AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 27, Number 00, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/aid.2011.0246

HIV Type 1 Subtypes Based on the pol Gene and Drug Resistance Mutations Among Antiretroviral-Naive Patients from , Southern

Qijian Su,1 Hao Liang,2 Ping Cen,3 Zhiyou Bi,3 and Ping Zhou4

Abstract

We investigated the distribution of HIV-1 subtypes and the prevalence of HIV-1 drug resistance among HIV- infected antiretroviral-naive patients in Guangxi, southern China. A total of 144 subjects from or , two cities having the most HIV-infected cases in Guangxi, were enrolled. HIV-1 pol fragments were amplified and sequenced from plasma of all patients. In all, 124 sequences were obtained, with 67 from Liuzhou and 57 from Nanning. All sequences were subtyped by phylogenetic analysis and analyzed for antiretroviral resistance using the HIVdb program. Our data showed that the sequences from Liuzhou were subtyped as CRF01_AE (77.6%), CRF07_BC(20.9%), and BC (1.5%), respectively, and the sequences from Nanning as CRF01_AE (78.9%), CRF08_BC (15.8%), B (3.5%), and C (1.8%), respectively. Of the sequences 11.9% from Liuzhou and 28.1% from Nanning harbored drug resistance-associated mutations, but there were only two sequences with mutations associated with significantly reduced phenotypic susceptibility to antiretroviral drugs.

uangxi Zhuang Autonomous Region, a province lo- and limited data concerning HIV-1 drug resistance are Gcated in southern China, has experienced a rapid rise in available for the province, we also analyzed the prevalence of the number of HIV/AIDS cases in recent years. Since 2009, antiretroviral drug resistance mutations in this population. Guangxi has risen to the second position among all 31 prov- In 2008, a total of 144 HIV-infected antiretroviral-naive inces and municipalities of mainland China in terms of the patients were enrolled, of whom 80 were from Liuzhou and 64 number of HIV/AIDS cases; its north-central city, Liuzhou, from Nanning; of these 94 were males and 50 were females, 65 has been the most seriously affected with approximately were infected through the use of injected drugs, and 79 were 11,000 cumulative cases by 2010, and the central city Nanning infected through heterosexual sex. All patients were adults followed up with approximately 9000 cumulative cases, al- and were receiving HIV care at local clinics. together accounting for nearly one-third of all cases in the Informed consent was given before their personal infor- province, which may significantly influence the molecular mation and blood samples were collected. RNA was extracted epidemiology of HIV-1 in Guangxi. from plasma by the QIAAMP Viral RNA mini kit (Qiagen, Several studies reported that CRF01_AE and CRF08_BC Germany), in accordance with the manufacturer’s protocol. are the dominant HIV-1 subtypes in Guangxi,1,2 however, cDNA synthesis from HIV-1 RNA templates was carried out these studies sampled mainly from Binyang county of Nan- by the RevertAid First Strand cDNA Synthesis Kit (Fermen- ning, Pingxiang county of , or , rather than tas, USA) with a gene-specific reverse transcription primer (5¢- from Liuzhou or other regions in Nanning with more HIV GCTTGCCAATACTCC R TCCAC CA-3¢,R= A/G). cases than Binyang. Thus, we recruited HIV/AIDS patients Approximately 1330-bp gag-pol fragments (HXB2 positions from Liuzhou and Nanning, including Binyang county, to 2149–3477) were amplified by nested reverse transcriptase better understand the current molecular epidemiology of polymerase chain reaction (nested RT-PCR) using two exter- HIV-1 in Guangxi. In the meantime, considering that insight nal primers (forward outer primer: 5¢-GGGTTTTGGCTGA into the epidemiology of HIV-1 drug resistance is important GGCAATGAG-3¢, reverse outer primer: 5¢-GCTTGCCAA for guiding the optimal selection of antiretroviral drugs,3–5 TACTCCRTCCACCA-3¢,R= A/G) and two internal primers

1Center for AIDS Research, the Affiliated Ruikang Hospital of Guangxi Traditional Chinese Medical University, Nanning, China. 2College of Public Health, Guangxi Medical University, Nanning, China. 3Department of AIDS Control and Prevention, Nanning Center for Disease Control and Prevention, Nanning, China. 4Department of Tuberculosis Control and Prevention, Liuzhou Center for Disease Control and Prevention, Liuzhou, China.

