Identification of a complex second-generation HIV-1 circulating recombinant form (CRF158_0107) among men who have sex with men in China

Dear Editor, Recent correspondence published in this journal has highlighted that significant and ongoing HIV-1 recombination events are occurring among men who have sex with men (MSM) in China.1Li Y. Chang W. Liang J. Liu Y. Li L. Liu L. et al.Characterization of a new HIV-1 second-generation circulating recombinant form (CRF170_0107) among men who have sex with men in Yunnan, China.J Infect. 2024; 88: 206-209Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar, 2Chen M. Ma Y. Chen H. Dai J. Dong L. Jia M. Identification of a newly emerging second-generation HIV-1 circulating recombinant form (CRF145_0755) among men who have sex with men in China.J Infect. 2024; 88106126Abstract Full Text Full Text PDF Scopus (0) Google Scholar Viral recombination, a key mechanism in HIV variation and evolution, complicates HIV genetics and has the potential to modify the biological properties of the virus, particularly its replication capacity, pathogenicity and drug resistance. Initially, HIV-1 recombination in Chinese MSM occurred mainly between subtype B and CRF01_AE, resulting in the emergence of circulating recombinant forms (CRFs) such as CRF55_01B, CRF59_01B, CRF67_01B and CRF68_01B. However, in recent years, with the emergence of CRF07_BC as the predominant circulating strain among Chinese MSM, recombination between CRF01_AE and CRF07_BC has become a prevalent mode, resulting in novel CRF_0107. The study of prevalent HIV-1 recombination among MSM provides invaluable insights into the recombination properties of the HIV-1 genome. In this study, we identified a novel HIV-1 CRF, designated CRF158_0107, from MSM in Kunming City, Yunnan Province. This novel CRF is composed of CRF01_AE and CRF07_BC genetic elements. We also analyzed its evolutionary history, shedding light on the dynamic and complex nature of HIV-1 recombination in the Chinese MSM population. During an earlier HIV-1 molecular epidemiological survey conducted among MSM in Kunming City, Yunnan Province, China, four partial pol gene sequences were found to form a unique cluster distinct from the previously known subtypes/CRFs, indicating a possible novel CRF. Of the corresponding samples, two (20M082 and 20M108) were collected in 2020, and two (21M114 and 21M115) were in 2021. To further confirm whether they represented a new CRF, these samples were further analyzed. No direct epidemiological link was found between these cases. Demographic data are shown in Table S1. Written informed consent for sample collection and subsequent analysis was obtained from all participants. The HIV-1 near full-length genome (NFLG) sequences from the four subjects were amplified and sequenced as previously described. The NFLG sequences from subjects 20M082, 20M108, 21M114 and 21M115 were 8871 (620–9513 in HXB2), 8851 (649–9513 in HXB2), 8475 (674–9174 in HXB2) and 8317 (673–9036 in HXB2) nt in size, respectively, and ranged from part of the 5′ long terminal repeat (LTR) to part of the 3′ LTR. The sequences were submitted to GenBank under accession numbers PP977572-PP977575. When phylogenetic analysis was performed using NFLG sequences, the four sequences formed a monophyletic cluster with a bootstrap value of 100%, separated from other known subtypes and CRFs (Fig. 1A). To further investigate their recombination structure, recombination analysis was performed using RIP and Bootscaning analysis, which suggested that they were composed of CRF01_AE and CRF07_BC with the same recombination pattern (Fig. 1B). A total of 15 recombination breakpoints divided the genome into eight CRF01_AE subregions and eight CRF07_BC subregions (Fig. 1C), including ICRF01_AE (761–2655), IICRF07_BC (2656–3058), IIICRF01_AE (3059–3217), IVCRF07_BC (3218–3533), VCRF01_AE (3534–3690), VICRF07_BC (3691–3810), VIICRF01_AE (3811–4013), VIIICRF07_BC (4014–4834), IXCRF01_AE (4835–5147), XCRF07_BC (5148–5907), XICRF01_AE (5908–6300), XIICRF07_BC (6301–8599), XIIICRF01_AE (8600–9096), XIVCRF07_BC (9097–9227), XVCRF01_AE (9228–9389), XVICRF07_BC (9390–9513). The subregion Phylogenetic analyses showed that subregions I, III, V, VII, IX, XI, XIII and XV were related to the counterpart of CRF01_AE, and subregions II, IV, VI, VIII, X, XII, XIV and XVI were related to the counterparts of CRF07_BC, confirming the origin of each subregion (Fig. 2A). According to the CRF naming criteria, these sequences represent a novel CRF and were therefore named CRF158_0107.Fig. 2Phylogenetic and evolutionary analysis of concatenated CRF01_AE and concatenated CRF07_BC subregions from CRF158_0107. (A) The maximum likelihood phylogenetic trees of the 16 mosaic fragments. The reliability of tree branches was assessed by 1000 bootstrap replicates. The scale bar indicates 5% nucleotide sequence divergence. (B) The maximum clade credibility (MCC) trees of combined CRF01_AE segments (I+III+V+VII+IX+XI+XIII+XV) and combined CRF07_BC segments (II+IV+VI+VIII+X+XII+XIV+XVI).