HIV-1 Subtype C Drug-Resistance Background Among ARV-Naive Adults in Botswana

Antiviral Chemistry & Chemotherapy 16:103–115 HIV-1 subtype C drug-resistance background among ARV-naive adults in Botswana

Hermann Bussmann1, Vladimir Novitsky2, William Wester1, Trevor Peter1, Kereng Masupu3, Lesego Gabaitiri4, Soyeon Kim5, Simane Gaseitsiwe1, Thumbi Ndung’u1, Richard Marlink6, Ibou Thior1 and Max Essex2*

1Botswana–Harvard School of Public Health AIDS Initiative Partnership, Gaborone, Botswana 2Harvard School of Public Health, Department of Immunology & Infectious Diseases and the Harvard School of Public Health AIDS Initiative, Boston, MA, USA 3National AIDS Coordinating Agency, Gaborone, Botswana 4University of Botswana, Gaborone, Botswana; Harvard School of Public Health, Department of Biostatistics, Boston, MA, USA 5Botswana–Harvard School of Public Health AIDS Initiative Partnership, Gaborone, Botswana; Harvard School of Public Health, Department of Biostatistics, Boston, MA, USA 6Harvard School of Public Health AIDS Initiative, Boston, MA, USA

*Corresponding author: Tel: +1 617 432 2334; Fax: +1 617 739 8348; E-mail: [email protected]

Current HIV-1 antiretroviral (ARV) drug resistance HIV-1C–specific polymorphic sites were found knowledge is limited to HIV-1 subtype B (HIV-1B). across the pol gene. Northern and southern We addressed whether unique genetic and pheno- Botswana viral sequences showed no significant typic properties of HIV-1 subtype C (HIV-1C), differences from each other. Population geno- southern Africa’s most prevalent subtype, may typing shows that, without countrywide ARV foment earlier and/or distinct resistance muta- treatment, HIV-1C–infected Batswana harbour tions. Population-level HIV-1C genotypes were virtually no primary mutations known to confer evaluated with respect to drug resistance preva- resistance to the three major HIV-1B ARV drug lence before Botswana’s public ARV treatment classes. Some secondary PI mutations and poly- programme began. Viruses were genotyped from morphic sites in the protease enzyme necessitate 11 representative districts of northern and continuous population monitoring, particularly southern Botswana, and consensus sequences after introduction of countrywide ARV treatment from these 71 individuals and 51 previously in Botswana. Although its PI resistance develop- reported sequences from HIV-positive blood ment rate and kinetics are not known, our data donors were constructed. Phylogenetic analysis may suggest increased susceptibility and readi- classified all 71 sequences but one, which exhib- ness of HIV-1C to develop resistance under drug ited pol gene mosaicism, as HIV-1C. The protease pressure when the PI class of drugs is used. and reverse transcriptase coding region had no detectable known primary mutations associated Keywords: ARV drug resistance, sub–Saharan with HIV-1B protease inhibitor (PI) drug resistance. Africa (Botswana), HIV-1 subtype C, genotype Secondary mutations associated with PI drug polymorphisms resistance were found in all sequences. Several

Introduction

Transmission of drug-resistant virus in the AIDS epidemic ment-naive, chronically-infected individuals have been has been increasingly documented in different regions in estimated in cohorts from the UK (UK Collaborative the world (Geretti et al., 2001, 2002; Boden et al., 1999; Group, 2001), Spain (Briones et al., 2001) and Belgium Salomon et al., 2000; Frater et al., 2001). An increase in (Van Vaerenbergh et al., 2001). Significantly lower rates antiretroviral (ARV) drug resistance, from 8% to 22.7% were reported from other cohorts (Descamps et al., 2001; among recently infected individuals in the USA, has been Perno et al., 2002; Yerly et al., 1999; Bennett et al., 2003; reported from 1995–2000 (Little et al., 2002). Drug-resis- Jayaraman et al., 2003; Chaix et al., 2003), including a large tance rates of up to 29% among seroconvertors or treat- European multi-country cohort (Wensing et al., 2003).

Little information is available on baseline drug resistance in Figure 1. Map of Botswana depicting the 11 national non-industrialized countries, especially from regions of the health districts used in this study* world where HIV-1C infection predominates (Vergne et al., 2003; Adje-Toure et al., 2003; Gordon et al., 2003; ANGOLA ZAMBIA Shafer et al., 1997). Specific biological characteristics of HIV-1C, including high genetic diversity, may potentiate the emergence and spread of ARV drug-resistant HIV Kasane strains (Novitsky et al., 1999; Montano et al., 1997; ZIMBABWE Montano et al., 1998; Montano et al., 2000), which warrants further studies. Maun Botswana has one of the highest rates of HIV-1 infec-

Rakops Francistown NAMIBIA tion prevalence in the world, with HIV-1C being the most Ghanzi prevalent subtype. The 2003 Botswana Sentinel Selebe-Phikwe Surveillance documented an HIV prevalence of 37.4% Serowe/Palapye among pregnant women presenting for routine antenatal care (NACA, 2003). The 2003 estimate of HIV seropreva- lence in Botswana is consistent with those obtained during Molepolole the previous 4 years (35.4–38.5%). In response to the Gaborone epidemic, the Botswana government began comprehensive Kanye educational and primary prevention activities in the early Tsabong 1990s and, in January 2002, it initiated its national ARV SOUTH AFRICA treatment programme, providing public highly active antiretroviral therapy (HAART) to all qualifying citizens. It was estimated that approximately 110000 HIV-infected Kasane, Francistown and Gaborone are major international individuals in Botswana urgently need HAART. The border posts. In italics, northern Botswana; in Roman type, south- national ARV treatment programme began at Princess ern Botswana. Marina Hospital, in the capital Gaborone, and has expanded to 21 sites; as of September 2004, approximately 21000 persons were receiving publicly funded HAART. mitted infections from 22 health districts of Botswana. A Adults eligible for HAART must have symptomatic HIV total of 71 samples from documented HIV-1 infected disease (AIDS-defining conditions) and/or a CD4+ cell adults were randomly selected from 11 representative count of less than 200 cells/mm3. Current first-line treat- health districts, as shown in Figure 1. Although the status ment consists of zidovudine plus lamivudine, given together of ARV use was not documented among the 2001 Sentinel as Combivir™, with either efavirenz (males) or nevirapine Surveillance participants, it is highly unlikely that partici- (females with reproductive potential). Adults who fail first- pants knew their HIV status or were receiving HAART at line treatment are presently offered didanosine, stavudine or before the time of blood collection. The availability of and nelfinavir in combination. HAART was very limited in Botswana prior to the official The purpose of this study was (i) to analyse the baseline opening of the national ARV programme in January of ARV drug-resistance patterns in Botswana before wide- 2002. Sample collection for the purposes of this analysis spread use of ARVs, (ii) to determine potential regional was completed by September 2001. Therefore, the proba- differences in drug resistance and (iii) to further charac- bility that analysed samples represented circulating ARV- terize the biological and virological properties of the HIV- naive HIV-1 strains is relatively high. The Institutional 1C virus. We analysed 2001 countrywide HIV Sentinel Review Board of the Ministry of Health, Botswana, Surveillance samples for baseline drug resistance of HIV- approved the study. 1C by genotyping a representative number of samples from Screening for antibodies to HIV-1 was performed by the northern and southern parts of the country. double EIA testing using Murex HIV 1.2.0 (Abbott Pharmaceuticals, Abbott Park, IL, USA) and Ortho HIV- Materials and methods 1/HIV-2 AB-Capture ELISA Test System (Ortho- Clinical Diagnostics, Rochester, NY, USA) assays. Study subjects From July to September of 2001, the 2001 Botswana HIV Resistance testing Sentinel Surveillance enrolled women attending antenatal Plasma virus was analysed for the presence of drug-resis- clinics (ANC) and men with symptoms of sexually trans- tant mutations. Viral genotyping was performed by using

