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 Zolta´n Gyori,} 1 Ferenc Ba´na´ti,1 Ma´rta Marschalko´,3 Sarolta Ka´rpa´ti,3 and Ja´nos 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 prev- alence 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.