Multiple HIV-1 Subtypes Were Found Circulating in Shijingshan District of Beijing,China

Abstract

High level of HIV prevalence was found in some specific areas or populations in China. Shijingshan district was one of the areas with rapid increase of newly reported HIV cases in Beijing, China. Comprehensive investigation on HIV genetic characteristics is necessary in the district. In this study, we enrolled 223 newly identified HIV-1 sero-positive drug-naive patients from Shijingshan district. The full-length gag, pol, and partial env gene sequences were amplified and sequenced. Genetic characterization and transmission of drug resistance of HIV-1 were further investigated. Many stains, including CRF01_AE (48.25%), CRF07_BC (31.47%), subtype B (4.9%), 55_01B (1.40%), 59_01B (0.70%), 65_cpx (2.10%), 79_0107 (0.70%), and A (0.70%) and unique recombinant forms (URF) of 0107 (6.29%), 01B (1.40%), and 01BC (2.10%), were identified. Among 106 strains that harbor full-length PR gene, 10 (9.43%) strains contain mutations associated with drug resistance to protease inhibitors. Among 59 strains that harbor full-length reverse transcriptase (RT) gene, no strains contained mutations conferred to nucleoside reverse transcriptase inhibitors resistance and 4 strains (6.78%) contained drug-resistant mutations to non-nucleoside reverse transcriptase inhibitors. Among 58 sequences harboring full-length IN gene, 1 stain (1.72%) contained a mutation causing drug resistance to integrase strand transfer inhibitors. Our results provided detailed genetic data on HIV prevalent in Shijingshan district of Beijing, which would be helpful for understanding HIV-1 transmission among local populations.

The first HIV-positive case in China was identified in 1985.¹ Subsequently, two outbreaks of HIV infections happened among intravenous drug users in Yunnan province^1
–3 and former blood and plasma donors in central China, respectively.⁴ After that, HIV infections due to sexual transmission have increased dramatically and have become the primary route of transmission after 2007. Accordingly, the reported HIV-positive cases attributed to sexual transmission keep increasing in China.⁵ Without doubt, HIV has spread out from initial high-risk population to general population in China.

Beijing, the capital of China, is a metropolitan area with high proportion of floating population, which always conferred to HIV transmission. In recent years, the number of newly reported HIV cases in the city increased dramatically, especially in the MSM (men who have sex with men) population.⁶ Initially, all of HIV infection confirmation tests were fulfilled in the city CDC (Center of Disease Control and Prevention). After the middle of 2010s, the local district CDC has started HIV antibody confirmation test, which would be convenient for district CDC to monitor HIV epidemic in a local area.

Shijingshan district is one of the six central districts of Beijing and contains 639,000 people. The first HIV infection was identified in 2001. After that, HIV epidemic in the district has spread quickly with newly reported HIV-positive cases increasing from 1 in 2001 to 223 in 2016. However, no comprehensive study has been fulfilled on the HIV epidemic in the district. It is important to characterize the HIV epidemic in the area and investigate the possible reason on rapid spreading of HIV. This study enrolled all newly reported HIV-positive cases in 2016 in Shijingshan district. HIV genetic characterizations and drug resistance were analyzed based on full-length gag, pol, and partial env genes.

A total of 223 individuals who were confirmed as HIV antibody positive in Shijingshan district CDC in 2016 were enrolled into the study with informed consent. Demographic information is summarized in Table 1. The average age of all HIV-positive cases was 37.14 years (ranging from 0.67 to 91 years). Male cases (197, 88.34%) are obviously more than female cases (26, 11.66%). The transmission routes were complicated. The most dominant transmission route was MSM (134, 60.09%), followed by heterosexual transmission (5, 2.24%), intravenous drug use (1, 0.45%), and blood-borne transmission (1, 0.45%). The transmission routes of the other cases (82, 36.77%) were unknown.

Table 1.

Sociodemographic Characteristics of Participants

Characteristics	Case no. and percentage
Age (years old)
2–14	0 (0.0)
15–19	1 (0.4)
20–25	36 (14.4)
26–29	41 (16.4)
30–34	65 (26.0)
35–39	40 (16.0)
40–49	48 (19.2)
≥50	18 (7.2)
Unknown	1 (0.4)
Gender
Male	197 (88.34)
Female	26 (11.66)
Unknown	0 (0)
Route of transmission
Heterosexual	5 (2.24)
MSM	134 (60.09)
IDU	1 (0.45)
Blood borne	1 (0.45)
Unknown	82 (36.77)

MSM, men who have sex with men; IDU, injection drug user.