ViroSeq HIV-1 Genotyping System (Celera Diagnostics, model. Both DNADist and PROTDist are components of formerly Applied Biosystems, Alameda, CA, USA), the PHYLIP package (phylogeny inference package, according to the manufacturer’s instructions. Briefly, HIV- versions 3.52c and 3.572c; University of Washington, 1 RNA was extracted from viral particles pelleted from Seattle, WA, USA). The neighbour-joining method was plasma by isopropanol/ethanol precipitation, converted to employed for tree-building. For the recombinant analysis, cDNA and amplified in PCR. The amplicon represented we employed the neighbour-joining bootscan analysis with the HIV-1 pol region spanning the entire protease (PR) and a sliding window of 300 bp incremented by 10 bp with 335 codons of the reverse transcriptase (RT). The PCR reference sequences of HIV-1 subtypes A1, A2, B, C, D, product was then sequenced (both strands) using Big Dye F1, F2, G, H, J, K and known recombinants, using SimPlot chemistry on an ABI 3100 Genetic Analyzer. (Lole et al., 1999). The possibility of unidentified recombinant viruses cannot be excluded with certainty because only Consensus sequences the pol region was analysed in this study. HIV-1C RT and PR extended consensus sequences (Novitsky et al., 2002) were built for the following subsets of Accession numbers sequences: northern Botswana 2001 (BW North); southern The 71 new HIV-1C nucleotide sequences from Botswana Botswana 2001 (BW South); a total Botswana consensus were deposited in GenBank under accession numbers sequence (BW) constructed from 121 sequences, including AY829268 to AY829338. 70 sentinel sequences plus 51 nearly full-length genome sequences from ARV treatment-naive blood donors Results (Novitsky et al., 2002); and South African, KwaZulu-Natal sequences (ZA) (Gordon et al., 2003). HIV-1 subtyping Of the 71 samples, 70 (36 from northern Botswana and 34 Analysis from southern Botswana) were identified as HIV-1 subtype Comparison of amino acid residue frequencies at polymor- C strains based on their phylogenetic relationship (Figure phic sites on different sequences was performed using SAS 2). One sample (01BW2413) from the southern part of statistical software with Fisher’s Exact test for R×C tables. Botswana did not cluster within HIV-1C. We performed All tests used a significance level of 0.10 percent level neighbour-joining bootscan analysis for this sample within (P=0.001). No adjustments were made for multiple the available 1 302 bp that spanned the entire PR and 5′- comparisons. We used Monte-Carlo methods to estimate P portion of RT (data not shown). The bootscan analysis values using StatXact software (Cytel Software, revealed that sample 01BW2413 could be classified as a Cambridge, MA, USA) for amino acid residue at PR posi- complex A/J recombinant virus similar to a viral isolate tion 63 and RT positions 135, 207 and 334. described elsewhere (Novitsky et al., 2000). Within the new The HIV-1 subtype B and C reference sequences from set of 70 HIV-1C from Botswana, no subcluster(s) of the HIV-1 infected treatment-naive patients (HIV-1B ref and geographically different sequences (BW north and BW HIV-1C ref, respectively) were retrieved from the Stanford south) was found (Figure 2). According to our results, 70 HIV RT and Protease Sequence Database (http:// (98.6%) of 71 new sequences belong to HIV-1C, which hivdb.stanford.edu/hiv/). confirms our previous finding (Novitsky et al., 2002) that Phylogenetic analysis was performed to determine the HIV-1C is the predominant circulating viral strain in HIV-1 subtype and to characterize the phylogenetic rela- Botswana. tionship between sequences. The HIV-1 subtyping was performed using a set of reference sequences obtained from Analysis of genetic distances the HIV Sequence Database (http://www.hiv.lanl.gov/content/hiv- The pairwise nucleotide and translated amino acid distances db/SUBTYPE_REF/align.html). We also compared the for the Botswana and South Africa HIV-1C PR and RT are pol regions of the 71 HIV-1C 2001 Botswana sequences to shown in Table 1. We compared genetic distances within the isolates from South Africa, Ethiopia, India, Brazil and entire set of 196 HIV-1C sequences (121 from Botswana HIV-1 subtype references. The multiple alignment of RT and 75 from South Africa) (Gordon et al., 2003). The and PR sequences was performed by ClustalX (version Botswana sequences included subsets of 34 new sequences 1.81) (Thompson et al., 1997), accompanied by manual from the south of Botswana, 36 sequences from the north of editing using BioEdit (Novitsky et al., 2000). Determination Botswana, and 51 previously described isolates (Novitsky et of the pairwise distances of nucleotide alignment was al., 1999; Novitsky et al., 2002; Ndung’u et al., 2000). The performed by DNADist with the Kimura two-parameter mean value of genetic distances within PR and RT was about model. Pairwise distances between translated amino acid 5% on both nucleotide and amino acid levels, with minimal alignments were computed by PROTDist with the PAM variation between the subsets (Table 1). Interestingly, genetic

Antiviral Chemistry & Chemotherapy 16:2 105 H Bussmann et al.

Figure 2. Phylogenetic analysis of the pol region of HIV-1 sequences from 71 Botswana 2001 Sentinel Surveillance sequences