1 2 SU ET AL.

(forward inner primer: 5¢-GAGCCAACAGCCCCACCA-3¢, reverse inner primer: 5¢-GAATCTCCCTGTTTTCTGCCA-3¢). The PCR products were purified by the TIANgel Midi Pur- ification Kit (Tiangen, China), and then sent to Beijing Sun- biotech Co. Ltd. for DNA sequencing with four primers: 5¢-GAGCCAACAGCCCCACCA-3¢ (forward), 5¢-GCCTGAA AATCCATACAATACTCC-3¢ (forward), (5¢-GAATCTCCCT GTTTTCTGCCA-3¢ (reverse), and 5¢-GGATGCGGTATTCC TAATTGAACTTC-3¢ (reverse). The results from sequencing were aligned and assembled manually into contiguous sequences using Contig Express Project, a component of the Vector NTI Suite 6 software. Fi- nally, 124 sequences were obtained, including 67 from Liuz- hou and 57 from Nanning. The other 20 samples could not be successfully amplified or sequenced. Phylogenetic analysis was carried out by using MEGA version 5 software. Subtype reference sequences used in phylogenetic analysis were re- trieved from the Los Alamos National Laboratory HIV data- base (http://www.hiv.lanl.gov/). On the basis of phylogenetic analysis, 66 out of the 67 se- quences from Liuzhou, except for the one named LZ2094, could be identified, of which 52 (77.6%) were subtyped as CRF01_AE and 14 (20.9%) as CRF07_BC. All 57 sequences from Nanning could be identified, of which 45 (78.9%) were subtyped as CRF01_AE, nine (15.8%) as CRF08_BC, two (3.5%) as subtype B, and one (1.8%) as subtype C (Fig. 1). To obtain more information about LZ2094, we conducted a blast search using HIV Blast tool from the Los Alamos Na- tional Laboratory HIV database (http://www.hiv.lanl.gov/) and found that LZ2094 had the highest homology with a B/C

recombinant strain 06CN_YUN01_24 (96%) from Yunnan province, China. Moreover, we compared LZ2094 to a set of background sequences known as subtype consensus using Recombinant Identification Program 3.0 (RIP 3.0).6 The results revealed that LZ2094 was a B/C recombinant, having a backbone similar to CRF07_BC, but distinctly different breakpoint positions from CRF07_BC. It has been reported that CRF07_BC is one of the most widespread isolates of HIV-1 throughout China.7–10 Epide- miologists have speculated that Guangxi should be in the transmission line of CRF07_BC that has spread from the northwest to the southeast across the country, however, there was no substantial evidence to support this speculation, ex- cept for a CRF07_BC strain named CNGL179 (GenBank ac- cession number AF503396), which was identified in 2002 by McCutchan et al.11 In the present study, we showed that CRF01_AE and CRF08_BC are still the most common isolates in Nanning, accounting for 78.9% (45/57) and 15.8% (9/57) of the se- quences from the city, respectively. CRF01_AE is also the most common isolate in Liuzhou, accounting for 77.6% (52/

67). Unexpectedly, we did not find any CRF08_BC strain in ‰

FIG. 1. Phylogenetic analysis of HIV-1 pol sequences. A neighbor-joining tree based on Kimura’s two-parameter distances was generated using MEGA 5 software. The reli- ability of tree topologies was assessed by bootstrapping with 500 replicates. A bootstrap support of 70%, or greater, was required to define a phylogenetic cluster, and was shown at nodes on the tree. (:) Highlights a reference sequence; (C) indicates an undetermined sequence. HIV SUBTYPES AND RESISTANCE MUTATIONS IN GUANGXI 3

Table 1. Drug Resistance Mutations in Antiretroviral-Naive HIV-Infected Patients from Guangxi

City Isolate Subtype Major PI mutations Minor PI mutations NRTI mutations NNRTI mutations

