Sequence Note. The Molecular Epidemiology and Drug Resistance Determination of HIV Type 1 Subtype B Infection in Barbados ABSTRACT

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1 AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 19, Number 4, 2003, pp Mary Ann Liebert, Inc. Sequence Note The Molecular Epidemiology and Drug Resistance Determination of HIV Type 1 Subtype B Infection in Barbados MARQUITA V. GITTENS, 1 WILLIAM W. ROTH, 2 TIMOTHY ROACH, 1 H. GENE STRINGER, JR., 2 DANUTA PIENIAZEK, 3 VINCENT C. BOND, 2 and PAUL N. LEVETT 1 ABSTRACT To better understand the emergence of HIV-1 variants in Barbados and the association with transmission modes, we analyzed phylogenetic relationships and genetic variability among HIV-1 strains collected in 1996 from 36 antiretroviral therapy-naive patients. Only subtype B variants were present in this sampling, based on analysis of HIV-1 envelope (env) C2V3, protease (PR), and reverse transcriptase (RT) sequences. The genetic diversity of env sequences was broad (13.9%; range, %), suggesting multiple introductions of distinct HIV-1 strains to the island. The frequency of subtype B HIV-1 variants with similar env V3 features, including the tetrameric tips, GPGR and GPGK, the threonine deletion at position 23, and the substitution of threonine to arginine at position 22, was comparable in heterosexual, bisexual, and homosexual patients. Analyses of amino acid variations in PR sequences revealed a lack of major drug resistance-conferring mutations and a high (90%) prevalence of secondary mutations at positions 36, 63, 71, and 77. While the occurrence of 36I, 63P, and 71T mutations in Barbadian strains was similar to the global prevalence for subtype B variants, the frequency (64%) of the V77I mutation was more than three times that seen worldwide. Only two RT antiretroviral resistance mutations (M41L and T215Y) were observed, both from a single patient. This comprehensive genetic analysis documents a broad diversity within HIV-1 subtype B in Barbados and suggests a lack of association between particular subtype B variants and transmission modes. BARBADOS, an English-speaking Caribbean country of 268,000 people, is located on a 21-mile by 14-mile island. The Barbadian Ministry of Health has reported 2415 HIV-positive cases as of 2000, following the first 2 recorded cases in Although the first HIV infections were recognized in homosexual men, more recent data indicate that heterosexual contact is the predominant mode of HIV transmission in Barbados. 2 Previous limited molecular analysis of HIV-1 strains from seroconverting Barbadian patients revealed distinct HIV- 1 env subtype B variants in two of three individuals analyzed. 3 It was hypothesized that local subtype B variants might influence the spread of HIV-1 infection within different risk populations. 4 Examples include subtype B variants with the GPGR motif at the tip of the env V3 loop, which are predominant within two major high-risk groups, homosexual men and intravenous drug users in the United States and Europe, but less common in heterosexual populations. In contrast, in Latin America, where distinct subtype B variants circulate, heterosexual transmission is common. 5 For instance, 46% of all HIV- 1 infections in Brazil were caused by subtype B variants with the GWGR domain, of which at least 50% have been found among heterosexuals. 6 Moreover, data from Trinidad and Tobago have revealed the presence of genetically unique subtype B variants with deletion of threonine at position 22 in the env 1 School of Clinical Medicine and Research, University of the West Indies, Barbados. 2 Morehouse School of Medicine, Atlanta, Georgia Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, Atlanta, Georgia

2 314 V3 loop, predominantly among heterosexuals. 4 These data suggest that either a high number of distinct HIV-1 subtype B variants were initially introduced in the population and a founder effect was established and perpetuated in all risk groups, or observed variation in HIV-1 sequences may be due to biological properties of serotype B variants. It is believed that the molecular characterization of circulating HIV-1 variants among different risk groups allows improving knowledge on pathogenesis of HIV-1. Therefore, to better understand the emergence of HIV-1 variants in Barbados, we characterized the gp120 C2V3 region of env and the protease (PR) and reverse transcriptase (RT) of pol from 36 Barbadian heterosexual, bisexual, and homosexual HIV-1-positive patients, and compared them with sequences from other parts of the world. Barbadian patients entering a clinical trial 7 were selected for inclusion in the study on the basis of two consecutive HIV-positive tests, using standard enzyme-linked immunoabsorbent assay (ELISA) or ELISA and Western blot procedures, and of not having received antiretroviral therapy at any time. The study received ethical approval from the Medical Research Advisory Board in the Ministry of Health. After obtaining informed consent, 20 ml of blood was collected in 1996 from 43 HIVseropositive patients attending the Queen Elizabeth Hospital in Bridgetown. Demographic and clinical information about patients was obtained during their medical evaluation at the time of blood collection. The processing of viral RNA, conditions for reverse transcriptase-polymerase chain reaction (RT-PCR), and sets of primers used for PCR amplification have been previously described. 