3383.N 00BW1811.3 4009.S 1432.N 1934.S HIV-1 Protease & 00BW2128.3 00BW1759.3 1451.N 96BW16.26 2673.S Reverse Transcriptase 00BW1773.2 96BW0502 1392.S 4136.N 00BW3842.8 2793.S 4292.N 2433.N 3605.N NJ tree 00BW2036.1 96BW15B03 2414.S 96BW11.06 3538.S 3931.N 1780.S 97 2844.N 96BW01B21 2210.N 00BW3819.3 3368.N 81 00BW2127.2 99BW3932.1 C.IN.95.95IN21068 00BW3886.8 Subtype C (India) 4011.S 91 2614.N 00BW3876.9 2064.S 3381.N 00BW1859.5 99BW4642.4 4755.N 3895.N 3388.N 00BW0768.2 96BWMO3.2 4012.S 0800.N 0797.N 93 4902.N 4271.N 3582.N 2206.N 00BW3970.2 2508.S 1386.S 98BWMO37.d 4330.N 00BW2276.7 00BW2087.2 98BWMO14.1 3772.S 100 00BW0874.2 98BWMC12.2 3374.N 94 00BW3891.6 99BW4745.8 3845.N 2065.S 98BWMO36.a 99 2053.S 96BW1210 1900.S 3389.N 2528.S 4013.S 3226.N 00BW5031.1 00BW1783.5 00BW1880.2 1387.S 3920.N 00BW0762.1 00BW1795.6 99BW4754.7 3726.N 1557.S 98BWMC13.4 2796.S 00BW2063.6 96BWMO1.5 2165.S 1374.S 99BWMC16.8 3930.N 93 0971.S 98BWMO18.d 1187.S 1375.S 96BW0407 C.ET.86.ETH2220 Subtype C (Ethiopia) C.BR.92.92BR025 4789.N Subtype C (Brazil) 1025.N 4127.N 0972.S 80 4589.N 4309.N 98BWMC14.a 1549.S 3224.N 00BW3871.3 00BW1686.8 2790.S 0961.S 99 96BW06.J4 1899.S 00BW1616.2 100 1378.S 87 0498.S 95 00BW1471.2 96BW17A09 00BW1921.1 99 H.CF.90.90CF056 H.BE.93.VI991 Subtype H 96 F2.CM.95.MP257 98 F2.CM.95.MP255 100 F1.BR.93.93BR020.1 F1.BE.93.VI850 Subtype F 100 D.CD.83.NDK 100 D.CD.83.ELI Subtype D 100 B.US.83.RF B.FR.83.HXB2 Subtype B 100 A2.CD.97.97CDKTB48 99 A2.CD.97.97CDKFE4 100 A1.SE.94.SE7253 Subtype A A1.KE.93.Q23-17 G.SE.93.SE6165 G.NG.92.92NG083 Subtype G 100 K.CM.96.MP535 K.CD.97.EQTB11C Subtype K 100 BW2413 A/J recombinant 100 J.SE.94.SE7022 J.SE.93.SE7887 Subtype J

0.01

Tree topology was inferred by neighbour-joining method and was based on the alignment of 1302 bp that spanned the entire PR and 5′-portion of the RT. Sequences from northern Botswana are designated by open circles; sequences from southern Botswana are designated by shaded circles.

Table 1. Nucleotide (Nt) and translated amino acid (Prot) distances within the subsets of HIV-1C protease (PR) and reverse transcriptase (RT) sequences, mean values (%)

Distances BW ZA HIV-1C* BW south BW north n=121 n=75 n=196 n=34 n=36

Protease Nt 5.6 4.9 5.4 5.9 5.0 Prot 5.8 5.6 5.8 5.8 4.8 RT Nt 5.6 5.0 5.5 5.6 5.0 Prot 4.8 4.5 4.7 4.6 4.3

BW, Botswana; ZA, South Africa; Nt, nucleotide distance; Prot, protein distance. *Combined total for BW and ZA. Comparison of Nt and Prot distances between the subsets of HIV-1C PR and RT sequences revealed significantly higher diversity in Botswana than in South Africa, and in southern Botswana as compared with the northern Botswana subset (P<0.001 using two-tailed tests for all comparisons). The pairwise distances of Nt alignment was performed by DNADist with the Kimura two-parameter model. Pairwise distances between Prot alignments were computed by PROTDist with the PAM model.

distances were significantly greater in Botswana samples Polymorphism in the PR and RT of Botswana than in South African samples (P<0.001). Distances among sequences the southern Botswana subset were greater than those of the The Botswana consensus sequence was built using 121 northern Botswana subset (P<0.001). HIV-1C translated amino acid sequences. The PR coding region displayed multiple amino acid residues, in low Presence of mutations known to cause drug- frequency, which were unique when compared with other resistance in HIV-1 subtype B subtype C sequences (Figure 3). The PR coding region also No primary drug-resistance mutations (mutations that displayed a significant number (high frequency) of HIV- reduce drug susceptibility by themselves) (Shafer, 2002) to 1C amino acid substitutions at the following PR positions: the PR inhibitors (PIs) were detected in any of 70 BW 12S (77%), 15V (91%), 19I (82%), 41K (93%), 69K (99%) north and BW south samples. Secondary mutations (muta- and 89M (89%) (Table 2). tions that are associated with additional drug resistance in Similarly, within the RT coding region we found multiple isolates containing primary drug resistance mutations) low-frequency polymorphic sites (Figure 4) containing were found at the following positions in the PR enzyme: amino acid residues unique to subtype C sequences. In addi- L10I, K20R, M36I, L63P/A/Q/S/H/C/T/I, V77I and tion, significant numbers (high frequency) of HIV-1C- I93L. Each sequence had at least one known secondary PI specific amino acid substitutions were found at the drug-resistance mutation. Thirty-one sequences (44.3%) following RT positions: 35T (93%), 36A (81%), 39E had two secondary resistance mutations, of which 26 had (92%), 48T (93%), 122E (87%), 123G (31%), 173A (71%), the combination of M36I+I93L. Thirty sequences had 177E (78%), 200A (94%), 207E (48%), 211K (70%), 245Q three or four secondary mutations. Two mutations (D30N (75%), 272P (77%), 277R (61%), 286A (59%), 291D and V82A) were found within the previously reported (96%), 292I (94%) and 293V (89%) (Table 3). sequences from Botswana (Novitsky et al., 1999; Novitsky et al., 2002; Ndung’u et al., 2000) that have been primarily Comparison of polymorphic sites in different reported among HIV-1B-infected individuals treated with sets of sequences the PIs nelfinavir (D30N), indinavir, ritonavir or saquinavir We compared sequences from the north (BW north) to (Shafer, 2002). those from the south (BW south) of Botswana. No signif- No primary and/or secondary nucleoside RT inhibitor icant differences were observed at any position within the (NRTI) drug-resistance mutations were found in the RT PR region. However, there was a significant difference coding region in any of the 70 BW north or BW south between the north and south at amino acid position 27 samples. At known HIV-1B resistance mutation sites in within the RT region (P=0.02), where a substitution from RT, amino acid residues not specifically associated with threonine (T) to serine (S) was found in 17% of northern non-NRTI (NNRTI) drug resistance were found, namely sequences. This substitution was found in none of the one A98S (BW north), one V179T (BW south), and one southern Botswana sequences, and in only 3% of the ZA G333E (BW south). One mutation (G190E) that confers sequences (Gordon et al., 2003). resistance to efavirenz (NNRTI) was found in the set of 51 We also compared HIV-1C reference sequences to the blood donors. BW and ZA sequences. Within the PR coding region, amino acid residues at positions 19, 37, 41, 61, 63

Antiviral Chemistry & Chemotherapy 16:2 107 H Bussmann et al.

