Characterization of a New HIV-1 CRF01_AE/B Recombinant Virus Form Among Men Who Have Sex with Men in Shanghai,China

Abstract

To date, there are 16 types of CRF01_AE/B circulating recombinant forms identified, and most of them are distributed in Asian countries such as China, Malaysia, and Singapore. Previous HIV molecular epidemiological surveys showed that CRF01_AE (27.6%) and B (9.6%) subtypes are predominant strains in mainland of China. At the same time, the HIV-1 virus spreads faster in the men who have sex with men (MSM) population than in other risk groups. In Shanghai district, ∼66.0% of newly reported cases were infected through homosexual transmission. In this study, we report a novel recombinant strain of CRF01_AE/B. The near full-length genome phylogenetic tree showed that the strain clustered with the CRF01_AE reference sequence and placed in the peripheral position within the branch of the CRF01_AE strain. Subregional evolutionary results indicated that the CRF01_AE subtype was derived from cluster 4 of CRF01_AE, which is mainly distributed in northern China. The subtype B was correlated with the U.S./Europe B, which are widely prevalent in the Chinese MSM population. In recent years, a large number of recombinant forms between CRF01_AE and B strains are continuously emerging in China. Therefore, understanding the current epidemic recombinant forms will have significant implications for prevention and treatment of HIV/AIDS.

Widely circulating HIV-1 strains can be seen in various regions of Asia, where various strains are cocirculated within and among different risk populations,^1,2 such as CRF07_BC³ and CRF08_BC.⁴ Meanwhile, many new circulating recombinant forms (CRFs) and unique recombinant forms have been reported in several regions in Asia.² In addition, CRF_01AE and subtype B are the two major strains circulating in Southeast and East Asia.^5,6 For instance, CRF15_01B in Thailand,⁷ CRF53_01B in Malaysia,⁸ and CRF55_01B was detected in men who have sex with men (MSM) population in China.⁹

CRF01_AE played an important role in regional epidemics. Around the world, ∼9% of CRF01_AE was found in East Asia.¹⁰ In the early 1990s, CRF01_AE strains were transmitted into southwest of China through sexual transmission and injection drug users (IDUs).^11,12 Over the past decades, CRF01_AE epidemic has been expanding from eastern and southwestern areas to almost the entire country.¹³ Although the proportion of subtype B was decreasing continually, it remains to play an important role in the HIV-1 epidemic in Shanghai. By the end of 2013, 8.3% of HIV-1 patients in Shanghai were infected with subtype B.¹⁴

Shanghai is one of the economically developed provinces in China, attracting large number of migrants from all parts of the world. HIV-1 among migrants have been a major issue being largely responsible for the HIV-1 epidemic; however, the newly reported HIV-1 infections among Shanghai residents remained at low levels (about 36%) in recent years compared with the migrants (∼64%).¹⁵ Furthermore, about 66.0% of HIV-1 new cases were infected through homosexual transmission.¹⁶

In this study, we detected a novel HIV-1 recombinant form (CRF01_AE/B), designated as SH150507. The sample was collected from an MSM in Shanghai, China, a 26-year-old unmarried Han ethnicity citizen with undergraduate degree. He was first diagnosed as HIV positive on April 15, 2015. His CD4+ T cell counts were 49 cells/μL on April 28, 2015 when the sample was collected. In addition, the Coreceptor (http://coreceptor.geno2pheno.org/index.php) and Web PSSM (https://indra.mullins.microbiol.washington.edu/webpssm) results show that this is an X4 Tropism (FPR = 5% and PRE = 1). The study was approved by the institutional review board of the National Center for AIDS/STD Control and Prevention of China.