3 Briefly, RNA was extracted from 280 ml of concentrated plasma, using a QIAamp-viral RNA purification kit (Qiagen, Valencia, CA) with the following modifications to the basic protocol: 60 ml of buffer AVE preheated to 80 C was added to silica columns followed by incubation for 5 min at 80 C. RNA was eluted in 60 ml of diethylpyrocarbonate (DEPC)-treated water (Amresco, Solon, OH). Reverse transcription of RNA was performed with random hexamer primers. 3 Fifty- and 10-ml volumes of cdna products were used as templates for PCR amplification of C2V3 and PR/RT regions, respectively. The following sets of primers were used for nested PCR amplifications of a 375-bp env amplicon, C207/C072 (outer) and 207S/V3S (nested) 3 ; a 297-bp PR amplicon, DP10/DP11 and DP16/DP17 8 ; and a 320-bp RT amplicon, RT9/RT12 and RTRT1/RT4. 9 Duplicate patient samples were analyzed to ensure reproducibility. To assure that crosscontamination of PCRs had not occurred, all reactions were carried out with appropriate negative controls. PCR-amplified products were purified and sequenced directly in both directions, using fluorescent dye-labeled sequencing terminators on automated ABI 373XL and 377 DNA sequencers (Applied Biosystems, Foster City, CA) according to the manufacturer recommendations. Sequences were resolved on the ABI 373 XL sequencer after electrophoresis of PR and GITTENS ET AL. C2V3 sequencing reactions on a 4.75% polyacrylamide gel and on the ABI 377 sequencer after electrophoresis of RT on a 4% polyacrylamide gel. Nested PCR primers were used as sequencing primers in all reactions. However, for better resolution of some RT sequences two other sequencing primers were applied: RT-18M, 59-GCAGTATACTTTCTGAAG-3 9 (corresponding to HIV-1 HXB2, nucleotides ) and RT- 20M, 59-GACTTCAGGAAGTATACTGC-3 9 (nucleotides ). Sequence editing and assembly were performed by the Autoassembler and Sequence Navigator programs included in the ABI sequencer software package. The sequences were aligned by the CLUSTAL V multiplesequence alignment program. 10 After elimination of regions containing gaps and primer regions, the aligned sequences were analyzed by the neighbor-joining method with nucleotide distance calculated by the Kimura-two parameter approach included in the PHYLIP package version 3.5c. 11 The stability of the tree topology was tested by pruning, which consists of removing one species from the alignment and rerunning the phylogenetic analysis. The following reference sequences were included in the phylogenetic analysis: subtype A, 92UG032.1; subtype B, MN and SF2; subtype C, 92BR025.8; subtype D, 84ZR085.1; subtype E, CRF01-AE/93TH253.3; subtype F, subsubtypes F1 93BR020.1 and F2: MP255; subtype G, LBV217; subtype H, 90CF56.1; subtype J, SE9173; and subtype K, MP535. SIVcpz sequences were used as outgroups. The aligned DNA sequences were translated to amino acids by the Genetic Data Environment (GDE) package. 12 The GenBank accession numbers are AF AF for env C2V3, AY AY for PR, and AY AY for RT sequences, respectively. Of the 43 samples, 36 specimens were amplified for the env region, of which 24 were also successfully amplified for PR and RT. Phylogenetic analysis classified all Barbadian env and PR sequences as HIV-1 subtype B (Fig. 1A and B). Moreover, Barbadian RT sequences clustered into subtype B viruses (data not shown), further indicating a lack of intersubtype recombination within patient sequences. A comparison between subtype B C2V3 sequences from Barbados and 80 reference env sequences, including variants from Brazil, Argentina, Puerto Rico, Trinidad and Tobago, the United States, Lebanon, and Thailand, revealed a lack of clustering between Barbadian and worldwide env sequences (Fig. 1A). Likewise, analyses of the more highly conserved PR gene, which was previously used in studies of the origin, spreading, and evolution of HIV-1 worldwide, 13 revealed a lack of definitive grouping between Barbadian and 71 reference PR sequences from Brazil, Argentina, Puerto Rico, the United States, Spain, Lebanon, Uganda, and Thailand (Fig. 1B). Phylogenetic analysis revealed that Barbadian env and PR sequences were spread across the branches of the trees. Although some of them created multiple Barbadian-like minisubclusters that were shown to be stable by pruning analysis, they FIG. 1. Phylogenetic classification and relationships of HIV-1 sequences isolated from Barbadian individuals: (A) env; (B) PR. HIV- 1 subtypes are marked with prefixes A, B, C, D, CRF01-AE, F1, F2, G, H, J, and K. Country codes: Barbados (BB), Argentina (AR), Brazil (BR), Lebanon (LE), Puerto Rico (PR), Spain (SP), Uganda (UG), United States (US), and Thailand (TH); abbreviation for samples from Trinidad and Tobago: QH and QZ. Barbadian sequences are in boldface. Numbers represent the bootstrap values for subtype B. The scale bar indicates an evolutionary distance of 0.10 (A) and 0.01 (B) nucleotide per position in the sequence.

