The Epidemic History of HIV-1 CRF07_BC in Hetian Prefecture and the Role of It on HIV Spreading in China

CRF07_BC is one of the four predominant strains circulating in China. ¹ It originated from subtypes B' and C prevailing in Yunnan province located in southwest China in the 1980s. ^{2 –5} After that, Yunnan province became the first epicenter of CRF07_BC spreading to other regions of China, especially CRF07_BC spreading to the west of China along with the drug trafficking route. ⁶ In 1997, CRF07_BC was first detected in Xinjiang Uygur Autonomous Region, ⁴ then in Gansu and Sichuan province. In recent years, some studies show that Xinjiang is the second epicenter of CRF07_BC spreading to other regions of China based on temporal–spatial dynamics analysis of B'/C recombinants circulating in China. ^7,8

Hetian Prefecture, located in the south of Xinjiang Uygur Autonomous Region of China, has more than 2.12 million people. By the end of 2014, 1956 HIV and AIDS patients were accumulatively reported. Previous studies showed the Xinjiang Uygur Autonomous Region was the second epicenter of CRF07_BC spreading to other regions of China. To know the role of Hetian Prefecture on HIV spread in China, we investigated the molecular epidemiological characteristic of HIV circulating in Hetian Prefecture.

In total, 208 HIV-1-positive samples were collected from Hetian Prefecture. All subjects were diagnosed as HIV-1 infected from 2006 to 2014. After ELISA-positive screening, each patient was confirmed to be infected by Western blot (HIV blot 2.2; Genelab Technologies, Inc., Singapore). Whole blood samples were collected using sterile ethylenediaminetetraacetic acid tubes. The plasma was centrifugally separated at 3,000 rpm within 6 h, and then kept at −80°C for viral RNA extraction. The study was reviewed and approved by the ethical committee at the Anhui Center for Disease Control and Prevention. Written informed consent was obtained from all participants after we informed them of the objective of this study.

Viral RNA was extracted from 140 μl of plasma using the QIAamp Viral RNA Mini kit (Qiagen, Valencia, CA). HIV-1 segments gag (p17 and partial p24) and pol (protease and p51RT) were amplified using reverse transcriptase (RT)-nested polymerase chain reaction (PCR). The first PCRs were performed using the Superscript TM III one-step RT-PCR system with platinum Taq DNA polymerase (Invitrogen) with outer primer pair GAG-L/GAG-E2 or MAW26/RT21. The second PCRs were performed using the TaKaRa ExTaq kit (TaKaRa Biotechnology Co. Ltd., Dalian, China) with inner primer pair GAGF2/c-gag or PRO-1/RT20. The sequences of primers used in this study have been previously described in detail. ⁹ Amplified PCR products were separated on an agarose gel and purified using the QiaQuick gel extraction kit (Qiagen, Valencia, CA). Purified products were subjected to direct DNA sequencing using an automated ABI 3730/3730xl DNA analyzer by Shanghai GeneCore BioTechnologies Co., Ltd.

To avoid potential laboratory errors, all the nucleotide sequences obtained in this study were screened using the online HIVBLAST Search tool (www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html) to search for sequences similar to previously reported sequences in the HIV database. Nucleotide sequences were aligned with reference strains using the ClustalW program implemented in MEGA 6.0, and were further adjusted manually. All sequences were submitted to online server using REGA HIV-1 Subtyping Tool—Version 3.0(http://dbpartners.stanford.edu:8080/RegaSubtyping/stanford-hiv/typingtool) to conduct subtype analysis.

Maximum clade credibility trees were constructed using Markov Chain Monte Carlo (MCMC) inference implemented in the BEAST v1.82 package using the relaxed log-normal molecular clock according to that previously described. ¹⁰ The MCMC chains were run 20 million times and sampled every 1,000 steps. Bayesian MCMC output was analyzed using TRACER v1.5 and all parameters were estimated from an ESS >200.

As shown in Table 1, 91.95% of people with HIV infection are between 20 and 49 years old. Of the participants, 56.73% are male and 43.27% are female. A total of 39.42% of them were divorced and 40.87% were single. Only 19.71% were married. Of them 99.52% belong to the Uygur ethnic group. Most of them received primary school or middle school level education, which accounted for 33.17%, and 49.52%, respectively. There are four transmission routes, that is, men who have sex with men, heterosexual behavior, intravenous drug user (IDU), and mother to infant. Among transmission routes, heterosexuality accounts for 87.03%. Among 208 HIV infectors, 32 are AIDS patients.

