Identification of an Emerging HIV-1B/C Circulating Recombinant Form (CRF176_BC) in Heterosexual Populations in Yunnan Province,China

The persistent emergence of novel circulating recombinant forms (CRFs) of HIV-1 was recently reported in China, attributed to prolonged co-circulation of divergent subtypes/CRFs among high-risk populations.^1,2 HIV-1 recombination results from a template switching event that occurs during viral replication when host cells are co-infected with different viral strains. Dual or multiple infections with the same subtype are difficult or impossible to detect and are therefore underreported. However, when two or more distinct subtypes or CRFs co-circulate in the same high-risk group, multiple infections lead to inter-subtype recombinants, which are easily identified. In the 1980s, HIV-1 subtypes B and C were first introduced into Yunnan Province—historically a major epicenter of HIV-1 transmission in China—primarily through intravenous drug use networks. ³ Subsequently, widespread recombination between these two subtypes gave rise to various recombinant B/C forms. ⁴ The HIV-1 circulating B/C recombinant forms identified in China show a unique geographic and epidemiological distribution, with Yunnan province representing the primary hotspot, accounting for 11 of the 13 documented CRF_BC strains. In this study, we reported the discovery and comprehensive genomic characterization of HIV-1 CRF176_BC, a recombinant composed of subtypes B and C, isolated from individuals who identified as heterosexual in Yunnan.

Plasma specimens were obtained from four treatment-naïve individuals with HIV-1 infection (ages 42–72 years) in Honghe Prefecture, Yunnan Province, in 2022. The demographic details were showed in Table 1. All cases represented heterosexual transmission without identified epidemiological linkages. The study received ethical approval from the Yunnan Provincial Center for Disease Control and Prevention Institutional Review Board, with written informed consent obtained from all participants. Near full length HIV-1 genomes were successfully amplified using a nested PCR protocol from the previous study. The obtained sequences (GenBank accession numbers: PV548826–PV548829) demonstrated lengths of 8706–8827 nucleotides. These sequences encompassed critical structural elements from the 5′ long terminal repeat (LTR) to the 3′ LTR region, corresponding to nucleotide positions 719–9501 in the HXB2 genome.

OPEN IN VIEWER

Table 1.

Demographic Characteristics of HIV-1 Infected Participants

Sample number	Sampling year	Sex	Age	Occupation	Education level	Marital status	Infection route
22HH158	2022	Male	61	Farmer	High school	Married	Heterosexual contact
22HH275	2022	Male	72	Farmer	Primary School	Married	Heterosexual contact
22HH789	2022	Male	42	Farmer	Primary School	Unmarried	Heterosexual contact
22HH825	2022	Female	68	Farmer	Illiterate	Married	Heterosexual contact

Phylogenetic analysis revealed that the four strains formed a monophyletic cluster with 100% bootstrap support, phylogenetically distinct from all established subtypes and CRFs (Fig. 1A). Bootscanning analysis revealed that these sequences retained a subtype C backbone with two inserted subtype B fragments, resulting in five subregions designated as follows: I_C (744–1210), II_B (1211–1305), III_C (1306–2806), VI_B (2863–3167), and V_C (3208–9386) (Fig. 1B and C). Phylogenetic validation of individual subregions showed that subregions I, III, and V were closely related to subtype C references, whereas subregion IV showed exclusive clustering with subtype B references (Fig. 2A). However, due to insufficient sequence length of subregion II, phylogenetic analysis failed to resolve its clustering with subtype B reference strains (Supplementary Fig. S1), precluding definitive assignment of its origin. These phylogenetically coherent sequences from epidemiologically unrelated individuals met the criteria for a novel CRF, and were designated as CRF176_BC.

OPEN IN VIEWER

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 CRF176_BC are highlighted in red. Values on the branches represent the percentage of 1,000 bootstrap replicates, and the scale bar indicates 5% nucleotide sequence divergence. (B) Bootstrap analysis of the candidate CRF. Conditions used for this analysis: Window: 250 bp, step: 20 bp, GapStrip: on, replicates: 100, Kinura (2-parameter), T/t: 2.0. The subtype B reference group included AY173951, U71182, JF932495, JF932471, DQ354118, AY945711, and MG989506. The subtype C reference group included AF067155, AY967806, KF835522, KC898995, KF250403, AB023804, and AF067158. The subtype A4 reference group included AM000053 and AM000054. (C) Genomic structure of CRF176_BC. The mosaic map was generated using the Recombinant HIV-1 Drawing Tool. CRF, circulating recombinant form.

