Identification of a novel second-generation HIV-1 circulating recombinant form, CRF182_0708, in Yunnan, China

Abstract

Background

To report a novel HIV-1 circulating recombinant form (CRF) identified in Yunnan Province, China, through a molecular epidemiological study based on near-full-length HIV-1 genome sequencing and phylogenetic analyses.

Methods

Plasma samples were collected from five HIV-1 seropositive individuals. Viral RNA was extracted, and near-full-length genomes (NFLG) were amplified and sequenced. Maximum likelihood (ML) phylogenetic analysis was performed using IQ-TREE v2.4.0. Recombination patterns were characterized using SimPlot v3.5.1. The time to the most recent common ancestor (tMRCA) was estimated using Bayesian phylogenetic analysis in BEAST v1.10.5.

Results

In the ML tree, the five NFLG sequences clustered into a distinct monophyletic clade. Recombination analyses revealed a mosaic genome composed of multiple fragments derived from subtype B and subtype C. Comparative genomic mapping and subregion phylogenetic analyses further confirmed that this strain represents a second-generation recombinant form derived from CRF07_BC and CRF08_BC, with CRF08_BC serving as the backbone and incorporating several fragments from CRF07_BC. The novel CRF was designated CRF182_0708. Bayesian analysis estimated that CRF182_0708 originated around 2002.7–2003.1.

Conclusion

We identified a novel second-generation HIV-1 CRF derived from CRF07_BC and CRF08_BC, designated CRF182_0708, among HIV-1-positive individuals in relatively developed cities in Yunnan Province, China.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12879-026-13194-w.

Background

Human immunodeficiency virus type 1 (HIV-1) has developed remarkable genetic diversity through global propagation and continuous evolution. HIV-1 can be classified into four distinct groups, designated as M, N, O, and P. Within the M group, further diversification exists, with multiple subtypes, including A, B, C, D, F, G, H, J, K, and L [1]. Co-infection of a host with different subtypes and subsequent viral recombination gives rise to numerous circulating recombinant forms (CRFs) and unique recombinant forms (URFs) [2, 3]. To date, more than 180 distinct CRFs have been reported worldwide, whose continuous emergence poses significant challenges to precise epidemic surveillance, prevention strategies, and vaccine development [4]. Yunnan Province, China, which is bordered by Myanmar, Laos, and Vietnam, was the first region in China to report cases of HIV-1 infections [5]. The region’s distinctive geographical and epidemiological characteristics have rendered it a focal point for the emergence and identification of novel CRFs. To date, the province has identified and named 29 novel CRFs for the first time, including 13 CRFs originating from recombination between subtypes B and C, with the remainder being CRFs arising from recombination between CRF01_AE and subtypes B or C [6]. These findings underscore the complexity and dynamic nature of the local HIV-1 epidemic. CRF07_BC and CRF08_BC, first reported in China, have long been the dominant strains in this region [7, 8]. The long-term co-circulation of these two predominant CRFs has created a unique epidemiological landscape conducive to the emergence of second-generation recombinant forms [9–12]. In light of the aforementioned context, the present study identified a novel second-generation CRF, CRF182_0708, of HIV-1 among individuals in Yunnan Province, China. Phylogenetic and evolutionary analyses were conducted to characterize its recombinant structure and infer its origin.

Methods

The study participants and ethical approval

A total of 7553 whole blood samples were collected between 2021 and 2022 as part of a molecular epidemiological survey focused on the partial pol gene. Participants were recruited from the Yunnan Provincial Infectious Disease Hospital. The inclusion criteria encompassed confirmed HIV-1 antibody positivity and confirmed virological failure (plasma viral load > 1,000 copies/mL after ≥ 6 months of therapy). Subsequent screening of this cohort identified five pol sequences that shared identical recombination breakpoints. Phylogenetic analysis of these pol sequences demonstrated that they formed a distinct monophyletic cluster, separate from all known subtypes and CRFs. To confirm the uniqueness of this recombination pattern, we compared these sequences against the Los Alamos HIV Sequence Database and GenBank using BLAST. No matches to any known CRF or URF were found. These five sequences were derived from epidemiologically unlinked patients. To fully characterize these potential novel viruses, near-full-length genomes (NFLGs) were amplified from the corresponding plasma samples.

Amplification of near full-length HIV-1 genomes

Viral RNA was extracted from plasma samples using the TianGen Viral RNA Mini-Extraction Kit (China), in accordance with the manufacturer’s protocol. As described previously [13], two overlapping fragments of the HIV-1 genome were amplified by nested reverse transcription polymerase chain reaction (RT-PCR). Purified amplicons were subjected to bidirectional Sanger sequencing (Qingke Biotechnology Co., Ltd.). The resulting sequences were then assembled and analyzed using ContigExpress v 9.1.

