Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Gonorrhea as a risk factor for HIV acquisition.

OBJECTIVE: The role of gonorrhea in facilitating acquisition of HIV infection has only recently been studied. A previous nested case-control analysis in a cohort of female sex workers in Zaïre found a strong association between HIV seroconversion and prior gonorrheal infection. The objective of this study was to replicate the Zaïre study analysis in a cohort of 273 Cameroonian sex workers to determine whether gonorrhea increased the risk of HIV acquisition, and if the crude association between gonorrheal infection and HIV acquisition was weakened when the level of unprotected coitus was more carefully controlled. METHODS: We conducted a nested case-control study of initially HIV-1-negative women (n = 273) followed prospectively (with monthly sexually transmitted disease check-ups and 3-monthly HIV-1 serology). As in Zaïre, cases (seroconverters, n = 17) were compared with controls (women who remained HIV-1-negative, n = 68) for incidence of gonorrhea and sexual exposure during the presumed period of HIV-1 acquisition. RESULTS: The association between gonorrheal infection and subsequent HIV acquisition was stronger in Zaïre than in Cameroon [crude odds ratios (OR), 6.3 versus 2.2]. In both the Zaïre and Cameroon data the crude OR were reduced (6.3 to 4.8, and 2.2 to 1.7, respectively) by controlling for risk factors including a dichotomous variable indicating irregular or no condom use. When this variable was replaced in the Cameroon data with a more precise continuous variable indicating the percentage of unprotected coital acts, the gonorrhea OR was further reduced to 1.4 (95% confidence interval, 0.4-4.9). CONCLUSION: These results suggest that in the Cameroon cohort, gonorrheal infection did not facilitate HIV acquisition, but that having gonorrhea was a marker for unprotected coitus that facilitated HIV acquisition. The data demonstrate how OR can be overestimated when imprecise dichotomous measures of unprotected coitus are used. Future studies should plan for better control of self-reported condom use.

PIP: The objective was to replicate a Zaire study with a cohort of 273 Cameroonian sex workers to determine whether gonorrhea increased the risk of HIV acquisition, and whether the crude association between gonorrheal infection and HIV acquisition was weakened when unprotected coitus was more carefully controlled. 303 Cameroonian female sex workers were enrolled in Yaounde between 1989 and 1990. Eligibility criteria included age 18 years or older; no pregnancy during the previous 42 days; no history of adverse reaction to a spermicidal product; negative enzyme-linked immunosorbent assay (ELISA) HIV-antibody test; and monthly follow-up visits for 1 year. Women were asked to use condoms and suppositories containing N-9 at every sexual activity and coital logs were reviewed monthly. 17 women were identified as cases and 68 as controls. Unlike in Zaire, where 8% of the cases and controls reported never using condoms, none of the women reported unprotected coitus more than 50% of the time in Cameroon. Almost 30% of both cases and controls in Cameroon had evidence of genital ulcers, compared with less than 5% of the cases and controls in Zaire. The crude OR of HIV infection among those who had gonorrhea during the exposure period was much higher in Zaire than in Cameroon (6.3 vs. 2.2). In both the Zaire and Cameroon data the crude OR were reduced (6.3 to 4.8 and 2.2 to 1.7, respectively) by controlling for risk factors of young age, number of partners per week, trichomoniasis, genital ulcers, and a dichotomous variable indicating irregular or no condom use. In the 1st alternative model, the OR for gonorrhea was 2.0 when the dichotomous measure of unprotected coitus (i.e., more than 25% of coital acts were unprotected) was used. When a more precise continuous estimate of level of unprotected coitus was used (i.e., the percentage of acts where neither condoms nor N-9 was used) the OR for gonorrhea was reduced to 1.4.

Barrier contraceptive use and HIV infection among high-risk women in Cameroon.

