Identification of Two Novel HIV-1 Second-Generation Recombinant Forms (CRF01_AE/CRF07_BC) in Men Who Have Sex with Men in Hebei,China

Introduction

Since the first case of HIV/AIDS was identified in the 1980s, the rapid replication and high mutation rate of HIV type 1 (HIV-1) has led to an epidemic of strains of multiple subtypes and their recombinant forms. There are four groups of HIV-1: M, N, O, and P, respectively. M group, consisted of 10 subtypes (A, B, C, D, F, G, H, I, J, K, and L), was more popular than others and caused global HIV-1 pandemic. Meanwhile, the recombination of different subtypes formed a large number of circulating recombinant forms (CRFs) and unique recombinant forms (URFs). ¹ Up to now, 132 CRFs and a large number of URFs have been reported. ²

Hebei Province is a northern Chinese province that surrounds Tianjin and Beijing. The HIV-1 transmission mechanism and the level of social development in the surrounding areas will affect the HIV-1 prevalence in Hebei due to its unique geographical location. The first case of HIV-1 infection in Hebei Province was reported in 1989, and it soon became an epidemic, mainly through sexual transmission. In Hebei, the first recombinant form (CRF07_BC) was found in 2002. ³ The first recombinant form of CRF01_AE and CRF07_BC was found in 2008, ⁴ and URFs were found in 2011. ⁵ CRF07_BC, CRF01_AE, and B subtypes were the most common genotypes. ⁶ HIV-1 was introduced into the men who have sex with men (MSM) population in Hebei from neighboring metropolises in recent years and quickly spread among the population.

The proportion of MSM among newly reported HIV-1 infections increased from 4.9% in 2005 to 62.6% in 2013. ⁷ MSM has obviously become the main population of HIV-1 infection in Hebei. Furthermore, high-risk sexual behaviors in MSM caused the AIDS epidemic and increased HIV-1 genetic diversity, which may lead to a high level of recombination. ⁸ A previous study published in 2019 found that 77.5% of HIV-1 infections in Hebei were MSM, with 49.6% infected with CRF01_AE strains and 29.7% infected with CRF07_BC strains. ⁹ As a result, the co-circulation and dual infections of CRF01_AE and CRF07_BC in MSM will certainly facilitate the emergence of the second-generation recombinant strain. Besides, new recombinants are not inferior to other HIV-1 M strains in pathogenicity and therapeutic efficacy. The appearance of new recombinants suggests that dual infections or superinfections occur in MSM.

In this study, two new URFs derived from CRF01_AE and CRF07_BC in MSM from Hebei Province were detected by near full-length genome (NFLG) sequence analysis. Plasma samples were collected from two HIV-1 positive individuals (HB070052 and HB070056) of 867 samples infected through homosexual contacts in Tangshan, Hebei. The patients HB070052 and HB070056, a 31-year-old male and a 62-year-old male, were both diagnosed as HIV-1 positive in October 2020 The NFLG sequences of HB070052 and HB070056 were amplified based on subtype inconsistencies in gag, pol, and env refer to Table 1.

The RNA was extracted from 200 μL plasma samples, then complementary DNA was reverse transcribed from the extracted RNA using the Superscript IV First Strand Synthesis System (Invitrogen). After that, both halves of the NFLG were amplified by nested PCR using ExTaq reagent (TaKaRa). Two rounds of PCR were performed under the following conditions: 94°C for 5 min, 94°C for 30 s, 60°C for 30 s, 72°C for 6 min for a total of 30 cycles, and 72°C for a 10 min extension. PCR products were detected by 1% agarose gel electrophoresis, then purified and sequenced by SinoGenoMax (China) with a series of special primers.

In addition, chromatographic data were assembled by ContigExpress software (a component of Vector NTI version 11.5.1; Invitrogen). Then, the two NFLG sequences were aligned with subtype reference sequences and the CRFs' sequences in China (http://hiv.lanl.gov/components/aequnce/HIV/search/search.html) by Mafft (http://mafft.cbrc.jp/alignment/server/index.html) and adjusted manually using BioEdit (version 7.2.5.0).

Furthermore, the phylogenetic tree and subregion tree were constructed using the neighbor-joining method based on the Kimura two-parameter model with 1,000 bootstrap replications by Mega6. Recombination form and breakpoints were identified by the Recombination Identification Program (RIP) (https://hiv.lanl.gol/contents/sequence/RIP/RIP.html), jpHMM (http://jpHMM.gobics.ed/submission_hiv.html) and bootscanning using Simplot (version 3.5.1). The NFLG phylogenetic tree analysis (Fig. 1) suggested the two sequences formed a distinct monophyletic cluster separately from other subtypes and CRFs.

