit for transmission via breast milk in comparison with non-recombinant subtypes A, C, and D.     

Generation of a consensus sequence from prevalent and incident HIV-1 infections in West Africa to guide AIDS vaccine development

We considered several key issues regarding the development of a DNA-based human immunodeficiency virus type 1 (HIV-1) vaccine: (1) should the candidate vaccine construct be derived from incident or prevalent HIV-1 strains; and (2) should circulating plasma virus, archived HIV-1 provirus recovered from peripheral blood mononuclear cells, or both be included? To address these questions, we collected circulating HIV-1 strains from infected individuals residing in Abidjan, C?te d'Ivoire. From a panel of 23 strains, 22 were HIV-1 subtype A in gag, 19 of which phylogenetically clustered with the recombinant HIV-1, CRF02-AG strains from West Africa. The mosaic genome of CRF02-AG was confirmed by sequencing the protease gene. A consensus gag p24 protein sequence was generated and 147 of 148 codons were identical to CRF02-AG (IbNG). Regardless of the sequence origin (RNA, provirus, incident, or prevalent), the gag p24 consensus sequences were highly representative of these distinct virologic compartments. These data suggest that the consensus sequence generated from incident and prevalent infections may provide an appropriate sequence for a DNA vaccine and is largely representative of the major circulating viral strain.     

Presence of multiple HIV subtypes and a high frequency of subtype chimeric viruses in heterosexually infected women

The HIV-1 subtype distribution was determined in 41 HIV-positive women (-8% of all HIV-infected women in Denmark) belonging to different risk groups. HIV p17 gag and env gene subtypes were determined by DNA sequence analysis. Five different HIV subtypes were detected across all patients. Most HIV-1-positive women of Danish origin carried subtype B viruses, and a minority had virus belonging to subtypes A or C. All injecting drug users (IDUs) were infected with HIV subtype B viruses, whereas all non-B subtypes were present in cases linked to heterosexual transmission. In contrast, all women of African origin carried non-B HIV subtypes (subtypes A, C, D, or G) regardless of transmission mode. Of these women, 21% infected with non-B HIV subtypes appeared to be infected by subtype chimeric viruses and 7% were jointly infected with viruses belonging to two different subtypes (A and C). Data demonstrate a preferential representation of non-B HIV subtypes in women infected through heterosexual contact, as well as a high degree of recombination between viruses derived from endemic areas in which several HIV subtypes predominate. Combined with the increased incidence of heterosexual transmission of HIV, the results imply that an increased subtype diversity can be anticipated in newly infected individuals.     

A gag gene heteroduplex mobility assay for subtyping HIV-1

A heteroduplex mobility assay (HMA) using 753 and 446 base pair (bp) amplicons of the p17/p24 region of the gag gene of HIV-1 has been developed and validated with reference clones and clinical samples representative of subtypes A, B, C, D, E, G, and H. There was complete concordance between the gag HMA assigned subtype and the subtype known from gag or env sequence data or env HMA. The heteroduplexes from both amplicons can be clearly resolved on either MetaPhor XR agarose or MDE polyacrylamide gels. The MetaPhor XR gel system was the more convenient and is the preferred choice for routine HMA subtyping. This gag HMA provides a rapid, simple and inexpensive method for subtyping HIV-1 based on a genomic region other than the commonly used env gene target. The incorporation of gag HMA into subtype determination algorithms should allow the detection of gag/env recombinant strains of HIV-1.     

Characterization of human immunodeficiency virus type-1 from HIV-1 seropositive cases with undetectable viremia.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) viral load has become a standard of care among HIV-1-infected patients; however, a small number of patients have undetectable viral load even though they have never been treated. METHODS: By using RT-PCR and DNA-PCR, and followed by sequencing and phylogenetic analyses, a detailed molecular characterization was carried out from five HIV-1-seropositive patients who had undetectable viral load by commercially available ultrasensitive viral load assays. RESULTS: Of the four patients whose plasmas were available, viral RNAs were detected in three of them by using an in-house RT-PCR in at least one of the three regions (integrase, protease or envgp41). The fourth patient had positive RT-PCR signals in these regions only when RNA isolated from the supernatant of cocultivated patient PBLs with PHA-stimulated HIV-1 negative donor PBLs was used. Further analysis of DNA extracted from the PBMCs revealed that four of the five patients had detectable proviral sequences in at least two of the three regions. The fifth patient had only positive PCR results in all three regions when DNA isolated from PHA-stimulated patient's PBLs was used. Phylogenetic analysis of protease and envgp41 regions revealed that three patients were infected with subtype B viruses while the remaining two patients were infected with subtype C and CRF02_AG viruses. These subtypes coincided with geographic origin and known molecular epidemiology of HIV-1 infection. CONCLUSION: These data provide evidence that both subtype B and non-B HIV-1 infection can result in undetectable viral load in HIV-1-infected patients and that efforts should continue to further characterize these viruses.     

Genotypic resistance profile of HIV-1 protease gene: a preliminary report from Vellore, south India

HIV-1 subtypes other than B are responsible for most new HIV infections worldwide; virus sequence data for drug resistance is described only from a limited number of non-B subtype HIV-1. This study is on mutations and polymorphisms of HIV-1 protease gene that can predict drug resistance in subtype C. The genotypic resistance assay was carried out on 38 HIV-1 strains with their plasma RNA and in nine, the proviral protease gene was sequenced. The treatment naïve strains showed minor resistance mutations, there were no major resistance mutations in the protease gene. We suggest the use of resistance testing to monitor individuals on therapy and also before initiation of therapy, gathering more sequence information for a data bank of Indian strains.     

In-utero transmission of quasispecies among human immunodeficiency virus type 1 genotypes

Samples from infants infected in-utero by the human immunodeficiency virus type 1 (HIV-1) subtypes A, C, D, and recombinants from Dar es Salaam, Tanzania, were examined for the presence of viral genetic quasispecies. HIV-1 envelope diversity was measured on peripheral blood mononuclear cells collected within the first 48 h of life from 53 infants. Phylogenetic analysis of C2-C5 envelope nucleotide sequences was used for HIV-1 subtype classification. Forty-two of 53 samples (79%) showed a heteroduplex mobility assay (HMA) suggestive of transmission of a single quasispecies, while 21% showed infection with multiple quasispecies. No differences among HIV-1 subtypes were found in the proportion of single to multiple quasispecies transmitted in-utero (Likelihood ratio test, P = 0.83), nor were differences found among single to multiple quasispecies transmitted and maternal viral load (Mann-Whitney test, P = 0.44). This suggests that differences in perinatal transmission between subtypes we previously observed in this cohort could not be associated with the likelihood for multiple independent infections during in-utero infections.