OBJECTIVES: To measure the association between spermicide use and HIV infection, adjusting for condom use, and to measure the association between condom use and HIV infection, adjusting for spermicide use. DESIGN: Prospective study of women using nonoxynol-9 (N-9) spermicides and latex condoms, with up to 12 monthly clinic visits for interviews, examinations and tests, and re-supply. METHODS: A total of 273 HIV-negative women with multiple sexual partners were enrolled, given latex condoms and N-9 vaginal spermicidal suppositories, and advised to use both every time they had sexual intercourse. Participants recorded data on sexual activity on pictorial coital logs. New HIV infections were detected and confirmed by quarterly enzyme-linked immunosorbent assays and Western blots, respectively. RESULTS: Nineteen HIV infections occurred during mean follow-up of 8.1 months (an incidence rate of 10.4 infections per 100 women-years). The adjusted HIV rate ratio (RR) was 0.1 [95% confidence interval (CI), 0.1-0.6] for more consistent compared with less consistent spermicide users; and 1.1 (95% CI, 0.4-2.9) for more consistent compared with less consistent condom users. Among the subgroup of experienced condom users, the RR for more versus less consistent condom use was 0.3. CONCLUSIONS: This is the first epidemiological evidence that N-9 spermicides can reduce the incidence of HIV infection. A more definitive randomized clinical trial is urgently needed.

PIP: In a prospective study, the association between spermicide use and HIV infection was measured, adjusting for condom use, the association between condom use and HIV infection, and spermicide use. A total of 273 HIV-negative female prostitutes, the majority of them in their twenties, were enrolled in the study in Yaounde, Cameroon in 1989-1990. They made up to 12 monthly clinic visits for interviews, examinations, tests, and re-supply of condoms and spermicide. When they were admitted to the study, they were given plain lubricated latex condoms and N-9 vaginal spermicidal suppositories containing 100 mg N-9, and they were advised to use both every time they had sexual intercourse. Participants recorded data on sexual activity and barrier method use on pictorial coital logs (calendars on which coitus, condom use, and spermicide use could be marked). New HIV infections were detected and confirmed by quarterly enzyme-linked immunosorbent assays and Western blots, respectively. Both consistent spermicide use and consistent condom use were associated with lower incidences of gonorrhoea and genital ulcers. 19 HIV-seroconversions (7.0% of the 273 women) occurred during a mean follow-up of 8.1 months (an incidence rate of 10.4 infections per 100 woman-years). The HIV rate ratio (RR) for more consistent spermicide users ( or = 67% use) compared with less consistent users was 0.2 (95% CI, 0.1-0.7), indicating substantial protection against HIV. The HIV RR for more consistent condom users ( or = 63% use) compared with less consistent users was 0.8 (95% CI, 0.3- 2.0), suggesting slight protection. Adjusting for condom use, the RR for more versus less consistent spermicide use was 0.1 (95% CI, 0.1- 0.6). Adjusting for spermicide use, the RR for more versus less consistent condom use was 1.1 (95% CI, 0.4-2.9). Among the subgroup of experienced condom users, the RR for more versus less consistent condom use was 0.3. These findings prove that N-9 spermicides can reduce the incidence of HIV infection.

The epidemiology of gonorrhoea, chlamydial infection and syphilis in four African cities.

OBJECTIVES: To compare the epidemiology of gonorrhoea, chlamydial infection and syphilis in four cities in sub-Saharan Africa; two with a high prevalence of HIV infection (Kisumu, Kenya and Ndola, Zambia), and two with a relatively low HIV prevalence (Cotonou, Benin and Yaoundé, Cameroon). DESIGN: Cross-sectional study, using standardized methods, including a standardized questionnaire and standardized laboratory tests, in four cities in sub-Saharan Africa. METHODS: In each city, a random sample of about 2000 adults aged 15-49 years was taken. Consenting men and women were interviewed about their socio-demographic characteristics and their sexual behaviour, and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection, and (women only) Trichomonas vaginalis infection. Risk factor analyses were carried out for chlamydial infection and syphilis seroreactivity. RESULTS: The prevalence of gonorrhoea ranged between 0% in men in Kisumu and 2.7% in women in Yaoundé. Men and women in Yaoundé had the highest prevalence of chlamydial infection (5.9 and 9.4%, respectively). In the other cities, the prevalence of chlamydial infection ranged between 1.3% in women in Cotonou and 4.5% in women in Kisumu. In Ndola, the prevalence of syphilis seroreactivity was over 10% in both men and women; it was around 6% in Yaoundé, 3-4% in Kisumu, and 1-2% in Cotonou. Chlamydial infection was associated with rate of partner change for both men and women, and with young age for women. At the population level, the prevalence of chlamydial infection correlated well with reported rates of partner change. Positive syphilis serology was associated with rate of partner change and with HSV-2 infection. The latter association could be due to biological interaction between syphilis and HSV-2 or to residual confounding by sexual behaviour. At the population level, there was no correlation between prevalence of syphilis seroreactivity and reported rates of partner change. CONCLUSION: Differences in prevalence of chlamydial infection could be explained by differences in reported sexual behaviour, but the variations in prevalence of syphilis seroreactivity remained unexplained. More research is needed to better understand the epidemiology of sexually transmitted infections in Africa.