Figure 3. Frequency and type of amino acid substitutions in PR genes from the Botswana consensus sequences built from 121 HIV-1 subtype C sequences

1 10 20 30 40

P99 Q 100 I100 T 100 L 99 W 100 Q 99 R 100 P10000 100 S77 I98 K 96 V 91 G 96 G 98 Q 100 I82 K 92 E 100 A 100 L 100 L 100 D 99 T 98 G 100 A 100 D 100 D 100 T 99 V 100 L 100 E 100 E 72 I79 N 63 L 99 P97 G 99

L 1 P1 R 1 T 19 V 2 R 2 I9 E 4 D 2 V 9 R 8 H 1 P1 N 1 A 1 D 28 M 15 S29 M 1 S2 R 1

Q 1 T 2 T 7 I1 L 5 T 2 L 1

P1 L 1 V 1 K 2

N 1 M 1 D 2

A 1 E 1 A 1

41 50 60 70 80

K 93 W 100 K 98 P100 K 98 M 100 I99 G 100 G 100 I100 G 100 G 100 F 99 I100 K 100 V 100 R 97 Q 100 Y 100 D 90 Q 83 I94 L 43 I96 E 99 I100 C 98 G 99 K 99 K 93 A 96 I97 G 100 T 90 V 100 L 99 V 88 G 100 P100 T 100

R 7 R 2 R 2 M 1 L 1 K 2 E 10 E 12 V 6 P27 L 2 D 1 E 1 E 1 Q 1 R 7 T 4 M 2 S7 S1 I12

G 1 N 2 V 10 V 1 Y 1 L 1 A 3

D 1 T 7 M 1

R 1 S6

H 1 H 2

A 2

Y 1 M 1 G 1

81 90 99

P100 V 97 N 99 I100 I99 G 100 R 100 N 100 M 89 L 100 T 99 Q 99 L 98 G 100 C 100 T 99 L 100 N 100 F 100

I2 D 1 V 1 L 10 A 1 P1 I1 I1

A 1 I1 F 1

(P<0.0001); 12 (P=0.0008); 14, 77 (P=0.003); 93 mainly from the Republic of South Africa, one might (P=0.005); and 36 (P=0.011) of the BW sequences were expect to see distinct differences in the HIV-1C epidemic significantly different from the HIV-1C reference within Botswana itself. Results obtained from phylogenetic consensus sequences of the Stanford Database. BW analysis of the pol region in this study, however, do not sequences also differed significantly from ZA sequences at suggest any regional clustering of viruses within Botswana. positions 19 (P=0.0049), 37 (P=0.0071), 14 (P=0.011), and No phylogenetic distinctions within HIV-1C pol were found 61 (P=0.019). See Table 2. in samples from northern Botswana, southern Botswana, or Within the RT coding region, amino acid residues at viruses reported from neighbouring countries. Therefore, positions 39, 60, 135, 173, 200, 207, 250, 334 (all P- our data suggest that the AIDS epidemic in southern Africa values<0.0001), 286 (P=0.0004), 48 (P=0.0012), 211 is caused by a heterogeneous swarm of HIV-1C. (P=0.0014), 248 (P=0.004), 174 (P=0.014), 36 (P=0.024) Interestingly, one sequence showed a pol gene mosaicism and 122 (P=0.027) of the BW sequences were significantly that resembled the structure of a previously described HIV- different than HIV-1C reference consensus sequences of 1 A/J recombinant found in a patient with advanced AIDS the Stanford Database (Table 3). BW sequences also in Botswana in 1998 (Novitsky et al., 2000). differed significantly at amino acid positions 60 (P<0.0001), We studied the intra-subtype diversity of HIV-1C 286 (P=0.0012), 250 (P=0.0018), 311 (P=0.0033), 174 isolates from Botswana and South Africa (KwaZulu-Natal). (P=0.0054), 207 (P=0.013), 251 and 293 (P=0.019) and 177 Sequences from southern Botswana had increased sequence (P=0.031) as compared to ZA sequences. variability when compared to those from northern Botswana. Overall sequence diversity of samples from Discussion Botswana was higher when compared to South African isolates. These differences may suggest a more advanced and We confirmed that the predominant HIV-1 virus in the established HIV-1 epidemic in southern Botswana. Botswana epidemic is HIV-1C. Based on the geography of We also compared sequences obtained from ARV-naive Botswana and the resulting transnational migratory patterns HIV-1C-infected individuals with geographically distinct of populations, dividing the country into a northern zone sequences from Botswana and South Africa (Gordon et al., with active border crossings to and from Zambia and 2003). Several significant polymorphisms in the PR and RT Zimbabwe, and a southern zone with population influx encoding region were found. This may suggest the evolution

Table 2. Comparison of polymorphic codon sites (greater than 10%) in PR encoding region from treatment- naive HIV-1B ref with HIV-1C consensus sequences from Botswana (BW), KwaZulu-Natal, South Africa (ZA) and a set of subtype C ref sequences

PR position HIV-1B ref HIV-1C ref BW ZA Significance§ n=(1715)–(1947)‡ n=(341)–(362)‡ n=121 n=75

† 12 T88 S4 A3 S59 T36 P3 S77 T19 Q1 S73 T19 P4 P=0.0008

I2 N1 P2 A2 P1 N1 A1 A4 *NS

† 14 K89 R11 K90 R10 K96 R2 T2 K83 R13 T3 P=0.0028

S1 *P=0.011

† 15 I84 V16 V84 I16 V91 I9 V93 I7 NS *NS

† 19 L91 I7 Q1 I55 L28 V9 I82 V9 T7 I59 V23 T12 P<0.0001

V1 T7 Q1 L1 M1 L4 E1 A1 *P=0.0049

† 36 M87 I13 I77 M11 V8 I78 M16 L5 I85 M9 L4 P=0.011

T2 L2 V1 V1 T1 *NS

† 37 N65 S18 D9 T3 N60 K27 S9 N63 S29 T2 K2 N78 S11 D5 P<0.0001

H2 E2 C1 K0 D3 A1 D2 E1 A1 A4 T1 E1 *P=0.0071

† 41 R77 K23 K66 N26 R8 K93 R7 K91 R9 P<0.0001 *NS

† 61 Q98 E2 Q96 E4 Q83 E12 N2 Q98 H1 E1 P<0.0001

D1 R1 H1 *P=0.019

† 63 P53 L33 S5 A3 L54 P28 V6 T5 L43 P27 V10 T7 L61 P22V7 T5 P<0.0001

H2 T2 Q1 C1 S4 A1 H1 I1 S6 H2 A2 Y1 S1 Q1 I1 H1 *NS

M1 G1 D1

† 77 V76 I24 V95 I5 V87 I13 V92 I8 P=0.003 *NS

† 89 L99 M1 M85 L13 I2 M89 L10 I1 M88 L11 I1 NS *NS

† 93 I76 L24 L94 I6 L98 I1 F1 L97 I3 P=0.005 *NS

*Difference between BW and ZA. †Difference between HIV-1C ref and BW. ‡Range of sequences used to build consensus sequence for each position. §P values computed between frequencies of overall distribution of amino acid residues. NS, not significant. Bold: codon positions carrying mutations known to confer secondary drug resistance. Note: positions 35 and 60 were polymorphic in both HIV-1B and HIV-1C consensuses.

Antiviral Chemistry & Chemotherapy 16:2 109 H Bussmann et al.