Phylogenetic analyses

Multiple sequence alignment was performed with MAFFT v 7.310 between the obtained NFLG sequence and a set of reference sequences. These reference sequences, retrieved from the Los Alamos HIV Sequence Database, comprised representatives of HIV-1 subtypes B and C, and their associated recombinant forms, as well as outgroups O and P. A maximum likelihood (ML) phylogenetic tree was constructed using IQ-TREE v2.4.0 under the best-fit substitution model (GTR + F+R6), which was selected by ModelFinder implemented in IQ-TREE. Branch support was assessed using 1,000 ultrafast bootstrap replicates, and nodes with bootstrap values ≥ 90% were designated as well-supported. The final tree was then subjected to visualization and annotation using the FigTree v 1.4.4.

Recombination analyses

Initial screening for recombination in the five NFLG sequences was conducted using the Recombination Identification Program (RIP) and the Jumping Profile Hidden Markov Model (jpHMM), both available from the Los Alamos HIV Sequence Database. The putative recombination breakpoints suggested by these tools were subsequently confirmed using bootscanning analysis implemented in SimPlot v3.5.1. The reference sequences for Bootscan analysis included HIV-1 subtypes B and C, CRF07_BC, CRF08_BC, and groups O and P. To confirm the genotype of each recombinant region, ML phylogenetic trees were constructed for the corresponding subgenomic fragments. The reference datasets for these trees encompassed HIV-1 pure subtypes, CRF07_BC, CRF08_BC, and outlier groups N, O, and P. Finally, a schematic diagram of the recombinant genome structure was generated based on the confirmed breakpoints, using the Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper. html).

Evolutionary analyses

Bayesian phylogenetic analyses were conducted separately for parental-derived genomic regions to estimate the time of origin while accounting for recombination. The CRF07_BC-derived region (nt 2676–6613) was analyzed as a single continuous fragment together with a dataset of pure CRF07_BC sequences. The CRF08_BC-derived regions (nt 790–2675, 6614–7610, and 7836–8867) were concatenated and partitioned by genomic region in BEAST, and analyzed together with a dataset of pure CRF08_BC sequences. To construct the reference datasets, all available NFLG sequences of CRF07_BC and CRF08_BC were retrieved from the Los Alamos HIV Sequence Database. Temporal signal was assessed using TempEst v1.5.3, and sequences identified as outliers were excluded from subsequent analyses. The final datasets comprised 161 CRF07_BC and 57 CRF08_BC sequences, respectively. The most suitable nucleotide substitution model (GTR + G+I) was identified for each dataset using jModelTest2. Time-calibrated phylogenies were inferred using BEAST v1.10.5 under an uncorrelated lognormal relaxed molecular clock and a Bayesian Skyline coalescent model. For each analysis, Markov Chain Monte Carlo (MCMC) runs were performed for a minimum of 1.5 million generations, with a sampling frequency of 1,000 generations. To ensure the robustness of the analysis, the CRF07_BC analysis involved three independent MCMC runs, which were subsequently combined using LogCombiner v1.10.4. The convergence assessment indicated that a single run was sufficient for the CRF08_BC dataset. It was observed that all parameters converged, with effective sample sizes (ESS) > 200. Subsequent to the elimination of the initial 10% of samples as burn-in, maximum clade credibility (MCC) trees were summarized using TreeAnnotator v1.10.4 and subsequently rendered visually in FigTree.

Results

This study included five subjects from Yunnan Province, comprising three individuals from Kunming city and two from Qujing city. Sample collection dates spanned from 2021 to 2022 (2021: n = 4; 2022: n = 1). Among the five individuals, the median age was 53 years. The group consisted of three females and two males. The route of transmission was identified as heterosexual contact for four participants and remained undetermined for the other one (Table 1).

Table 1.

