Identification of Two Novel HIV-1 Unique Recombinant Forms Among Men Who Have Sex with Men in Northern China

Introduction

Since the discovery of HIV in the 1980s, a significant number of HIV-1 subtypes have been reported continuously. To date, 160 distinct HIV-1 subtypes, including circulating recombinant forms (CRFs), have been reported, and over 60 of them originate from China (https://www.hiv.lanl.gov/components/sequence/HIV/crfdb/crfs.comp). Among the prevalent subtypes in China, CRF07_BC, CRF01_AE, and CRF55_01B represent the three most dominant variants, collectively accounting for 74% of HIV-1 new infections in 2022. ¹

HIV-1 CRF01_AE was initially identified among sex workers and injection drug users (IDUs) in the provinces of Guangdong, Guangxi, and Yunnan during the period of 1996–1997. Subsequently, it disseminated rapidly to other provinces along the southeastern coast. ² In contrast, CRF07_BC emerged in northwestern China among IDUs along a drug trafficking route throughout the 1990s. ³ Furthermore, CRF55_01B experienced significant growth from 2005 to 2009, following its emergence in Shenzhen, and its expansion accelerated markedly after 2010. This particular strain began to spread to other provinces in 2007, and post-2010, CRF55_01B demonstrated a trend of rapid dissemination and epidemic expansion from the Guangdong-Shenzhen region to additional provinces across China. ⁴

The extensive prevalence of various subtypes of HIV facilitates recombination between them. In recent years, second-generation recombinants have emerged frequently, and most of them are CRF01_AE/CRF07_BC recombinants. The initial identification of the CRF79_0107, which was combined with the CRF01_AE/CRF07_BC recombinant, was documented in 2017, ⁵ followed by a rapid emergence of 22 additional cases within an 8-year period. No recombinants of CRF07_BC/CRF55_01B had been reported until 2024; however, in 2024, six CRF07_BC/CRF55_01B recombinants were identified from men who have sex with men (MSM). (https://www.hiv.lanl.gov/components/sequence/HIV/crfdb/crfs.comp).

In this study, we identified two URFs that comprise CRF01_AE/CRF07_BC and CRF07_BC/CRF55_01B, respectively, in Jilin province and Xinjiang province, northern China.

Materials and Methods

Donors XJWQ230011 (Urumqi, Xinjiang Province) and JLCC230106 (Changchun, Jilin Province) are 26 years old and 19 years old, respectively. Both were infected with HIV-1 through MSM. Additional information about the patients is provided in Table 1. The studies involving human subjects were approved by the Institutional Review Boards of the National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention (X140617334).

The NFLG sequences of two HIV-1 isolates were successfully obtained from plasma samples using a two-half-molecule approach. ⁶ The two NFLG sequences were then aligned with reference sequences and circulating recombinant forms (CRFs) in China using Aliview (version 1.28) and manually adjusted with BioEdit (version 7.2.5.0). Additionally, phylogenetic trees of the NFLG sequences and subregional trees were constructed using the maximum likelihood (ML) method with IQ-TREE 2 software. The recombinant breakpoints of the sequences were identified using the online Jumping Profile Hidden Markov Models (jpHMM), the Recombinant Identification Program (RIP 3.0), and SimPlot 3.5.1. The final recombination pattern for each recombinant strain was illustrated using the Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

The two NFLG sequences were submitted to the Stanford Drug Resistance Database (https://hivdb.stanford.edu/hivdb/by-sequences/), and the HIVdb program was utilized to assess drug resistance-related mutations. The Ge-no2pheno (coreceptor) 2.5 online tool was used to predict the use of coreceptors by the corresponding strains according to the sequence of V3 loop amino acids, and the false positive rate was set to 10.00%.

