Characterization of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant Strain Among Men Who Have Sex with Men in Liaoning,China

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) is due to its high evolutionary variability and recombination rate. Recombinants readily arise when different HIV strains co-circulate in the same area or within a population. To date, >100 circulating recombinant forms (CRFs) and large number of unique recombinant forms (URFs) have been reported in the Los Alamos HIV sequence database (www.hiv.lanl.gov). This increasing number of new recombinants has brought new challenges to the monitoring, diagnosis, treatment, and prevention of the HIV epidemic. ^1,2

China is among the countries that have suffered most from the emergence of complex HIV-1 subtypes and recombinants worldwide. In recent years, HIV-1 infection has spread rapidly among men who have sex with men (MSM) in China, ³ where multiple HIV-1 subtypes have been reported, with CRF01_AE and CRF07_BC domination strains among MSM. Moreover, the CRF01_AE strain has been reported to be further subdivided into at least five to seven distinct lineages because of multiple historical introductions of CRF01_AE strains into China, which subsequently spread in specific populations and regions. ^{4 –6} Among these, two lineages have been reported to be circulating among MSM in China. ⁴ The CRF07_BC strain is reported to have emerged in western Yunnan, China, in the mid-1990s, as a result of recombination between Thai B and Indian C HIV strains, and later spread rapidly in China, mainly through drug trafficking activity ^7,8 and subsequently through the heterosexual route later. ⁹ CRF07_BC strains were introduced into the MSM population in around the early 2000s, forming a unique lineage that has spread among MSM around China. ^3,10 The co-epidemic of CRF01_AE and CRF07_BC strains enables the generation of secondary recombination events.

In this study, a novel CRF01_AE/CRF07_BC HIV-1 recombinant was detected in Liaoning province, northeast China, with three fragments of CRF07_BC inserted into a CRF01_AE backbone, designated as 0107.CN.2015.LN321945, which was phylogenetically close but had a distinct recombination structure from another CRF01_AE/CRF07_BC recombinant isolated in Shaanxi province, northwest China.

The subject, LN321945, was diagnosed with HIV infection in 2015 and self-reported as infected with HIV through homosexual behavior. At the time of diagnosis, the CD4⁺ T cell count and viral load were 615 cells/μL and 34,900 copies/mL, respectively. Routine drug resistance genotyping test showed that the pol gene sequence was CRF01_AE/CRF07_BC recombination structure. To explore its recombinant structure in detail, we further amplified the near full-length genome (NFLG) from plasma viral RNA. The study was approved by the Medical Research Ethics Committee of the First Hospital of China Medical University.

Viral RNA was extracted from plasma and subsequently reverse-transcribed into cDNA. The amplification and sequencing procedures were performed as described previously. ¹¹ Finally, we obtained an NFLG sequence of 8,859 bp (HXB2:790-9611), designated as 0107.CN. 2015.LN321945, spanning the gag, pol, vif, vpr, tat, vpu, rev, env, nef genes, and a part of 3′-LTR. This NFLG sequence was aligned using Clustal W with reference sequences of CRF01_AE, CRF08_BC, CRF07_BC, CRF01_AE/CRF07_BC recombinant forms and pure genotypes of HIV-1 available in the Los Alamos HIV database, then manually adjusted using BioEdit (version 7.0.9.0). Neighbor-joining phylogenetic trees were reconstructed using MEGA 7.0.21 under the Kimura two parameter model with 1,000 bootstrap replications. The online software tools, jpHMM and RIP (www.hiv.lanl.gov), were used to conduct a preliminary analysis of recombination structure, and then the recombination breakpoints defined using SimPlot (version 3.5.1).

