Identification of a Novel HIV-1 Circulating Recombinant Form (CRF181_BC) with a Unique Subtype B Backbone from the China-Myanmar Border Region

Due to its proximity to the Golden Triangle, Yunnan Province has historically been a gateway for HIV-1 transmission into China. Subtypes B and C, which originated in Thailand and India, respectively, were introduced to Yunnan through injection drug users. ¹ Extensive local recombination subsequently occurred, resulting in multiple circulating recombinant forms (CRFs). According to existing data, Yunnan has reported over 10 distinct CRF_BC variants, including CRF07_BC, CRF08_BC, CRF57_BC, CRF61_BC, CRF62_BC, CRF64_BC, CRF86_BC, CRF110_BC, CRF118_BC and CRF178_BC.^2–4 From a global perspective, Yunnan and the neighboring northern Myanmar region have emerged as major hotspots for HIV-1 BC recombination.^5,6 These recombinant viruses increase HIV-1 genetic diversity and pose persistent challenges for diagnosis, antiretroviral treatment, and vaccine development. ⁷ In this study, we identified a novel CRF, CRF181_BC. Unlike previously reported CRF_BCs, which have subtype C as the backbone, CRF181_BC has subtype B as the backbone, as it has a higher proportion of fragments derived from subtype B.

Plasma samples were collected from three individuals who were positive for HIV-1 (case numbers 19BS001, 19BS006, and 20BS178) in Baoshan, Yunnan Province, between 2019 and 2020. As shown in Table 1, all three infections were transmitted through heterosexual contact, and epidemiological investigations revealed no links between the cases. The study was approved by the Research Ethics Review Committee of the Yunnan Provincial Center for Disease Control and Prevention, and written informed consent was obtained from all participants. Amplification and sequencing of the near-full-length genome (NFLG) were performed according to established methodologies. ⁸ The three final NFLG sequences obtained were 8,849, 8,874, and 8,425 nucleotides in length. These sequences spanned from the 5′ long terminal repeat (LTR) to the 3′ LTR and corresponded to nucleotide positions 673–9,479 in the HXB2 reference strain. These sequences have been deposited in the GenBank database under the accession numbers PV781616 to PV781618.

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Table 1.

Demographic Characteristics of HIV-1 Infected Participants

Sequence number	Sampling year	Sex	Age	Education	Marital status	Infection route
19BS001	2019	Male	74	Primary School	Divorced/widowed	Heterosexual contact
19BS006	2019	Male	42	Illiteracy	Unmarried	Heterosexual contact
20BS178	2020	Female	52	Junior middle school	Divorced/widowed	Heterosexual contact

Phylogenetic analysis revealed that the three NFLG sequences constituted a distinct monophyletic clade that was clearly distinct from all recognized HIV-1 subtypes and CRFs, with substantial bootstrap support (99%; Fig. 1A). Further analysis using Bootscanning revealed a conserved recombination pattern characterized by alternating segments from multiple segments from subtypes B and C (Fig. 1B). The genome structure was divided into 12 subregions as follows: I_B (790–4115), II_C (4116–4331), III_B (4332–4467), IV_C (4468–4687), V_B (4688–5080), VI_C (5081–5758), VII_B (5759–6455), VIII_C (6456–7371), IX_B (7372–7487), X_C (7488–8295), XI_B (8296–8982), and XII_C (8983–9479) (Fig. 1C). Phylogenetic analysis of each subregion confirmed the lineage origins of each fragment. Subregions I_B, III_B, V_B, VII_B, IX_B, and XI_B clustered closely with subtype B reference sequences, whereas subregions II_C, IV_C, VI_C, VIII_C, X_C, and XII_C clustered with subtype C reference sequences (Fig. 2A). Based on the criteria for naming novel CRFs, these epidemiologically unrelated sequences met the requirements for defining a new CRF, designated CRF181_BC. Within its chimeric genome structure, pol, env, vif, vpr, and nef gene fragments all exhibited B/C hybrid characteristics.

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FIG. 1.