Results and Discussion

The NFLG sequences derived from JLCC230106 and XJWQ230011 were 8,893 bp and 9,029 bp in length, respectively, spanning from the gag region to the U3 region of the 3' long terminal repeat. These two sequences formed a unique monophyletic branch with a bootstrap value of 95% and 85%, respectively, indicating a distant relationship to all known HIV-1 CRFs, as determined by phylogenetic analysis of the NFLG (Fig. 1). The mosaic structures were further analyzed using RIP 3.0, jpHMM, and BootScanning analyses individually. As shown in Figure 2, JLCC230106 is a recombinant of CRF01_AE/CRF07_BC, while XJWQ230011 is a recombinant of CRF07_BC/CRF55_01B. The subregions of the two NFLG sequences are as follows: JLCC230106: I_{CRF01_AE}: HXB2, 633-1283; II_{CRF07_BC}: HXB2, 1284-6252; III_{CRF01_AE}: HXB2, 6253-9601; XJWQ230011: I_{CRF55_01B}: HXB2, 633-1309; II_{CRF07_BC}: HXB2, 1310-2622; III_{CRF55_01B}: HXB2, 2623-4113; IV_{CRF07_BC}: HXB2, 4114-4428; V_{CRF55_01B}: HXB2, 4429-4697; VI_{CRF07_BC}: HXB2, 4698-4904; VII_{CRF55_01B}: HXB2, 4905-9081; VIII_{CRF07_BC}:HXB2, 9082-9479. The subregion trees were also constructed for each fragment (Fig. 3).

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FIG. 1.

A neighbor-joining phylogenetic tree was constructed for JLCC230106 and XJWQ230011 based on the NFLG sequences using IQ-TREE 2. Red line denotes study subject.

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FIG. 2.

Bootscanning analyses (A) and recombinant maps (B) of JLCC230106 and XJWQ230011. Bootscanning analysis was performed with reference sequences by using SimPlot. The analysis is set as a default setting except for the window size of 400 bp and step size of 50 bp. GapStrip: on, Kimura (2-parameter), T/t: 4.0. The x-axis represents the nucleotide position referred to as HXB2, and the y-axis represents the bootstrap value (percentage). The CRF01_AE reference group includes JX112859, EF036527, and AY358049. The CRF07_BC reference group includes JX392381, HQ215552, and AF286230. The CRF55_01B reference group includes JF340054, KF927151, and JX574662. The subtype J reference is SE9280. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). CRF, circulating recombinant form.

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FIG. 3.

Subregion tree analysis of two HIV-1 URFs. Phylogenetic analyses of different segments of JLCC230106 (A) and XJWQ230011 (B) were conducted using IQ-TREE 2, employing the neighbor-joining method with 1,000 bootstrap replications. The results were visualized using iTOL.

Drug resistance analysis indicated that the two NFLGs did not exhibit drug resistance-related mutations in the protease and integrase regions. However, in XJWQ230011, V179D (a non-nucleoside reverse transcriptase inhibitors-related mutation) presented in the reverse transcriptase region. This mutation conferred intermediate resistance to efavirenz, emtricitabineand nevirapine, as well as potential resistance to rilpivirine. In JLCC230106, D67N (a nucleoside reverse transcriptase inhibitor-related mutation) presented in the reverse transcriptase region. This mutation resulted in the development of intermediate resistance to zidovudine (AZT) and stavudine, characterized as low-level resistance. Both sequences are predicted to use CCR5 as a coreceptor.

In this study, we identified two HIV-1 URFs from Jilin province and Xinjiang province. In recent years, the HIV-1 diversity prevalent in China has gradually increased, particularly with the continuous emergence of second-generation recombinants, including CRFs and URFs. ^{7 –9} This trend may be attributed to rapid economic development and increased population mobility within China, which have facilitated the HIV transmission. ⁴ The occurrence of superinfection with different viral subtypes in the same geographic area is a critical precursor for viral recombination. ¹⁰ Given that CRF01_AE and CRF07_BC are the most prevalent subtypes, many second-generation recombinants are new strains derived from CRF01_AE and CRF07_BC. ^11,12 Conversely, CRF55_01B, a more recently identified strain, has exhibited low pathogenicity coupled with high transmissibility, leading to its rapid spread and the emergence of numerous second-generation recombinants, including CRF55_01B, within the current year. ^{13 –15} The emergence of these second-generation recombinants may complicate the genomic structure of HIV-1, potentially posing significant challenges to HIV prevention and control efforts, as well as to the development of effective treatments and vaccines. Therefore, it is crucial to monitor the novel HIV-1 subtypes, particularly in the MSM population, to formulate more effective prevention strategies.