Figure 4. Frequency and type of amino acid substitutions in RT gene from the Botswana consensus sequences built from 121 HIV-1 subtype C sequences

110203040

P100 I100 S100 P98 I100 E 96 T 100 V 98 P100 V 98 K 98 L 100 K 99 P100 G 100 M 100 D 99 G 100 P100 K 98 V 100 K 96 Q 100 W 99 P99 L 100 T 94 E 98 E 100 K 100 I100 K 95 A 100 L 100 T 93 A 81 I100 C 100 E 92 E 92

S2 K 3 I2 X 1 N 1 N 1 N 1 R 2 R 2 R 1 T 1 S5 K 1 Q 2 K 5 E 17 D 3 D 7

D 1 L 1 R 1 N 2 A 1 A 1 R 1 M 1 V 1 A 2 G 1

L 1 I1 G 1 K 2

E 1 T 1

41 50 60 70 80 M 100E 100 K 97 E 100 G 100 K 100 I99 T 93 K 100 I99 G 100 P100 E 98 N 100 P100 Y 99 N 100 T 100 P99 V 93 F 99 A 100 I100 K 100 K 100 K 100 D 100 S100 T 100 K 100 W 100 R 99 K 100 L 100 V 99 D 100 F 100 R 97 E 100 L 100

R 2 V 1 E 5 V 1 K 1 H 1 S1 I7 S1 K 1 L 1 G 2

E 1 S2 D 1 K 1

81 90 100 110 120 N 100 K 100 R 100 T 100 Q 100 D 100 F 100W 100 E 99 V 100 Q 100 L 100 G 100 I100 P100 H 100 P100 A 98 G 100 L 99 K 99 K 99 K 100 K 97 S100 V 99 T 99 V 100 L 100 D 100 V 99 G 100 D 100 A 100 Y 100 F 100 S100 V 98 P100 L 100

K 1 V 1 S1 X 1 Q 1 R 2 L 1 A 1 M 1 I2

S1 N 1

121 130 140 150 160 D 97 E 87 G 31 F 100 R 100 K 100 Y 100 T 100 A 100 F 100 T 100 I100 P100 S100 I77 N 100 N 100 E 92 T 98 P100 G 100 I95 R 99 Y 99 Q 100 Y 100 N 100 V 100 L 100 P99 Q 100 G 100 W 100 K 99 G 100 S100 P100 A 94 I99 F 100

H 2 K 11 D 26 T 10 A 7 A 2 V 3 T 1 H 1 S1 N 1 S6 V 1

Y 1 R 1 S22 V 7 S1 T 2

P1 N 19 R 3

E 2 M 1

L 1

K 1

161 170 180 190 200 Q 100 S75 S98 M 100 T 97 K 80 I100 L 100 E 95 P99 F 100 R 100 A 71 Q 61 N 100 P100 E 78 I84 V 98 I99 Y 100 Q 100 Y 100 M 100 D 100 D 99 L 100 Y 100 V 100 G 98 S99 D 98 L 99 E 96 I100 G 94 Q 98 H 98 R 98 A 94

C 17 T 2 I3 R 19 K 2 L 1 T 24 K 28 D 20 M 9 T 1 V 1 N 1 R 1 X 1 N 2 F 1 D 2 E 2 K 2 R 2 T 2 E 3

A 2 X 1 D 2 V 2 R 9 G 2 L 6 L 1 E 1 Q 1 V 1 T 2

Y 1 G 1 L 1 N 1 V 1 K 1 T 1 V 1

N 1 I1 L 1 R 1

H 1 E 1 K 1

G 1

F 1

D 1

201 210 220 230 240 K 98 I96 E 98 E 94 L 100 R 97 E 48 H 100 L 100 L 100 K 70 W 100 G 100 F 91 T 100 T 100 P100 D 100 K 100 K 100 H 100 Q 100 K 100 E 99 P100 P100 F 100 L 98 W 100 M 100 G 100 Y 100 E 100 L 99 H 100 P100 D 100 K 99 W 99 T 99

R 1 V 3 Q 1 K 3 K 2 D 16 R 26 L 9 D 1 M 1 P1 Q 1 G 1 A 1

Q 1 L 1 D 1 Q 1 T 1 A 10 Q 2 H 1

G 1 K 8 I1

D 1 N 7 G 1

G 4

R 3

T 2

Q 1

241 250 260 270 280 V 99 Q 100 P92 I99 Q 75 L 100 P97 E 80 K 99 D 67 S91 W 100 T 100 V 100 N 100 D 99 I98 Q 100 K 98 L 100 V 100 G 100 K 100 L 100 N 100 W 100 A 99 S100 Q 100 I99 Y 100 P77 G 98 I98 K 95 V 98 R 61 Q 93 L 97 C 100

I1 T 4 M 1 K 12 T 1 D 10 Q 1 E 31 D 5 G 1 V 1 R 1 V 1 N 1 A 11 R 1 V 2 R 3 I2 K 35 H 5 M 2

S2 V 4 I1 T 7 N 1 N 3 L 1 E 1 S7 K 1 Q 2 T 3 N 2 I1

K 1 E 3 A 1 N 2 A 1 I1 K 3 Q 1

A 1 H 2 A 1 T 1

N 1 Q 1

M 1

L 1

281 290 300 310 320 K 93 L 99 L 97 R 100 G 100 A 59 K 100 A 97 L 100 T 100 D 96 I94 V 89 P98 L 100 T 98 E 92 E 99 A 100 E 99 L 99 E 100 L 100 A 99 E 100 N 99 R 100 E 99 I99 L 100 K 88 E 98 P99 V 100 H 99 G 99 V 93 Y 100 Y 100 D 100

R 7 I1 I2 T 39 V 1 E 3 V 5 I10 T 2 P1 D 3 A 1 A 1 V 1 E 1 D 1 K 1 L 1 R 12 G 2 Q 1 Q 1 E 1 A 7 V1 V 2 S1 V1 R1 G1 N 1 K 2 K 1 A 2

