Characterization of a Novel HIV-1 CRF01_AE/CRF07_BC Recombinant Virus Form in Guizhou,China

Since 1980, HIV-1 has been an important public health problem around the world. During the global epidemic, HIV-1 evolved into various subtypes and their hybrids, including many unique recombinant forms (URFs), ¹ and the so-called circulating recombinant forms (CRFs), ² including CRF07_BC, ³ CRF08_BC, ⁴ and CRF01_AE.

CRF01_AE was the major strain circulating in Southeast and East Asia, and ∼9% of CRF01_AE was found in East Asia. ^5,6 Since CRF01_AE was introduced into China after the 1990s, it has been expanding from eastern and southwestern areas to almost all of China. ^{6 –9} In addition, CRF07_BC was found gradually predominating among injection drug users (IDUs) in southern China, ⁴ and then it was transmitted to other provinces among the IDUs along the drug-trafficking route. ¹⁰ In the global transmission, the epidemic is dominated by CRF07_BC and CRF08_BC, CRF01_AE, and subtype B′ (Thailand variant of subtype B), which were also predominant in China, ^5,8 thus they provide opportunities for HIV-1 second generation recombinant forms among various high-risk groups or subtypes (sub-subtypes). In recent years, there have been reported some second generation recombinants for CRF01-AE and CRF07_BC, such as in Jining, ¹¹ Tianjin, ¹² and Zhejiang. ¹³

In this study, we first detected a novel HIV-1 recombinant form (CRF01_AE/CRF07_BC), which was designated as GZ18021. The sample was collected from an HIV-positive woman in Guizhou, southwestern China. The patient was first diagnosed as HIV-positive on October 28, 2018, when she came back to her hometown from eastern China. Subsequently, she was confirmed on November 10, 2018, and was recruited. The patient was a 30-year-old, married woman, and self-reported that she used to be a waiter in a night club. Her CD4+ T cell count was 316 cells/μL when the HIV-1 antibody was confirmed positive. The study was approved by the Institutional Review Boards of the Second Affiliated Hospital of Guizhou Medical University.

The near full-length genome (NFLG) sequence (9,002 bp) was amplified as described previously. ¹⁴ First, the sequence was uploaded to BLAST to detect whether it had cross-contamination. Then, we uploaded the sequence to RIP (www.hiv.lanl.gov/content/sequence/RIP/RIP.htmld) and jpHMM (http://jphmm.gobics.de/submission_hiv) to determine the potential recombination breakpoints. Reference sequences were downloaded from Los Alamos HIV Sequences Database (www.hiv.lanl.gov/components/sequence/HIV/search/search.html). Phylogenetic trees were constructed by the FastTree based on the approximately-maximum-likelihood method with the general time-reversible model for the NFLG alignment tree. ¹⁵ Branches with bootstrap values >0.8 were considered as phylogenetic clusters. As shown in Figure 1, the strain formed a distinct and well-supported (bootstrap values = 1) monophyletic cluster, distantly related to subtype CRF01_AE. Simplot version 3.5.1 was used to detect more accurate breakpoint positions, which are composed of Bootscan and similarity plot analysis. Then, each fragment was analyzed by subregion phylogenetic trees. Similarity plot showed that this was a CRF01_AE/CRF07_BC recombinant strain (Fig. 2A), and Bootscan demonstrated that the NFLG was divided into seven sections based on the HXB2 nucleotide positions (Fig. 2B), which were described as follows: I_{crf01_AE} (HXB2, 789–3,500 nt), II_{CRF07_BC} (HXB2, 3,501–4,500 nt), III_{CRF01_AE} (HXB2, 4,501–6,040 nt), IV_{CRF07_BC} (HXB2, 6,041–6,190 nt), and V_{CRF01_AE} (HXB2, 6,191–6,550 nt). For more intuitive results, the final map results of the schematic structure are shown in Figure 2C.

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FIG. 1.

Phylogenetic tree analysis of the NFLG sequence of GZ18021. The approximately-ML phylogenetic tree was constructed with the GTR model. Reference sequences were downloaded from HIV databases. The branches of GZ18021 were displayed as a bold line and circle. GTR, general time-reversible; ML, maximum-likelihood; NFLG, near full-length genome.

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FIG. 2.

Recombinant analysis of the novel identified CRF07_BC/CRF01_AE. (A) The similarity between GZ18021 and the reference sequences was plotted using SimPlot 3.5.1 software. (B) Bootscanning analyses of the NFLG of GZ18021, CRF07_BC, and CRF01_AE were used as parental reference sequences, and subtype N as a background group. (C) The recombinant map of GZ18021. Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) was used to drawn the schematic structure.

The subregion phylogenetic analysis of GZ18021 (Fig. 3) showed that the regions I, III, and V greatly related to the CRF01_AE subcluster 4 lingeage. However, the regions II and IV clustered with the CRF07_BC reference sequences.

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FIG. 3.

Subregion tree analysis of the novel identified GZ18021. The phylogenetic trees of the five mosaic regions were identified by bootscanning plot analysis, and the distinct subregion (from I to V) were constructed with FastTree using the approximately-ML method with the GTR model, and bootstrap values of 0.80 and higher were shown at the corresponding nodes. Reference sequences were labeled on the right of the trees. The sequences of GZ18021 were bold line and circle. The scale bars were shown at the bottom of each tree.

At present, up to 99 types of CRFs have been confirmed in the HIV strains, about 20.2% are closely correlated with those of China. However, only two types of CRF01_AE/CRF07_BC CRFs were identified, and named CRF79 0107 ¹⁶ and CRF80 0107 ¹⁷ (www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html), respectively, and the rest were URFs. In the past 30 years, the HIV epidemic has become increasingly genetically complex in China. ¹⁸ CRF07_BC was initially detected among Xinjiang, Sichuan IDUs, Shao et al. ¹⁹ Previous researches showed that CRF07_BC originated from Yunnan, and transmitted to other provinces among the IDUs along the drug-trafficking route. ^10,18 Interestingly, some studies have reported that CRF07_BC has also been found in men who have sex with men (MSM) population. ^18,20 In addition, a previous study reported a CRF01_AE/CRF07_BC unique recombinant circulating in Zhejiang province, where the CRF01_AE fragments were very close to cluster 4. ¹³ However, earlier research showed that cluster 4 is predominant in northeastern China (68.6%), especially in the MSM population. ^6,9

Migration and travel are the two major drivers of the spread of infectious diseases. ²¹ And the spread and epidemic of HIV in Guizhou Province is highly correlated with the route of transmission and social activities. ²² Guizhou is partially surrounded by Yunnan and Guangxi, and the northwest is bordered by Sichuan Province. Meanwhile, as a part of the drug-trafficking route, there was a large number of HIV-positive IDUs belonging to CRF07_BC and CRF08_BC. ²² In addition, as a major labor-exporting province, many immigrants work in the eastern region each year, and this population has an important impact on infectious disease transmission. ²³

In summary, many factors are associated with high numbers of recombinant viruses and increase the complexity of HIV-1 prevention. Therefore, we need to strengthen the monitoring of HIV-1 strain trends and facilitate the development of more effective preventive interventions.