Molecular Epidemiological Analysis of Env and Pol Sequences in Newly Diagnosed HIV Type 1-Infected, Untreated Patients in Hungary

AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 27, Number 00, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/aid.2011.0077

Molecular Epidemiological Analysis of env and pol Sequences in Newly Diagnosed HIV Type 1-Infected, Untreated Patients in Hungary

Ma´ria Mezei,1 E´ va A´ y,1 Anita Koroknai,1 Rena´ta To´th,2 Andrea Bala´zs,2 A´ gnes Bakos,1 Zoltań Gyori,} 1 Ferenc Bańa´ti,1 Ma´rta Marschalko´,3 Sarolta Ka´rpa´ti,3 and Jańos Mina´rovits1

Abstract

The aim of our study was to monitor the diversity of HIV-1 strains circulating in Hungary and investigate the prevalence of resistance-associated mutations to reverse transcriptase (RT) and protease (PR) inhibitors in newly diagnosed, drug-naive patients. A total of 30 HIV-1-infected patients without prior antiretroviral treatment diagnosed during the period 2008–2010 were included into this study. Viral subtypes and the presence of RT, PR resistance-associated mutations were established by sequencing. Classiﬁcation of HIV-1 strains showed that 29 (96.6%) patients were infected with subtype B viruses and one patient (3.3%) with subtype A virus. The prevalence of HIV-1 strains with transmitted drug resistance mutations in newly diagnosed individuals was 16.6% (5/30). This study showed that HIV-1 subtype B is still highly predominant in Hungary and documented a relatively high transmission rate of drug resistance in our country.

he high genetic variability of HIV-1 allows the virus median age of 34 years and a mean viral load of 127,109 copies/ Tto escape from the immune system and resist anti- ml. The main route of transmission was homosexual contact in retroviral therapy. The genetic variation in HIV pol and env 27 cases (90%) and heterosexual contact in three cases (10%). genes plays a major role in the rapid development of resis- The clinical data of patients are presented in Table 1. tance to current drugs. This variation has influenced disease For env subtyping, proviral DNA was extracted from pa- progression among the infected individuals and necessitated tients’ peripheral blood mononuclear cells (PBMCs) using the the search for alternative drugs with novel targets. QIAamp blood DNA isolation kit (Qiagen) and was subjected In the present study we examined the prevalence of anti- to nested polymerase chain reaction (PCR) to amplify the env retroviral drug resistance mutations and the subtype distri- V3 region. The outer primers1 were ED5/ED12 and the inner bution of HIV-1 isolates from untreated patients in Hungary. primers were ADP826: 5¢-CGCTAGGAATTCGGCCAGTAG In Hungary, a country with a relatively low prevalence of HIV TATCAACTCAA-3¢ and ADP828: 5¢-GTACACAAGCTTTCT infection, the estimated prevalence is 0.02% in the total pop- GGGTCCCCTCCTGAGGA-3¢. ulation. The first HIV-positive persons were detected in 1985 For drug resistance study viral RNA was isolated from and by the end of September 2010 their number increased patients’ plasma samples using the Nuclisens magnetic ex- to 1911. The first patient with AIDS was diagnosed in 1986. By traction reagents (Biome´rieux). The pol gene region, encoding the end of September 2010 a total 617 HIV-infected persons the complete protease (PR) (amino acids 1–99) and partial developed AIDS. In our country the most common route of reverse transciptase (RT) (amino acids 41–237), was amplified HIV infection is homosexual contact (Department of Epide- by one tube reverse transcription PCR using the RobusT II RT- miology, National Center for Epidemiology, Budapest). PCR kit (Finnzymes) followed by a nested PCR. The outer Blood samples were obtained in 2008–2010 from 30 HIV-1- protease primers2 were PR1/RT3303. The outer reverse infected antiretroviral therapy-naive, newly diagnosed pa- transcriptase primers were K1: 5’-CCACCAGAAGAGAGC tients of the Dermatology, Venereology and Dermatooncology TTCAGG - 3’ 2161–2181 nt, HXB2 and K2: 5’-CTAGCTCT Clinic of Semmelweis University, Budapest. The studied pa- GCTTCTTCTGTTAGTGG-3’, 3429–3453 nt, HXB2. The in- tients included 29 (96.6%) males and one (3.3%) female, with a ner protease primers2 were PR3/PR4. The inner reverse