Liuzhou LZ2009 CRF01_AE L10V LZ2015 CRF01_AE V75LV LZ2069 CRF07_BC V179E LZ2094 CRF_BC V179D LZ2151 CRF01_AE L10V T69N LZ2164 CRF01_AE L10V GXCDCM 6116 CRF07_BC V179D GXCDCM 6543 CRF07_BC A71T Nanning GXNN1256 CRF01_AE M46L GXBY2058 CRF01_AE T69N GXNN2170 B V179E GXNN0282 CRF08_BC T69S GXBY5005 CRF08_BC T69S GXHX0144 CRF08_BC T69S GXNN1027 CRF08_BC T69S GXBY0040 CRF08_BC T69S GXNN2172 CRF08_BC T69S GXNN0073 B A71T GXHX7505 CRF01_AE V75L GXNN1508 CRF08_BC T69S GXNN7219 CRF01_AE E35G, A71T D67N, M184V GXNN0019 CRF08_BC T69S GXHX6917 CRF01_AE T69NT GXBY7291 CRF01_AE T69N

PI, protease inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, nonnucleoside reverse transcriptase inhibitor.

Liuzhou, but another BC recombinant, CRF07_BC, in 20.9% mutations, E35G and A71T, were present in two (3.5%)

(14/67) of the sequences. of the sequences. NRTI-related mutations, T69S, T69N, Our findings suggest that CRF07_BC has become one of the T69NT,V75L, D67N, and M184V, were present in 13 (22.8%) most common HIV isolates in Liuzhou, and may have been of the sequences. Notably, T69S was present in eight se- circulating in its surrounding cities, such as , , quences and all these sequences were identified as CRF08_BC. , and especially Guiling, where the first HIV-1 NNRTI-related mutations (V179E) were present in one (1.8%) CRF07_BC strain in Guangxi was identified. More studies are sequence (Table 1). There were only two sequences, needed to support this speculation and to further describe the GXNN1256 and GXNN7219, with mutations associated with molecular epidemiological profile of HIV-1 in the areas and significantly reduced phenotypic susceptibility to anti- throughout Guangxi. In addition to the three dominant sub- retroviral drugs. M46L found in GXNN1256 can confer in- types, other subtypes including B, C, and BC were found, termediate-level phenotypic resistance to a protease inhibitor, which may indicate a more complicated and diverse trend of nelfinavir (NFV). M184V found in GXNN7219 can confer HIV-1 epidemiology in the province in the future. high-level phenotypic resistance to NRTIs, such as lamivu- To understand the prevalence of HIV-1 drug resistance, dine (3TC) and emtricitabine (FTC). each sequence was submitted to the HIVdb program in the In summary, HIV-1 subtypes CRF01_AE, CRF07_BC, HIV Drug Resistance Database from Stanford University CRF08_BC, B, and C are circulating in Guangxi, of which (http://hivdb.stanford.edu/); as a result, it returned the mu- CRF01_AE is the most common isolate, followed by tation patterns and the inferred levels of resistance to 20 FDA- CRF07_BC and CRF08_BC. This is for the first report indi- approved ARV drugs including eight protease inhibitors cating that the HIV-1 CRF07_BC virus is circulating in Liuz- (PIs), seven nucleoside analogue reverse transcriptase inhib- hou, Guangxi. The prevalence of HIV-1 drug resistance itors (NRTIs), and four nonnucleoside analogue reverse mutations in antiretroviral-naive patients is 11.9% (8/67) in transcriptase inhibitors (NNRTIs). Liuzhou and 28.1% (16/57) in Nanning. The results showed that 11.9% (8/67) of the sequences from Liuzhou harbored drug resistance-associated mutations. Acknowledgments Major PI mutations were not found. Minor PI mutations, L10V and A71T, were present in four (6.0%) of the sequences. This work was supported by the National Nature Science NRTI-related mutations, T69N and V75LV, were present in Foundation of China (No. 30760218) and research fund of two (3.0%) of the sequences. NNRTI-related mutations, the Affiliated Ruikang Hospital of Guangxi Traditional V179D and V179E, were present in three (4.5%) of the se- Chinese Medical University (No. RKZ201004). We are grate- quences (Table 1). ful to Linda Frisse and Lori Black for assigning GenBank ac- Among 57 sequences from Nanning, 16 (28.1%) harbored cession numbers, HQ588180–HQ588303, for the fragments of drug resistance-associated mutations. A major PI mutation, HIV-1 genomes. Finally, we thank all volunteers for their M46L, was present in one (1.8%) of the sequences. Minor PI participation. 4 SU ET AL.

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