The epidemiology of trichomoniasis in women in four African cities.

OBJECTIVES: To describe the epidemiology of Trichomonas vaginalis infection and its association with HIV infection, in women in four African cities with different levels of HIV infection. DESIGN: Cross-sectional study, using standardized methods, including a standardized questionnaire and standardized laboratory tests, in four cities in sub-Saharan Africa: two with a high prevalence of HIV infection (Kisumu, Kenya and Ndola, Zambia), and two with a relatively low prevalence of HIV (Cotonou, Benin and Yaoundé, Cameroon). METHODS: In each city, a random sample of about 2000 adults aged 15-49 years was taken. Consenting men and women were interviewed about their socio-demographic characteristics and their sexual behaviour, and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection, and (women only) T. vaginalis infection. Risk factor analyses were carried out for trichomoniasis for each city separately. Multivariate analysis, however, was only possible for Yaoundé, Kisumu and Ndola. RESULTS: The prevalence of trichomoniasis was significantly higher in the high HIV prevalence cities (29.3% in Kisumu and 34.3% in Ndola) than in Cotonou (3.2%) and Yaoundé (17.6%). Risk of trichomoniasis was increased in women who reported more lifetime sex partners. HIV infection was an independent risk factor for trichomonas infection in Yaoundé [adjusted odds ratio (OR) = 1.8, 95% confidence interval (CI) = 0.9-3.7] and Kisumu (adjusted OR = 1.7, 95% CI = 1.1-2.7), but not in Ndola. A striking finding was the high prevalence (40%) of trichomonas infection in women in Ndola who denied that they had ever had sex. CONCLUSION: Trichomoniasis may have played a role in the spread of HIV in sub-Saharan Africa and may be one of the factors explaining the differences in levels of HIV infection between different regions in Africa. The differences in prevalence of trichomoniasis between the four cities remain unexplained, but we lack data on the epidemiology of trichomoniasis in men. More research is required on the interaction between trichomoniasis and HIV infection, the epidemiology of trichomoniasis in men, and trichomonas infections in women who deny sexual activity.

Factors influencing the difference in HIV prevalence between antenatal clinic and general population in sub-Saharan Africa.

OBJECTIVE: To compare HIV prevalence in antenatal clinics (ANC) and the general population, and to identify factors determining the differences that were found. DESIGN: Cross-sectional surveys in the general population and in ANC in three cities. METHODS: HIV prevalence measured in adults in the community was compared with that measured by sentinel surveillance in ANC in Yaoundé, Cameroon, Kisumu, Kenya, and Ndola, Zambia. RESULTS: In Yaoundé and Ndola, the HIV prevalence in ANC attenders was lower than that in women in the population overall, and for age groups over 20 years. In Kisumu, the HIV prevalence in ANC attenders was similar to that in women in the population at all ages. The only factors identified that influenced the results were age, marital status, parity, schooling, and contraceptive use. The HIV prevalence in women in ANC was similar to that in the combined male and female population aged 15-40 years in Yaoundé and Ndola, but overestimated it in Kisumu. In Yaoundé and Ndola, the overall HIV prevalence in men was approximated by using the age of the father of the child reported by ANC attenders, but this method overestimated the HIV prevalence in Kisumu, and did not give good age-specific estimates. CONCLUSION: Few factors influenced the difference in HIV prevalence between ANC and the population, which could aid the development of adjustment procedures to estimate population HIV prevalence. However, the differences between cities were considerable, making standard adjustments difficult. The method of estimating male HIV prevalence should be tested in other sites.

