HIV-1 Diversity and Drug-Resistant Mutations in Infected Individuals in Changchun, China

Objectives Human immunodeficiency virus type 1 (HIV-1) infection has been detected in all provinces of China. Although epidemiological and phylogenetic studies have been conducted in many regions, such analyses are lacking from Jilin province in northeastern China. Method Epidemiological and phylogenetic analyses, as well as detection of drug-resistant mutations, were conducted on 57 HIV-1 infected patients from Changchun city identified and confirmed through annual surveillance by local Centers for Disease Control in Jilin province of northeastern China in 2012. Results Sexual contact was determined to be the major pathway for HIV-1 transmission in Jilin, where hetero- and homosexual activities contributed almost equally. Phylogenetic analyses detected multiple subtypes of HIV-1 including subtype G circulating in Jilin, with multiple origins for each of them. Both subtype B and CRF01_AE were dominant, and evidence of subtype B transmitting between different high-risk groups was observed. Mutations in the viral protease at position 71 indicated the presence of a selective pressure. Several drug-resistant mutations were detected, although they were predicted with low-level resistance to antiviral treatments. Conclusions Information from this study fills the gap in knowledge of HIV-1 transmission in Changchun city, Jilin province, China. By revealing the origin and evolutionary status of local HIV-1 strains, this work contributes to ongoing efforts in the control and prevention of AIDS.


Introduction
Human immunodeficiency virus type 1 (HIV-1) has continued to cause a global epidemic since its discovery in the early 1980s [1]. The prevalence of HIV-1 in China has also been increasing in recent years [2]. Although many HIV-1 surveillance studies have been conducted in different regions of China [3][4][5][6][7][8][9], only a few cases in northeastern China were reported [10,11], and the current transmission status of HIV-1 in Jilin province remains unknown.
Several pathways are associated with HIV-1 transmission. Injection drug use (IDU) was once the dominant route for the spread of HIV-1 in China [3,12], especially between the adjacent Yunnan and Guangxi provinces. However, recent studies revealed that sexual activities have become the major pathway for HIV-1 transmission [13][14][15], particularly among homosexual partners (or men who have sex with men, MSM) due to their secrecy, low awareness of the need for protection and difficulties in receiving intervention. In addition, infected paid blood donors (PBD) were discovered in Henan and Hubei provinces [14,16], and subtype B HIV-1 strains were found to be highly dominant among this highrisk group throughout the country [8,17]. Previous studies suggested that HIV-1 has been cross-transmitted among high-risk groups [18], which provides greater opportunities for the generation of circulating recombinant forms (CRF) of the virus and increases difficulties for control and prevention efforts.
In this study, an epidemiological survey of 57 HIV-1 infected individuals from Changchun city in Jilin province of northeastern China indicated that sexual contact was the major pathway for local HIV-1 transmission, while phylogenetic analyses on 35 HIV-1 strains revealed that subtype B and CRF01_AE were dominant. The sequences were also screened for drug-resistant mutations. Information gained from the observed transmission patterns among high-risk groups in this region and evidence of selective pressure may be applied towards improving treatment and prevention strategies.

Ethics Statement
Written informed consent was obtained from each participant involved in the study. This study was reviewed and approved by the institutional review board of the First Hospital of Jilin University, Changchun city, Jilin province, China.

Study Subjects
HIV-1-positive subjects from Changchun city in Jilin province, China, were identified and confirmed through annual surveillance by the local Centers for Disease Control in 2012. After pretest counseling, 57 of them participated in this study with informed consent, and their personal identifiable information was encoded. Questionnaires completed by the participants provided demographic and behavioral information. All enrolled patients had previously received antiviral treatments. Blood samples were collected in 2012 and stored at 270uC.

DNA Purification and Sequencing
PCR products were purified for sequencing by using the QIAquick PCR purification kit (Qiagen) according to the manufacturer's protocols. Sequencing of PCR products was performed by Sangon Biotech Ltd. Co.(Shanghai, China) with an automated sequencer (PRISM automated sequencer, version 3100; ABI, Foster City, CA). For an internal control, a random selection of 25% of the samples was re-amplified, sequenced and analyzed. Identical sequences were retrieved from the control group and compared to previous sequencing results.

Alignment and Phylogenetic Analysis
DNA alignments were performed by the ClustalW method under MEGA5 [19], followed by manual adjustment. Reference sequences were retrieved from the Los Alamos National Laboratory HIV Sequence Database (http://www.hiv.lanl.gov). Phylogenetic analysis was also conducted with MEGA5. Neighbor-joining trees were constructed using a Kimura 2-parameter model and tested by the bootstrap method with 1,000 replicates. Bootscanning analysis was also performed with Simplot 3.5.1 [20] to detect potential recombination events.

