Characterization of Two Novel HIV-1B/CRF01_AE/CRF07_BC Recombinant Forms Among Men Who Have Sex with Men in Hebei Province,China

Human immunodeficiency virus (HIV) exhibits remarkable genetic diversity which has significant implications for its evolution and epidemiology. Co-infection with multiple HIV-1 subtypes facilitates viral recombination. ¹ Globally, HIV-1 is classified into four groups based on the homology of the env gene (encoding envelope proteins) and the gag gene (encoding capsid proteins), including groups M, N, O, and P. Group M is the most prevalent worldwide and further divided into 10 subtypes (A-D, F-H, J-L) and 2 sub-subtypes (A1/A2 and F1/F2). ² In addition, 158 HIV circulating recombinant forms (CRFs) (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html) and numerous unique recombinant forms (URFs) have been documented, reflecting extensive recombination events.

In China, the transmission dynamics of HIV-1 have shifted in recent years, and sexual transmission has become the most dominant infection pathway. Molecular epidemiological investigation revealed that CRF07_BC and CRF01_AE were the predominant subtypes driving sexual transmission networks, especially among men who have sex with men (MSM), followed by CRF08_BC and subtype B.^3,4 In Hebei Province, a region in Northern China characterized by relatively low HIV-1 prevalence, the recent molecular surveillance has identified the presence of four CRFs (CRF80_0107, CRF103_01B, CRF123_0107, and CRF159_01103) and several URFs (URFs_0107 and URFs_01B).^5–8 These findings underscore the evolving molecular diversity of HIV-1 and its epidemiological implications in this region. In this study, we identified two new HIV-1 URFs_B/CRF01_AE/CRF07_BC (sample IDs: BD076A and BDL161), among MSM in Hebei Province, China, through near-full-length genome (NFLG) analysis.

Both BD076A, aged 64 years, and BDL161, aged 23 year, were two MSM and diagnosed as HIV-1 positive through Western blot testing in August 2021 and April 2023, respectively. Both were infected with HIV-1 via homosexual sexual contact. This study was conducted in strict accordance with ethical guidelines. And it was approved by the Medical Ethics Committee of Baoding People’s Hospital (protocol number 2023-04). Prior to sample collection, written informed consent was obtained from both participants.

We carried out HIV-1 RNA extraction, amplification, sequencing, and sequence assembly of NFLGs in accordance with methods described in the reference. ⁹ In brief, viral RNA was extracted from participants` plasma (280 μL) using the QIAamp Viral RNA Mini Kit (Catalog no. 52904, Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. Then, following the method described in the previous report, we used PrimeScript™ III Reverse Transcriptase (Code No. TCH0 13, TaKaRa Biotechnology, Dalian, China) and TaKaRa PrimeSTAR® GXL DNA Polymerase (Code No. R050A, TaKaRa Biotechnology, Dalian, China) for RT-PCR (reverse transcription nested polymerase chain reaction). PCR products were detected using 1.0% agarose gel electrophoresis, purified from the corresponding electrophoretic bands, and sequenced using Sanger sequencing technology by Tianyi Huiyuan Bioscience & Technology Inc. (Beijing, China). Standard reference sequences for HIV-1 subtypes were downloaded from HIV databases (https://hiv.lanl.gov/components/sequence/HIV/search/search.html). Sequences were aligned using MAFFT version 7, followed by terminal sequence trimming and quality refinement using BioEdit v7.2.5. The neighbor-joining (N-J) phylogenetic trees were constructed using Mega 6.0. Subsequently, online tools such as jpHMM and SimPlot 3.5.1 were used to identify the recombination breakpoints.

The lengths of NFLG sequences obtained from BD076A and BDL161 were 8718 bp (HXB2: 772–9490) and 8851 bp (HXB2: 759–9610), respectively. The NFLG phylogenetic tree analysis demonstrated that the NFLGs BD076A and BDL161 form a distinct cluster with a bootstrap of 100% (Fig. 1). However, both recombination breakpoint and bootscanning analyses revealed minor genetic variations between these sequences within the env region (Figs. 2 and 3). As shown in Figs. 2A and 3A, the recombinant structure of BD076A was based on the CRF07-BC backbone, with the insertion of a subtype B and a CRF01_AE gene fragment, containing four subregions. As shown in Figs. 2B and 3B, BDL161 contained a CRF07_BC backbone, within which one subtype B fragment and two CRF01_AE fragments were identified, containing seven subregions. The recombinant mosaic structures of two NFLGs were described as follows: ICRF07_BC (HXB2, 772-1396nt), IIB (HXB2, 1397-6131nt), IIICRF01_AE (HXB2, 6132-6554nt), IVCRF07_BC (HXB2, 6555-9490nt), BD076A, ICRF07_BC (HXB2, 759-1466nt), IIB (HXB2, 1467-5044nt), IIICRF07_BC (HXB2, 5045-5849nt), IVCRF01_AE (HXB2, 5850-6120nt), VCRF07_BC (HXB2, 6121-7396nt), IVCRF01_AE (HXB2, 7397-7627nt), VCRF07_BC (HXB2, 7628-9610nt), BDL161. Subregion N-J trees of BD076A and BDL161 analyzed the genetic origin of each fragment (Fig. 4A and 4B). Figure 4A showed the clustering of two CRF07-BC fragments, one CRF01_AE, and one subtype B fragment of BD076A with their respective reference sequences. Figure 4B showed the clustering of four CRF07_BC fragments, one subtype B fragment, and two CRF01_AE fragment of the BDL161 with their respective reference sequences.

