Characterization of a New HIV-1 CRF01_AE/CRF07_BC Recombinant Virus in Tianjin,China

HIV, the pathogen of AIDS, was divided into types HIV-1 and HIV-2. HIV-1 contains M, N, O, and P groups, and M group viruses, consisting of nine major subtypes (subtypes A to D, F to H, J, and K), and prevalent circulating recombinant forms (CRFs), are responsible for global pandemic. ¹ To date, at least 96 CRFs have been reported (https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html) and numerous unique recombinant forms (URFs) were identified worldwide. In China, HIV-1 CRF01_AE and CRF07_BC viruses are currently predominant. HIV-1 CRF01_AE was first detected among sex workers and intravenous drug users (IDUs) in Guangdong, Guangxi, and Yunnan provinces around year 1996–1997 and rapidly spread to other provinces along the southeast coast. ^{2 –5} In recent years, CRF01_AE strains have become the most widespread HIV-1 strain in China. ⁶ In contrast, CRF07_BC spread to northwestern China in IDUs along a drug traffic route in the 1990s. ^{7 –9} However, the recombinant forms between CRF07_BC and CRF01_AE are only found sporadically in different regions of China. ¹⁰ In this study, we identified a new HIV-1 recombinant virus between CRF07_BC and CRF01_AE in Tianjin, China.

Samples were obtained from an HIV-positive 24-year-old woman (TJ20170316) and her 1-year-old boy (TJ20170317) in 2017 in Tianjin, China. The mother did not know her HIV-positive status until she went to the hospital for baby delivery. Any intervention for prevention of HIV mother-to-child transmission was too late. The mother was not a sex worker, only stayed at home, and got infected from her HIV-positive husband, a man who has sex with men. The mother was diagnosed in 2017 and CD4⁺ T cell count in baseline was 327 cells/μL. Informed consent was signed before collecting patient's blood samples. Research Ethics Community of School of Medicine, Nankai University, reviewed and proved this study.

HIV-1 proviral DNA was extracted from the whole blood samples using QIAamp^® DNA Blood Mini Kit. Nested polymerase chain reaction (PCR) with the gradient DNA template to amplify full-length genome was conducted in the conditions for both of the two rounds, including an initial denaturation 94°C for 3 min followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, 68°C for 4 min, and final extension at 68°C for 10 min. The positive PCR products were purified (QIAquick Gel Extraction Kit; Qiagen) and sequenced, and the sequences were spliced and assembled using Sequencher v4.10.1 (Gene Codes).

The near full-length genome (NFLG) sequences of TJ20170316 and TJ20170317 were aligned with HIV-1 group M reference of different subtypes and CRFs in China, obtained from the Los Alamos HIV Sequence Database (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html), using software Clustal W and BioEdit (version 7.0.5.3) for analysis. The neighbor-joining trees were constructed using MEGA 7 with the Kimura two-parameter model and 1,000 bootstrap replications. Simplot (version 3.5.1) and jpHMM (http://jphmm.gobics.de/submission_hiv.html) determined the recombinant structures and recombination breakpoints. The segments between breakpoints were used to build subregion phylogenetic trees to confirm the origin of the different segments. The Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) was used to illustrate the structure of the new HIV-1 recombinant genome.

We finally obtained two 8,975 bp near full-length sequences from TJ20170316 and TJ20170317 (relative position to reference HIV-1 HXB2 760-9342). Both sequences are very close, except some point mutations. Phylogenetic analyses showed that the NLFG of TJ20170316 was closely related to CRF01_AE reference sequences, but forming a monophyletic branch with a bootstrap value of 100% (Fig. 1). Recombinant identification program and Simplot identified that the genome of TJ20170316 was composed of CRF01_AE and CRF07_BC (Fig. 2). Bootscan results showed that TJ20170316 consisted of at least five interlaced mosaic segments, including CRF01_AE (I, III, V) and CRF07_BC (II, IV) and four unique recombination breakpoints (Fig. 2A). Similar results were obtained using online software jpHMM (http://jphmm.gobics.de/submission_hiv.html). Thus, the map of the new recombinant virus TJ20170316 is as follows: I _{CRF01_AE} (760-1164 nt), II _{CRF07_BC} (1165‐2579 nt), III _{CRF01_AE} (2580-5178 nt), IV _{CRF07_BC} (5179-5844 nt), and V _{CRF01_AE} (5845-9342 nt) referenced by HXB2 positions (Fig. 2B). We further constructed phylogenetic trees of every segment using MEGA to identify those subtypes. Segments I, III, and V were clustered with reference CRF01_AE, and segments II and IV were clustered with reference CRF07_BC (Fig. 3). Phylogenetic analyses confirmed the recombinant breakpoints in TJ20170316, and showed that the breakpoints were totally different from reported CRFs and URFs.

OPEN IN VIEWER

FIG. 1.

Phylogenetic tree analysis of the NFLG sequences of TJ20170316 and TJ20170317. All M group reference sequences were constructed using the neighbor-joining tree by MEGA 7 with the Kimura two-parameter model and 1,000 bootstrap replications test. Bootstrap values >0.7 are shown at the corresponding nodes, and sequences TJ20170316 (from the mother) and TJ20170317 (from the child) are marked in solid circle. NFLG, near full-length genome.

OPEN IN VIEWER

FIG. 2.

Recombination breakpoint analyses of TJ20170316. (A) Bootscan plots of TJ20170316 using CRF01_AE, CRF07_BC, and subtype J as genotype references. The parameters of SimPlot bootscan analysis were set as the default (a window size of 300 and step size of 20). (B) Genome map of the NFLG of TJ20170316 (based on HXB2 numbering). The mosaic map was generated using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

OPEN IN VIEWER

FIG. 3.

Phylogenetic analyses of five mosaic segments (I–V) identified by bootscanning. Plot analyses were constructed by the neighbor-joining trees using MEGA 7 with the Kimura two-parameter model and 1,000 bootstrap replicates. Bootstrap values >0.7 are shown at the corresponding node. The sequences of TJ20170316 are marked in the solid circle. The scale bars are shown at the bottom of each tree. Reference sequences of CRF01_AE lineages detected in China are divided into seven groups (g1 to g7), and CRF07_BC lineage identified among men who have sex with men. Figure 3 can be viewed in greater detail online at www.liebertpub.com/aid

In 2014, an estimated 501,000 people in China were infected with HIV, and 0.9% of them were infected with HIV through mother-to-child transmission. ¹¹ There are relatively few cases due to mother-to-child transmission of HIV in China, ¹² because the Chinese government initiated programs for the prevention of mother-to-child transmission in 2004. ¹³ However, the possibility of increasing HIV prevalence through vertical transmission still exists, since the increasing number of HIV-positive women in reproductive ages, such as the case in this study. ¹³ The emergence of a new HIV-1 recombinant form reflects the HIV diversity in Tianjin. Therefore, the effective HIV-1 molecular epidemiologic investigation characterizing potential recombinant forms and their transmissions is necessary to control and prevent HIV-1 epidemic.

In summary, we identified a novel CRF01_AE/CRF07_BC recombinant form TJ20170316 from the pair of mother and child in Tianjin in this study. The NFLG of TJ20170316 contains four breakpoints. It was the first time that the URF of CRF01_AE and CRF07_BC recombinant was identified in Tianjin. The novel recombinant form suggested the local genetic complexity. Thus, a more effective strategy for preventing HIV-1 transmission is needed in Tianjin, China.