Genomic Characterization of a Novel HIV-1 Second-Generation Recombinant Form (CRF01_AE/07_BC) from a Married Man Who Has Sex with Men in Zhejiang,China

Since the first case of HIV infection through sexual transmission in mainland China was reported in 1985, ¹ the HIV epidemic has become increasingly genetically complex, including various recombinant forms of strains cocirculating across the country. So far, there are four predominant subtypes, circulating recombinant forms, CRF01_AE, CRF07_BC, CRF08_BC, and B (Thai B), estimated to have contributed to >90% of the total HIV-positive cases in China. ² In the early 1990s, CRF01_AE strains were first identified among populations at risk of sexual transmission and injection drug users (IDUs) in southwest China. ^3,4 Recently, CRF01_AE strains have also become the predominant strain among men who have sex with men (MSM) in China, ⁵ among which clusters 4 and 5 are prevailing in northern provinces. ⁶ In the late 1990s, CRF07_BC was initially found among the IDU population in western China. ⁷ However, in recent years, CRF07_BC strain began to expand to the entire country, and has been detected in various high-risk populations, especially among MSMs. ⁸

Despite progress on issues such as marriage equality and decriminalization of same-sex behavior in some countries, there has been a marked increase in antigay legislation in many countries. In China, owing to traditional thoughts and family values, Chinese MSM often choose to marry to conceal their homosexuality behavior from society. It was estimated that ∼26.3% of MSM in China have had sex with females in the past 6 months and 17.0% are currently married to a woman. This indicated that bisexual behaviors among Chinese MSM are common. ⁹

Sample 17ZJ075 was collected from a male patient residing in Zhejiang Province who was confirmed as HIV positive on January 25, 2017, and was recruited by the Zhejiang Provincial Center for Disease Control and Prevention (ZJCDC). Sample 17ZJ075 is a Chinese citizen of Han ethnicity, 35 years old, residing in Lin'an District. He presented with headache, nausea, and vomiting for about 10 days, then went to a local hospital for help. Epidemiological survey data showed that the patient was married, however, had been infected through homosexual behavior. By near full-length genome (NFLG) analyses, we detected a novel HIV-1 second-generation recombinant form (CRF01_AE/CRF07_BC), which is highly different from previously reported CRF01_AE/CRF07_BC recombinant viruses in Zhejiang province. Written informed consent was obtained from the subject before sample collection, and was approved by the institutional review board of the First Affiliated Hospital, School of Medicine, Zhejiang University.

The amplification and sequencing analyses of the new generated NFLG were performed as previously described. ¹⁰ Nucleotide sequences were first aligned using HIV Align (www.hiv.lanl.gov) and then adjusted by using BioEdit. An approximately maximum likelihood (ML) phylogenetic tree was built using software FastTree 2.3¹¹ under the generalized time-reversible (GTR)+G+I nucleotide substitution model, in comparison with the standard subtype reference alignment file, including all HIV-1 group M, group P, CRF01_AE, CRF07_BC, and CRF08_BC, downloaded from the Los Alamos National Laboratory HIV Database (www.hiv.lanl.gov). The final tree was visualized in Figtree v1.4.2. To analyze the recombination form of 17ZJ075 strain, the sequence was submitted to RIP and jpHMM (www.hiv.lanl.gov). Similarity plot analysis was performed with SimPlot (version 3.5.1). Bootscan analysis was carried out using the same software to position the recombination breaking point. Breakpoint confirmation and origin of each segment were then analyzed by phylogenetic trees in FastTree+FigTree using the ML method with GTR model and 1,000 bootstrap replications to confirm the subtype of mosaic fragments. ¹² Branches with bootstrap values higher than 0.7 were considered as phylogenetic clusters.

