Identification of a New HIV-1 CRF68_01B/CRF01_AE Recombinant Form Among Men Who Have Sex with Men in Hebei Province,China

A global survey of 383,519 samples from 116 countries found that the three most common human immunodeficiency virus type one (HIV-1) subtypes globally were C, B, and A, accounting for 69% of isolates. ¹ However, CRF01_AE, CRF07_BC, and B are the most frequent strains in China. Compared with global patterns of HIV-1 genetic diversity, HIV-1 recombinant strains are more frequently observed in China. To this date, at least 123 HIV-1 circulating recombinant forms (CRFs) have been observed in the HIV Databases (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html) and numerous unique recombinant forms (URFs) in the epidemic have been also identified.

In China, the sexual contact transmission especially men who have sex with men (MSM) has been the most frequent route of HIV-1 transmission. The co-circulation and dual infections of the CRF01_AE, CRF07_BC, and subtype B strains in the sexual contact population will undoubtedly provide opportunities for the occurrence of their recombinant strains. In particular, more and more recombination strains were identified among MSMs (https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/crfs.comp), ^{2 –5} such as CRF79_0107, CRF67_01B, CRF69_01B, CRF59_01B, CRF80_0107, CRF102_0107, CRF104_0107, CRF55_01B, CRF68_01B, CRF109_0107, CRF113_0107, CRF117_0107, CRF103_01B, and CRF123_0107. Of the aforementioned 14 CRFs, 13 were identified in China except CRF69_01B from Japan.

In this study, we identified a new HIV-1 second-generation recombinant form originated from CRF68_01B and CRF01_AE among the recently infected MSMs by the near full-length genome (NFLG) analysis in Hebei province, China. Furthermore, we analyzed its genetic characterization and the parental linkage. In Hebei, CRF68_01B was minor subtype, and CRF01_AE was the most frequent subtype.

Blood plasma sample was collected from a man (named 242) in Cangzhou city of Hebei province, North China. Subject 242 was confirmed as HIV-1 positive using western blot on November 17, 2020, and was diagnosed as the recent HIV-1 infection using limiting antigen avidity enzyme immunoassay. He was a 65-year-old man infected with HIV-1 through homosexual contact. Written informed consent was obtained from subject 242 before blood collection. This study was approved by the Local Ethics Committee of Hebei Provincial Center for Disease Control and Prevention [No. IRB(S)2020-031].

HIV-1 NFLG extraction, amplification, and sequencing were implemented as previously described. ⁶ Raw sequences were assembled using Contig Express 9.1. Multiple sequences alignment with Clustal W and manual editing were performed using Bio-Edit 7.0 software. Finally, the NFLG sequence isolated from subject 242 was 8,943 bp (HXB2:649-9 599) in size. The subtype reference sequences and CRFs were downloaded from the HIV Databases (www.hiv.lanl.gov/content/index). The phylogenetic trees were constructed using the Kimura two-parameter model with 1,000 bootstrap replicates in MEGA 6.0. To analyze recombination breakpoints of the NFLG, the online jumping profile Hidden Markov Model (http://jpHMM.gobics.de/submission_hiv.htm), the online RIP 3.0 (https://www.hiv.lanl.gov/content/sequence/RIP/RIP.html) and SimPlot v3.5.1 were used.

Figure 1A indicated that subject 242 formed a distinct monophyletic branch separately from other subtypes and CRFs in the NFLG N-J phylogenetic tree, suggesting that this NFLG sequence should be a novel recombinant form. According to the results of the online jpHMM (Fig. 1B) and RIP (Fig. 1C), the breakpoints analysis identified that this NFLG sequence was originated from CRF68_01B and subtype A1 or CRF01_AE. The subtype of subregion II segment was confirmed as CRF01_AE (Fig. 1D) using SimPlot v3.5.1 (Fig. 2A) and the subregion phylogenetic trees (Fig. 2B). Figures 1D and 2A indicated that the NFLG of subject 242 contained a recombinant pattern with a CRF01_AE gene fragment inserted to a CRF68_01B backbone, spanning from the pol gene position 3802 to 4076 based on HXB2 numbering. The position in the original sequence was 3197 to 3471.

OPEN IN VIEWER

FIG. 1.

The phylogenetic tree and recombinant analysis based on HIV-1 NFLG sequences. (A) The NFLG phylogenetic tree. An N-J tree was constructed using MEGA 6.0 with 1,000 bootstrap replicates. The standard reference sequences of HIV-1 subtypes were downloaded from the HIV Database (www.hiv.lanl.gov/content/index). Bootstrap values ≥70% are shown in the tree. The scale length indicates 5% nucleotide sequence divergence. Black dot (•) denotes study subject. (B) The jpHMM analysis of the NFLG sequences. The NFLG mosaic maps of subject 242 and the CRF68_01B reference from Anhui province were obtained using the online jpHMM (http://jphmm.gobics.de). (C) The RIP analysis of the NFLG sequence of subject 242. Similarity distance analysis was performed using RIP (version 3.0; Siepel AC, Halpern AL, Macken C, Korber BT, http://hiv-web.lanl.gov) from the Los Alamos National Laboratory HIV Database with default setting except for the window size of 300. (D) Genetic map of CRF68_01B/CRF01_AE. The mosaic map was obtained using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov./content/sequence/DRAW_CRF/recom_mapper.html). The CRF68_01B and subtype CRF01_AE segments are shown in grey and black, respectively. CRF, circulating recombinant form; HIV-1, human immunodeficiency virus type one; NFLG, near full-length genome; N-J, neighbor-joining.

