CRF01_AE/B/C,a Novel Drug-Resistant HIV-1 Recombinant in Men Who Have Sex with Men in Beijing,China

HIV-1 group M strains, which consist of nine subtypes (A, B, C, D, F, G, H, J, and K) and at least 72 circulating recombinant forms (CRFs), play a major role in the global HIV-1 HIV/AIDS pandemic. Furthermore, innumerable unique recombinant forms (URFs) have been reported, especially in regions where multiple subtypes and/or CRFs cocirculate in the same high-risk population. ¹

The number of reported HIV infections has been increasing rapidly among men who have sex with men (MSM) in China in recent years, especially in large and medium sized cities. Of note, the origins of the HIV-1 strains circulating among MSM are remarkable complex. Three predominant strains (subtype B, CRF01_AE, and CRF07_BC) and many other lineages and sublineages of HIV-1 strains are cocirculating together. Two new CRFs (CRF55_01B and CRF59_01B) and many URFs have been characterized among MSM in this population. ²

As the largest city in China, Beijing becomes a microcosm of the HIV-1 epidemic among MSM in China. The prevalence of HIV among MSM in Beijing increased from 3.1% in 2002 to 7.8% in 2010, ³ which is much higher than that found in most of the other cities in China (1.3–1.6%). ^{4 –6} The HIV-1 genotype distribution among MSM in Beijing was first studied in 2005–2006. ⁷ Subtype B was predominant among MSM (71.1%), followed by CRF01_AE (24.4%) and CRF07_BC (4.4%). Surveys that followed have shown that subtype B infections decreased to 41.9% in 2007 and to 30.8% in 2010. In contrast, CRF01_AE increased from 3.7% in 2005 to 56.0% in 2010 and CRF07_BC increased from <5% in 2005 to 12.6% in 2010. ⁸ The cocirculation of viruses from different subtypes and/or CRFs in the same region and risk groups fosters the emergence of new intersubtype recombinant strains. ⁹ In the present study, a novel near full-length genome (NFLG) sequence of an HIV-1 URF consisting of gene regions from CRF01_AE and subtypes B and C has been characterized in Beijing MSM.

This sample was collected from an MSM Beijing resident in September 2014. He was diagnosed as HIV-1 positive in April 2014 and began highly active antiretroviral therapy (HAART) 2 months later. RNA was exacted and then the NFLGs were amplified as we have done previously. ^{10 –12} Because of the short time (3 months) between the start of therapy and obtaining this sample for sequencing, we believe the patient was infected with a drug-resistant strain rather than believing that drug resistance arose in this short period of time in this patient. The patient's viral load was 15,600 copies/ml at the time of sampling, and this in part motivated the sequencing of his virus. The positive polymerase chain reaction (PCR) products were purified by the QIAquick Gel Extraction Kit (QIAGEN, Germany) and sequenced by an ABI 3730XL sequencer using BigDye terminators (Applied Biosystems, Foster City, CA). The sequenced data were cleaned and assembled with Sequencher v.4.9 (Gene Codes, Ann Arbor, MI). To avoid possible cross-contamination events, the NFLG was queried against all sequences obtained in the laboratory.

The NFLG sequence was then aligned against standard Los Alamos National Laboratory (www.hiv.lanl.gov/content/sequence/HIV.html) subtype reference sequences, which include subtype A1, A2, B, C, D, F, G, H, J, P, CRF01_AE, CRF07_BC, and CRF08_BC, and was edited manually using BioEdit v.7.1. ¹³ Subgenomic phylogenetic analyses were performed using the neighbor-joining method based on the Kimura two-parameter model implemented in MEGA v.6.06. ¹⁴ To analyze the recombinant structure of the strain, the sequence was submitted to the recombination identification program (RIP) (www.hiv.lanl.gov/content/sequence/RIP/RIP.html) using all default settings except a window size of 300, and a similarity plot analysis was performed using SimPlot (v.3.5.1). ¹⁵