3 SUBTYPE B VARIANTS IN BARBADOS 315 A B

4 316 were not strongly supported by bootstrap values (ranging from 40 to 73%), indicating a lack of geographic clustering. However, there were two distinct env subclusters of two strains each with bootstrap values of 85 and 98%, respectively, isolated from the married couple BB30/BB31 and from two patients BB9 and BB19 with unknown relationship. The nucleotide divergence between BB30/BB31 and BB9/BB19 subcluster sequences was 15.3% (from 12.6 to 18.26%), indicating distinct HIV-1 variants in each group. In contrast, when pairwise analysis was done on sequences within the same cluster, the range of divergence was 5.9% for BB30/BB31 sequences and 6.5% for BB9/BB19 sequences. These values implied that HIV-1 variants within each subcluster were related, further supporting the existence of two independent epidemiological links. We next examined the impact of Barbadian DNA sequences on specific changes at the protein level. The deduced Barbadian amino acid sequences of the V3 loop, an immunologically and functionally important env domain and a target for neutralizing antibodies, 14 were aligned with consensus subtype B sequence (Table 1). Several observations can be derived from this analysis. First, GPGR was the predominant (64%, 23 of 36) tetrameric tip motif for the Barbadian sequences, whereas the remaining 36% (13 of 36) included a variety of substitutions including GPGK (17%, 6 of 36), GPGS (6%, 2 of 36), and GPGG, GPLR, GQGR, APGR, and APGS (3%, 1 of 36 for each). Interestingly, Barbadian env variants did not contain the GWGR motif, which is widely distributed among HIV-1 strains in Brazil. 15 Second, GPGK, which was present in 17% of Barbadian env sequences, was not reported in Brazil and is infrequently (,8%) observed worldwide, with the exception of the United States, where it is common. 16 Third, a threonine deletion at position 23, which was found in more than one-third of Barbadian env sequences, was not reported in subtype B sequences from Brazil, Argentina, Puerto Rico, Trinidad and Tobago, or the United States, and was found in less than 0.5% of all V3 sequences recorded globally. 16 Fourth, substitution of alanine for threonine at position 22, a critical region for the syncytium-inducing phenotype of HIV-1 strains, 14 was present in 50% of Barbadian sequences. These data contrasted with the occurrence of a deletion at this position in more than 80% of HIV-1 strains from Trinidad and Tobago. 4 The apparent lack of greater epidemiology linkage events occurring between Barbados and Trinidad or Venezuela is of special note because of the high frequency of travel within these areas. Analysis of Barbadian PR protein sequences revealed differences from the worldwide consensus subtype B sequence at positions 41 (R R K), 72 (I R V), and 77 (V R I). 17 None of the 27 PR sequences harbored major drug resistance-conferring mutations. These mutations usually lead to a severalfold decrease in sensitivity to protease inhibitors (PIs). In contrast, 23 of 27 (85%) sequences contained at least one secondary mutation. These mutations may be associated with an increase in viral replication capacity (fitness) when a major mutation is selected during drug therapy. Such secondary mutations were identified at four positions: 36I, 63P, 71T, and 77I (Table 1). The 77I substitution (seen in 18 of 27, or 67% of patients) was the most common mutation, followed by 63P (17 of 27, 63%), 71T (2 of 27, 6%), and 36I (1 of 27, 3%). The prevalence of 63P, 71T, and 36I was similar to the global prevalence reported from HIV-1 subtype B variants. In contrast, the prevalence of GITTENS ET AL. the 77I substitution, which is associated with nelfinavir resistance, was more than three times higher in Barbadian (67%) than worldwide (19%) PR sequences. 18 Moreover, 56% of PR sequences harbored dual PI-associated substitutions comprising mainly 77I and 63P, which contrasts with a prevalence of less than 25% of dual substitutions reported for global subtype B sequences. 