Identification of a Novel HIV-1 Recombinant Form (CRF01_AE/07_BC) from an Elderly Individual in Hangzhou,Eastern China

The human immunodeficiency virus 1 (HIV-1) is characterized by extremely high genetic variability and rapid evolution.^1,2 In addition to its naturally high mutation rate, HIV-1 further generates new variants through genetic recombination, particularly when an individual is coinfected with two or more distinct strains.^3,4 To date, more than 160 circulating recombinant forms (CRFs) and countless of unique recombinant forms (URFs) have been reported globally (https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html). Given the ongoing prevalence of HIV-1 coinfection and viral evolution, the number of recombinant HIV-1 strains is expected to continue increasing.

Since the mid-1990s, CRF07_BC and CRF01_AE have been the dominant HIV-1 subtypes in China.^5,6 The cocirculation of these two CRFs in various high-risk populations unavoidably creates more opportunities to generate further complex recombinants. To date, a total of 19 CRFs derived from CRF07_BC and CRF01_AE have been identified across multiple provinces in China, with the majority being identified among men who have sex with men, followed by heterosexual transmission groups. ⁷ In recent years, the number of newly reported HIV-1 cases among people aged ≥50 years has been on the rise in China. The majority of these cases are males infected via heterosexual contact, highlighting an urgent need for strengthened monitoring in this population.^8,9

Here, we report the near full-length genome (NFLG) characterization of a novel HIV-1 URF, designated 24HZ0410, from a 79-year-old male infected via nonmarital commercial heterosexual contact. The patient was diagnosed as HIV-1 at a sexually transmitted disease clinic in 2024. At baseline (pretreatment), his CD4⁺ T-cell count and viral load were 287 cells/μL and 23,400 copies/mL, respectively. Following 1 year of antiretroviral therapy, his viral load was suppressed to undetectable levels, but his CD4⁺ count remained low (256 cells/μL). This study was approved by the Hangzhou Center for Disease Control and Prevention, and oral informed consent was obtained before sample collection.

Viral RNA was extracted from 140 µL plasma using the QIAamp® Viral RNA Mini Kit (Qiagen, Hilden, Germany), and then reversely transcribed into cDNA using a Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, USA). The NFLG was amplified in two halves by nested PCR with ExTaq reagent (TaKaRa, Dalian, China) under conditions previously described. ¹⁰ The target PCR products were purified and commercially sequenced by Hangzhou TsingKe Bio-Tech Co., Ltd. Chromatogram data were manually spliced and assembled using Sequencer® v5.4.6 (Gene Codes). The assembled NFLG sequence of 24HZ0410 had a length of approximately 9.0 kb. It was further validated using the HIV Sequence Database’s Quality Control tool (http://www.hiv.lanl.gov/content/sequence/LOCATE/locate.html). Subsequent analysis confirmed that this genome encodes all canonical HIV-1 structural and regulatory genes, including gag, pol, vif, vpr, tat, rev, vpu, env, and nef.

The NFLG sequence of 24HZ0410 was aligned with reference sequences using MAFFT v7.4.8. Reference sequences comprised HIV-1 group M subtypes and major CRFs prevalent in China, which were obtained from the Los Alamos National Laboratory HIV Database (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html). The alignment sequences were trimmed to an identical lengths (HXB2: 633–9,614 nt) using BioEdit v7.2.5.0. Subsequently, maximum-likelihood（ML） phylogenetic trees of the NFLG and subgenomic regions were constructed with IQ-TREE v2.3.2. The resulting trees were visualized and annotated using FigTree v1.4.4 and the Interactive Tree of Life (iTOL) v6.0 (https://itol.embl.de/itol.cgi). The ML phylogenetic tree of the NFLG showed that the 24HZ0410 sequence forms a distinct, highly supported monophyletic cluster (100% bootstrap) with the CRF07_BC reference strain. However, its position at the periphery of this cluster suggests that 24HZ0410 may represent a newly emerging lineage, closely related to but genetically distinct from classical CRF07_BC strains (Fig. 1A).

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FIG. 1.

Phylogenetic analysis of the NFLG and recombinant segments of 24HZ0410. (A) The maximum-likelihood (ML) phylogenetic trees of 24HZ0410 based on the currently known HIV-1 subtype NFLG reference sequences. (B–D) The ML phylogenetic trees of three recombinant segments of 24HZ0410 based on the main HIV-1 subtype reference sequences and some selected CRF01_AE and CRF07_BC reference sequences. The ML trees were reconstructed by IQ-TREE program v2.3.2. Only bootstrap values ≥80% were displayed on the corresponding nodes. NFLG, near full-length genome.