Demographic characteristics of participants with HIV-1

Sequence ID	GenBank accession number	Sampling year	Sex	Age	Marital status	Sample city
YN364	PX459504	2022	Female	53	Heterosexual contact	Kunming
YN269	PX459505	2021	Female	86	Heterosexual contact	Kunming
YN214139	PX459506	2021	Female	49	Unknown	Kunming
YN210253	PX459507	2021	Male	62	Heterosexual contact	Qujing
YN210154	PX459508	2021	Male	44	Heterosexual contact	Qujing

The phylogenetic analysis revealed that the five NFLG sequences formed a single, distinct clade with 100% bootstrap support, clearly distinguishing them from all other known HIV-1 subtypes and CRFs (Fig. 1A). Independent recombination analyses using jpHMM and RIP indicated the presence of recombination patterns and identified potential recombination breakpoints (Supplementary Fig. S1). Bootscanning analysis using HIV-1 subtypes B and C as reference sequences revealed that four subtype B fragments were embedded within a subtype C backbone, resulting in a mosaic genome composed of nine subregions: 1 C (nt 790–1234), 2B (nt 1235–1680), 3 C (nt 1681–3010), 4B (nt 3011–3310), 5 C (nt 3311–5699), 6B (nt 5700–6310), 7 C (nt 6311–8863), 8B (nt 8864–9021), and 9 C (nt 9022–9417) (Fig. 1B). However, the phylogenetic trees of each subregion demonstrated that subregions 4B, 5 C, 6B, 7 C, 8B, and 9 C were associated with the corresponding segments of CRF07_BC, while subregions 1 C, 2B, and 3 C were associated with that of CRF08_BC. These findings indicate that the novel strains resulted from recombination between CRF07_BC and CRF08_BC.

Fig. 1.

Phylogenetic and recombinant analyses were based on NFLG sequences. (A) Phylogenetic analyses were performed based on NFLG sequences covering HXB2 positions 790–9417. A total of 88 NFLG sequences were included in the analysis, comprising the study sequences and reference strains representing subtype B, subtype C, and BC-related CRFs, with HIV-1 group O and group P sequences included as outgroups. (B) Similarity plot and bootscanning analyses of the NFLG were performed using SimPlot v3.5.1. Bootscanning analysis was conducted under the Kimura two-parameter substitution model (transition/transversion ratio = 2.0), with a sliding window of 300 bp, a step size of 50 bp, and 1,000 bootstrap replicates. The subtype B reference group included AB097870, AB750367, and DQ990880. The subtype C reference group included AY967806, KF835522, and MG989567. The CRF07_BC reference group included JX392380, EF368372, EF368370, EF420986, JX392378, KF835494, KF835510, KF835532, KF250375, KF250377, OQ513528, OQ513533, OQ513538, OQ513481, OQ513489, OQ513595, and OQ513597. The CRF08_BC reference group included KF835493, KF835507, KF835497, KF835508, KF835534, KF835500, KF835501, KF835512, and AY008717. HIV-1 group O (KU168286) and group P (GU111555) were used as outgroups

To confirm the parental origin and delineate the recombination pattern, bootscanning analysis was performed using CRF07_BC and CRF08_BC as reference sequences. The results confirmed that this recombinant consisted of genes from both CRF07_BC and CRF08_BC (Fig. 1B). Bootscanning analyses of all five NFLGs demonstrated identical mosaic structures (Supplementary Fig. S1). The viral genome was composed of six recombinant segments, with CRF07_BC-derived fragments embedded within a CRF08_BC backbone (Fig. 1B). Across the six identified subregions, ML phylogenetic tree analysis confirmed that subregions II (nt 2676–6613) and VI (nt 8868–9417) were related to CRF07_BC, whereas subregions I (nt 790–2675), III (nt 6614–7610), and V (nt 7836–8867) were related to CRF08_BC (Fig. 2). However, it was unclear whether subregion IV (nt 7611–7835) was derived from CRF07_BC or CRF08_BC. In accordance with the established criteria for classifying new CRFs, these novel viruses were designated as CRF182_0708.

Fig. 2.

Phylogenetic analysis of the CRF182_0708 subregions. The construction of ML phylogenetic trees for the six mosaic segments within the CRF182_0708 genome was achieved by utilising the IQ-TREE v2.4.0 software, with HIV-1 subtypes, CRF07_BC, and CRF08_BC reference sequences serving as anchors. The CRF182_0708 sequence is highlighted with a red branch

To further explore the origin of CRF182_0708, Bayesian evolutionary analyses were conducted to estimate the time of the most recent common ancestor (tMRCA) for the concatenated CRF07_BC subregions II and the concatenated CRF08_BC subregions (I + III + V). The CRF07_BC and CRF08_BC datasets showed R² values of 0.61 and 0.88, respectively (Supplementary Fig. S2), indicating adequate temporal structure for molecular clock analyses. The estimated median tMRCA for the CRF07_BC subregions was 2002.7 (95% interval of the highest probability density [HPD]: 1998.1–2007.3), while that for the CRF08_BC subregions was 2003.1 (95% HPD: 2000.3–2008.5). These results suggested that CRF182_0708 probably emerged between 2002 and 2003 (Fig. 3).