R 1

321 330 335 P99 S100 K 98 D 93 L 99 I91 A 98 E 98 I95 Q 100 K 100 Q 100 G 99 H 48 D 88

S1 E 2 E 7 I1 V 7 V 1 D 2 V 4 E 1 N 25 G 8

T 1 T 1 L 1 Q 9 E 2

K 1 D 9 Y 1

Y 2 C 1

C 2

R 1

L 1

G 1

E 1

of geographically distinct strains or the influence of selective ground polymorphisms (2002). Brenner et al. have shown pressure from factors other than ARV treatment. that a common HIV-1C polymorphism at RT codon posi- Our sequence data, obtained before Botswana initiated tion 106 facilitates high-level multi-NNRTI resistance its public national ARV treatment program, document that mutations (V106M) among patients under selective drug there is virtually no known primary drug resistance to the pressure, specifically those patients being treated with three major classes of ARV drugs. Review of HIV-1 efavirenz (2003). A London cohort, evaluating HIV-1 subtype C isolates obtained from ARV-naive persons in non-subtype-B-infected, ARV-naive African patients, Israel (Grossman et al., 2001), Ethiopia (Loemba et al., showed that the presence of specific baseline polymor- 2002), Zambia (Handema et al., 2003), Zimbabwe (Kantor phisms did not negatively influence future response to PI- et al., 2002) and South Africa (Gordon et al., 2003) corrob- containing HAART, but this is still preliminary as subjects orate our findings. have only had 1 year of follow-up (Frater et al., 2001). None of the mutations known to cause primary resis- Additional research to elucidate the impact of HIV-1C tance to zidovudine, lamivudine, didanosine, stavudine or variability on the development of ARV drug resistance is abacavir among HIV-1B-infected individuals were found needed. in the RT coding region of Botswana sequences. A few In summary, our primary objective was to establish the amino acid substitutions known to facilitate resistance background ARV drug-resistance profile for Botswana were found at positions 211, 214 and 333. The F214L prior to the initiation of its large-scale national public mutation in combination with R211K has been described ART programme. Our results provide a rationale for to facilitate zidovudine and lamivudine resistance (Shafer, offering PI-sparing (dual NRTI, single NNRTI) HAART 2002), and it was fairly common in our Botswana HIV-1C as a first-line treatment in Botswana. Our data also sequences [five (7.1%) of 70 in our samples] (Loemba et emphasize the importance of monitoring ARV drug-resis- al., 2002). The G333E mutation, reported in 6% of ARV- tance patterns over time, especially as the public ARV treatment-naive HIV-1B-infected individuals, facilitates programme is rapidly scaling up. Although genotypic zidovudine resistance in the presence of nucleotide exci- resistance testing may become affordable in some settings, sion mutations (Shafer, 2002). its large-scale use in sub-Saharan Africa for individual Additionally, none of the mutations known to cause patient management is not at present economically primary resistance to nevirapine and efavirenz (mutations feasible, thereby stressing the need for longitudinal popu- located between codons 98–108 and 179–190) were found lation-based surveillance efforts guiding first- and second- in the RT coding region of our sequences. Only one line HAART choices. However, it will also be important sequence showed a substitution at position 190 (G190E); to characterize trends in the emergence of drug resistance this is a mutation that has been associated with reduced among ARV-naive and ARV-experienced individuals NNRTI susceptibility (Bacheler et al., 2001). (Havlir et al., 2002; Wainberg & Friedland, 1998), as well Two mutations associated with primary resistance to as monitor drug-resistance trends among recently infected PIs (one D30N and one V82A) were found in the PR individuals in this region of the world. Our data serve as gene. However, a significant number of sequences an important benchmark for future regional population- harboured multiple secondary drug-resistance mutations based ARV resistance studies. in the PR coding region, namely L10I, K20R, M36I, L63P/A/Q/S/H/C/T/I, V77I and I93L (Shafer et al., Acknowledgements 1997; Grossman et al., 2001). Drug resistance to PIs typi- cally develops in a gradual fashion from the additive accu- We recognize and acknowledge all 2001 Botswana mulation of primary and/or secondary mutations. Sentinel Surveillance participants. We also acknowledge Although the rate and kinetics of PI resistance are not the following organizations within Botswana for their known for HIV-1C, our findings may suggest an increased collaboration: National AIDS Coordinating Agency susceptibility and readiness of HIV-1C to develop resis- (NACA), Ministry of Health, and collaborating referral tance under selective drug pressure. and district hospital teams. We thank Bristol-Myers While the effects of naturally occurring substitutions in Squibb’s ‘Secure the Future’ for their significant laboratory HIV-1C (mean of 10 PR and 14 RT substitutions per and clinical staff support. We thank AM Reich and S sequence in this study) on the kinetics and patterns of Gaolekwe for overseeing sample collection, transport and drug-resistance development are largely unknown, several processing. We also thank R Shafer for his assistance in studies have attempted to address their in vitro and obtaining drug-naive reference sequences, as well as M biochemical significance. Velazquez-Campoy et al. Gordon and S Casol from the University of Natal for suggested that the effect of drug-resistance mutations providing South African sequences. within PR may be enhanced by subtype-specific back-

Antiviral Chemistry & Chemotherapy 16:2 111 H Bussmann et al.

Table 3. Comparison of polymorphic codon sites (greater than 10%) on RT encoding region from treatment- naive HIV-1B ref with HIV-1C consensus sequences from Botswana (BW) and KwaZulu-Natal South Africa (ZA) and a set of subtype C ref sequences; continued on following page

RT position HIV-1B ref HIV-1C ref BW ZA Significance§ n=(91)–(1074)‡ n=(89)–(436)‡ n=121 n=75

† 35 V82 I9 T5 M3 L1 T93 V2 K3 T93 K5 M1 I1 T94 K3 Q1 M1 NS

M2 I1 *NS

† 36 E99 K1 A75 E25 A81 E17 V1 G1 A77 E21 V1 T1 P=0.024 *NS

† 39 T97 A3 E65 D27 T5 K3 E92 D3 A2 K2 E83 D9 K4 T1 P<0.0001

T1 Q1 N1 A1 *NS

† 48 S98 T2 T89 S10 E1 T93 E5 S2 T92 S7 E1 P=0.0012 *NS

† 60 V91 I9 V67 I33 V93 I7 I63 V37 P<0.0001 *P<0.0001

† 122 K68 E31 P1 E83 K17 E87 K11 R1 P1 E92 K7 Q1 P=0.027 *NS

† 123 D71 E24 N3 S2 G27 S22 N15 D36 G31 D26 S22 N19 G35 D28 S25 N12 NS

E2 *NS

† 135 I57 T33 V8 R1 I67 T15 V11 R6 I77 T10 V7 R3 I70 V15 R7 M4 P<0.0001

M1 M1 M1 K1 L1 T3 L1 *NS

† 173 K96 E2 R2 A58 K21 T19 I2 A71 T24 V2 L1 A69 T21 K5 V3 P<0.0001

I1 E1 I1 G1 *NS

† 174 Q96 H2 K2 Q72 K22 R4 N2 Q61 K28 R9 N1 Q53 K35 N7 E3 P=0.014

L1 T1 R1 *P=0.0054

† 177 D79 E20 N1 E70 D27 G3 E78 D20 G2 E84D9 G4 N3 NS *P=0.031

† 200 T79 A14 I6 E1 A81 T17 E2 A94 E3 T2 V1 A94 T4 I1 E1 P<0.0001 *NS

† 207 Q82 E11 H2 K2 E58 Q13 D7 G7 E48 D16 A10 K8 E64 A11 K9 G9 P<0.0001

A2 R1 A6 K6 N2 R1 N7 G4 R3 T2 D3 S1 R1 Q1 *P=0.013

Q1 I1 N1

† 211 R54 K43 G2 S1 K68 R32 K70 R26 Q2 I1 K55 R44 Q1 P=0.0014

G1 *NS

† 245 V61 M15 E10 K6 Q77 K10 V5 L3 Q75 K12V4 E3 Q74 K19 L4 V1 NS

T2 Q2 I2 L2 E2 H2 N1 H2 N1 M1 L1 H1 E1 *NS

† 248 E96 D4 E91 D7 T2 E80 D10 T7 N2 E87 D9 T3 N1 P=0.004

A1 *NS

† 250 D96 E4 D86 E14 D67 E31 N1 A1 D88 E12 P<0.0001 *P=0.0018

Table 3. Continued from previous page

RT position HIV-1B ref HIV-1C ref BW ZA Significance§ n=(91)–(1074)‡ n=(89)–(436)‡ n=121 n=75