HIV-1 subtypes and the HIV epidemics in four cities in sub-Saharan Africa.

OBJECTIVE: To describe the distribution of HIV-1 subtypes in two cities with high HIV prevalence (Kisumu, Kenya and Ndola, Zambia) and two with relatively low prevalence (Cotonou, Benin and Yaoundé, Cameroon), and to examine whether the differences in prevalence of HIV infection could be due to the predominance within the infected populations of subtypes with differing efficiency of heterosexual transmission. METHODS: For around 100 randomly selected HIV-positive sera from the general population and 60 from sex workers in each city, the HIV-1 subtype was determined in the envfragment. For between 19 and 52 of the sera from the general population and 20-32 sera from sex workers, the subtype was also determined in the gag fragment. RESULTS: Over 70% of infections in Cotonou, Yaoundé and Kisumu were with subtype A (by env). However, around one-half of subtype A infections in Cotonou and Yaoundé were found to be the circulating recombinant form CRF02_AG when the gag fragment was also examined. A large number of different HIV strains were found in Yaoundé, including some belonging to group O. Over 20% of infections in Kisumu and around 10% in Yaoundé were with isolated intersubtype recombinant forms. All but a few infections in Ndola were with subtype C and no recombinants were found. CONCLUSIONS: The pattern of distribution of subtypes that we found does not suggest that differences in circulating subtypes play a major role in explaining the differences in prevalence of HIV-1 infection between the four cities. The emergence and spread of recombinants requires close surveillance to adapt testing strategies if needed, to inform vaccine development and to ascertain their role in the future spread of HIV.

The epidemiology of HSV-2 infection and its association with HIV infection in four urban African populations.

OBJECTIVES: To estimate age- and sex-specific herpes simplex virus type-2 (HSV-2) prevalence in urban African adult populations and to identify factors associated with infection. DESIGN AND METHODS: Cross-sectional, population-based samples of about 2000 adults interviewed in each of the following cities: Cotonou, Benin; Yaoundé, Cameroon; Kisumu, Kenya and Ndola, Zambia. Consenting study participants were tested for HIV, HSV-2 and other sexually transmitted infections. RESULTS: HSV-2 prevalence was over 50% among women and over 25% among men in Yaoundé, Kisumu and Ndola, with notably high rates of infection among young women in Kisumu and Ndola (39% and 23%, respectively, among women aged 15-19 years). The prevalence in Cotonou was lower (30% in women and 12% in men). Multivariate analysis showed that HSV-2 prevalence was significantly associated with older age, ever being married, and number of lifetime sexual partners, in almost all cities and both sexes. There was also a strong, consistent association with HIV infection. Among women, the adjusted odds ratios for the association between HSV-2 and HIV infections ranged from 4.0 [95% confidence interval (CI) = 2.0-8.0] in Kisumu to 5.5 (95% CI = 1.7-18) in Yaoundé, and those among men ranged from 4.6 (95% CI = 2.7-7.7) in Ndola to 7.9 (95% CI = 4.1-15) in Kisumu. CONCLUSIONS: HSV-2 infection is highly prevalent in these populations, even at young ages, and is strongly associated with HIV at an individual level. At a population level, HSV-2 prevalence was highest in Kisumu and Ndola, the cities with the highest HIV rates, although rates were also high among women in Yaoundé, where there are high rates of partner change but relatively little HIV infection. The high prevalence of both infections among young people underlines the need for education and counselling among adolescents.

Multicentre study on factors determining differences in rate of spread of HIV in sub-Saharan Africa: methods and prevalence of HIV infection.