Study Population
In this study, 57 HIV-1-positive patients, including 47 males and 10 females from Changchun city in Jilin province, China, were identified and confirmed through annual surveillance by local Centers for Disease Control in 2012. Among all 57 patients, 27 were infected through MSM, 23 through heterosexual activity, 6 through PBD and 1 through IDU. Thus, sexual activity (87.72% in total) was the major pathway for HIV-1 transmission in Changchun. Unlike in southern cities, IDU did not contribute significantly to HIV-1 transmission, which was similar to other northern areas [10].

Phylogenetic Analysis of Jilin HIV-1 Strains
Overall, 35 HIV-1 gag/pol sequences (positions 1831-3025 corresponding to HXB2) were successfully retrieved from the 57 samples. Phylogenetic analysis of these amplified gag/pol sequences revealed several subtypes circulating in Changchun ( Figure 1). Subtype B and CRF01_AE were both dominant, each with the same ratio of 37.14% (13 out of 35) ( Table 1). Other HIV-1 subtypes were also detected in Changchun ( Figure 1 and Table 1). Four sequences were identified as CRF07_BC, three as subtype C and one as subtype CRF08_BC, leaving sequence JL2012 confirmed as subtype G.

Origin Analysis for each HIV-1 Subtype in Changchun City, Jilin Province
From the phylogenetic analysis, subtype B strains formed two clusters, B1 and B2, with 100% bootstrap value for each ( Figure 1). All HIV-1 strains retrieved from PBDs were clustered in B1, suggesting that a transmission pathway still exists among this highrisk group. Consistently, BLAST analysis (http://blast.ncbi.nlm. nih.gov/Blast.cgi) indicated that this cluster originated from Henan province where an outbreak of HIV-1 infection once occurred among PBDs (data not shown). However, three strains also were transmitted through heterosexual activities in this same B1 cluster, indicating the presence of HIV-1 transmission between PBDs and people infected through heterosexual contact.
Besides subtype B, CRF01_AE was the other dominant subtype in Changchun city ( Figure 1). It was also separated into two clusters, AE1 and AE2, each of which indicated transmission between people participating in homo-or heterosexual activities. Since the transmission pattern was similar for AE1 and AE2 (both through sexual activities), the formation of different clusters for this one subtype possibly indicated different origins for these strains. Indeed, BLAST analysis of strains from cluster AE1 revealed a high level of similarity (.97%) with other HIV-1 sequences from the neighboring province of Liaoning, which is located to the south of Jilin province. On the other hand, analysis of AE2 strains suggested an HIV-1 origin from a more distant area in China, such as Guangxi province or even from regions abroad (i.e., Vietnam or Thailand), with a maximum identity greater than 96%.
Previously, a unique in-frame deletion was found in the p6 domain of CRF07_BC sequences that was absent from CRF08_BC and CRF01_AE sequences [21,22]. In this study, among all four gag/pol sequences retrieved from CRF07_BC strains in Changchun city, a 7-aa deletion (IDKELYP, according to the HXB2 sequence) could be detected in three of them ( Figure 2). JL2076 as the only CRF07_BC sequence with an intact p6 region and that was transmitted through IDU, clustered with the reference sequence 98CN009, suggesting that it originated from Xinjiang province [23]. Furthermore, JL2036 and JL3085 formed a cluster with high bootstrap value (.93%) ( Figure 1) and were found to originate from Beijing based on the BLAST results. JL3028 did not show a clear origin, but it was from either Beijing or Liaoning. Thus, the analysis indicated that CRF07_BC was introduced to Jilin province from multiple sources.
Interestingly, JL2048 (subtype C) was found to originate from Yunnan, where HIV-1 subtype C is currently no longer dominant and rarely detected. However, BLAST results of JL2005 and JL2006, which were classified as subtype C during the initial phylogenetic analysis, showed extremely high similarity (.98% identity) to recently reported sequences of CRF61_BC [24]. The classification of JL2005 and JL2006 as this newly emerged circulating recombinant form CRF61_BC was further confirmed with phylogenetic tree construction (Figure 3).
Several subtype G strains previously were detected in the provinces of Yunnan, Shandong, Guangdong, Guangxi and Liaoning [25][26][27][28]. However, BLAST analysis of JL2012 showed 95% similarity with multiple sequences from Liberia and Ghana, and only 89% with the one sequence from Liaoning province, which is adjacent to Jilin province. Phylogenetic analysis with subtype G sequences retrieved from GenBank also confirmed that JL2012 had no closer relationship with other Chinese subtype G strains (Figure 4).

