Identification of Two Novel HIV-1 Unique Recombinant Forms (CRF01_AE/CRF07_BC) and Genomic Characterization in Tongzhou District of Beijing,China

Introduction

Acquired immune deficiency syndrome (AIDS) is a chronic infectious disease caused by human immunodeficiency virus (HIV) infection of CD4⁺T lymphocytes, resulting in a decline in the humoral immunity functions. According to newly published data from the Joint United Nations Program on HIV/AIDS (UNAIDS) in 2022, 85.6 million people have been infected with HIV-1 globally since the first case of AIDS was detected. ¹ The rapid evolution and genetic diversity of HIV-1 were caused by high rates of replication, base mismatching, and recombination. ^2,3 The predominant strain currently circulating in the world derived from HIV-1 group M, which has evolved into 10 subtypes including A-D, F-H, and J-L. ³ Simultaneously, the high rate of recombination among the different subtypes had developed 145 circular recombinant phenotypes (CRFs) (https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/crfs.comp) and a significant number of unique recombinant forms (URFs). In China, an increasing number of infections living with HIV and the complexity of HIV-1 subtypes made it difficult to prevent and control the spread of the virus. ⁴ The fourth national HIV-1 molecular epidemiological survey in 2016 revealed a rapid increase in the variety of HIV-1 subtypes, which had become a priority for AIDS prevention and control in China. Multiple HIV-1 subtypes are prevalent in China, such as CRF01_AE, CFR07_BC, CRF08_BC, subtype B, and so on. ^5–6 Since the 1990s, Subtype B had been more popular than others, then subtypes CRF01_AE and CRF07_BC replaced it gradually as changing of transmission routines and floating of popuation. ^7–8 With a growing genetic heterogeneity of HIV-1, ⁹ it is possible that more and more URFs could be generated in future, so there is a necessity to conduct the surveillance of novel CRF and URF of HIV-1 to prevent them spreading further in China.

As the urban sub-center of Beijing, Tongzhou plays an important role in all aspects. Meanwhile, the favorable geographic and transportation location facilitated the transmission of the AIDS epidemic in the area to neighboring areas. Since the first case was reported in 1998 in Tongzhou, individuals infected with HIV-1 had amounted to 2,405 till 2023. A survey conducted among the MSM population in Beijing showed that 69.81% (349/500) of sexual partners were unfixed in the past six months. The high-risk sexual behavior and unprotected sexual behavior with multiple partners could easily infect HIV-1 and lead to recombination of different subtypes of HIV. ⁹ In this study, we analyzed near full-length genome (NFLG) characterization of two novel HIV-1 URFs obtained from two individuals infected with HIV-1 in 2021–2022 in the Tongzhou Center for Disease Control and Prevention (CDC). Written informed consent was obtained from the subjects before peripheral blood samples were collected.

The Tongzhou CDC collected plasma samples, along with demographic information, from two HIV cases (20110561 and 21110743). We extracted RNA using the MagNA-Pure LC Total Nucleic Acid Isolation Kit by MagNA-Pre LC 2.0 Instrument (Roche), followed by one-step reverse transcription with the PrimeScript^TM one-step RT-PCR Kit Version NO. RR055A) and nested PCR [TaKaRa Premix Taq^TM (Ex-Taq^TM Ver 2.0 plus dye) No. RR902A]. ¹⁰ Then, we sequenced the PCR product using the Sanger sequencing method of Beijing Biomed Gene Technology Co. ¹¹ The sequencing files were analyzed using Sequencher 5.4.5 and CExpress 6.0 software to assess the quality of the original peak maps. The HIV database Quality Control tool was utilized for quality control to exclude sequences that contained >5% mixed bases and stop codons. Subtyping was performed using Comet, and we aligned the sequences 20110561 and 21110743 with the HIV-1 reference sequences of each subtype in MAFFT 7. The ends of the sequences were trimmed using BioEdit 7.2.5. We constructed Maximum-likelihood phylogenetic trees by Maximum-Likelihood using FAST Tree 2.1.10, with nucleotide substitution modeled as GTR+CAT, SPR = 4, and Shimodaira-Hasegawa test for calculating the branching nodes of the evolutionary tree. The constructed evolutionary tree was embellished using ITOL (iTOL: Interactive Tree Of Life [embl.de]). Further, we analyzed the recombination breakpoints using jpHMM and RIP tools (https://www.hiv.lanl.gov/content/sequence/HIV/HIVTools.html). And we constructed a Neighbor-Joining phylogenetic tree for each recombinant fragment using MEGA6.06 software to validate the genotype of each fragment and perform a homology analysis of each recombinant fragment. The aim was to obtain accurate recombination breakpoint results. The recombinant structural patterns of the virulent strains were mapped by Recombinant GenomeDrawingTool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html). Amplification of NFLG sequences from plasma samples due to different subtypes in the ltr, gag, pol, and env gene regions. NFLG phylogenetic tree analysis showed that these sequences form a unique monophyletic cluster independent of other subtypes and CRFs (Fig. 1). Simplot v3.5.1 analysis showed that the NFLG sequence of 20110561 and 21110743 consisted of CRF01_AE and CRF07_BC (Fig. 2 and 3). The chimeric recombinant structures of two sequences obtained by the recombination identification program RIP with jpHMM were described as follows: I_{CRF01_AE}(HXB2, 989-1230nt); II;_{CRF07_BC}(HXB2, 1237-1867nt); III_{CRF01_AE}(HXB2,1891-9214nt), 20110561. I_{CRF01_AE}(HXB2, 989-2658nt); II_{CRF07_BC}(HXB2, 2662-4835nt); III_{CRF01_AE}(HXB2, 5036-5287nt); IV_{CRF07_BC}HXB2, 52902-5788nt); V_{CRF01_AE}(HXB2, 5801-6556nt); VI_{CRF07_BC}(HXB2, 6569-8423nt); VII_{CRF01_AE}(HXB2, 8427-9214nt), 21110743 (Fig. 2). Neighbor-joining phylogenetic trees were constructed using the MEGA6.06 with 1,000 bootstrap replicates under the Kimura 2-parameter model. The results showed that parental origin of all CRF01_AE regions of the two NFLGs originated from the MSM-associated CRF01_AE Cluster 4 (Fig. 4). The CRF07_BC parental origin of 20110561 clustered with 07BC_N and the CRF07_BC parental origin of 21110743 clustered with 07BC_O (Fig. 4).