† 251 S100 S99 D1 S91 D5 N3 I1 S99 H1 NS *P=0.019

† 272 A52 P44 S4 P83 A9 S6 K1 P77 A11 S7 K3 P77 A11 S7 Q3 NS

Q1 T1 Q1 R1 K1 *NS

† 277 K57 R43 R69 K30 T1 R61 K35 T3 Q1 R66 K33 S1 NS *NS

† 286 T69 A28 P3 A76 T21 V3 A59 T39 V2 A76 T17 V7 P=0.0004 *P=0.0012

† 291 E96 D4 D97 E3 D96 E3 V1 D96 E4 NS *NS

† 292 V95 I5 I95 V5 I94 V5 R1 I97 V3 NS *NS

† 293 I52 V48 V94 I6 V89 I10 G1 V97 I3 NS *P=0.019

† 311 K95 R5 K93 R7 K87 R13 K99 R1 NS *P=0.0033

† 334 Q74 L10 E6 H5 H35 Q33 N16 D11 H48 N25 Q9 D9 – P<0.0001

Y5 Y3 E2 Y2 C2 S1 R1

L1 G1 E1

*Difference between BW and ZA. †Difference between HIV-1C ref and BW. ‡Range of sequences used to build consensus sequence for each position. §P values computed between frequencies of overall distribution of amino acid residues. NS, not significant. Note: positions 162, 166 and 178 were polymorphic in HIV-subtype B consensus and the three HIV-1C consensus sequences.

This research was supported in part by grants failing non-nucleoside reverse transcriptase inhibitor therapy. Journal of Virology 75:4999–5008. AI47067 and AI43255 from the National Institutes of Health, and grant TW00004 from the Fogarty Bennett DE, Zaidi IF, Heneine W, Woods T, Garcia-Lerma JG, Smith AJ, McCormick L & Weinstock HS (2003) Prevalence of International Center, National Institutes of Health, mutations associated with antiretroviral drug resistance among Bethesda, MD, USA. men and women newly diagnosed with HIV in 10 US cities, 1997–2001. Antiviral Therapy 8:S134. Boden D, Hurley A, Zhang L, Cao Y, Guo Y, Jones E, Tsay J, Ip J, References Farthing C, Limoli K, Parkin N & Markowitz M (1999) HIV-1 drug resistance in newly infected individuals. Journal of the Adje-Toure C, Celestin B, Hanson D, Roels TH, Hertogs K, Larder American Medical Association 282:1135–1141. B, Diomande F, Peeters M, Eholie S, Lackritz E, Chorba T & Nkengasong JN (2003) Prevalence of genotypic and phenotypic Brenner B, Turner D, Oliveira M, Moisi D, Detorio M, Carobene HIV-1 drug-resistant strains among patients who have rebound in M, Marlink RG, Schapiro J, Roger M & Wainberg MA (2003) A viral load while receiving antiretroviral therapy in the UNAIDS- V106M mutation in HIV-1 clade C viruses exposed to efavirenz Drug Access Initiative in Abidjan, Côte d’Ivoire. AIDS 17(Suppl confers cross-resistance to non-nucleoside reverse transcriptase 3):S23–29. inhibitors. AIDS 17:F1–5. Bacheler L, Jeffrey S, Hanna G, D’Aquila R, Wallace L, Logue K, Briones C, Perez-Olmeda M, Rodriguez C, del Romero J, Hertogs Cordova B, Hertogs K, Larder B, Buckery R, Baker D, Gallagher K & Soriano V (2001) Primary genotypic and phenotypic HIV-1 K, Scarnati H, Tritch R & Rizzo C (2001) Genotypic correlates of drug resistance in recent seroconverters in Madrid. Journal of phenotypic resistance to efavirenz in virus isolates from patients Acquired Immune Deficiency Syndromes 26:145–150.

Antiviral Chemistry & Chemotherapy 16:2 113 H Bussmann et al.