Analysis of Drug-resistant Mutations
All patients enrolled in this study had received antiviral treatments. Therefore, all sequenced gag/pol sequences were submitted to the HIV Drug Resistance Database at Stanford University (http://hivdb.stanford.edu/) to detect potential mutations conferring resistance against anti-HIV drugs. In total, ten retrieved sequences (28.57%) were detected with possible drugresistant mutations against either protease inhibitors (PI) or reverse transcriptase (RT) inhibitors (Table 2). Six sequences were detected with possible PI resistance mutations, five of which occurred at position 71 of the viral protease sequence and led to no obvious drug resistance; meanwhile, one sequence had a replacement of V10 with I, which is associated with low level resistance to nelfinavir. As for RT resistance mutations, two sequences were confirmed with mutations such as A62V or K70R against nucleoside reverse transcriptase inhibitors (NRTI), while several non-NRTI (NNRTI) resistance mutations were detected. JL2048 and JL3087 contained mutations at E138 of the viral RT, leading to drug resistance of varying degrees. Notably, due to the undetermined nucleotide, JL1009 contains a mutation K101DEHN, which is highly selected by NNRTI treatment. Other detected mutations, such as V106I and K103Q, are selected by some antiviral drugs, yet no drug resistance has been linked with these mutations.

Discussion
Due to the low rate of HIV-1 transmission in the region studied, only 57 HIV-1 patients from Changchun city were involved in this study. Nevertheless, it provides for the first time important information on the HIV-1 epidemiological pattern within the area of Jilin province. In addition to the most common subtype B in northern China, several other subtypes such as C, G, CRF01_AE, CRF07_BC, CRF08_BC and even the novel  CRF61_BC were detected among the HIV-1 samples collected in northeastern China. The predominant subtypes in Changchun city were B and CRF01_AE, which are different from those of a recent HIV-1 prevalence study in Heilongjiang province in the north of Jilin [10], but highly similar to viruses from Liaoning province in the south [11]. Surprisingly, multiple origins of some HIV-1 subtypes in Changchun were observed ( Figure 5): subtype B appeared to have originated in Henan and Liaoning, while subtype CRF01_AE came from Liaoning, Guangxi and even foreign countries such as Thailand. Multiple origins were also detected for CRF07_BC viruses. In addition, multiple pathways of transmission, such as through heterosexual activity, MSM, PBD and IDU, were found responsible for HIV-1 circulating in Changchun city, further increasing difficulties in prevention and control.
The geographical migration of HIV-1 in China was further demonstrated by the finding of Yunnan, where subtype C viruses are now rarely isolated, as the origin of the subtype C strain JL2048 from Jilin province. The re-classification of the two strains of JL2005 and JL2006 during our analysis from subtype C to CRF61_BC also suggested that circulating recombinant forms are replacing subtype C viruses which were once dominant in China.
HIV-1 subtype G strains are not commonly detected in China, as only 20 samples have been classified in China over the past 10 years (based on searches of the GenBank database). However, this subtype can be more easily detected in Africa, Europe and South America [29][30][31]. As indicated by both BLAST searches and phylogenetic analysis in the current study, JL2012 apparently originated directly from Africa and not within China even though subtype G strain was once detected in the neighboring province of Liaoning. As patient JL2012 had not traveled abroad, this subtype G strain potentially was imported through travelers or labor import, suggesting that increased attention is needed for monitoring international HIV-1 transmissions.
Viral DNA integrated into the human genome provides the template for replication and thus is responsible for passing on mutations, including those conferring antiviral drug resistance, to the next generation of HIV-1 virions. Therefore, we screened all Gag/Pol sequences from this study for antiviral drug resistance through the HIV Drug Resistance Database at Stanford University. All samples were retrieved from patients who had been treated with antivirals for one to three months. NRTI-and NNRTI-resistant mutations were expected to be found, as inhibitors targeting viral RT but not viral protease are widely used as anti-HIV-1 drugs in China. Indeed, we observed seven such mutations in six sequences which may be the result of selective pressure from antiviral drugs. JL1009 was possibly resistant to NNTRIs such as efavirenz (EFV), etravirine (ETR), nevirapine (NVP) and rilpivirine (RPV). However, this finding is inconclusive since multiple possible mutations were detected at JL2048 was predicted to have resistance against ETR and RPV. Notably, an intermediate level of resistance against zidovudine (AZT), along with low-level resistance against stavudine (D4T), was observed in the JL1013 sequence. Since this patient JL1013 was using AZT, these findings indicated that an adjustment in his antiviral therapy was required.
Although no PIs were used during prior treatment of the patients, six samples were detected with mutations in protease, leading to PI resistance of various degrees. Interestingly, five of these sequences contained a mutation at position 71, and all of these five sequences were located in the B1 cluster. Furthermore, A71T/V polymorphisms are expected at the frequency of 2,3% without selection, but the observed ratio was 14.29% (5 out of 35), indicating the existence of additional selective pressure. Another Transmission of HIV-1 in Northeastern China unusual phenomenon was that sequences such as JL1009 contained multiple nucleotide mutations, which could result in potential but still undetermined drug resistance. Nevertheless, all of these observations revealed that the strains were under selection by anti-viral drugs or other factors such as local cellular tropism. As the existence of such mutations certainly presents difficulties in  providing effective antiviral treatment, further confirmation of the drug resistance of these strains should be obtained in order to better control the infection in patients or prevent transmission.