To determine whether 24HZ0410 is a URF, we performed recombination analysis using two independent methods: Similarity Plot (SimPlot) v3.5.1 and the jumping profile Hidden Markov Model (jpHMM) (http://jphmm.gobics.de/submission_hiv). Bootscanning analysis with SimPlot (window: 400 bp; step: 20 bp) indicated that the 24HZ0410 genome is composed of CRF01_AE and CRF07_BC, featuring a short CRF01_AE fragment inserted into a CRF07_BC backbone (Fig. 2A). This recombinant structure was confirmed by jpHMM analysis. A schematic representation of the recombination pattern, mapped against the HXB2 coordinates, was generated using the HIV-1 Recombinant Drawing Tool to illustrate the genomic organization clearly (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) (Fig. 2B).

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FIG. 2.

Recombination breakpoints analysis of the NFLG sequence of 24HZ0410. (A) The bootscanning analysis of the NLFG of 24HZ0410. With the NLFG of 24HZ0410 as the query sequence, recombinant breakpoints were scanned by Simplot program v3.5.1. The x-axis represents each sequence position, and the y-axis represents the percentages of permuted trees. (B) Genomic structure map of the NFLG sequence of 24HZ0410 (HXB2:633–9614 nt). The red colorrepresents HIV-1 CRF07_BC subtype, and the blue color represents HIV-1 CRF01_AE subtype in the map. All structural genes and regulatory genes involved in the NFLG sequence of 24HZ0410 were labeled in detail. NFLG, near full-length genome.

These findings demonstrate that the 24HZ0410 NFLG was a recombinant derived from CRF07_BC and CRF01_AE. The genome was structured with CRF07_BC as the dominant backbone, into which a fragment of CRF01_AE was inserted. Recombination analysis delineated two breakpoints defining three segments: Segment I (HXB2: 633–5,945 nt) and Segment III (HXB2: 6,348–9,614 nt), both originating from CRF07_BC, span the gag-pol-vif-vpr-partial tat and partial env-nef-partial 3'-LTR regions, respectively. Segment II (HXB2: 5,946–6,347 nt), derived from CRF01_AE, located partial tat-rev-vpu-partial env region.

Phylogenetic analysis of the three individual recombinant segments corroborated the bootscanning results (Fig. 1B–D). The trees confirmed the distinct origins of each segment: both Segment I and Segment III of 24HZ0410 clustered with 100% bootstrap support within the CRF07_BC lineage, specifically with a newer cluster (CRF07_BC-N) predominantly found among MSM in northern China (Fig. 1B,D). The segment II formed a well-supported monophyletic cluster (bootstrap >80%) with reference sequences of the CRF01_AE subtype (Fig. 1C).

In conclusion, we identified a novel URF originating from CRF07_BC-N and CRF01_AE in an elderly individual from Hangzhou, eastern China. Against the backdrop of a rapidly rising HIV-1 incidence among older adults in China, ⁸ this case highlights that sexually active elderly individuals represent an emerging demographic in the dynamics of HIV-1 transmission and viral diversification. The case presented here exemplifies the clinical challenges in this group, where immunosenescence led to suboptimal CD4⁺ T-cell recovery despite effective virological suppression after 1 year of therapy. In the Chinese context, factors such as predominant sexual transmission, frequent late diagnosis, and a high burden of comorbidities collectively worsen health outcomes and complicate management for elderly people living with HIV. ¹¹ Therefore, alongside broader interventions targeting this vulnerable group, it is essential to strengthen molecular surveillance to accurately track emerging recombinant strains and gain a deeper understanding of their transmission dynamics among the elderly.

Importantly, CRF07_BC in China is divided into two distinct clusters: the original CRF07_BC-O, circulating mainly among people who inject drugs and heterosexual populations in southwestern and northwestern regions, and the more recently identified CRF07_BC-N, which is predominantly found among MSM in northern China and exhibits high transmission potential, gradually displacing CRF07_BC-O.^12,13 The identification of a CRF07_BC-N-derived recombinant in a heterosexual individual in eastern China suggests the potential geographic and demographic expansion of this cluster. Furthermore, as a major economic hub in the Yangtze River Delta with substantial population mobility, Hangzhou provides favorable conditions for the co-circulation and recombination of diverse HIV-1 strains. ¹⁴ Previous surveillance based on the pol gene region identified at least 20 HIV-1 genetic subtypes, with URFs accounting for approximately 5.0%, ¹⁵ significantly lower than the 19.6% URF rate reported from three Chinese provinces based on near-full-length genome sequencing. ¹⁶ Notably, the recombination event described here occurred outside the pol region, indicating that pol-based surveillance alone may fail to capture the full spectrum of recombinant viruses. Therefore, our study underscores the urgent need to develop and implement more comprehensive approaches, such as near-full-length genomic sequencing, for detecting and characterizing HIV-1 recombinants to accurately elucidate their molecular epidemiology and evolutionary dynamics.

Sequence Data

The nucleotide sequence of 24HZ0410 has been submitted to GenBank with accession no. PX377366.

Authors’ Contributions

K.X. and Q.F. designed the study. L.Y., W.L., and S.W. performed experiments. M.Z. collected the sample. L.Y. and S.W. analyzed the data. L.Y. wrote the main article and prepared all figures. Z.S. and X.Z. collected sample information.