Fig. 3.

The most recent common ancestor time (tMRCAs) origin analysis for CRF182_0708 is presented herewith. Generated using BEAST v1.10.5 for the CRF07_BC region (nt 2676–6613) and the merged CRF08_BC region (nt 790–2675 + nt 6614–7610 + nt 7836–8867). Annotated tMRCA and 95% HPD intervals for key nodes. CRF182_0708 sequences are highlighted with red and green branches, respectively

Discussion

This study identified a novel second-generation recombinant HIV-1 strain, CRF182_0708, in an HIV-1–positive population in Yunnan Province, China. The strain is a mosaic of six genomic fragments from CRF07_BC and CRF08_BC, originating around 2002–2003. Following the identification of CRF160_0708, CRF174_0708, and CRF177_0708, CRF182_0708 represents the fourth such CRF07_BC/CRF08_BC second-generation recombinant detected in Yunnan [10–12]. Collectively, these successive discoveries affirm that recombination between the two predominant circulating strains in Yunnan is not an isolated historical phenomenon, but rather a persistently active local epidemiological feature that is continuously driving increasing complexity in viral genetic diversity.

Yunnan exhibits the highest HIV-1 genetic diversity in China. The evolution of HIV-1 in this region is closely linked to shifts in transmission routes and socio-demographic factors. In the early phase of the epidemic, HIV-1 was introduced primarily through individuals who inject drugs, with subtypes B and C predominating. With the shift toward sexual transmission after the beginning of the 21st century, along with intensified population mobility, CRF07_BC and CRF08_BC gradually became the dominant strains and have co-circulated over the long term [14]. Additional BC recombinants (CRF85_BC, CRF118_BC) and imported strains (CRF55_01B) further contribute to the complexity of the local viral gene pool [15–17]. The co-circulation of these diverse strains provides sustained opportunities for co-infection and subsequent second-generation, or even multi-generation, recombination events.

CRF182_0708 was predominantly identified in Kunming and Qujing, major economic hubs with high population mobility. This spatial aggregation suggests that high-density population networks, in conjunction with ongoing transmission flows, generate hotspots for the emergence of novel recombinant strains [18]. In these settings, distinct viral lineages are more likely to come into contact, thereby substantially increasing the likelihood of co-infection and genetic recombination within individuals.

Conclusions

In conclusion, this study delineates a novel second-generation recombinant, CRF182_0708, in Yunnan, China. This finding emphasizes the ongoing, active, and complex evolution of local HIV-1 lineages. The expanding genetic diversity poses sustained challenges to viral surveillance, drug resistance assessment, and vaccine development. Consequently, future prevention and research strategies should prioritize sustained and dynamic molecular epidemiological monitoring in such high-risk regions to effectively track and respond to the evolving viral diversity.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

CRF

Circulating recombinant form

ESS

Effective sample sizes

HIV-1

Human immunodeficiency virus type 1

jpHMM

Jumping Profile Hidden Markov Model

MCC

Maximum clade credibility

MCMC

Markov Chain Monte Carlo

ML

Maximum likelihood

NFLG

Near-full-length genome

RIP

Recombination Identification Program

RT-PCR

Reverse transcription polymerase chain reaction

tMRCA

Time to the most recent common ancestor

URF

Unique recombinant form

Author contributions

XD, XX, and YF conceived and designed the study. WC, HP, QY, and LG conducted the investigation and developed the methodology. Formal analysis was performed by WC, HP, and LG. Resources were provided by XD, XX, and YF. The manuscript was written by WC, HP, LG, QY, XD, XX, and YF. XD, XX, and YF acquired funding and supervised the project. All authors have read and agreed to the final version of the manuscript.

Funding

This research was supported financially by Yunnan Major Scientific and Technological Projects (202405AJ310002, 202502AA310014).

Data availability

The sequences have been deposited in the GenBank database, with the accession numbers listed below: https://www.ncbi.nlm.nih.gov/genbank/. The NFLG genomes are represented by the accession numbers PX459504, PX459505, PX459506, PX459507, and PX459508, respectively.

Declarations

Ethics approval and consent to participate

The present study was conducted in accordance with the tenets of the Declaration of Helsinki and received approval from the Yunnan Provincial Hospital of Infectious Diseases Ethics Committee, AIDS Care Center (Approval No. YNACCA [2015]-12). Written informed consent was obtained from all participants prior to sample collection.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.