According to the NFLG neighbor-joining tree, LN321945 showed a closer link with kang124 (accession no. KJ778897), a CRF01_AE/CRF07_BC URF previously reported from an MSM lived in Shaanxi province (bootstrap value 96%), rather than any previously published CRF01_AE/CRF07_BC URFs in HIV sequences database (www.hiv.lanl.gov) (Fig. 1). Recombination analyses showed that the NFLG sequences of both LN321945 and kang124 consisted of CRF01_AE and CRF07_BC segments (Fig. 2). LN321945 contained seven interlaced segments of CRF01_AE and CRF_07BC. The seven fragments and the positions of breakpoints (relative to the HXB2 genome) were as follows: I, CRF01_AE (790–2,525 nt); II, CRF07_BC (2,526–2,996 nt); III, CRF01_AE (2,997–4,984 nt); IV, CRF07_BC (4,985–8,460 nt); V, CRF01_AE (8,461–8,838 nt); VI, CRF07_BC (8,839–8,914 nt); and VII, CRF01_AE (8,915–9,181 nt). Whereas kang124 comprised three fragments with breakpoints as follows: I, CRF01_AE (790–4,658 nt); II, CRF07_BC (4,659–8,462 nt); and III, CRF01_AE (8,463–9,181 nt). Consistent results were also obtained using the online tools RIP and jpHMM. Furthermore, the breakpoint between LN321945 fragments IV and V, corresponding to the second exon of the tat/rev region in the HXB2 genome, was almost identical to the breakpoint between fragments II and III in kang124. Several studies have suggested that the recombination breakpoints are nonrandomly distributed, with clear regional recombination hotspots, such as the 5′ and 3′ ends of env, vpu, tat, and rev. ^12,13 The mechanism of the recombination hotspots may be attributed to vulnerable sites in the RNA genome, pause sites during reverse transcription, RNA secondary structures, sequence homology, immune selection, and so on. ^14,15

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FIG. 1.

Neighbor-joining tree of the NFLG sequence of LN321945 and publicly available CRF_01AE/CRF07_BC unique recombinant forms. All reference sequences were downloaded from the HIV database (www.hiv.lanl.gov). The solid circle indicates the LN321945 sequence and the solid upward-pointing triangle indicates the kang124 sequence. Bootstrap values (≥70%) are presented at the corresponding nodes. Scale bar represents 2% genetic distance. NFLG, near full-length genome.

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FIG. 2.

Bootscan analysis of the LN321945 (A) and kang124 (B) near full-length sequences. Publicly available CRF01_AE and CRF07_BC NFLG sequences, with the high similarity to the recombinant fragments, were selected as reference sequences based on the results of the HIV database online program, BLAST (www.hiv.lanl.gov/content/sequence/BASIC_BLAST). SimPlot (version 3.5.1) parameter selection criteria were 200 bps window size and 20 bps step size.

To further explain the close relationship between LN321945 and kang124 in the phylogenetic tree, subregion trees were reconstructed using MEGA 7.0.21 (Fig. 3), including seven CRF01_AE lineage, ⁴ CRF07_BC, and pure genotypes of HIV-1 group M as reference sequences. The results demonstrated that the fragment spanning the vif, vpr, vpu, env, tat, and rev genes of LN321945 and kang124 (corresponding to fragment IV of LN321945 and fragment II of kang124) clustered with the CRF07_BC lineage predominant in MSM, rather than those from intravenous drug users in China. Four fragments of LN321945 (I, III, V, and VII) clustered together with CRF01_AE lineage 4, but the corresponding regions of kang124 clustered with CRF01_AE lineage 5. Both lineage 4 and lineage 5 of CRF01_AE have been reported to be widespread among MSM in China; however, lineage 5 is more common in northern China, including Liaoning province. ^10,11 These results show that the CRF01_AE fragments of LN321945 have a distinct origin from kang124, although their NFLG sequences were close together in the phylogenetic tree.

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FIG. 3.

Subregion trees analysis for five fragments of LN321945 and kang124. Solid circles indicate LN321945 fragments and solid upward-pointing triangles indicate kang124 fragments. CRF01_AE L1 to CRF01_AE L7 represent seven lineages of CRF01_AE in China. ⁴ CRF07_BC Lineage1 and CRF07_BC Lineage2 are spread among the intravenous drug users and men who have sex with men populations, respectively, in China. Scale bars are presented at the bottom right of each tree. Bootstrap values ≥80% are presented at the corresponding nodes.

In summary, we detected a novel HIV-1 CRF01_AE/CRF07_BC URF from an MSM lived in Liaoning province, northeast China, which is a second-generation recombinant from two predominant strains (CRF01_AE and CRF07_BC) among MSM in China. These findings imply a more complicated trend of HIV-1 epidemic in China.

Sequence Data

The nucleotide sequence of LN321945 has been submitted to GenBank with the accession no. KX434796.