Identification of a Novel HIV-1 Circulating Recombinant Form (CRF184_BC) Among Heterosexual Populations Along the China–Myanmar Border Region

Recently, the high frequency of HIV-1 recombination events was reported in China.^1,2 The emergence of novel HIV-1 recombinant viruses could alter the virus’s phenotypic characteristics, such as the accumulation of drug-resistant mutations or enhanced immune evasion capabilities. ³ Since current vaccine designs are primarily based on the conserved sequences of dominant subtypes, recombination could make vaccine research more challenge. ⁴ The China–Myanmar border region has high population mobility and limited public health resources. Historically, injecting drug use has been prevalent in this area, creating conditions conducive to viral recombination among different subtypes. ⁵ Several BC recombinant strains have previously been reported. ⁶ This study identified a novel circulating recombinant form (CRF) of the virus in this area, designated CRF184_BC.

Samples were obtained from four individuals (22BS143, 22BS144, 23BS055, and 23BS056) who were positive for HIV-1 in Baoshan Prefecture, Yunnan Province, China. As summarized in Supplementary Table S1, all four subjects were reported to have become infected through heterosexual contact. Three of them were Burmese nationals (22BS143, 22BS144, and 23BS055), and one was a Chinese citizen (23BS056). No epidemiological connections were identified among them. The study was approved by the Research ics Review Committee of the Yunnan Center for Disease Control and Prevention. Written informed consent was obtained from the participants. Near full-length genome (NFLG) sequences were amplified and sequenced as described in a previous study. ⁷ The NFLG sequences of strains 22BS143, 22BS144, 23BS055, and 23BS056 were 8845 (644–9481 in HXB2), 8830 (644–9468 in HXB2), 8820 (644–9492 in HXB2), and 8830 (648–9485) nucleotides in size, ranging from part of 5′ long terminal repeat (LTR) to part of 3′ LTR. Phylogenetic trees were constructed with MEGA 12 software. Recombination breakpoints were analyzed using SimPlot 3.5.1 software. Bayesian Markov chain Monte Carlo analysis was performed using BEAST v 1.8.2.

Phylogenetic analysis demonstrated that the four NFLG sequences constituted a distinct monophyletic clade with strong bootstrap support, clearly separate from all previously identified HIV-1 subtypes and CRFs (Fig. 1A). Bootscanning analysis further indicated a conserved recombination profile characterized by three subtype B fragments embedded within a subtype C genomic backbone (Fig. 1B). The genome structure was segmented into seven regions as follows: I_C (790–2897), II_B (2898–3208), III_C (3209–5983), IV_B (5984–6435), V_C (6436–8821), VI_B (8822–8969), and VII_C (8970–9468) (Fig. 1C). Subsequent subregion phylogenetic analyses confirmed the lineage origins of each segment: regions IC, IIIC, VC, and VIIC clustered closely with subtype C references, while regions IIB, IVB, and VIB grouped within subtype B (Fig. 2A). In accordance with the criteria for designating novel CRFs, these epidemiologically unlinked sequences fulfill the requirements for classification as a new CRF, designated CRF184_BC. In mosaic architecture, the pol, tat, rev, env, and nef regions display a hybrid B/C signature. Gag, vif, and vpr remain subtype C lineages, while the vpu region is replaced by a B lineage.

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FIG. 1.

Phylogenetic and recombinant analyses based on the near-full-length genome sequences. (A) A neighbor-joining phylogenetic tree was constructed using the reference sequences of HIV-1 CRFs identified in China. The sequences of CRF184_BC are highlighted in red. Values on the branches represent the percentage of 1000 bootstrap replicates, and the scale bar indicates 5% nucleotide sequence divergence. (B) Bootstrap analysis of the candidate CRF. Conditions used for this analysis: Window: 350 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 KF835522, KC898995, KC898996, KF250403, AB023804, AF067158, and AF067157. The subtype A4 reference group included AM000053 and AM000054. (C) Genomic structure of CRF184_BC. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool.

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FIG. 2.