18 In addition, R8Q, R57K, D60E, and N88S substitutions, associated with resistance in vitro to experimental PI, 19 were more common in Barbadian (33%) than global (8%) PR sequences. Of the 24 RT sequences, 23 did not carry any drug-resistant substitutions. These findings support previous observations of a low prevalence of such mutations within RT in antiretroviral drug-naive individuals worldwide. However, one sequence harbored dual mutations at positions M41L and T215Y, which are responsible for resistance to zidovudine (ZDV) and also to zalcitabine and didanosine. These drugs have not been available to most Barbadian HIV-1-infected patients. It is possible that a ZDV-resistant HIV-1 variant was transmitted to a Barbadian individual; transmission of such strains has been described previously. 20 Overall, the results of distinct approaches indicate differences between recent Barbadian and worldwide HIV-1 subtype B strains, including those from neighboring countries. However, a relatively high intrasubtype genetic diversity among Barbadian env C2V3 sequences collected in 1996 (13.9%; range, %), together with the first report on HIV infection in Barbados in 1984, 1,21 may contradict the model of a long existence of HIV-1 in Barbados, based on an annual nucleotide divergence of 0.5 1% in the env C2V3 region. 22 Whether Barbadian-like subtype B variants have been and still continue to be introduced from outside of the country or whether they evolved faster than predicted remains to be determined. The molecular signature patterns identified in this study of Barbadian env C2V3 and PR sequences may be useful information for identification and monitoring transmission of such variants. The epidemiologic characteristics of Barbadian HIV-1-infected patients demonstrated that the frequency of infections was higher in heterosexuals (56%, 20 of 36) than bisexuals (22%) and homosexuals (14%) (Table 2), further supporting the serologic findings of increased spread of subtype B infections among this risk group in Barbados. 2 The median age of 20 HIV- 1-infected heterosexual patients (37.4 years; range, years) was higher than for 8 bisexual patients (35.1 years; range, years) and 5 homosexual patients (32.8 years; range, years), but this difference was not significant (Table 1). Both patients with AIDS and asymptomatic HIV-1-positive individuals were observed in all three categories of risk patients. Whereas the majority of heterosexual (14 of 20) and bisexual (6 of 8) patients had AIDS, the majority of homosexual patients (3 of 5) remained without symptoms, although these data are not conclusive because of the small numbers of patients in the latter two groups. A combination of molecular and epidemiological results documented that each of three Barbadian risk groups was infected with a broad spectrum of HIV-1 variants sharing similar characteristics in the loop such as the presence of GPGR and GPGK domains, threonine deletion at position 23, and substitution of arginine for threonine at position 22 (Table 2). Briefly, 23 HIV- 1 env sequences with the GPGR motif were present in hetero-

5 SUBTYPE B VARIANTS IN BARBADOS 317 TABLE 1. ALIGNMENT OF DEDUCED AMINO ACID SEQUENCES CORRESPONDING TO THE V3 LOOP OF THE env REGION OF CONSENSUS SUBTYPE B AND 36 BARBADIAN SEQUENCES Dots (.) indicate concordance with the upper sequence in the alignment; dashes ( ) indicate gaps; GPGR tip is shown in boldface; an asterisk (*) indicates deletion at the threonine position. Numbering of amino acid residue is as in subtype B consensus. Accession numbers AF AF for env sequences. sexual (65%, 13 of 20), bisexual (50%, 4 of 8), homosexual (60%, 3 of 5), and risk-unknown (100%, 3 of 3) patients. Other diverse tetrameric tip motifs including GPGK, GPGS, APGR, APGS, GPLR, GQGR, or GPGG were found in heterosexuals (35%, 7 of 20), bisexuals (50%, 4 of 8), and homosexuals (40%, 2 of 5). Thirteen strains with threonine deletion at position 23 of the V3 loop were identified in heterosexual (30%, 6 of 20), bisexual (37%, 3 of 8), homosexual (40%, 2 of 5), and risk-unknown (67%, 2 of 3) patients. Also, a similar number of AIDS and asymptomatic patients, respectively, were infected with threonine-deleted strains (61%, 8 of 13 and 39%, 5 of 13) and strains without this deletion (65%, 15 of 23 and 35%, 8 of 23). The exact dates of patient seroconversions are not known. However, the earliest seropositive result was reported in 1988 for a heterosexual male patient (BB26) infected with the HIV-1 threonine-deleted variant (Table 2). While these data may indicate that such an HIV-1 variant was present in Barbados as early as 1988, this patient may have been infected with a classic subtype B strain that diversified as the result of subsequent introduction of newer HIV-1 strains, particularly the threonine-deleted subtype B variant. The presence of dual infections caused by subtype B variants has been reported. 