1National Center for Epidemiology, Microbiological Research Group, Budapest, Hungary. 2Eo¨tvo¨s Lora´nd University, Department of Microbiology, Budapest, Hungary. 3Semmelweis University, Department of Dermatology,Venereology and Dermatooncology, Budapest, Hungary.

1 2 MEZEI ET AL.

Table 1. HIV-1 Subtypes and Drug Resistance Mutations from Hungarian Patients

Year of Plasma HIV-1 HIV-1 subtype HIV DRM Sample HIV-1 Route of RNA level ID Sex Age diagnosis infection (copies/ml) env pol PI NRTI NNRTI

2999 Male 29 2008 Homosexual 1,200,000 B B A71V —— 3000 Male 40 2008 Homosexual 74,000 B B — — V179E 3008 Male 24 2008 Homosexual 290,000 B B — V118I, L210M — 3010 Male 32 2008 Homosexual 52,000 B B — — — 3011 Male 38 2008 Homosexual 240,000 B B — — V179E 3012 Male 36 2008 Homosexual 27,000 B B Q58E, A71T L210F/L — 3014 Male 33 2008 Homosexual 1,100 B B — — — 3029 Male 33 2009 Homosexual 31,000 B B — — K101Q, K103N 3033 Male 41 2009 Homosexual 95,000 B B L10I, A71V —— 3037 Male 23 2009 Homosexual 250,000 B B A71T —— 3038 Female 56 2009 Heterosexual 25,000 B B — — — 3039 Male 28 2009 Homosexual 21,000 B B — — — 3043 Male 21 2009 Homosexual 6,400 B B — — — 3056 Male 36 2009 Homosexual 150,000 B B A71T —— 3062 Male 32 2010 Homosexual 2,100 B B A71V M41L, T215E — 3065 Male 58 2010 Homosexual 560,000 B B — — V179E 3066 Male 31 2009 Homosexual 6,200 B B A71V V118I E138A 3067 Male 53 2010 Homosexual 68,000 B B — — — 3071 Male 31 2010 Homosexual 370 B B — — — 3072 Male 34 2009 Homosexual 8,400 B B — — V179E 3073 Male 34 2010 Homosexual 10,000 B B A71V M41L, T215E — 3074 Male 26 2010 Homosexual 78,000 B B — V118I, L210M — 3077 Male 29 2010 Heterosexual 12,000 B B A71V M41L, T215E — 3078 Male 36 2010 Homosexual 56,000 B B — — — 3079 Male 29 2010 Heterosexual 48,000 A A — — — 3084 Male 28 2010 Homosexual 9,700 B B — — K103N 3090 Male 29 2010 Homosexual 370,000 B B L10I —— 3094 Male 37 2010 Homosexual 20,000 B B — — V90I, E138AE 3095 Male 33 2010 Homosexual 85,000 B B — — — 3097 Male 31 2010 Homosexual 17,000 B B A71T ——

Mutations causig intermediate or high level resistance are in italics.