Viral RNA was extracted from plasma. Then HIV full-length gag (nucleotide 790–2,292 by using HXB2 as calibrator), pol (nucleotide 2,085–5,096 by using HXB2 as calibrator), and partial env (C2V3 region) genes were amplified and sequenced as described before.⁷ Possible contaminations were excluded by using BLAST search (http://hiv-web.lanl.gov/content/index). Contiguous sequences were assembled with sequenced segments from the same sample and edited manually according to the graph of ABI files.⁸ Finally, 114 full-length gag genes (51.12%), 110 pol genes (49.33%, including 58 full-length and 52 partial genes), and 110 env C2V3 genes (49.33%) were successfully obtained.

The inconsistency in obtaining different genes was probably due to the sequence variations at the primer binding sites or genetic diversity of HIV quasi-species in different gene regions. All assembled sequences were submitted to the HIV-1 Sequence Quality Control Tool (https://www.hiv.lanl.gov/content/sequence/QC/index.html) to confirm sequence quality. The results showed that all of the gene structures were normal with the correct open reading frames.

All assembled sequences were submitted to the HIV-1 Sequence Quality Control Tool (https://www.hiv.lanl.gov/content/sequence/QC/index.html) and REGA^9,10 to determine HIV subtypes. Quality control tool uses both Basic Local Alignment Search Tool (BLAST) and Recombinant Identification Program (RIP) to look for the best subtype. The REGA tool is designed to use phylogenetic methods to identify the subtype of a specific sequence. Despite phylogenetic methods have been used during subtyping by REGA, these results were further confirmed by the phylogenetic tree using the reference sequences representing subtypes A–D, F–H, J, K, CRF01_AE, CRF07_BC, CRF55_01B, and CRF65_cpx (www.hiv.lanl.gov) and all 114 full-length gag genes (Fig. 1). To construct the phylogenetic trees, all of our sequences were aligned together with reference sequences. Phylogenetic trees were generated using maximum likelihood (ML) methods in the MEGA6 software package.¹¹ The reliability of topologies was estimated by performing bootstrap analysis with 1,000 replicates. In the phylogentic tree, the samples were tightly clustered within their respective subtypes. Finally, a total of 143 strains successfully obtained their subtypes. Sixty-nine of them were CRF01_AE (48.25%), 45 were CRF07_BC (31.47%), and 7 strains were subtype B (4.9%). In addition, there existed other stains including 55_01B (1.40%), 59_01B (0.70%), 65_cpx (2.10%), 79_0107 (0.70%), subtype A (0.70%), and URFs of 0107 (6.29%), 01B (1.40%), and 01BC (2.10%). Among 110 subtyped stains from MSM, CRF01_AE accounts for 48.18%, CRF07_BC accounts for 30%, and subtype B accounts for 4.55%. In earlier studies exploring HIV subtypes in MSM in China, subtype B was always the dominant strain.^12,13 In this study, we found that CRF01_AE strains have become the dominant strain in Shijingshan MSM, which is similar to the change found in MSM of the whole Beijing city.¹⁴ In China, CRF01_AE was initially associated with heterosexual transmission, the dramatic increase of CRF01_AE strains in MSM indicates that blend of MSM and heterosexual transmission population might be happening in China.

FIG. 1.

The ML phylogenetic tree was built using 114 HIV-1 full-length gag sequences from treatment-naive HIV-1 infectors in Beijing Shijingshan district. The reference gene sequences of subtypes A–D, F–H, J, K, CRF01_AE, CRF07_BC, CRF55_01B, CRF65_cpx, and group O were downloaded from the HIV database (www.hiv.lanl.gov/content/index). Each reference sequence is labeled with the HIV-1 subtype, followed by country, sampling year, name, and accession number. Bootstrap probabilities are indicated at the corresponding nodes of the tree. ML, maximum likelihood.

To explore the potential origins of three dominant subtypes prevalent in Shijingshan district, including CRF07_BC, CRF01_AE, and subtype B, further analysis was performed to investigate the phylogenetic relationship of our sequences with those from other regions of China. BLAST search was used to screen sequences most similar to ours. All sequences were further aligned and manually edited. ML phylogenetic trees were constructed using full-length gag gene. For all three subtypes, our strains intermixed with those from Beijing, Shijiazhuang, and Liaoning regions in the ML tree (Fig. 2). Considering that most of our strains were from MSM, the results strongly supposed that there were a close behavior contact between MSM populations from Beijing, Shijiazhuang, and Liaoning regions. Therefore, behavior interventions should be considered to prevent HIV transmission among MSM residing in these areas.