23 Therefore, the exact time of appearance of such variants in Barbados is unknown but our data suggest a time as early as Although the sample size was

6 318 GITTENS ET AL. TABLE 2. CHARACTERISTICS OF THE STUDY POPULATION V3 loop characteristics Tetrameric tip Patient Age Year of first CD4 1 cell count HIV RNA Risk code Sex (years) serodiagnosis Status (cells/ml) (copies/ml) group Thr GPGK GPGR Other BB2 M HIV Het 1 BB9 F AIDS Het 1 BB10 M AIDS Het GPGS BB11 F AIDS Het 1 BB13 M AIDS Het 1 BB19 F AIDS Het GPLR BB20 F AIDS Het GQGR BB21 F HIV Het 1 BB23 F HIV Het 1 BB25 M AIDS Het 1 1 BB26 M AIDS Und Het 1 1 BB27 F AIDS Het 1 BB30 M AIDS Het 1 1 BB31 F HIV Het 1 1 BB32 F AIDS Het GPGG BB33 M AIDS Het 1 BB34 F HIV Het 1 1 BB38 F AIDS Het 1 1 BB39 F HIV Het 1 1 BB40 M AIDS Het GPGS BB3 F HIV Bi 1 BB5 M AIDS Bi 1 1 BB6 M AIDS Bi 1 1 BB7 M AIDS Bi 1 APGS BB12 M HIV Bi 1 BB15 M AIDS Bi 1 BB18 F AIDS Bi 1 BB36 M AIDS Bi 1 BB1 M HIV Homo APGR BB8 M HIV Homo 1 BB17 M HIV Homo 1 BB29 M AIDS Homo 1 1 BB41 M 42 Unk AIDS Homo 1 BB16 M HIV Unk 1 1 BB37 M AIDS Unk 1 BB42 M 56 Unk HIV Unk 1 1 Abbreviations: M, male; F, female; Het, heterosexual; Homo, homosexual; Bi, bisexual; Unk, unknown; Und, undetectable; Thr, threonine (deletion at position 23 is marked 1); 1, presence of the specific tetrameric tip. not large, the comparable distribution of subtype B variants showing similar molecular features within the V3 region among heterosexuals, bisexuals, and homosexuals suggests a lack of association between particular subtype B variants and these risk groups. Therefore, our findings support a founder effect rather than a genetic selection according to transmission modes. In summary, the results presented in this report provide important information about HIV-1 genetic variation in Barbados. A high genetic diversity of subtype B env sequences may indicate multiple introductions of distinct viral variants into the island. Given a limited number of samples tested, finding subtype B variants with similar molecular characteristics in the V3 region among heterosexual, bisexual, and homosexual populations is remarkable and suggests that Barbadian viruses infected different risk groups equally. Detection of HIV-1 subtype B predominantly in heterosexuals indicates that such a demographic pattern of subtype B infection resembled that of non- B heterosexual epidemics in Africa and Asia. Finally, this report provides new information about HIV-1 variation associated with RT and PR inhibitor resistance. These data provide important baselines prior to the anticipated widespread introduction of triple antiretroviral therapy, or HAART, in Barbados. ACKNOWLEDGMENTS This work was supported by grants from the School of Graduate Studies and Research, the University of the West Indies,

7 SUBTYPE B VARIANTS IN BARBADOS 319 and from Advanced Viral Research Corporation, Yonkers, New York. This work was also supported in part by MBRS/NIGMS/ NIH grant S06-GM , and by RCMI/NCRR/NIH grant G12-RR SEQUENCE DATA The new sequences have been deposited in GenBank under the following accession numbers: AF AF for the envelope, AY AY for the protease region, and AY AY for the reverse transcriptase region. REFERENCES 1. Anonymous: Update on HIV Infection and AIDS in Barbados for the Period April June Ministry of Health of Barbados, Bridgetown, Barbados, 2000, pp UNAIDS/WHO Working Group on Global HIV/AIDS and STI Surveillance: Barbados: Epidemiological Fact Sheet on HIV/AIDS and Sexually Transmitted Infections 2000 Update. World Health Organization, Geneva, Switzerland, Roth WW, Levett PN, Hudson CP, et al.: HIV type 1 envelope sequences from seroconverting patients in Barbados. 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