transcriptase primers2 were RT1/RT4 and B3: 5’-CCTGAA subtype A (Fig. 1). The average intrasubtype nucleotide di- AATCCATACAATACTCC-3’, 2703–2725 nt, HXB2/B4: 5’- vergence among the Hungarian env sequences with subtype B GGTATTACTTCTGTTAGTGCTTTGG-3’, 3406–3430 nt, was 18.3%. HXB2. The V3 loop of HIV-1 is known to play a critical role in the All env and pol PCR fragments were purified by the QIA- biology of the virus in determining infectivity, cell tropism, quick purification kit (Qiagen). Purified PCR products were coreceptor usage, and immunogenicity. To analyze the V3 subjected to direct sequencing using the DYEnamic ET Dye loop sequences of Hungarian isolates, the predicted amino Terminator Cycle Sequencing kit. Electrophoresis and data acid sequences of the C2V3 region of the env gene were collection were accomplished with the MegaBACE 1000 ge- aligned and compared with the consensus sequences (Fig. 2). netic analyzer system (Amersham Biosciences). Within the subtype B cluster the prevalent crown tetrapeptide Subtype sequence homologies were examined with the motif in the V3 loop was GPGR, which is the most common REGA subtyping tool (http://bioafrica.net). The env se- motif found in subtype B isolates.5 This motif was found in 16 quences were aligned with reference sequences obtained from isolates (53.3%). The GPGK occured in five cases (16.6%), the Los Alamos HIV Database using CLUSTAL W3 and GPGG in four cases (13.3%), GPGQ in three cases (10%), and subtype determination was further confirmed by phyloge- GPGS in two cases (6.6%). These motives are also common in netic analysis, using programs from the Molecular Evolution B subtype isolates. The subtype A isolate contained the highly Genetic Analysis (MEGA5) software (Kimura distance esti- conserved GPGQ motif, which is the most prevalent one mation model, pairwise gap deletion, neighbor-joining within subtype A strains5 (Fig. 2). method, and bootstrap analysis with 1000 replications).4 The Different amino acid patterns in V3 region may have a pol sequences were submitted to the Stanford HIV Sequence clinical significance by influencing the effectiveness of CCR5 Database (http://hivdb.stanford.edu/hiv)for drug resistance inhibitors. V3 sequences with basic amino acids R or K at interpretation. positios 11 and 25, and an overall V3 loop net charge The phylogenetic analysis based on env C2V3 sequences + 5.5 – 1.3, predict X4 virus and V3 sequences with no basic revealed that 29 of the 30 samples were subype B and one was amino acid at this positions predict R5 virus.6,7 The presence HIV-1 DIVERSITY IN HUNGARY 3

a swich in tropism, from CCR5 to CXCR4 usage in about 50% of patients, whereas CXCR4 usage among subtype C viruses is less frequent even in later stage patients. Subtype D has a higher prevalence of X4 tropism than subtype A, which is mostly R5-tropic.8 Uncharged or negatively charged amino acid residues at positions 11 and 25 were identiﬁed in most of the Hungarian isolates, suggesting R5 viruses. One isolate, HU3084, pos- sessed a positively charged amino acid at position 25 and had an overall V3 loop charge of + 4, suggesting an X4 virus (Fig. 2). Drug resistance mutations in the 30 HIV-1 pol sequences were analyzed by examining amino acid mutations at the PR and RT sites associated with antiretroviral drug resistance. There was no major mutation associated with drug resistance in the protease gene. Minor mutations that might contribute to protease inhibitor (PI) resistance were detected in 11 of 30 (36.6%) Hungarian drug-naive patients. The most frequent substitutions were A71V in 6/30 (20%), A71T in 4/30 (13.3%), L10I in 2/30 (6.6%), and Q58E in 1/30 (3.3%). Q58E is a mutation associated with a potential low level resistance to ti- pranavir (Table 1). Nucleoside reverse transcriptase inhibitor (NRTI) resistance mutations M41L and T215E, associated with intermediate resistance to zidovudine/stavudine, were observed in 3/30 (10%) cases. Nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutation K103N, which is associated with a high level resistance to dela- virdine/efavirenz/nevirapine, was detected in 2/30 (6.6%) patients. Two patients, 3073 and 3077, had the same PI and NRTI mutation pattern, and their env sequences were also