OBJECTIVE: The objective of this study was to explore whether the differences in rate of spread of HIV in different regions in sub-Saharan Africa could be explained by differences in sexual behaviour and/or factors influencing the probability of HIV transmission during sexual intercourse. METHODS: A cross-sectional, population-based study was conducted in two cities with a high HIV prevalence (Kisumu in Kenya and Ndola in Zambia) and two cities with a relatively low HIV prevalence (Cotonou in Benin and Yaoundé in Cameroon). In each of these cities, approximately 1000 men and 1000 women, aged 15-49 years, were randomly selected from the general population. Consenting men and women were interviewed and were tested for HIV, syphilis, herpes simplex virus type 2 (HSV-2), gonorrhoea, chlamydial infection and trichomoniasis (the latter for women only). In addition, a survey was conducted on a random sample of 300 sex workers in each city. The research instruments, including the questionnaires and the laboratory procedures, were standardized to permit comparison of results. RESULTS: The numbers of men interviewed were 1021 in Cotonou, 973 in Yaoundé, 829 in Kisumu, and 720 in Ndola. The corresponding figures for women were 1095, 1116, 1060 and 1130. In Yaoundé, Kisumu and Ndola, the response rates for men were lower than for women due to failure to make contact with eligible men. The proportion of eligible women who were interviewed was 86% in Yaoundé, and 89% in Kisumu and Ndola. In Yaoundé, 76% of eligible men were interviewed, along with 82% in Kisumu and 75% in Ndola. The prevalence of HIV infection in men was 3.3% in Cotonou, 4.1% in Yaoundé, 19.8% in Kisumu and 23.2% in Ndola. For women, the respective figures were 3.4, 7.8, 30.1 and 31.9%. The prevalence of HIV infection among women aged 15-19 years was 23.0% in Kisumu and 15.4% in Ndola. Among women in Kisumu who had their sexual debut 5 years before the interview, the prevalence of HIV infection was 46%; in Ndola, it was 59%. Among sex workers, the prevalence of HIV infection was 57.5% in Cotonou, 34.4% in Yaoundé, 74.7% in Kisumu and 68.7% in Ndola. CONCLUSIONS: The HIV prevalence rates in the general population confirmed our preliminary assessment of the level of HIV infection in the four cities, which was based on estimates of HIV prevalence from sentinel surveillance among pregnant women. The very high prevalence of HIV infection among young women in Kisumu and Ndola calls for urgent intervention.

African origin of GB virus C/hepatitis G virus.

Ninety-four GB virus C/hepatitis G virus (GBV-C/ HGV) RNA-positive serum samples were obtained from all over the world. We found that all 15 GBV-C/HGV isolates from the Pygmies and the Bantu in the Central African region had a 12-amino acid indel (i.e. insertion or deletion) in the non-structural protein (NS) 5A region. Phylogenetic analyses of the NS5A region, using GBV-A as an outgroup, showed that these 15 isolates had diverged from the common ancestor much earlier than the remaining isolates, indicating an African origin of GBV-C/HGV.

HIV type 1 genetic variability in the northern part of Cameroon.

In 1995, 53 blood samples from Muslim patients with AIDS, or who were thought to have AIDS, were collected in the main hospitals of Adamaoua Province, in the northern part of Cameroon. The variable env C2V3 region of HIV-1 was amplified by nested PCR and phylogenetically analyzed. The results indicated that of 15 amplified samples, 1 belonged to HIV-1 group O, 1 to HIV-1 subtype D, 1 to subtype G, 2 to subtype H, and 10 to subtype A. Furthermore, the northern Cameroonian subtype A could be divided into at least two subclusters as shown by the env tree as well as by two remarkably conserved hexameric amino acid sequences in the apex of V3 (GPGQAF in one subcluster and GPGQTF in the other). This distinction suggests that the HIV-1 subtype A circulating in northern Cameroon evolved from two main sources. More recently, three HIV-1 strains from Nigeria (IBNG) and Djibouti (DJ263 and DJ264), previously reported on the basis of their env C2V3 sequences as subtype A, were found to have a similar A/G mosaic structure alongside their full-length sequence and were tentatively designated as members of a new subtype called "IBNG." Interestingly, within the northern Cameroonian subtype A described, the isolates of the second subcluster clustered distinctly with these A/G mosaic strains, strongly suggesting that they may be members of the IBNG subtype.

Coreceptor requirements of primary HIV type 1 group O isolates from Cameroon.