Phylogenetic and evolutionary analysis of subtype B and C subregions from CRF184_BC. (A) The maximum likelihood phylogenetic trees of the seven mosaic fragments. The reliability of tree branches was assessed by 1000 bootstrap replicates. The scale bar indicates 1% nucleotide sequence divergence. (B) The maximum clade credibility (MCC) trees of combined subtype C segments (I+III+V+VII) and combined subtype B segments (II+IV+VI).

To elucidate the evolutionary origin of CRF184_BC, a Bayesian evolutionary analysis was conducted with concatenated subtype C subregions (I, III, V, and VII) and concatenated subtype B subregions (II, IV, and VI) (Fig. 2B). The analysis estimated the time of the most recent common ancestor (tMRCA) for the subtype C-derived segments to be around 2005.0 (95% highest probability density [HPD]: 2000.2–2009.2]), while the tMRCA for subtype B-derived segments to be approximately 2012.1 (95% HPD: 1997.4–2020.9) (Fig. 2). Based on these tMRCA estimates, the formation of the CRF184_BC likely occurred during a window between the mid-2000s and the early 2010s.

Due to its unique geographical location, Yunnan Province has consistently been at risk of HIV-1 importation. Initially, HIV-1 entered Yunnan’s border regions through cross-border drug trafficking and injection drug use. ⁸ Clusters of HIV-1 infections were first identified among injection drug users in Dehong Prefecture in 1989, making Yunnan one of the earliest areas in China to experience an HIV-1 epidemic. ⁸ The early circulating strains included subtype C from India and subtype B from Thailand. These two subtypes underwent widespread recombination among injecting drug users (IDUs), generating numerous BC recombinant forms, including CRF07_BC and CRF08_BC, which gradually spread to other regions of China via drug trafficking routes.^6,9 Neighboring countries bordering Yunnan, particularly Myanmar, exhibit complex HIV transmission dynamics. Related reports indicate that Myanmar was one of only three countries worldwide where URF accounts for over 30% of infections. ¹⁰ A study from 2009 found that recombinant strains accounted for up to 86.1% of cases among IDUs in northern Myanmar. The B/C and CRF01_AE/B/C recombinants were the two most prevalent, accounting for 54.4% and 42.6% of intersubtype recombinants, respectively. ¹⁰ Meanwhile, a high prevalence of HIV-1 recombinants was also found in Dehong Prefecture and Baoshan City, which border Myanmar.^11,12 A series of novel CRFs have been identified there, including CRF57_BC, CRF62_BC, CRF64_BC, CRF65_cpx, CRF78_cpx, CRF86_BC, CRF110_BC, CRF118_BC, and CRF178_BC. Consequently, the China–Myanmar border region has long been recognized as a hotspot for HIV-1 recombination.^11,12

This study involved three Myanmar nationals and one Chinese individual, suggesting that CRF184_BC may have established latent transmission in the China–Myanmar border region. All infected individuals reported heterosexual transmission routes, which is consistent with the current primary transmission route and suggests that it is prevalent within the general population. ¹³ The ancestral time of the subtype C and B subtype fragments within CRF184_BC spans a certain period. This suggests that the evolutionary history of this recombinant strain may be more complex than initially thought. Given the region’s complex epidemiological situation and high frequency of viral genetic recombination, it is possible that the subtype C fragment serving as the backbone has undergone additional recombination events, incorporating parental strains from different time periods into its sequence. In other words, subtype C may not be the original subtype C, but rather a recombinant product within the same genotype.^14,15 Such recombination is generally difficult to distinguish, posing challenges for tracing its origins. Furthermore, the hybrid structures of key genes such as env and pol may impact the virus’s ability to evade the immune system and develop drug resistance. Further validation through functional experiments is therefore required.

The identification of CRF184_BC not only enriches the map of HIV-1 genetic diversity but also highlights the public health risks posed by the China–Myanmar border region as a hotbed for viral recombination. Enhanced surveillance is required to provide the molecular epidemiological evidence needed to develop regional prevention and control strategies.

Sequence Data

The sequences have been submitted to GenBank under accession numbers PV690538 to PV690541.