The near full-length genome (NFLG) sequence (8,871 bp) was amplified as described previously,¹⁷ and a BLAST search was performed to exclude the possibility of cross-contamination. Reference sequences were downloaded from Los Alamos HIV Sequences Database (https://www.hiv.lanl.gov/components/sequence/HIV/search/search.html). Phylogenetic tree was constructed by the randomized axelerated maximum likelihood (RAxML) based on the approximately maximum-likelihood method with the general time reversible model for the NFLG alignment tree.¹⁸ 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 B. SimPlot3.5.1 version was used to detected recombination breakpoints sites (Reference sequence used for recombination site detection are shown in Supplementary Data, which contains BootScan and similarity plot analysis. Then, each fragment was analyzed by subregion phylogenetic trees. Similarity plot revealed that this was a CRF01_AE/B recombinant strain (Fig. 2A), and, BootScan revealed that the NFLG was divided into seven sections based on the HXB2 nucleotide positions (Fig. 2B), which are described as follows: I_B(HXB2,789-3500 nt), II_{CRF01_AE} (HXB2,3501-4500 nt), III_B(HXB2,4501-6040 nt), IV_{CRF01_AE} (HXB2,6041-6190 nt), V_B(HXB2,6191-6550 nt), VI_{CRF01_AE} (HXB2,6551-8572 nt), and VII_B(HXB2,88573-9412 nt). To make the breakpoints position more intuitive, the final map results of the schematic structure are shown in Figure 2C.

FIG. 1.

Phylogenetic tree analysis of the NFLG sequences of SH150507. The approximately ML phylogenetic tree was constructed with the GTR model by the RAxML. HIV-1 group M and group P sequences were used as reference sequences. The branches of SH150507 are displayed as a bold line and solid circle (). The scale bars are shown at the bottom of tree. GTR, general time reversible; ML, maximum-likelihood; NFLG, near full-length genome; RAxML, randomized axelerated maximum likelihood.

FIG. 2.

Recombinant analysis of the novel identified CRF01_AE/B. (A) The similarity between SH150507 and the reference sequences was plotted using SimPlot3.5.1 software. (B) BootScan analysis of the NFLG of SH150507, CRF01_AE groups, and subtype B groups were used as putative parental reference sequences, and subtype P groups were used as out-group (Supplementary Data). (C)The recombinant map of SH150507. The schematic structure was created using the Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

The subregion phylogenetic results (Fig. 3) showed that the sections of I, III, V, and VII were much closer to subtype B (a variant of subtype B prevalent in North America/Western Europe) than other subtype B clades. In addition, II, IV, and VI fragments were clustered with the prevalent cluster 4 of CRF01_AE, which was dominant in Northeast China (68.6%) and North China (17.5%), especially in MSM populations.^10,19

FIG. 3.

Subregion tree analysis of the novel identified SH150507. The phylogenetic trees of the seven mosaic segments defined by BootScan plot analysis were constructed with RAxML using the approximately ML method with the GTR model, and bootstrap values of 0.8 and higher are shown at the corresponding nodes. Reference sequences are labeled on right of the trees, respectively. The branches of SH150507 are displayed as a bold line and solid circle (). The scale bars are shown at the bottom of each tree.

To date, up to 16 different types of CRF01_AE/B CRFs are identified in many regions (https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html), such as in China, Japan, Malaysia, and Singapore. The most potential reason is that the CRF01_AE was increasing in predominance and intermingled with the subtype B lineage among MSM. This phenomenon provided an opportunity for generating novel recombinant form of HIV-1. In addition, widespread cocirculation and dual infection of CRF01_AE and subtype B in various regions have led to the emergence of CRFs comprising subtype B and CRF01_AE. Meanwhile, many factors associated with population migration, urbanization, and patterns of sexual activity can spread high numbers of recombinant viruses.

In conclusion, different HIV-1 subtypes coinfection from MSM populations of domestic areas and foreign regions could generate recombinant virus and spread into other lower risk groups²⁰ and geographic regions. In addition, other transmission routes could also have helped to create and spread recombinant viruses.²¹ This may increase the complexity of HIV-1 prevention and molecular epidemiological surveillance. However, it will provide important insights on local and cross-border transmission of HIV-1 molecular epidemiology. Also, this realization could affect our current understanding of the range of diversity within the HIV-1 epidemics and vaccine design, especially in light of superinfection involving multiple subtypes.²¹

Sequence Data

The NFLG sequences of SH150507 have been deposited in GenBank with the accession number MH615836.

Footnotes

Acknowledgments

This study was supported by grants from the Guangxi Bagui Honor Scholars, Ministry of Science and Technology of China (2017ZX10201101). Additional support was provided by the National Natural Science Foundation of China (81471962) and the Beijing Science and Technology Commission 2016 S&T Plan Major Project (SCW 2016-07).

Author Disclosure Statement

No competing financial interests exist.

Supplementary Material

Supplementary Data

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Supplementary Material

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