HIV-1 group O has its epicenter in Cameroon and neighboring countries and is responsible for 3 to 5% of all HIV infections in this region. It is believed that HIV-1 group O was introduced into the human population by a separate cross-species transmission, occurring independently of the HIV-1 (group M and group N) and HIV-2 transmissions. We have studied the coreceptor requirements of 12 primary HIV-1 O-type isolates from individuals with different clinical symptoms. Only 2 of these 12 viruses showed a syncytium-inducing phenotype after infection of primary peripheral blood mononuclear cells (PBMCs) and were infectious for the T cell line C8166. These isolates used CXCR4 as a coreceptor for entry, whereas the remaining isolates used only CCR5 efficiently. One isolate was able to use BOB and CCR8 as coreceptors in addition to CXCR4. All group O isolates tested were efficiently inhibited by SDF-1 or RANTES, the natural ligands of CXCR4 and CCR5, respectively. These results indicate that CXCR4 and CCR5 are the principal coreceptors for HIV-1 O-type viruses. Most of the HIV-1 group O isolates studied were derived from patients at later stages of the disease. Although HIV-1 group O and group M infections do not differ in their pathogenesis, the studied isolates did not evolve to use a broad range of coreceptors as described for HIV-1 group M and HIV-2.

Envelope sequence variability and serologic characterization of HIV type 1 group O isolates from equatorial guinea.

Four sera from Equatorial Guinea (EG) suspected to contain antibody against HIV-1 group O-related viruses were identified on the basis of unusual and differential serologic reactivity in selected commercial assays and Western blot. Degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences from the suspect EG sera. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. Analysis (PHYLIP package of programs) of Env amino acid sequences (translated from nucleotide sequences) indicated that the amino acid sequences obtained from EG sera clustered more closely with HIV Env sequences of group O compared to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY (one isolate), RIGPMAWY (two isolates), or GLGPLAVY (one isolate). The V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV (Los Alamos) database, but was present in two of our group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from three of the sera, RLLALETLIQNQQLLNLWGCKGR(K)L(I)VCYTSVK(T)W, whereas sequence from the fourth serum contained three changes as noted in parentheses. IDR sequences derived from EG sera were unique compared to those reported for other HIV-1 group O isolate ANT70, VAU, or MVP5180. Antibody in each EG serum directed against the IDR could be detected using synthetic peptides comprising sequences from the ANT70 or MVP5180 IDRs, but were most reactive against the sequences derived from the samples themselves. Little or no serologic reactivity was detected when EG sera were reacted against peptides comprising the IDR of HIV-1 group M (subtype B consensus) or HIV-2 (consensus).

PIP: The genetic variation and epidemiology of HIV-1 group O isolates are of considerable importance to the design of HIV-1 diagnostic and screening assays, especially since current serologic and genetic methods to detect HIV-1 have been developed mainly on the basis of sequences from isolates belonging to HIV-1 group M. The HIV envelope protein, especially the gp41 immunodominant region, plays a major antigenic role in the detection of HIV infection and for discriminating HIV-1 from HIV-2 antibody. This paper reports upon genetic variation and the serologic characterization of env sequences from 4 people living in Equatorial Guinea (EG) who were infected with HIV-1 group O. Selected commercial assays and Western blot were first used to identify the sera, then degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. The env amino acid sequence analysis found the EG sera sequences to be clustered more closely with the HIV env sequences of group O rather than to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY, RIGPMAWY, or GLGPLAVY. Although the V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV database, it was present in 2 of the group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from 3 of the sera. IDR sequences derived from the EG sera were unique compared to those reported for other HIV-1 group O isolates ANT70, VAU, or MVP5180. Other findings are discussed in detail.

Phylogenetic analysis of protease and transmembrane region of HIV type 1 group O.

The molecular diversity and phylogenetic relationship of 22 HIV-1 group O strains were examined on the basis of the protease gene and the N-terminal region of gp41env. Analysis of the newly characterized protease sequences with 12 reference sequences revealed no specific clustering patterns, despite the distinct geographic locations of the specimens. In contrast, analysis of the newly sequenced gp41 sequences with 34 published sequences revealed two distinct clusters, each represented by one full-length sequence (MVP5180 and ANT-70). Further, four of the specimens classified as group O in the protease region clustered with group M in the gp41 region (three subtype A and one subtype G, respectively), suggesting dual and/or recombinant infections with HIV-1 groups M and O. The presence of two distinct clusters in the gp41 region indicates at least two possible subtypes within group O viruses, and this may provide useful information regarding molecular epidemiological studies of group O infections.