closely related (Fig. 1). In accordance with the env sequence data (Fig. 1), all of the PR and RT sequences belonged to subtype B, except HU3079, which proved to be subtype A in the analyzed regions. Our results are in agreement with our earlier study showing the predominance of HIV type 1 subtype B strains in Hungary among AIDS patients undergoing antiretroviral treatment.9 HIV-1 subtype B strains are also dominant in most of the neighboring countries (Slovakia, Serbia, Croatia, Slovenia, and Austria), except for Romania where subtype F1, and Ukraine, where subtype A strains are the most prevalent ones.10–16 We noticed a significant sequence het- erogeneity in the C2V3 region of env among the Hungarian subtype B isolates included in this study. This finding sug- gests that subtype B HIV-1 strains were transmitted to Hungary during the study period (2008–2010) by multiple sources. Four patients included in our study had been infected abroad, patients 3033, 3071, 3078 in Romania and patient 3079 in Africa. A recent study reported a 8.4% overall prevalence of transmitted drug resistance associated with newly diagnosed 17 FIG. 1. Phylogenetic tree based on the env C2V3 region of HIV-1 infection in Europe. The most common NRTI- related HIV-1 strains from Hungary with reference sequences of mutations was T215Y/F or 215 revertant and the most com- representative subtypes (http://hiv.lanl.gov). Phylogenetic mon NNRTI-related mutation was K103N, which is in relationships were determined using MEGA5.4 Bootstrap agreement with our results. Vercauteren et al. noticed that values higher than 50% are shown at the corresponding men who have sex with men infected with a subtype B virus nodes. The scale bar represents the evolutionary distance of had a higher chance of being infected with drug-resistant 0.05 nucleotides per position in the sequence. HIV-1 than other patient groups.17 The majority of newly diagnosed patients included in our study belonged to the of viruses that utilize CXCR4 will influence the clinical utility former category, because homosexual and bisexual trans- of CCR5-targeted inhibitors, and this property appears to mission represents the major routes of HIV infection in differ between subtypes. The subtype B V3 domain facilitates Hungary. 4 MEZEI ET AL.

FIG. 2. Amino acid sequence alignment of the env C2V3 regions of Hungarian HIV-1 strains compared with consensus

sequences from the Los Alamos database (http://hiv.lanl.gov). The nucleotide sequences were translated by the program TRANSLATE (EXPASY: www.expasy.org/) and aligned by CLUSTAL W.3 Dots indicate amino acid identity with the reference sequence and dashes correspond to gaps introduced to maintain the correct alignment. Residues involved in potencial N-linked glycosylation sites are marked with a caret (^). Asterisks represent the terminal cysteine residues of the V3 loop region.

Although the prevalence of transmitted HIV-1 drug re- crobiological Research Group, National Center for sisance was higher (16.6%) in Hungary than the overall Epidemiology, Budapest). prevalence reported for Europe, this discrepancy may be related to the relatively low number of samples analyzed in our Author Disclosure Statement study. Further efforts are needed for the continuous moni- No competing ﬁnancial interests exist. toring of the HIV pandemic in Hungary. References Sequence Data 1. Delwart EL, Shpaer EG, Louwagie J, et al.: Genetic rela- The sequences described in this article have been deposited tionships determined by a DNA heteroduplex mobility in the GenBank Nucleotide Sequence Database under acces- assay: Analysis of HIV-1 env genes. Science 1993;262:1257– sion numbers JF419364–JF419394 (env sequences), JF419394– 1261. JF419424 ( pol protease sequences), and JF419424–JF419453 2. Steegen K, Demecheleer E, Cabooter N, et al.: A sensitive in- ( pol reverse transcriptase sequences). house RT-PCR genotyping system for combined detection of plasma HIV-1 and assesment of drug resistance. J Virol Acknowledgments Methods 2006;133:137–145. 3. Thompson JD, Higgins DG, and Gibson TJ: CLUSTAL W: This study was supported by Norway Grants (Contract No. Improving the sensitivity of progressive multiple sequence ¨ 0094/NA/20008-3/OP-5). We are grateful for the expert ad- alignment through sequence weighting, positions-speciﬁc vice of Ga´bor To´th (Agricultural Biotechnology Center, gap penalties and weight matrix choice. Nucleic Acids Res Bioinformatics Group, Go¨do¨llo,} Hungary). We also highly 1994;22:4673–4680. appreciate the excellent technical assistance provided by 4. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, and Ma´ria Ackermann (Semmelweis University, Department of Kumar S: MEGA5: Molecular evolutionary genetics analysis Dermatology, Venereology and Dermatooncology, Buda- using maximum likelihood, evolutionary distance, and max- pest), and A´ gnes Hortoba´gyi Pocskay and Ilona Ve´gh (Mi- imum parsimony methods. Mol Biol Evol 2011 (accepted). HIV-1 DIVERSITY IN HUNGARY 5