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FIG. 1.

Phylogenetic tree analysis. A Neighbor-joining phylogenetic tree of 20110561 (8202 bp) and 21110743 (8231 bp) was constructed based on the NFLG sequences using Mega6.0. Bootstrap tests with 1,000 replicates assessed the stability of each node, and Bootstrap Value below 75 is no markings, while a value between 0.75 and 1.0 is displayed in five sizes of purple circles. Pure subtypes are represented with a light purple background, B and C subtype recombination with a yellow background; B, C, and CRF_01AE recombination with a light blue background; CRF_0107 are represented with a light green background, and CRF_0108 are represented with a light orange background.

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FIG. 2.

Genome maps of the NFLG of 21110743 (A) and 20110561 (B) are indicated with recombinant breakpoints (based on HXB2 numbering). The recombinant HIV-1 drawing tool is available at the Los Alamos National Laboratory HIV Database (www.hiv.lanl.gov./content/sequence/DRAW_CRF/recom_mapper.html). NFLG, near full-length genome.

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FIG. 3.

Bootscanning plot (up panel) and similarity plot (below panel) of 20110561 and 21110743. These analyses were performed using SimPlot v.3.5.1 with subtypes CRF07_BC and CRF01_AE as reference sequences. The bootscan window was 200 bp with a step size of 20 bp.

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FIG. 4.

Subregion phylogenetic tree. The subregion tree analyses of 20110561 and 21110743 were constructed by Mega6 through the Neighbor-joining method with 1,000 bootstrap replications. The 20110561 and 21110743 are labeled with the red solid circle (). Bootstrap values ≥75% were shown at the corresponding nodes. The scale bar represents 5% genetic distance.

In this study, two new recombinant strains of HIV-1 (20110561 and 21110743) were isolated from MSM in Tongzhou. The recombination patterns of the two NFLG sequences differed significantly from the URF sequences published in open reports and papers. Previous recombination breakpoints in URF mostly occurred in structural genes such as gag, pol, and env gene regions. However, the NFLG sequence reported in this study (21110743) with CRF01_AE subtype as the genomic backbone showed a segment was inserted from CRF07_BC subtype in the regulatory gene region (vif-vpu) (about 600 bp). The two URF source subtypes and populations were consistent. Therefore, the strains CRF01_AE and CRF07_BC were co-prevalent among MSM. Cluster 4 was the primary cluster for CRF01_AE, which has higher levels of X4 phagocytosis than cluster 5. As the viral infection progresses, there was the possibility that viral tropism would shift from R5 to X4 phagocytosis. CRF07_BC was widely spread in China due to migration for economic, cultural, and technological. The emergence of these new complex recombinant forms had increased the diversity of HIV-1 prevalence in the region. Therefore, it is necessary to focus on monitoring the evolution of the HIV-1 gene at the molecular level in Tongzhou. Strengthening infection control measures in MSM populations to reduce HIV-1 infection in other at-risk groups.