OPEN IN VIEWER

FIG. 2.

Bootscanning analysis and subregion phylogenetic trees of CRF68_01B/CRF01_AE. (A) Bootscanning analysis of CRF68_01B/CRF01_AE. Bootscanning analysis was conducted using SimPlot 3.5.1 software along with reference sequences of subtype C (the light grey line), CRF68_01B (the dark grey line) and CRF01_AE (the black line). (B) Subregion phylogenetic trees of subject 242. Subregion phylogenetic analysis of three segments of subject 242 were constructed by Mega 6.0 using the N-J method with 1,000 bootstrap replications. Bootstrap values ≥70% are shown at the corresponding nodes. The scale bar represents 5% genetic distance. Black dot (•) denotes study subject. The HIV-1 M group subtype reference set including just the nonrecombinant A-L genomes and the CRF01_AE and CRF68_01B reference genomes were downloaded from HIV Databases (www.hiv.lanl.gov/content/index).

Moreover, a total of three recombinant segments were identified by recombination analysis. According to position based on HXB2 numbering, its three subregions were as follows: I_{CRF68_01B} (790–3,801 nt), II_{CRF01_AE} (3,802–4,076 nt), and III_{CRF68_01B} (4,077–9,412 nt). The subregion phylogenetic tree (Fig. 2B) revealed that all the subregions clustered with their respective reference sequences, which identified the NFLG mosaic structure of the novel second-generation recombinant form too. Figure 2B also indicated that CRF68_01B fragment and CRF01_AE fragment within the NFLG of this recombinant form should be originated from Anhui province and neighboring provinces of Hebei, respectively.

Since the first acquired immunodeficiency syndrome (AIDS) case was found in 1981 in America, four decades have gone. HIV-1 has spread to all countries worldwide and caused serious global public health problem. According to an official report released by the Joint United Nations Programme on HIV/AIDS (UNAIDS), 37.7 million people were infected with HIV worldwide in 2020. By the end of 2021 October, estimated 1.14 million people were living with HIV/AIDS reported in China.

Currently, a total of 17,891 individuals with HIV-1/AIDS were reported in Hebei province. The most frequent HIV-1 subtype prevalent in Hebei has changed from subtype B before 2005 to CRF01_AE and CRF07_BC after 2005 because of the shift of HIV-1 transmission routes from blood to sexual contact. ^7,8 In addition, Cangzhou city shares a border with Tianjin, and was severely affected by HIV-1. Since the first individual infected with HIV-1 in 2003, the number of HIV-1 infections in eight counties of Cangzhou were >100. Homosexual contact plays a key role in HIV-1 spread in Cangzhou city.

With the shift of HIV-1 transmission routes, HIV-1 genetic diversity is more and more complex and constantly evolving. In past years, numerous HIV-1 recombinant strains were formed due to the co-circulation and dual infections of the main prevalent strains. In Hebei, several CRFs and URFs have been identified continually such as CRF103_01B, ⁵ CRF123_0107² and CRF01_AE/CRF07_BC. ⁹ In our study, a new CRF68_01B/CRF01_AE recombinant form was identified in Cangzhou city. It is well known that the prevalence rates of CRF68_01B are very poor.

Currently, CRF68_01B has been reported in Zhangjiakou of Hebei, ¹⁰ suggesting that this subtype is circulating in Hebei. CRF68_01B was first described in Anhui province, China, in 2013, and its prevalence rate was only 1.44% in 2020. ¹¹ To date, CRF68_01B is circulating in seven provinces of China, with a prevalence rate of 0.2% to 2.79%. In Hebei, CRF01_AE is the most frequent subtype circulating in the MSM population, according for 57.6%. ¹² However, subtype A1 was an imported strain, and the overall prevalence rate of subtype A1 and other subtypes such as C, F, G, and CRF02_AG was <5% according to the fourth National Molecular Epidemiological Survey of HIV-1.

We find through literature searches that this is the first report of HIV-1 CRF68_01B\CRF01_AE form in the world. We infer that factors associated with the occurrence of this CRF68_01B\CRF01_AE form are as follows: first, subject 242 is a MSM recently infected with HIV-1; this CRF68_01B\CRF01_AE form may be the original strain that infected him, suggesting that this recombinant form has been prevalent in China, and it may be a potential CRF. Second, our previous report indicated that CRF68_01B circulating in the Hebei province was minor subtype, and the prevalence of CRF01_AE is the most common, accounting for >50.0%. So we infer that this MSM infected with CRF68_01B was reinfected with CRF01_AE. The identification of this CRF68_01B/CRF01_AE form suggested that further dynamic surveillance of new recombinant forms related to not only major strains but also minor strains is necessary.

In conclusion, we identified a novel HIV-1 second-generation recombinant form originated from CRF68_01B and CRF01_AE among the recently infected MSMs in Hebei, China. To our knowledge, MSM has become the most frequent transmission route in the Beijing–Tianjin–Hebei region, as there is a close transmission relationship of HIV-1 among MSMs in this area. Therefore, we should constantly monitor HIV-1 diversity in Hebei and take further effective measures to control the recombination of not only HIV-1 major strains but also minor strains.

Sequence Data

The sequence reported in this study has been submitted to GenBank with the accession number ON007374.