17ZJ075 was 8,983 nucleotides in size (relative to the HXB2 nucleotide numbering system: positions 631 to 9719) and the blast search data showed that no evidence of sample contamination was detected. 17ZJ075 clustered with CRF07_BC subtype reference sequences (bootstrap value 100%), but formed a distinct monophyletic branch different from CRF07_BC subtype reference sequences (Fig. 1). SimPlot analysis showed that the NFLG sequence of 17ZJ075 contained at least three interlaced mosaic segments, with one region of CRF01_AE inserted into a CRF07_BC backbone (Fig. 2A). Bootscanning analysis of the NFLG sequence reveals that the two unique recombination breakpoints corresponded to HXB2 nucleotide positions 6060 and 8350, located in tat/vpu and env gene regions, respectively, and divided the NFLG into three regions. The map of the new recombinant virus MSM0720 is as follows: region I (HXB2, 631-6059), region II (HXB2, 6060-8349), and region III (HXB2, 8350-9719) (Fig. 2B). Similar results were obtained using online software RIP (www.hiv.lanl.gov/content/sequence/RIP/RIP.html) and jpHMM-HIV (http://jphmm.gobics.de/submission_hiv.html). The final map results of the schematic structure are shown in Figure 2C. Subregion tree analysis of 17ZJ075 further confirmed the two recombinant breakpoints identified by bootscanning analysis (Fig. 3). It also demonstrated that the CRF01_AE region (II) in 17ZJ075 belonged to CRF01_AE subcluster 4 lineage. The CRF07_BC regions (I and III) in 17ZJ075 clustered with CRF07_BC reference sequences (bootstrap value 97.7% and 100%). In summary, the parental origins of the CRF01_AE and CRF07_BC regions of novel second-generation recombinant (17ZJ075) could be from CRF01_AE cluster 4 and CRF07-BC.

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FIG. 1.

Phylogenetic tree analysis of the near full-length genome nucleotide sequence of the subject, 17ZJ075. The tree was constructed through the ML method with 1,000 bootstrap replicates. The solid black circle denotes 17ZJ075. All reference sequences of the HIV-1 group M subtypes and CRFs were downloaded from the HIV database (www.hiv.lanl.gov/). The scale bar represents a 0.04 genetic distance. CRF, circulating recombinant form; ML, maximum likelihood. Figure 1 can be viewed in greater detail online.

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FIG. 2.

Recombinant analyses of the near full-length genome nucleotide sequence of 17ZJ075. (A) The simplot analysis between 17ZJ075 and the reference sequences. (B) Bootscanning analysis of 17ZJ075 using CRF01_AE, CRF07_BC, and subtype J as reference sequences. The bootscan window was 300 bp with a step size of 20 bp using SimPlot version 3.5.1. (C) The results of the Recombinant HIV-1 Drawing Tool with the two breakpoints of 17ZJ075 according to HXB2 nucleotide sequence numbering.

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FIG. 3.

The subregion trees of one CRF01_AE and two CRF07_BC segments of 17ZJ075 were established by the methods shown in Figure 1 and based on the recombinant breakpoints shown in Figure 2C. Figure 3 can be viewed in greater detail online.

CRF01_AE and CRF07_BC are two major HIV-1 circulating strains in Zhejiang Province, whose proportion of the HIV-1 subtypes among MSM was 64.2% and 31.7%, respectively. ¹³ In the study, a novel unique recombinant form (URF) strain 17ZJ075 was characterized with a CRF01_AE fragment inserted into a CRF07_BC parental backbone. Although the three novel second-generation recombinants are unrelated variants, they may indicate that diverse recombination forms exist between CRF01_AE and CRF07_BC prevailing in Zhejiang Province. In China, due to high social stigma, discrimination, and family pressure, large number of MSM conceal their sexual orientation and have multiple sex partners. Moreover, because of China's one-child policy, they are often the only son in their families, they have to marry to satisfy their parents' expectation to have children to continue the family line. Furthermore, according to recent analysis, low condom usage was common between married MSM and their wives, hence posing a higher risk of transmitting HIV. ⁹ Such a phenomenon facilitates the transmission of HIV-1 and increases the difficulty of HIV/AIDS prevention and control work. Therefore, harm-reduction programs targeting married MSM and their female partners and an effective and continuous HIV-1 molecular epidemiologic monitoring are necessary to curb the further spread of HIV infection to the general female population.