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FIG. 1.

Phylogenetic tree analysis. A neighbor-joining phylogenetic tree of HB070052 (8,771 bp, ●) and HB070056 (8,937 bp, ▲) was constructed based on the NFLG sequences using Mega6.0 with 1,000 bootstrap replications. Bootstrap values ≥90% were shown at the corresponding nodes. The scale bar represents 5% genetic distance. CRF, circulating recombinant form; NFLG, near full-length genome.

The analyses of RIP, jpHMM, and Boostcan demonstrated that both NFLG sequences were composed of CRF01_AE and CRF07_BC. Three CRF07_BC fragments inserted into the CRF01_AE backbone in the NFLG of HB070052. HB070056 was made up of three CRF01 AE fragments inserted with CRF07 BC (Figs. 2 and 3). Subsequently, the mosaic recombinant structure of two sequences was described as follows: ICRF01_AE (HXB2, 776–1,197 nt); IICRF07_BC (HXB2, 1,197–1,858 nt); IIICRF01_AE (HXB2, 1,858–2,065 nt); IVCRF07_BC (HXB2, 2,065–2,462 nt); VCRF01_AE (HXB2, 2,462–4,138 nt); VICRF07_BC (HXB2, 4,138–5,750 nt); VIICRF01_AE (HXB2, 5,750–9,511 nt), HB070052; ICRF01_AE (HXB2, 776–1,177 nt); IICRF07_BC (HXB2, 1,177–1,854 nt); IIICRF01_AE (HXB2, 1,854–4,044 nt); IVCRF07_BC (HXB2, 4,044–4,583 nt); VCRF01_AE (HXB2, 4,583–9,513 nt); HB070056 (Fig. 4).

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FIG. 2.

RIP analysis of the NFLG sequence of HB070052 (A) and HB070056 (B). Similarity distance analysis was performed using RIP (version 3.0; Siepel AC, Halpern AL, Macken C, Korber BT, http://hiv-web.lanl.gov) from the Los Alamos National Laboratory HIV Database with default setting except for the window size of 300. RIP, Recombination Identification Program.

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FIG. 3.

Bootscanning analysis of the NFLG sequence of HB0700052 (A) and HB070056 (B). Bootscanning analysis was performed with reference sequences of subtype G, CRF01_AE, and CRF07_BC by using SimPlot (version 3.5.1). The analysis is set as default setting except for the window size of 400 bp and step size of 100 bp. The x-axis represents the nucleotide position referred to HXB2 and the y-axis represents the bootstrap value (percentage).

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FIG. 4.

Genetic maps of HB070052 (A) and HB070056 (B). The mosaic map was generated using the Recombination HIV-1 Drawing Tool at the Los Alamos National Laboratory HIV Database (http//www.hiv.lanl.gov./content/sequence/DRAW_CRF/recom_mapper.html).

Subregion tree analysis of two NFLGs revealed that the parental origin of all CRF01_AE fragments was from the MSM-related CRF01_AE cluster 4 linkage and cluster 6 linkage (Fig. 5). CRF07_BC reference sequences clustered with CRF07_BC regions in HB070052 (II, IV, and VI) and HB070056 (II and IV). In conclusion, the parental origins of the two novel second-generation URFs were CRF01_AE and CRF07_BC.

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FIG. 5.

Subregion phylogenetic tree. Subregion phylogenetic analysis of different segments of HB070052 (A) and HB070056 (B) were constructed by Mega6 using the neighbor-joining method with 1,000 bootstrap replications. The solid circle (●) marks HB070052 and HB070056. Bootstrap values ≥90% were shown at the corresponding nodes. The scale bar represents 5% genetic distance.

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In this study, we reported two novel CRF01_AE/CRF07_BC recombinant forms from MSM in Hebei Province, China. MSM has become the main source of HIV-1 transmission in Hebei, and the diversity of HIV-1 among MSM is more complex than in other provinces. ¹⁰ A previous study found that the URF, composed of CRF01_AE and CRF07_BC, is the most common second-generation recombination modes of HIV-1 in MSM. ¹¹

Therefore, the two novel URFs in Hebei Province provided the complexity of high-risk behaviors and HIV-1 genetic diversity in MSM. It is critical to continuously survey the prevalence of recombinant HIV-1 subtypes among MSM to control and prevent the HIV-1 epidemic in this region. Although the collected information indicates that the patients were infected through homosexual contact, the actual cause of the infection may still be open to question. Currently, we are developing a sequence analysis-based method to identify the real source of the infected strains, distinguish whether they are from the MSM populations or not, and this may be helpful for finding the most appropriate strategy to interrupt the local transmission of the strains.