5. Vasil S, Thakallapally R, Korber B, and Foley B: Global var- 1 isolates from children in Romania: Identification of a new iation in the HIV-1 V3 region. In: Korber B, Kuiken C, Foley B, envelope subtype. J Infect Dis 1994;168:281–288. et al.: Human Retroviruses and AIDS 1998: A Compilation and 13. Stanojevic M, Papa A, Papadimitriou E, et al.: HIV-1 sub- Analysis of Nucleic Acid and Amino Acid Sequences. Theoretical types in Yugoslavia. AIDS Res Hum Retroviruses 2002; Biology and Biophysics Group, Los Alamos National La- 18:519–522. boratory, Los Alamos, NM, 1998, pp. III118–III265. 14. Zidovec Lepej S, Baca Vrakela I, Poljak M, Bozicevic I, and 6. Xiao L, Owen SM, Goldman I, et al.: CCR5 coreceptor usage Begovac J: Phylogenetic analysis of HIV sequences obtained of non-syncytium-inducing primary HIV-1 is independent of in a respondent-driven sampling study of men who have phylogenetically distinct global isolates: Delineation of sex with men. AIDS Res Hum Retroviruses 2009;25:1335– consensus motif in the V3 domain that predicts CCR5-usage. 1338. Virology 1998;240:83–92. 15. Mezei M, Balog K, Babic DZ, et al.: Genetic variability of gag 7. De Jong JJ, De Ronde A, Keulen W, Tersmette M, and and env regions of HIV type 1 strains circulating in Slovenia. Goudsmit J: Minimal requirements for the human immu- AIDS Res Hum Retroviruses 2006;22:109–113. nodeficiency virus type 1 V3 domain to support the syncy- 16. Kessler HH, Deuretzbacher D, Stelzl E, et al.: Determination tium-inducing phenotype: Analysis by single amino acid of human immunodeficiency type 1 subtypes by a rapid substitution. J Virol 1992;66:6777–6780. method useful for the routine diagnostic laboratory. Clin 8. Lynch RM, Shen T, Gnanakaran S, and Derdeyn CA: Ap- Diagn Lab Immunol 2001;8:1018–1020. preciating HIV type 1 diversity: Subtype differences in Env. 17. Vercauteren J, Wensing AMJ, van de Vijver DAMC, et al.: AIDS Res Hum Retroviruses 2009;3:237–248. Transmission of drug-resistant HIV-1 is stabilizing in Eur- 9. Mezei M, Balog K, Takaćs M, et al.: Genetic subtypes of ope. J Infect Dis 2009;200:1503–1508. HIV type 1 in Hungary. AIDS Res Hum Retroviruses 2000;16:513–516. 10. Habekova M, Takacova M, Lysy J, et al.: Genetic subtypes of Address correspondence to: HIV type 1 circulating in Slovakia. AIDS Res Hum Retro- Ma´ria Mezei viruses 2010;26:1103–1107. National Center for Epidemiology 11. Pukish NS, Shcherbinska AM, Babij NO, and Polishchuk VP: Microbiological Research Group Analysis of molecular features of the ukrainian isolates of Pihen}ou´t1 HIV-1. Biopolymers Cell 2009;25:50–55. [Translated from H-1529 Budapest Ukranian.] Hungary 12. Dumitrescu O, Kalish ML, Kliks SC, Bandea CI, and Levy JA: Characterization of human immundeficiency virus type E-mail: [email protected]