In the phylogenetic tree reconstructed with NFLG reference sequences, the URF clustered with a 01BC recombinant URF reference sequence sampling from Anhui province in 2011 with bootstrap value (86%) (Fig. 1). ¹⁶ Rip, Bootscan, and similarity plot analysis of the NFLG sequence show a very complex mosaic structure composed of six CRF01_AE fragments arranged with three B and C intervals (Fig. 2). The breakpoints of the NFLG sequence are described as follows (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/aid): I (790–1196 nt) CRF01_AE, II (1197–1817 nt) C, III (1818–2823 nt) CRF01_AE, IV (2824–3340 nt) B, V (3341–3731 nt) CRF01_AE, VI (3732–4121 nt) B, VII (4122–5317 nt) C, VIII (5318–6049 nt) CRF01_AE, IX (6050–6544 nt) B, X (6545–6933 nt) CRF01_AE, XI (6934–7687 nt) C, and XII (7688–9411 nt) CRF01_AE. The Map-Draw Tool available at the Los Alamos HIV sequence database was used to map the breakpoints and illustrate the genomic structure of the novel HIV URF (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) (Fig. 3).

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FIG. 1.

A neighbor-joining phylogenetic tree of BJ.2014.MSM.SP01 with reference sequences constructed using MEGA 6.06. A solid circle (●) marks BJ.2014.MSM.SP01 throughout the article. The stability of each node was assessed by bootstrap analyses with 1,000 replicates and only significant bootstrap values of 70% or greater are shown at the corresponding nodes. The scale bar represents 5% genetic distance.

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FIG. 2.

Bootscan analysis was performed using CRF01_AE, B, and C reference sequences. The bootscan window was 300 bp with a step size of 10 bp using SimPlot 3.5.1 software. The x-axis is the nucleotide position in the HIV genomic sequence; the y-axis indicates the percentage supporting the clustering with reference sequences.

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FIG. 3.

The recombinant mapper results of BJ.2014.MSM.SP01. The schematic structure was created using the Recombinant HIV-1 Drawing Tool (www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html).

The mosaic structure of the recombinant genome is distinct from Anhui URF and any other known CRF or URF. Analysis of the subregion phylogenetic trees confirms the breakpoints described above and indicates that the parental origin of all CRF01_AE regions of the NFLG derived from the MSM-related CRF01_AE cluster 4 lineage, which is circulating primarily among MSM in Beijing ¹⁷ (Supplementary Fig. S1). The subtype B regions clustered with B (United States–European origin) reference sequences and the three subtype C regions clustered with the strain that was detected in China in 1998 among injection drug users (IDUs). ¹⁸

Multiple drug-resistant mutations were identified in this NFLG based on the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu/). One drug-resistant mutation to nucleoside reverse transcriptase inhibitors (NRTIs) at M184V and three mutations resistant to nonnucleoside reverse transcriptase inhibitors (NNRTIs) at V106M, V179E, and F227L were observed. The HIV gene map shows that this subtype B region covers a little more than codons 88 through 250 (refer to HXB2). Therefore all of the drug-resistant mutations are in the subtype B regions of the genome. Therefore it is most likely that the patient was infected with a drug-resistant strain, given the short time (3 months) of treatment before sampling.

The multiple drug resistance and recombination indicate the importance of this sample. The identification of this sample prompts us to pay more attention to the transmission of these new recombined strains. Increasing genetic diversity is a characteristic of HIV-1, resulting from both the accumulation of point mutations and recombination. HIV-1 recombination is an ongoing event and can significantly contribute to the epidemic in different regions. ^{19 –23} URFs are not strains of epidemic importance, but they hold the potential to enter high-risk transmission networks and become new CRFs. It is also of importance to note that recombination requires coinfection or superinfection of viral strains within an individual. Our detection found this NFLG had complicated breakpoints and insertions. In addition, multiple drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) is also illustrated in it. To our knowledge, what we detected (CRF01_AE/B/C) is a novel HIV-1 recombinant in MSM in Beijing, China. Both acquired and transmitted drug resistance of such a complicated URF necessitate more attention and further research.

Sequence Data

The NFLG sequence of BJ.2014.MSM.SP01 has been deposited in GenBank with accession number KP668994.