Naturally occurring sequence polymorphisms within HIV type 1 group O protease.

Mutations within the protease gene associated with reduced susceptibility to protease inhibitors have been well documented for HIV-1 group M subtype B strains. In contrast, limited genotypic and phenotypic information is available for the genetically diverse HIV-1 group O strains. Preexisting resistance-associated polymorphisms have the potential to contribute to a poor virological response to antiviral drug treatment in group O-infected patients. In the present study, the protease genes of 28 protease inhibitor-naive HIV-1 group O-infected patients were analyzed to identify any naturally occurring amino acid polymorphisms associated with drug resistance. Comparison of the consensus group O protease sequence with subtype B of group M indicated that both groups have almost identical sequences in the protease active site, the flap and the substrate-binding site. Analysis of the 28 individual protease sequences revealed polymorphisms at 34% of the positions within the protease gene, but no primary mutations associated with protease inhibitor resistance. In contrast, each of the strains harbored multiple secondary or accessory mutations associated with resistance to protease inhibitors in group M viruses. Residues 10I, 15V, 36I, 41K, 62V, 63T/A/K/I, 64V, 71V, and 93L were identified in most strains. The presence of multiple natural sequence polymorphisms associated with drug resistance in the protease gene of group O viruses may contribute to a more rapid emergence of drug resistance phenotype and treatment failure in group O-infected patients.

Sequence of gp41env immunodominant region of HIV type 1 group O from west central Africa.

PIP: HIV-1 group O is endemic in the west central region of Africa, where the frequency of infection is estimated to be 3-10% of all HIV-1-infected individuals. However, international travel and immigration have led to group O cases being identified in France, Germany, Belgium, Spain, and the US. With the exception of an infected French woman, all reported group O-infected individuals originate from or have a connection to west central Africa. Since most immunoassay reagents are based upon HIV-1 group M, many HIV immunoassays have lower sensitivity for the detection of group O infections. Serum samples were collected from patients at hospitals, tuberculosis (TB) clinics, and STD clinics in endemic regions of Cameroon and Equatorial Guinea in a study of the sequence divergence with group O isolate infections. Screening of the 1086 samples using a range of research and commercial immunoassays found 255 to be HIV-1 seropositive. On the basis of differential reactivity in the various immunoassays, 8 individuals were identified as potentially being infected with group O virus, of which 4 were drawn from TB patients. 7 of the group-O samples were then subjected to polymerase chain reaction (PCR) amplification to verify group O infection. The gp41(env) immunodominant region was successfully amplified and sequenced from 4 of the 7 samples, 2 of which were from the TB patients; 4 of 1086 samples were definitely infected with HIV-1 group O.^ieng

Evaluation of different V3 peptides in an enzyme immunoassay for specific HIV type 1 group O antibody detection.

Strategies to discriminate group O from group M infections need to be improved. We have developed and evaluated an HIV-1 group O V3 peptide-based enzyme immunoassay (PEIA) for specific HIV-1 group O antibody detection among HIV-1-infected patients. Synthetic peptides, derived from the amino acid sequences of the V3 loop of 15 different group O strains and 7 group O consensus sequences, were evaluated in a PEIA against a panel of genetically confirmed group O (n = 33), group M (n = 90), and HIV-1 antibody-negative sera (n = 17). The best-performing PEIA(s) were then used to screen 134 sera of European and 336 sera of Cameroonian origin for the presence of anti-HIV-1 group O antibodies. The reactivity of reference ("gold standard") sera to individual peptides in the PEIA resulted in the selection of five different peptides with sensitivities (sens), specificities (spec), and test efficiencies (TEs) in the range of 90 to 100%. Improvement of the PEIA was obtained with simultaneous reactivity of at least two different peptides in separate wells of an ELISA plate, together with stringent criteria for positivity. We were able to select seven peptide combinations each with a sens, spec, and TE of 96.9, 100, and 99.2%, respectively. None of the 134 European and 4 (1.2%) of the 336 Cameroonian samples sera were group O positive in the optimized HIV-1 group O PEIA; this was confirmed by the repeated presence of reactives, in agreement with the present knowledge of group O infection distribution. Finally, we were able to develop a strategy with a higher TE (99.2%) than the previously used ANT-70 (98.5%) and ANT-70/MVP5180 (95.7%). Our results show that optimal specificity rather than optimal sensitivity makes the V3 PEIA a sufficiently accurate epidemiological tool to be useful in estimating specifically group O infection among HIV-1-infected patients.

