Identification of a novel HIV-1 second-generation Circulating Recombinant Form CRF109_0107 in China
Authors: Xiaorui Wang, Jin Zhao, Xi Li, Hanping Li, Yu Zhang, Yongjian Liu, Lin Chen, Chenli Zheng, Lei Jia, Jingwan Han, Tianyi Li, Xiaolin Wang, Jingyun Li, Hongling Wen, Lin Li
Journal: Journal of Infection (2020)

Inter-subtype recombinants play an increasingly central role in the complex and dynamic HIV/AIDS epidemic. At present, 106 Circulate recombinant forms (CRFs) have been identified worldwide. In 2000, CRF07_BC and CRF08_BC were first reported in China, and novel CRFs identified in China has soared since 2013. Inter-subtype recombinant viruses, especially CRF01_AE and CRF07_BC, have become the predominant strains of the HIV-1 epidemic in China.1Li X. Li W. Zhong P. et al.Nationwide trends in molecular epidemiology of HIV-1 in China.AIDS Res Hum Retroviruses. 2016; 32 (09): 851-859Crossref PubMed Scopus (56) Google Scholar The double infection between CRFs established more optimal conditions for the second-generation recombination,2Luan H. Han X. Yu X. et al.Dual infection contributes to rapid disease progression in men who have sex with men in China.J Acquir Immune Defic Syndr. 2017; 75 (08 01): 480-487Crossref PubMed Scopus (17) Google Scholar and many second-generation recombinants were represented by the recombination of CRF01_AE and CRF07_BC fragments, such as CRF79_0107,3Li Y. Feng Y. Li F. et al.Genome sequence of a Novel HIV-1 circulating recombinant form (CRF79_0107) identified from Shanxi, China.AIDS Res Hum Retroviruses. 2017; 33: 1056-1060Crossref PubMed Scopus (32) Google Scholar CRF80_0107,4Zhang Y. Pei Z. Li H. et al.Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF80_0107) Among Men Who Have Sex with Men in China.AIDS Res Hum Retroviruses. Apr 2019; 35: 419-423Crossref PubMed Scopus (20) Google Scholar and CRF102_01075Li X. Wu J. Zhang Y. et al.Characterization Of A Novel HIV-1 S-generation circulating recombinant form(CRF102_0107) among men who have sex with men in Anhui, China.J Infect. 2019; 79: 612-625Scopus (11) Google Scholar. In the present study, we identified a newly emerging second-generation HIV-1 CRF called CRF109_0107, consisting of CRF01_AE and CRF07_BC fragments. Through the evolution analysis of all CRF01_AE and CRF07_BC recombinant fragments, we uncovered the evolutionary history of CRF109_0107 finally. Plasma samples were collected from two HIV-positive patients (LS11584 and LS14250) who were infected through sexual transmission in Shenzhen, China. The sequence BJSJS2016S195 is obtained from the Los Alamos HIV Sequence Database with the full-length gag (HXB2:790–2292 nt) and pol (HXB2:2085–5096 nt) gene sequences in the Shijingshan district of Beijing, China. There was no epidemiologic link among these three individuals. All participants gave a written, signed informed consent prior to plasma specimen collection and subsequent analyses. Basic epidemiological data are presented in Table 1.Table 1Demographic characteristics of study subjects infected with CRF109_0107.Strain nameSampling yearSampling regionGenderAgeEthnic groupMarriageRisk factorAccession no.LS115842014Shenzhen, GuangdongMale45HanunmarriedMSMMT919517LS142502014Shenzhen, GuangdongMale40HanmarriedHETEMT919518BJSJS2016S1952016BeijingMale33unknownunknownunknownMH921062.MH921177*MH921062: BJSJS2016S195 Gag Region; MH921177: BJSJS2016S195 Pol Region. Open table in a new tab *MH921062: BJSJS2016S195 Gag Region; MH921177: BJSJS2016S195 Pol Region. Viral RNAs were extracted from plasma samples using the High Pure Viral RNA Kit (Roche, Germany). Then, the near full-length genome (NFLG) was amplified through reverse transcription (RT)-nested polymerase chain reaction utilizing Superscript™ IV First-strand Synthesis System (Invitrogen) and Platinum™ Taq DNA Polymerase High Fidelity (Invitrogen). After PCR positive products were purified and sequenced, all sequence fragments were edited and assembled into contiguous sequences using the ContigExpress project. Finally, the near full-length genome (NFLG) of the LS11584 (HXB2:786–9604 nt) and the LS14250 (HXB2:771–9613 nt) were obtained. These two NFLG sequences were submitted to the Basic Local Alignment Search Tool (BLAST) analysis, and the most