OPEN IN VIEWER

FIG. 1.

Phylogenetic tree based on the NFLG sequences. The neighbor-joining tree based on the NFLG sequences of BD076A and BDL161was constructed using Mega 6.0 with a bootstrap value of 1000 replicates. Only bootstrap values ≥90% are shown in the corresponding nodes. The scale bar represents a 2% genetic distance. NFLG, near full-length genome. NFLG, near-full-length genome.

OPEN IN VIEWER

FIG. 2.

Bootscan analysis. A Bootscan analysis was conducted on two URFs, utilizing a window size of 500 bp and a step size of 120 bp. The analysis referenced sequences subtype B (green line), CRF01_AE (yellow line) and CRF07_BC (blue line), and the G subtype (black line). (A) BD076A. (B) BDL161.

OPEN IN VIEWER

FIG. 3.

Genetic map of two URFs. The Recombinant HIV-1 Drawing Tool was used, which is available at the HIV database: https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html.

OPEN IN VIEWER

FIG. 4.

The subregion trees of two URFs. The subregion phylogenetic trees were constructed using Mega 6.0 software and the neighbor-joining method with 1000 bootstrap replications. Bootstrap values ≥90% are shown at the corresponding nodes. The scale bars represent a 2% genetic distance. (A) BD076A. (B) BDL161.

OPEN IN VIEWER

FIG. 4.

(Continued).

CRF01_AE was first identified in Thailand during the 1980s and subsequently introduced to Yunnan, China, in the 1990s, where it rapidly disseminated across the country. ³ CRF07_BC, a recombinant strain, emerged in Yunnan during the 1990s. ⁴ Subtype B initially spread in the United States, Europe, and several Southeast Asian countries. Between the late 1980s and early 1990s, individuals with high-risk sexual behaviors brought these strains into China from Southeast Asia.^10,11 In recent years, CRF01_AE, CRF07BC, and subtype B have become the three predominant HIV-1 genotypes in the sexual contact population in China. ¹² In Hebei Province, MSM has been the most prevalent transmission route of HIV-1, and the main subtypes of HIV-1 are CRF01_AE, CRF07_BC, and subtype B among MSM. The recombinant forms of CRF01-AE and CRF07_BC, as well as CRF01_AE and B, are relatively common, while our study documents the first detection of triple-recombinant URFs (B/CRF01_AE/CRF07_BC) in Hebei’s MSM population. ¹³ BD076A and BDL161 reported that they were infected through MSM. However, it is certain that CRF01_AE, CRF07_BC, and subtype B will spread in the population, which indicates the complexity of the HIV-1 epidemic in Hebei Province and the arduousness of prevention and control efforts.

In this study, the participants were young adults aged 23 and older adults aged 64. Literature reports indicate that since 2010, the number of HIV/AIDS cases among young students and individuals aged ≥50 years in China’s HIV reporting system has been increasing annually,^12,14,15 highlighting the necessity for targeted and age-appropriate intervention measures. Educational and preventive efforts for young students should focus on reducing high-risk behaviors, and we should take health care services for older adults to address their growing vulnerability.

In summary, the co-circulation of different HIV-1 subtypes within the population has accelerated the spread of HIV-1 and resulted in a complex and diverse epidemic landscape. This situation has also created favorable conditions for the emergence of more HIV-1 recombinant strains. Therefore, it is necessary to utilize continuous molecular epidemiological surveillance to investigate the dynamic changes and distribution patterns of HIV-1 subpopulations and identify novel URFs and their populations of origin. This enables case tracing and the identification of high-risk groups and provides a scientific basis for relevant authorities to formulate targeted prevention measures and policy improvements. Furthermore, it facilitates the implementation of these measures to reach special populations requiring health care services and HIV/AIDS education.

Authors’ Contributions

F.Z., Z.L., and S.C.: Designed the study. Z.Z., W.F., and H.S.: Acquired the sequences. J.J., Z.Z., H.S., and W.F.: Analyzed and interpreted the data. F.Z., Z.L., and S.C.: Wrote the article. All authors read and agreed to the published version of the article.