Chaix ML, Descamps D, Mouajjah S, Deveau C, André P, reverse transcriptase (RT) and rapid development of resistance Cottalorda J, Ingrand D, Izopet J, Kohli E, Masqelier B, against non-nucleoside inhibitors of RT. Antimicrobial Agents & Parmentier K, Poggi C, Rogez S, Ruffault A, Schneider V, Chemotherapy 46:2087–2094. Schmück A, Tamalet C, Wirden M, Rouzioux C, Meyer L, Brun- Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Vézinet F, Costagliola D & the ANRS AC11 Resistance Group, Novak NG, Ingersoll R, Sheppard HW & Ray SC (1999) Full- Cohort PRIMO, INTERPRIM and PRIMSTOP Study Groups length human immunodeficiency virus type 1 genomes from sub- (2003) French National Sentinel Survey of antiretroviral resistance type C-infected seroconverters in India, with evidence of intersub- in patients with HIV-1 primary infection and in antiretroviral- type recombination. Journal of Virology 73:152–160. naive chronically infected patients in 2001–2002. Antiviral Therapy 8(S137): Abstract 123. Montano MA, Nixon CP & Essex M (1998) Dysregulation through the NF-kappaB enhancer and TATA box of the human immunod- Descamps D, Calvez V, Izopet J, Buffet-Janvresse C, Schmück A, eficiency virus type 1 subtype E promoter. Journal of Virology Colson P, Ruffault A, Maillard A, Masquelier B, Cottalorda J, 72:8446–8452. Harzic M, Brun-Vézinet F & Costagliola D; ANRS Antiretroviral Resistance Study Group (2001) Prevalence of resistance mutations Montano MA, Nixon CP, Ndung’u T, Bussmann H, Novitsky VA, in antiretroviral-naive chronically HIV-infected patients in 1998: a Dickman D & Essex M (2000) Elevated tumour necrosis factor- French nationwide study. AIDS 15:1777–1782. alpha activation of human immunodeficiency virus type 1 subtype C in Southern Africa is associated with an NF-kappaB enhancer Frater AJ, Beardall A, Ariyoshi K, Churchill D, Galpin S, Clarke JR, gain-of-function. Journal of Infectious Diseases 181:76–81. Weber JN & McClure MO (2001) Impact of baseline polymorphisms in RT and protease on outcome of highly active antiretro- Montano MA, Novitsky VA, Blackard JT, Cho NL, Katzenstein DA viral therapy in HIV-1-infected African patients. AIDS & Essex M (1997) Divergent transcriptional regulation among 15:1493–1502. expanding human immunodeficiency virus type 1 subtypes. Journal of Virology 71:8657–8665. Geretti AM, Smith M, Osner N, O’Shea S, Chrystie I, Easterbrook P & Zuckerman M (2001) Prevalence of antiretroviral resistance in NACA (2003) Botswana 2003 Second Generation HIV/AIDS a South London cohort of treatment-naive HIV-1-infected Surveillance. A Technical Report. Gaborone: National AIDS patients. AIDS 15:1082–1084. Coordinating Agency. Geretti AM, Smith M, Watson C, Mullen J, Osner N, O’Shea S, Ndung’u T, Renjifo B, Novitsky VA, McLane MF, Gaolekwe S & Chrystie I, Zuckerman M & Easterbrook P (2002) Primary anti- Essex M (2000) Molecular cloning and biological characterization retroviral resistance in newly diagnosed patients with established of full-length HIV-1 subtype C from Botswana. Virology heterosexually acquired HIV-1. AIDS 16:2358–2360. 278:390–399. Gordon M, De Oliveira T, Bishop K, Coovadia HM, Madurai L, Novitsky VA, Gaolekwe S, McLane MF, Ndung’u TP, Foley BT, Engelbrecht S, Janse van Rensburg E, Mosam A, Smith A & Vannberg F, Marlink R & Essex M (2000) HIV type 1 A/J recom- Cassol S (2003) Molecular characteristics of human immunodefi- binant with a pronounced pol gene mosaicism. AIDS Research & ciency virus type 1 subtype C viruses from KwaZulu-Natal, South Human Retroviruses 16:1015–1020. Africa: implications for vaccine and antiretroviral control strategies. Novitsky VA, Montano MA, McLane MF, Renjifo B, Vannberg F, Journal of Virology 77:2587–2599. Foley BT, Ndung’u TP, Rahman M, Makhema MJ, Marlink R & Grossman Z, Vardinon N, Chemtob D, Alkan ML, Bentwich Z, Essex M (1999) Molecular cloning and phylogenetic analysis of Burke M, Gottesman G, Istomin V, Levi I, Maayan S, Shahar E human immunodeficiency virus type 1 subtype C: a set of 23 full- & Schapiro JM; Israel Multi-Center Study Group (2001) length clones from Botswana. Journal of Virology 73:4427–4432. Genotypic variation of HIV-1 reverse transcriptase and protease: Novitsky V, Smith UR, Gilbert P, McLane MF, Chigwedere P, comparative analysis of clade C and clade B. AIDS 15:1453–1460. Williamson C, Ndung’u T, Klein I, Chang SY, Peter T, Thior I, Handema R, Terunuma H, Kasolo F, Kasai H, Sichone M, Foley BT, Gaolekwe S, Rybak N, Gaseitsiwe S, Vannberg F, Yamashita A, Deng X, Mulundu G, Ichiyama K, Munkanta M, Marlink R, Lee TH & Essex M (2002) Human immunodeficiency Yokota T, Wakasugi N, Tezuka F, Yamamoto N & Ito M (2003) virus type 1 subtype C molecular phylogeny: consensus sequence Prevalence of drug-resistance-associated mutations in antiretroviral for an AIDS vaccine design? Journal of Virology 76:5435–5451. drug-naive Zambians infected with subtype C HIV-1. AIDS Perno CF, Cozzi-Lepri A, Balotta C, Bertoli A, Violin M, Monno Research & Human Retroviruses 19:151–160. L, Zauli T, Montroni M, Ippolito G & d’Arminio-Monforte A; Havlir D, Vella S & Hammer S (2002) The Global HIV Drug ICONA Study Group (2002) Low prevalence of primary muta- Resistance Surveillance Program: a partnership between WHO tions associated with drug resistance in antiviral-naive patients at and IAS. International AIDS Society. AIDS 16:7–9. therapy initiation. AIDS 16:619–624. Jayaraman GC, Gleeson T, Sandstrom P & Archibald CP (2003) Salomon H, Wainberg MA, Brenner B, Quan Y, Rouleau D, Cote P, The Canadian HIV Strain and Drug Resistance Program—a pop- LeBlanc R, Lefebvre E, Spira B, Tsoukas C, Sekaly RP, Conway ulation-based effort to enhance HIV surveillance. Antiviral B, Mayers D & Routy JP (2000) Prevalence of HIV-1 resistant to Therapy 8:S135. antiretroviral drugs in 81 individuals newly infected by sexual con- tact or injecting drug use. Investigators of the Quebec Primary Kantor R, Zijenah LS, Shafer RW, Mutetwa S, Johnston E, Lloyd Infection Study. AIDS 14:F17–23. R, von Lieven A, Israelski D & Katzenstein DA (2002) HIV-1 subtype C reverse transcriptase and protease genotypes in Shafer RW (2002) Genotypic testing for human immunodeficiency Zimbabwean patients failing antiretroviral therapy. AIDS Research virus type 1 drug resistance. Clinical Microbiology Reviews & Human Retroviruses 18:1407–1413. 15:247–277. Little SJ, Holte S, Routy JP, Daar ES, Markowitz M, Collier AC, Shafer RW, Eisen JA, Merigan TC & Katzenstein DA (1997) Koup RA, Mellors JW, Connick E, Conway B, Kilby M, Wang L, Sequence and drug susceptibility of subtype C reverse transcriptase Whitcomb JM, Hellmann NS & Richman DD (2002) from human immunodeficiency virus type 1 seroconverters in Antiretroviral-drug resistance among patients recently infected Zimbabwe. Journal of Virology 71:5441–5448. with HIV. New England Journal of Medicine 347:385–394. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F & Higgins Loemba H, Brenner B, Parniak MA, Ma’ayan S, Spira B, Moisi D, DG (1997) The CLUSTAL_X windows interface: flexible strate- Oliveira M, Detorio M & Wainberg MA (2002) Genetic diver- gies for multiple sequence alignment aided by quality analysis gence of human immunodeficiency virus type 1 Ethiopian clade C tools. Nucleic Acids Research 25:4876–4882.

UK Collaborative Group on Monitoring the Transmission of HIV in HIV-1 protease from African subtypes. Biochemistry Drug Resistance (2001) Analysis of prevalence of HIV-1 drug 41:8613–8619. resistance in primary infections in the United Kingdom. British Vergne L, Kane CT, Laurent C, Diakhate N, Gueye NF, Gueye PM, Medical Journal 322:1074–1075. Sow PS, Faye MA, Liegeois F, Ndir A, Laniece I, Peeters M, Van Vaerenbergh K, Debaisieux L, De Cabooter N, Declercq C, Ndoye I, Mboup S & Delaporte E (2003) Low rate of genotypic Desmet K, Fransen K, Maes B, Marissens D, Miller K, HIV-1 drug-resistant strains in the Senegalese government initia- Muyldermans G, Sprecher S, Stuyver L, Vaira D, Verhofstede C, tive of access to antiretroviral therapy. AIDS 17(Suppl 3):S31–38. Zissis G, Van Ranst M, De Clercq E, Desmyter J & Vandamme Wainberg, MA & Friedland G (1998) Public health implications of AM (2001) Prevalence of genotypic resistance among antiretroviral antiretroviral therapy and HIV drug resistance. Journal of the drug-naive HIV-1-infected patients in Belgium. Antiviral Therapy American Medical Association 279:1977–1983. 6:63–70. Yerly S, Kaiser L, Race E, Bru JP, Clavel F & Perrin L (1999) Velazquez-Campoy A, Vega S & Freire E (2002) Amplification of the Transmission of antiretroviral drug-resistant HIV-1 variants. effects of drug resistance mutations by background polymorphisms Lancet 354:729–733.

Received 11 October 2004, accepted 7 December 2004

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