Phylogenetic and recombinant analyses for the near full-length genome (NFLG) sequences of CRF181_BC. (A) A neighbor-joining phylogenetic tree was constructed with the reference sequences of HIV-1 CRFs reported in China. The sequences of CRF181_BC are highlighted in red. The values on the branches represent the percentage of 1,000 bootstrap replicates, and the scale bar indicates 5% nucleotide sequence divergence. (B) Bootscannig analysis of NFLG sequences of CRF181_BC with the reference sequence of subtypes B and C. Conditions used in this analysis: Window: 200 bp, step: 20 bp, GapStrip: on, replicates: 100, Kinura (2-parameter), T/t: 2.0. The subtype B reference group included AY173951, JF932495, JF932471, JF932490, DQ354118, DQ354116 and DQ354114. The subtype C reference group included AF067155, KF835522, KC898995, KC898996, KF250403, AB023804, AF067158 and AF067157. The subtype A4 reference group included AM000053 and AM000054. (C) Genome map of CRF181_BC. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool.

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FIG. 2.

Phylogenetic analysis of individual subregions and Bayesian evolutionary analyses of concatenated subtype B and concatenated subtype C subregions from CRF181_BC. (A) The maximum likelihood phylogenetic trees of the 12 subregions. The reliability of the tree branches was assessed by 1,000 bootstrap replicates. The scale bar indicates nucleotide sequence divergence. (B) The maximum clade credibility (MCC) trees of the combined subtype B subregions (I+III+V+VII+IX+XI) and the combined subtype C subregions (II+IV+IV+IIIV+X+XII).

A Bayesian Markov chain Monte Carlo analysis was performed to investigate the evolutionary origins of CRF181_BC, using concatenated genomic segments derived from subtypes B (subregions I, III, V, VII, IX, and XI) and C (subregions II, IV, VI, VIII, X, and XII) (Fig. 2B). The results estimated the time to the most recent common ancestor (tMRCA) for subtype B fragments to be around 2006.0 (95% highest posterior density [HPD]: 2003.1–2008.8). In contrast, the tMRCA for the inserted subtype C fragments was estimated to be around 2005.0 (95% HPD: 2000.9–2008.7). The tMRCA estimates for the parental lineages indicated that the recombination event that resulted in CRF181_BC probably occurred between 2005 and 2006.

Frequent recombination between subtypes C and B characterized the early HIV-1 epidemic in Yunnan Province, driven primarily by transmission via injecting drug use during that period. ⁹ The reported CRF_BC strains in Yunnan were geographically concentrated along the province’s border with Myanmar, forming hotspots such as Dehong Prefecture and Baoshan City, where the multiple CRFs were first identified, such as CRF57_BC, CRF62_BC, CRF64_BC, CRF86_BC, CRF110_BC, CRF118_BC, and CRF178_BC.^2,4,10 Furthermore, BC recombinants exhibited potential cross-border transmission trends, as evidenced by the simultaneous detection of CRF178_BC in Chinese and Myanmar nationals. ² Retrospective analysis revealed that most CRF_BCs emerged from the late 1980s to the mid-2000s: Specifically, CRF110_BC emerged between 1986 and 1991, CRF142_BC between 1994 and 1997, CRF118_BC between 1996 and 1998, and CRF178_BC between 2000 and 2003. This highlighted a concentrated surge in recombination activity from the 1990s to the 2000s. This temporal distribution also closely correlated with Yunnan Province’s epidemiological transition: a gradual shift from an early transmission period dominated by injecting drug use to a period of expansion driven by heterosexual transmission. ¹¹

However, CRF181_BC differs significantly from other CRF_BC strains. In most CRF_BC strains, subtype C forms the backbone of the viral genome, accounting for proportions ranging from 63.6% to 93.7%. CRF181_BC is unique in having a subtype B backbone. Subtype B constitutes 55.1% of the CRF181_BC genome, suggesting that it may have evolved through a distinct pathway. Consequently, CRF181_BC may exhibit unique replication, cell tropism, or drug resistance profiles, which require validation through in vitro functional studies. Furthermore, CRF181_BC emerged around 2005–2006, which was during the late phase of the epidemic dominated by injecting drug use. This study primarily identified CRF181_BC among middle-aged and elderly heterosexual populations, indicating distinct demographic clustering.

In conclusion, we identified CRF181_BC, a novel HIV-1 CRF characterized by a unique subtype B backbone. This contrasts with the typical subtype C framework of other CRF_BCs. This virus likely emerged between 2005 and 2006, coinciding with a transition period in Yunnan Province during which the epidemic shifted from being dominated by injecting drug use to sexual transmission. The distinct genomic architecture and evolutionary origin of CRF181_BC highlight the ongoing genetic complexity and active recombination of HIV-1 in the China-Myanmar border region. Further research is needed to understand its biological properties and spread, including functional studies and expanded molecular surveillance.