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

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 Liuzhou or Nanning, 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.

G uangxi Zhuang Autonomous Region, a province located in southern China, has experienced a rapid rise in the number of HIV/AIDS cases in recent years. Since 2009, Guangxi has risen to the second position among all 31 provinces and municipalities of mainland China in terms of the number of HIV/AIDS cases; its north-central city, Liuzhou, has been the most seriously affected with approximately 11,000 cumulative cases by 2010, and the central city Nanning followed up with approximately 9000 cumulative cases, altogether accounting for nearly one-third of all cases in the province, which may significantly influence the molecular epidemiology of HIV-1 in Guangxi.

Several studies reported that CRF01_AE and CRF08_BC are the dominant HIV-1 subtypes in Guangxi,^1,2 however, these studies sampled mainly from Binyang county of Nanning, Pingxiang county of Chongzuo, or Baise, rather than from Liuzhou or other regions in Nanning with more HIV cases than Binyang. Thus, we recruited HIV/AIDS patients from Liuzhou and Nanning, including Binyang county, to better understand the current molecular epidemiology of HIV-1 in Guangxi. In the meantime, considering that insight into the epidemiology of HIV-1 drug resistance is important for guiding the optimal selection of antiretroviral drugs,^3
–5 and limited data concerning HIV-1 drug resistance are available for the province, we also analyzed the prevalence of antiretroviral drug resistance mutations in this population.

In 2008, a total of 144 HIV-infected antiretroviral-naive patients were enrolled, of whom 80 were from Liuzhou and 64 from Nanning; of these 94 were males and 50 were females, 65 were infected through the use of injected drugs, and 79 were infected through heterosexual sex. All patients were adults and were receiving HIV care at local clinics.

Informed consent was given before their personal information and blood samples were collected. RNA was extracted from plasma by the QIAAMP Viral RNA mini kit (Qiagen, Germany), in accordance with the manufacturer's protocol. cDNA synthesis from HIV-1 RNA templates was carried out by the RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA) with a gene-specific reverse transcription primer (5′-GCTTGCCAATACTCC R TCCAC CA-3′, R=A/G).

Approximately 1330-bp gag-pol fragments (HXB2 positions 2149–3477) were amplified by nested reverse transcriptase polymerase chain reaction (nested RT-PCR) using two external primers (forward outer primer: 5′-GGGTTTTGGCTGA GGCAATGAG-3′, reverse outer primer: 5′-GCTTGCCAA TACTCCRTCCACCA-3′, R=A/G) and two internal primers (forward inner primer: 5′-GAGCCAACAGCCCCACCA-3′, reverse inner primer: 5′-GAATCTCCCTGTTTTCTGCCA-3′). The PCR products were purified by the TIANgel Midi Purification Kit (Tiangen, China), and then sent to Beijing Sunbiotech 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. Finally, 124 sequences were obtained, including 67 from Liuzhou 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 retrieved from the Los Alamos National Laboratory HIV database (http://www.hiv.lanl.gov/).

On the basis of phylogenetic analysis, 66 out of the 67 sequences 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).

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 reliability 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; (●) indicates an undetermined sequence.

To obtain more information about LZ2094, we conducted a blast search using HIV Blast tool from the Los Alamos National 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 Epidemiologists 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, except for a CRF07_BC strain named CNGL179 (GenBank accession 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 sequences 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 Liuzhou, but another BC recombinant, CRF07_BC, in 20.9% (14/67) of the sequences.

Our findings suggest that CRF07_BC has become one of the most common HIV isolates in Liuzhou, and may have been circulating in its surrounding cities, such as Laibin, Hechi, Hezhou, and especially Guiling, where the first HIV-1 CRF07_BC strain in Guangxi was identified. More studies are needed to support this speculation and to further describe the molecular epidemiological profile of HIV-1 in the areas and throughout Guangxi. In addition to the three dominant subtypes, other subtypes including B, C, and BC were found, which may indicate a more complicated and diverse trend of HIV-1 epidemiology in the province in the future.

To understand the prevalence of HIV-1 drug resistance, each sequence was submitted to the HIVdb program in the HIV Drug Resistance Database from Stanford University (http://hivdb.stanford.edu/); as a result, it returned the mutation patterns and the inferred levels of resistance to 20 FDA-approved ARV drugs including eight protease inhibitors (PIs), seven nucleoside analogue reverse transcriptase inhibitors (NRTIs), and four nonnucleoside analogue reverse transcriptase inhibitors (NNRTIs).