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further investigate the origin of CRF158_0107, the concatenated CRF01_AE subregions (I+III+V+VII+IX+XI+XIII+XV) and the concatenated CRF07_BC subregions (II+IV+VI+VIII+X+XII+XIV+XVI) were used to estimate the most recent common ancestor (tMRCA) using Bayesian evolutionary analyses (Fig. 2B). According to maximum clade credibility (MCC) trees, the estimated tMRCAs for the concatenated CRF01_AE subregions and the concatenated CRF07_BC subregions were 2013.0 (95% highest probability density (HPD): 2010.5–2015.2) and 2015.3 (95% HPD: 2013.4–2016.9), respectively, suggesting that CRF158_0107 arose approximately between 2013 and 2015. Furthermore, the components of CRF01_AE were mainly derived from the CRF01_AE-C4 lineage, whereas the components of CRF07_BC were mainly derived from the CRF07_BC-N lineage. CRF01_AE was first introduced into southwest China from Thailand, including Yunnan Province.3Feng Y. He X. Hsi J.H. Li F. Li X. Wang Q. et al.The rapidly expanding CRF01_AE epidemic in China is driven by multiple lineages of HIV-1 viruses introduced in the 1990s.AIDS. 2013; 27: 1793-1802Crossref PubMed Scopus (179) Google Scholar At one time, it was the dominant strain in sexual transmission. Our previous study showed that the estimated time of introduction of CRF01_AE in Yunnan MSM was around 1996.4Chen M. Ma Y. Su Y. Yang L. Zhang R. Yang C. et al.HIV-1 genetic characteristics and transmitted drug resistance among men who have sex with men in Kunming, China.PLoS One. 2014; 9e87033Crossref Scopus (43) Google Scholar According to recent research, a total of 11 CRF01_AE lineages were circulating in China, of which C4 and C5 lineages were found in MSM.5Wang D. Feng Y. Ruan Y. Liao L. Hao J. Song C. et al.Criteria for classification, nomenclature, and reference sequence selection for HIV sub-subtypes of CRF01_AE and CRF07_BC strains in China.AIDS. 2024; 38: 427-430Crossref PubMed Scopus (1) Google Scholar In this study, the evolutionary analysis confirmed that the CRF01_AE segments in CRF158_0107 originated from the CRF01_AE-C4 lineage. On the other hand, CRF07_BC originated from recombination between subtype B and subtype C in intravenous drug users (IDUs) in the early 1990s in Yunnan.6Su L. Graf M. Zhang Y. von Briesen H. Xing H. Kostler J. et al.Characterization of a virtually full-length human immunodeficiency virus type 1 genome of a prevalent intersubtype (C/B′) recombinant strain in China.J Virol. 2000; 74: 11367-11376Crossref PubMed Scopus (0) Google Scholar Through the cross-risk behaviors, CRF07_BC transitioned to sexual transmission, including heterosexual and homosexual transmission. With increased transmissibility and decreased virulence, CRF07_BC spread rapidly in China,7Cheng Z. Yan H. Li Q. Ablan S.D. Kleinpeter A. Freed E.O. et al.Enhanced transmissibility and decreased virulence of HIV-1 CRF07_BC may explain its rapid expansion in China.Microbiol Spectr. 2022; 10e0014622Crossref Scopus (14) Google Scholar and became the predominant strain in China.8Liu X. Wang D. Hu J. Song C. Liao L. Feng Y. et al.Changes in HIV-1 subtypes/sub-subtypes, and transmitted drug resistance among ART-naive HIV-infected individuals – China, 2004-2022.China CDC Wkly. 2023; 5: 664-671Crossref PubMed Scopus (0) Google Scholar During the transmission, CRF07_BC evolved into two lineages, CRF07_BC-O and CRF07_BC-N.5Wang D. Feng Y. Ruan Y. Liao L. Hao J. Song C. et al.Criteria for classification, nomenclature, and reference sequence selection for HIV sub-subtypes of CRF01_AE and CRF07_BC strains in China.AIDS. 2024; 38: 427-430Crossref PubMed Scopus (1) Google Scholar, 9Ge Z. Feng Y. Zhang H. Rashid A. Zaongo S.D. Li K. et al.HIV-1 CRF07_BC transmission dynamics in China: two decades of national molecular surveillance.Emerg Microbes Infect. 2021; 10: 1919-1930Crossref PubMed Scopus (26) Google Scholar CRF07_BC-O is the original lineage that circulated mainly in IDUs and heterosexuals. In contrast, CRF07_BC-N is a new lineage found mainly in MSM. This study confirmed that the CRF07_BC segments in CRF158_0107 were confirmed to be derived from CRF07_BC-N. Kunming is the main cluster of MSM in Yunnan Province, and our previous research also suggested that MSM in Kunming were linked to MSM in the other provinces and were part of a domestic transmission network. Therefore, timely surveillance and intervention for new circulating strains is needed. In this study, we identified a complicated novel CRF in Chinese MSM, named CRF158_0107, which has 15 breakpoints and consists of 16 alternating segments of CRF01_AE and CRF07_BC. This suggests that HIV-1 is very capable of mutating and recombining, and may generate entirely new properties. It is therefore necessary to continuously monitor and study HIV mutation to better understand the mechanisms of HIV transmission and mutation, and to provide new approaches for vaccine and drug development. At the same time, interventions for MSM need to be further strengthened to reduce the potential risk of transmission. This work was supported by the National Natural Science Foundation of China (82160635) and the Revitalizing Yunnan Excellent Talents Support Plan (Famous Doctor Special Project).