Differential diagnosis of HIV type 1 group O and M infection by polymerase chain reaction and PstI restriction analysis of the pol gene fragment.

HIV-1 group O serological screening or confirmation strategies so far have not proved 100% sensitive and specific, indicating a lack of antibody reactivity or cross-reactivity with group O antigens. Therefore, genetic analysis currently represents the only method by which confirm presumed HIV-1 group O or group O/M infections. We have optimized the sensitivity (100%) and specificity (100%) of an HIV-1 group O/M-specific PCR of a pol gene fragment. In addition, we report on a highly sensitive (97.2%) and specific (100%) method for differentiation between HIV-1 group O and group M viruses, using PCR and PstI enzyme restriction fragment analysis of a pol fragment. Compared with sequencing, these methods are fast, inexpensive, and simple.

Natural infection of wild-born mandrills (Mandrillus sphinx) with two different types of simian immunodeficiency virus.

We found a novel primate lentivirus in mandrill (Mandrillus sphinx). To clarify the evolutionary relationships and transmission patterns of human/simian immunodeficiency virus (HIV/SIV), we screened blood samples from 30 wild-born healthy Cameroonian mandrills. Five (16.7%) of them were seropositive for SIV. Three SIV strains were isolated from the five seropositive mandrills by cocultivation of their peripheral blood mononuclear cells (PBMCs) with PBMCs of rhesus macaques, a human T cell line (M8166), and/or a cynomolgus macaque T cell line (HSC-F). One of the newly isolated SIV strains was intravenously inoculated into two rhesus macaques and resulted in chronic infection. In the SIV-infected macaques at 45 weeks after inoculation, we observed a mild decline in the number of peripheral CD4(+) lymphocytes, lymphadenopathy, and blastic follicular dendritic cells with mild follicular hyperplasia in the peripheral lymph nodes. A phylogenetic analysis based on the pol sequence showed that the newly found SIVs from Cameroonian mandrills did not cluster with SIVmndGB1, which is the former representative strain of SIVmnd. The SIVmnds from Cameroon formed a new, independent lineage that branched before the root of the HIV-1/SIVcpz lineage with 996 of 1000 bootstrap replications. They clustered host specifically, and exhibited about 16.9% diversity at the level of nucleotide sequence among Cameroonian SIVmnd strains. These results indicate that the SIVmnds isolated in Cameroon are a novel type of SIVmnd and have infected Cameroonian mandrills for a long time. We therefore designated the Cameroonian SIVmnd as SIVmnd type 2 and redesignated SIVmndGB1 as SIVmnd type 1. To date, M. sphinx is the only primate species other than humans that is naturally infected with two different types of SIV.

HIV-1 subtype O: epidemiology, pathogenesis, diagnosis, and perspectives of the evolution of HIV.

HIV-1 subtype O is a new HIV variant originating in the West-Central African region, with highest prevalences in countries such as Cameroon, Equatorial Guinea and Gabon. Detection of antibodies to HIV-1 subtype O can pose problems in unmodified ELISA tests, and confirmation of anti-HIV-1 subtype O in immunoblot may give false negative results in some specimens. Nucleic acid-based assays designed for HIV-1 detection do not amplify or detect sequences from HIV-1 subtype O. In their env sequences, HIV-1 subtype O strains show a higher heterogeneity than the classical HIV-1 subtypes, leading to the conclusion that HIV-1 subtype O has been introduced into the human population only recently. Further, unidentified subtypes are also likely to exist.