FIG. 2.

Evolutionary relationship between HIV-1 sequences including CRF01_AE, CRF07_BC, and subtype B from Shijingshan and other areas. The bootstrap probability (>70%, 1,000 replicates) is indicated at the corresponding nodes of the tree. The scale bar represents 2% genetic distance. The circle represents references of CRF01_AE. The triangle represents references of subtype B. The square represents references of CRF07_BC. Red indicates references from Beijing. Blue represents references from Liaoning. Green represents references from Shijiazhuang. Color images are available online.

The Chinese government started “Four Free and One Care” project in 2003, and since then highly active antiroviral therapy (HAART) has been widely used, which led to the prevalence of HIV-1 drug-resistant strains in some specific areas or populations.¹⁵ With the increasing level of HIV drug resistance in populations that undertook antiretroviral therapy (ART), transmission of drug-resistant strains has also become popular. An investigation in Liaoning province among ART-naive AIDS patients indicated that 4.4% HIV-1-infected people already contained drug-resistant strains even if they had never received a course of ART treatment.¹⁶ In this study, all participants were drug-naive individuals, which will be helpful to predict the level of HIV transmission of drug resistance. The 110 qualified pol genes obtained in this study were submitted to the online Calibrated Population Resistance tool (v5.0 beta)¹⁷ (http://hivdb.stanford.edu) to identify the transmitted drug resistance mutations. The detailed summary of drug resistance mutations is given in Table 2. Among 107 samples that harbor full-length PR region, 10 (9.43%) strains contained mutations associated with drug resistance. Among 59 samples that harbor full-length reverse transcriptase (RT) region, no mutations that conferred to nucleoside reverse transcriptase inhibitors (NRTIs) resistance were found, whereas 4 strains (6.78%) contained drug-resistant mutations related to non-NRTIs (NNRTIs). Among 58 samples that harbor full-length IN region, 1 stain (1.72%) contained a drug-resistant mutation conferred to integrase strand transfer inhibitors. In a previous study, high level of transmission of HIV drug-resistant strains as 15% was found in Beijing. Different to that study, much low level of HIV drug-resistant transmission was found in Shijingshan district of Beijing city. It might be because those high-risk populations of HIV infection in Beijing had changed over time. Therefore, patients with failing therapy are not a major source of new infections.

Table 2.

Drug-Resistant Mutations Detected in Newly Reported HIV-Infected Individuals in Shijingshan

ID	Drug-resistant mutations
ID	PIs	NRTIs	NNRTIs	INIs
67	N83D
98	M46L
107	L33F
120	V32VL, I47IM
60	M46L
61	M46L
103	K43T
163	Q58E
199	F53L		H221Y	E92K
215	V32VE
173			V179D
174			V179E
214			V179D

INI, integrase strand transfer inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; NNRTI, non-NRTIs; PIs, protease inhibitors

In this study, we found that MSM was the main population related to HIV infection in the Shijingshan district of Beijing city. In China, MSM are a special population who play more and more important roles in the spread of HIV. Owing to social discrimination and cultural stigma associated with homosexual behavior in China, 31.5% of HIV-positive MSM have been estimated to be engaged in sex with both men and women within and out of marriage.¹² This behavior facilitates the transmission of HIV from high-risk populations to the general population and contributes to the growing number of HIV-positive women infected through unprotected sex.⁷ Therefore, MSM will become the main population for behavior intervention for HIV prevention in China, especially in big cities. Genetic studies will provide detailed genetic data and would be helpful for prediction of transmission routes based on transmission network or phylodynamic analysis.

Sequence data

The gene sequences are deposited in the GenBank with accession numbers MH920854–MH921187.

Footnotes

Acknowledgments

This study was funded by the National Natural Science Foundation of China (no. 81773493), the National Key S&T Special Projects on Major Infectious Diseases (grant no. 2012ZX10002), and the State Key Laboratory of Pathogen and Biosecurity (Academy of Military Medical Science). This study was supported by the grant 2017YFC1200800, 16CXZ030.

Author Contributions

L.L. and H.L. designed the study; L.S., Y.L., and J.H. carried out the experimental data; L.J, J.L., and H.L. performed the analysis; and L.J. and L.L. wrote the article.

Author Disclosure Statement

No competing financial interests exist.

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