OPEN IN VIEWER

Table 1.

Basic Characteristics of the HIV-1-Positive Population Living in Hetian Prefecture, China

Variable	n	%
Age
<20	6	2.86
20–29	56	26.94
30–39	91	43.75
40–49	44	21.16
≥50	11	5.29
Sex
Male	118	56.73
Female	90	43.27
Marriage status
Divorced	82	39.42
Married	41	19.71
Single	85	40.87
Ethnic group
Uygur	207	99.52
Miao	1	0.48
Education level
Less than primary school	12	5.77
Primary school	69	33.17
Middle school	103	49.52
High school	11	5.29
College	13	6.25
Transmission route
MSM	2	0.96
Heterosexual	181	87.03
IDU	19	9.13
Unconfirmation	1	0.48
HIV	176	84.62
AIDS	32	15.38

IDU, intravenous drug user; MSM, men who sex with men.

Among 208 specimens, 199 were successfully amplified and sequenced. Among 199 sequences, 194(97.49%) belong to CRF07_BC, which is the predominant form of HIV circulating in this region (Fig. 1), and introduced to Hetian Prefecture before 1990 (shown in Fig. 2). Three CRF01_AEs, one subtype B, and one subtype C were identified, respectively.

OPEN IN VIEWER

FIG. 1.

The distribution of various HIV-1 subtypes and CRFs in Hetian Prefecture, China. Color images available online at www.liebertpub.com/aid

OPEN IN VIEWER

FIG. 2.

Maximum clade credibility trees based on partial pol gene segment of HIV-1. Red line indicates the sequence of HIV-1 isolated from Hetian Prefecture, blue line indicates sequences isolated from Xinjiang Uygur Autonomous Region except Hetian Prefecture, Black line indicates sequences isolated from other provinces of China. Color images available online at www.liebertpub.com/aid

To identify the origin and the role of CRF07_BC circulating in Hetian Prefecture on HIV-1 temporal spread in the whole of China, we estimated the dates of tMRCA based on a data set of partial nucleotide sequences of pol gene of CRF07_BC strains, which includes 199 sequences determined in this study in addition to 24 reference sequences with specific sampling times consisting of 8 from Xinjiang Uygur Autonomous Region and 16 from other provinces of China. As shown in Figure 2, CRF07_BC circulating in Hetian Prefecture formed two major clusters (cluster a and cluster b). Cluster b consists of 1 sequence from Xinjiang Uygur Autonomous Region and 15 sequences from Hetian Prefecture, they share the common ancestor, and the latter emerged earlier. This implies that one of the origins of HIV circulating in Hetian is the virus prevailing in other prefectures of Xinjiang Uygur Autonomous Region.

Cluster a consists of eight subclusters. Among subclusters 1, 2, and 3, the sequences isolated in Hetian Prefecture cluster closely with the sequences from other provinces of China. The estimated tMRCAs show that the sequences other than those from Xinjiang Uygur Autonomous Region prevailed earlier than those from Hetian. This could be one of the origins of HIV-1 circulating in Hetian Prefecture. However, in subcluster 8, the estimated tMRCAs indicate that the virus spread from Hetian Prefecture to other regions of China. Among subclusters 4,5, and 6, the sequences of Hetian Prefecture interwine with sequences from Xinjiang Uygur Autonomous Region; it is difficult to confirm the direction of spread between Hetian Prefecture and other regions in the Xinjiang Uygur Autonomous Region.

CRF07_BC has been the predominant HIV-1 among IDUs in China, especially in time when the IDUs were the major population of HIV-1 spreading in China. ^11,12 At that time, CRF07_BC was mainly spreading by IDUs. In this phase, the earlier CRF07_BC epidemic region may be a center of HIV spreading to other regions of China. ¹² About a decade ago, sexual behavior was the major transmission route spreading HIV-1 gradually, and HIV began to prevail among the general population in China. ^{13 –15} Moreover, migration of population takes place in all cities of China. Therefore, currently, every region may be only a point of HIV transmission net, no absolute epidemic center exists. In this point, controlling of HIV spread must cover all regions in the country, even throughout the world. The measure only to focus on the epidemic with high incidence fails to control global HIV prevalence.

Sequence Data

The sequences used in this study have been submitted to GenBank with accession numbers KX305973–KX306175 of partial pol gene fragment and KX306176–KX306371 of partial gag gene fragment.