closely related and highest similarity (>95%) partial sequence of BJSJS2016S195 gag-pol region (HXB2:790–5096 nt) was obtained. To demonstrate possible inter-subtype mosaicism, the jumping profile hidden Markov model (jpHMM) (http://jphmm.gobics.de/submission_hiv) was used to perform recombination breakpoint analysis with the default option on these three sequences. The three sequences were aligned with HIV reference sequences (https://www.hiv.lanl.gov) covering the major HIV-1 pure subtypes and CRFs using MAFFT v7.0. Neighbor-joining (NJ) method and the Kimura two-parameter model were used to build a phylogenetic tree by the MEGA 6.0 software. The time of the most recent common ancestor (tMRCA) of CRF109_0107 through the evolution analysis was uncovered by using BEAST v1.7.5. Phylogenetic analysis revealed that these three sequences formed a distinct monophyletic branch with a high bootstrap value of 100%, distantly related to all known HIV-1 subtypes/CRFs (Fig. 1A). The recombination analysis showed that the two NFLG sequences were composed of CRF01_AE and subtypes B and C (Fig. 1B). And the gag-pol region sequence of BJSJS2016S195 has the same breakpoints as the corresponding regions of LS11584 and LS14250. Subtype B and Subtype C fragments of CRF109_0107 were further phylogenetic analysis and submitted to BLAST, the results showed the highest similarity to (>95%) CRF07_BC (Fig. 1C). A total of 13 recombinant breakpoints were found at HXB2 positions according to the recombination analysis as follows: Ⅰ(790–1169 nt) CRF01_AE, Ⅱ(1170–1840 nt) Subtype B, Ⅲ(1841–2166 nt) CRF01_AE, Ⅳ(2167–2466 nt) Subtype B, Ⅴ(2467–3038 nt) CRF01_AE, Ⅵ(3039–3295 nt) Subtype B, Ⅶ(3296–4238 nt) CRF01_AE, Ⅷ(4239–4613 nt) Subtype C, Ⅸ(4614–5157 nt) CRF01_AE, Ⅹ(5158–5886 nt) Subtype C, Ⅺ(5887–6383 nt) CRF01_AE, Ⅻ(6384–8442 nt) Subtype C, XIII(8443–9411 nt) CRF01_AE, using HXB2 as a reference (Fig. 1C). Subregion phylogenetic analyses of 13 genomic segments were further conducted to explore their likely parental lineages. The high bootstrap values in the phylogenetic tree supported a close relationship with CRF01_AE or CRF07_BC subtype references respectively. Subregion phylogenetic analyses indicated that the segment Ⅰ, Ⅲ, Ⅴ, Ⅶ, Ⅸ, Ⅺ, and XIII of CRF109_0107 belonged to the CRF01_AE cluster 5,6Feng Y. He X. Hsi J.H. et al.The rapidly expanding CRF01_AE epidemic in China is driven by multiple lineages of HIV-1 viruses introduced in the 1990s.AIDS. 2013; 27: 1793-1802Crossref PubMed Scopus (147) Google Scholar which is mainly circulating among high-risk sexual behaviors in MSM and Heterosexual population in China (Fig. 2A). The segment Ⅱ, Ⅳ, Ⅵ, Ⅷ, Ⅹ, and Ⅻ of CRF109_0107 were clustered with the CRF07_BC cluster, which is also identified among high-risk sexual behaviors in the MSM population7Feng Y. Takebe Y. Wei H. et al.Geographic origin and evolutionary history of China's two predominant HIV-1 circulating recombinant forms, CRF07_BC and CRF08_BC.Sci Rep. 2016; 6: 19279Crossref PubMed Scopus (65) Google Scholar,8Wang X. Wu Y. Mao L. et al.Targeting HIV prevention based on molecular epidemiology among deeply sampled subnetworks of men who have sex with men.Clin Infect Dis. 2015; 61: 1462-1468Crossref PubMed Scopus (38) Google Scholar (Fig. 2B). The segments of CRF109_0107 are closely related to the clusters associated with the sexually transmitted population according to the sub-regional analysis, suggesting that CRF109_0107 may be predominantly prevalent in the MSM and Heterosexual population.Fig. 2Subregion tree analyses and maximum clade credibility (MCC) trees of CRF109_0107. (A) Segments Ⅰ, Ⅲ, Ⅴ, Ⅶ, Ⅸ, Ⅺ and XIII (HXB2: 790-1169nt, 1841-2166nt, 2467-3038nt, 3296-4238nt, 4614-5157nt, 5887-6383nt and 8443-9411nt) from CRF109_0107 genome map is the representative of all CRF01_AE segments inserted into the mosaic structure. (B) Segments Ⅱ, Ⅳ, Ⅵ, Ⅷ, Ⅹand Ⅻ(1170-1840nt, 2167-2466nt, 3039-3295nt, 4239-4613nt, 5158-5886nt and 6384-8442nt) from CRF109_0107 genome map is the representative of all CRF07_BC segments inserted into the mosaic structure. The subregion neighbor-joining tree was constructed based on Kimura 2-parameter model of nucleotide substitution with 1000 bootstrap replicates, and gamma distributed rates among sites were applied in MEGA6 software. The sequences of CRF109_0107 were marked with ''●''. Only bootstrap values >90% are presented at the corresponding nodes of the tree. (C) MCC trees display recombinant fragments CRF01_AE regions (segments Ⅰ+Ⅲ+Ⅴ+Ⅶ+Ⅸand segments Ⅺ+XIII). (D) MCC trees display recombinant fragments CRF07_BC regions (segments Ⅱ+Ⅳ+Ⅵ+Ⅷand segments Ⅹ+Ⅻ). Timescale is shown at the bottom of the tree. The mean tMRCA and 95% highest probability density (HPD) for the key nodes are displayed. CRF109_0107 strains are highlighted in yellow (CRF01_AE region) and blue (CRF07_BC region). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) The Bayesian analysis shows that the (tMRCA) of the CRF01_AE regions(segments Ⅰ+Ⅲ+Ⅴ+Ⅶ+Ⅸ and segments Ⅺ+XIII) from CRF109_0107 were predicted in 2012.4, [95% highest probability density (HPD): 2010.3, 2018.3] and 2013.8, [95% highest probability density (HPD): 2013.8, 2014.4], respectively (Fig. 2C). The (tMRCA) of the CRF07_BC regions (segments Ⅱ+Ⅳ+Ⅵ+Ⅷ and segments Ⅹ+Ⅻ) from CRF109_0107 were predicted in 2012.4, [95% highest probability density (HPD): 2010.4, 2018.4] and 2013.8, [95% highest probability density (HPD): 2013.8, 2014.5], respectively (Fig. 2D). The recombinant fragments (segments Ⅰ-Ⅸ and segments Ⅹ-XIII) originated around 2012.4 and 2013.8, respectively. Hence, it is inferred that the CRF109_0107 formed a recombination in two time periods, respectively. HIV-1 novel CRFs have been increased rapidly in individuals with high-risk sexual behaviors. CRF55_01B has been confirmed to be an epidemic in the MSM population in Shenzhen.9Zhao J. Cai W. Zheng C. et al.Origin and outbreak of HIV-1 CRF55_01B among MSM in Shenzhen, China.J Acquir Immune Defic Syndr. 2014; 66 (Jul 01): e65-e67Crossref PubMed Scopus (46) Google Scholar CRF65_cpx, which originated in Yunnan,10Feng Y. Wei H. Hsi J. et al.Identification of a novel HIV Type 1 circulating recombinant form (CRF65_cpx) composed of CRF01_AE and subtypes B and C in Western Yunnan, China.AIDS Res Hum Retroviruses. 2014; 30: 598-602Crossref PubMed Scopus (45) Google Scholar was also found to have spread to other parts of China after entering the MSM population.11Liu Y. Gui T. Jia L. et al.Phylogenetic analysis of HIV-1 CRF65_CPX Reveals Yunnan Province is still a source contributing to the spread of HIV-1 in China.J Acquir Immune Defic Syndr. 2015; 70 (Nov 01): e120-e122Crossref PubMed Scopus (10) Google Scholar Frequent high-risk sexual behavior led to the rapidly spread and to a higher genetic diversity of HIV, which may have caused the incidence of a high level of recombination.12Trujillo L. Chapin-Bardales J. German E.J. Kanny D. Wejnert C Trends in sexual risk behaviors among hispanic/latino men who have sex with men - 19 Urban Areas, 2011-2017.MMWR Morb Mortal Wkly Rep. 2019; 68 (Oct 11): 873-879Crossref PubMed Scopus (5) Google Scholar Second-generation recombinant virus, CRF79_0107, caused by double infection has also spread rapidly to 5 provinces in individuals with high-risk sexual behaviors in China. It is especially worth noting that, with the rapid spread of CRF01_AE and CRF07_BC among MSM, more and more new second-generation CRFs represented by the recombination of CRF01_AE and CRF07_BC will be identified and found spreading in high-risk populations in the future. In summary, a novel HIV-1 circulating recombinant form CRF109_0107 whose genome consists of CRF01_AE and CRF07_BC with 12 breakpoints and 13 segments was defined, which has a more complex recombinant form than others. The emergence of CRF109_0107 indicates that CRF01_AE and CRF07_BC have been spreading in the same population. The complex social behaviors of MSM and Heterosexual population will further complicate the epidemic of HIV-1 in China. More and more second-generation CRFs comprised of CRF01_AE and CRF07_BC in the future will emerge. Therefore, surveillance will be necessary in specific high-risk populations in China. No competing financial interests exist. This study was supported by the NSFC ( 81773493 , 31800149 , 31900157 ), the State Key Laboratory of Pathogen and Biosecurity ( AMMS ), the National 13th Five-Year Grand Program on Key Infectious Disease Control ( 2018ZX10721102 , 2018ZX10732101–001–003 ).