The results showed that 11.9% (8/67) of the sequences from Liuzhou harbored drug resistance-associated mutations. Major PI mutations were not found. Minor PI mutations, L10V and A71T, were present in four (6.0%) of the sequences. NRTI-related mutations, T69N and V75LV, were present in two (3.0%) of the sequences. NNRTI-related mutations, V179D and V179E, were present in three (4.5%) of the sequences (Table 1).

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.

Among 57 sequences from Nanning, 16 (28.1%) harbored drug resistance-associated mutations. A major PI mutation, M46L, was present in one (1.8%) of the sequences. Minor PI mutations, E35G and A71T, were present in two (3.5%) of the sequences. NRTI-related mutations, T69S, T69N, T69NT,V75L, D67N, and M184V, were present in 13 (22.8%) of the sequences. Notably, T69S was present in eight sequences and all these sequences were identified as CRF08_BC. NNRTI-related mutations (V179E) were present in one (1.8%) sequence (Table 1). There were only two sequences, GXNN1256 and GXNN7219, with mutations associated with significantly reduced phenotypic susceptibility to antiretroviral drugs. M46L found in GXNN1256 can confer intermediate-level phenotypic resistance to a protease inhibitor, nelfinavir (NFV). M184V found in GXNN7219 can confer high-level phenotypic resistance to NRTIs, such as lamivudine (3TC) and emtricitabine (FTC).

In summary, HIV-1 subtypes CRF01_AE, CRF07_BC, CRF08_BC, B, and C are circulating in Guangxi, of which CRF01_AE is the most common isolate, followed by CRF07_BC and CRF08_BC. This is for the first report indicating that the HIV-1 CRF07_BC virus is circulating in Liuzhou, Guangxi. The prevalence of HIV-1 drug resistance mutations in antiretroviral-naive patients is 11.9% (8/67) in Liuzhou and 28.1% (16/57) in Nanning.

Footnotes

Acknowledgments

This work was supported by the National Nature Science Foundation of China (No. 30760218) and research fund of the Affiliated Ruikang Hospital of Guangxi Traditional Chinese Medical University (No. RKZ201004). We are grateful to Linda Frisse and Lori Black for assigning GenBank accession numbers, HQ588180–HQ588303, for the fragments of HIV-1 genomes. Finally, we thank all volunteers for their participation.

Author Disclosure Statement

No competing financial interests exist.

References

Tee

, Pybus

, Li

et al. Temporal and spatial dynamics of human immunodeficiency virus type 1 circulating recombinant forms 08_BC and 07_BC in Asia. J Virol, 2008; 82:9206–9215.

Laeyendecker

, Zhang

, Quinn

et al. Molecular epidemiology of HIV-1 subtypes in southern China. J Acquir Immune Defic Syndr, 2005; 38:356–362.

Hirsch

, Brun-Vézinet

, Clotet

et al. Antiretroviral drug resistance testing in adults infected with human immunodeficiency virus type 1: 2003 recommendations of an International AIDS Society-USA Panel. Clin Infect Dis, 2003; 37:113–128.

Vandamme

, Sonnerborg

, Ait-Khaled

et al. Updated European recommendations for the clinical use of HIV drug resistance testing. Antivir Ther, 2004; 9:829–848.

Wensing

, van de Vijver

, Angarano

et al. Prevalence of drug-resistant hiv-1 variants in untreated individuals in Europe: Implications for clinical management. J Infect Dis, 2005; 192:958–966.

Siepel

, Halpern

, Macken

et al. A computer program designed to screen rapidly for HIV type 1 intersubtype recombinant sequences. AIDS Res Hum Retroviruses, 1995; 11:1413–1416.

Meng

, Xing

, He

et al. Genetic characterization of three newly isolated CRF07_BC near full-length genomes in China. AIDS Res Hum Retroviruses, 2007; 23:1049–1054

, Jia

, Ma

et al. The changing face of HIV in China. Nature, 2008; 455:609–611.

, Graf

, Zhang

et al. Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B′) recombinant strain in China. J Virol, 2000; 74:11367–11376.

10.

Rodenburg

, Li

, Trask

et al. Near full-length clones and reference sequences for subtype C isolates of HIV type 1 from three different continents. AIDS Res Hum Retroviruses, 2001; 17:161–168.

11.

McCutchan

, Carr

, Murphy

et al. Precise mapping of recombination breakpoints suggests a common parent of two BC recombinant HIV type 1 strains circulating in China. AIDS Res Hum Retroviruses, 2002; 18:1135–1140.