OPEN IN VIEWER

FIG. 2.

Phylogenetic and evolutionary analysis of subtype B and C subregions from CRF176_BC. (A) The maximum likelihood phylogenetic trees of the four mosaic fragments. The reliability of tree branches was assessed by 1,000 bootstrap replicates. The scale bar indicates 10% nucleotide sequence divergence. (B) The maximum clade credibility (MCC) trees of concatenated subtype C segments (I+III+V) and subtype B segment (IV).

To elucidate the evolutionary history of CRF176_BC, Bayesian Markov Chain Monte Carlo evolutionary analysis was further performed with the concatenated subtype C subregions (I+III+V) and the subtype B subregion (IV). The time of most recent common ancestor (tMRCA) for the concatenated subtype C subregions was estimated to be 2014.7 [95% highest probability density (HPD): 2011.6–2017.2], and the tMRCA for the subtype B subregion was estimated to be 2016.1 [95% HPD: 2010.2–202.4] (Fig. 2B). This temporal framework suggested a recombination event likely occurred between 2014 and 2016.

Previously reported CRF_BCs, whether discovered around 2000 (CRF07_BC and CRF08_BC) or only in recent years (CRF110_BC, CRF118_BC and CRF178_BC), were thought to have a high probability of originating from injecting drug users in the 1990s.^4–6 The four CRF176_BC genomes shared a common ancestor between 2014 and 2016, and they were identified within a heterosexual population. However, some regions of the genome are similar to those of CRF08_BC and other BC recombinants from China. Currently, it is not possible to establish a direct ancestral relationship between them. Therefore, as with other CRF_BC strains, there is a possibility that they emerged in injecting drug users around 2000 and subsequently spread to heterosexual populations as the transmission mode changed. ⁷

5In the context of local epidemiological characteristics, CRF08_BC and CRF07_BC were the most prevalent strains in Honghe Prefecture, where CRF160_0708 was also found.^8,9 However, we excluded the possibility that CRF176_BC was recombined from CRF08_BC and CRF07_BC. Since CRF08_BC and CRF07_BC both have a subtype B insertion in the similar region at the 3′ end of their respective genomes (Supplementary Fig. S2), if CRF176_BC was recombined from CRF08_BC and CRF07_BC, then regardless of how the recombination occurred, there should still be a subtype B insertion at the 3′ end of the genome. However, the analysis above suggested that there was no such piece of subtype B insertion sequence at the 3′ end of CRF176_BC. In terms of demographic characteristics, three of the four participants who carried CRF176_BC were over 60 years old, suggesting an age-specific sexual network that may act as a conduit for localized transmission of novel recombinants. This demographic pattern aligned with emerging epidemiological trends in China, where HIV incidence among older adults (≥50 years) has risen sharply over the past decade. ¹⁰ All of the above fully demonstrates the uniqueness of CRF176_BC. Further research is needed to understand its prevalence and evolutionary characteristics.

In conclusion, we have identified and characterized CRF176_BC, a recombinant form derived from subtypes B and C that emerged in the past decade. The emergence of CRF176_BC reflects the complexity of HIV-1 transmission dynamics in Yunnan Province, where divergent viral lineages and high-risk behaviors synergistically drive viral evolution. The identification of novel CRFs such as CRF176_BC advances our understanding of HIV-1 evolutionary mechanisms and mutation patterns while also providing critical insights into transmission networks to inform targeted public health strategies.

Sequence Data

The sequences have been deposited in GenBank (accession numbers: PV548826–PV548829).

Authors’ Contributions

M.C., W.D., and M.J. conceived and designed the project. Y.M. and H.C. collected the samples. M.C. performed the experiments. Y.M. and H.C. collected the epidemiological data. M.C. analyzed the data. M.C. prepared the article. All authors reviewed the article.