Human immunodeficiency virus (HIV) is the pathogen of Acquired Immune Deficiency Syndrome (AIDS), prevalent in low- and middle-income countries[1]. There have been 84.2 million individuals infected with HIV-1, according to data released from UNAIDS till 2020[2]. HIV-1 M group has evolved into ten subtypes: A-D, F- H, and J-L. In addition, 132 circulating recombinant forms (CRFs) (https://www.hiv.lanl.gov/content/sequence/HIV/CRFs/crfs.comp) and numerous unique recombinant forms (URFs) were determined because of the Co-circulation of different subtypes-and-high-recombinant-rate of HIV.
Hebei Province is located near the capital Beijing and shares borders with Tianjin in the east, Henan and Shandong in the southeast, and Shanxi in the west. Given its strategic geographical location, Hebei Province holds great significance. In 1989, the first case of AIDS was reported in Hebei and since then, the number of reported cases has been steadily increasing. The mode of transmission has also changed from blood transmission to male-to-male transmission[3]. Men who have sex with men (MSM) are at high-risk for unfixed sexual partners and rare condom use[6]. The ratio infected with HIV-1 among MSM rose rapidly from 41.8% in 2010 to 64.8% in 2019 in Hebei, according to the published papers[5, 9]. Such subtypes as CRF01_AE, CRF07_BC, and B were shared among individuals infected with HIV-1 through homosexual contact. A previous study published in 2019 found that 77.5% of HIV-1 infections in Hebei were MSM, with 50.5% infected with CRF01_AE strains, 30.4% infected with CRF07_BC strains, and 11.7% infected with CRF07_BC strains[8]. Co-circulation of those strains in MSM populations resulted in dual infection and the second recombinant strain. In recent years, novel complex CRFs and URFs were identified continually among the MSM population[4, 7, 10].
In this study, two novel unique recombinant forms originated from CRF01_AE, CRF07_BC, and B were determined from two HIV-1 positive individuals in Shijiazhuang City, Hebei Province. Plasma samples were collected from two HIV-positive MSM (HB010119, HB010132). HB010119 was a man aged 23 years old diagnosed as HIV positive in September 2020. The Baseline CD4 + T cell count was 657 cells/µl, and HB010132 was a 33-year-old male confirmed HIV positive in October 2020; baseline CD4 + T cell count was 657 cells/µl. They both have a college degree or above; their marital status is unmarried, and infected through homosexual transmission (Table.1). This study was approved by the Medical Ethics Committee of the Fifth Hospital of Shijiazhuang. Written informed consent was obtained from the subject before sample collection.
NFLG sequences of HB010119 and HB010132 were amplified to make up the discordant gag, pol, and env subtypes. RNA was extracted from 200µL of plasma samples by MAGNA Pure LC Total Nucleic Acid Isolation Kit ( No.03038505001 ). Extracted RNA was reversely transcribed into cDNA using the superscript Ⅳ First-strand synthesis system (Invitrogen No. 18091200). Furthermore, nested PCR amplification obtained the NFLG in two halves using ExTaq reagent (TaKaRa No. RR902A). The PCR conditions of both rounds were as follows: 95℃ 5 minutes followed by 30 cycles of 95℃ 30 seconds, 60℃ 30 seconds, and 72℃ 7 minutes, and an extension of 72℃ 10 minutes. The profitable products were detected by 1% agarose gel electrophoresis, then purified and sequenced by SinoGenoMax (China) with a series of special primers. Moreover, the chromatogram data were assembled by ContigExpress software (a component of Vector NTI version 11.5.1 Invitrogen) and Sequencher (version 5.4.5). Then, the two NFLG sequences were aligned with subtype reference sequences and CRFs in China ( https://hiv.lanl.gov/components/sequence/HIV/search/search.html) by Mafft (http://mafft.cbrc.jp/alignment/server/index.html) and adjusted manually using BioEdit (version 7.2.5.0). Additionally, the phylogenetic and subregion trees were constructed using the Neighbor-joining method based on Kimura two-parameter model with 1000 bootstrap replications by Mega6 and constructed using the Maximum Likelihood method based on GTR + G + I with Shimodaira-Hasegawa by FastTree 2.1.10. The phylogenetic and subregion trees were embellished by evolview ( http://www.evolgenius.info/evolview/ ), Recombination breakpoints were identified by Recombination Identification Program ( https://hiv.lanl.gov/contens/sequence/RIP/RIP.html), jpHMM (http://jpHMM.gobics.de/submission_hiv.html), and boost-canning using Simplot (version 3.5.1).
The analysis of the NFLG phylogenetic tree (Fig. 1) suggested that the two sequences formed a distinct monophyletic cluster from other subtypes and CRFs. Depending on the results of RIP, jpHMM, and BootScan, HB010119 was the CRF07_BC fragment inserted into the CRF01_AE backbone, and HB010132 was the B segment consisting of CRF07 BC(Fig S1, S2). In addition, the recombinant mosaic structure of two sequences was described as follows: ⅠCRF01_AE (HXB2, 711-1177nt); ⅡCRF07_BC (HXB2, 1177-1841nt); ⅢCRF01_AE (HXB2, 1841-9509nt), HB010119; ⅠCRF07_BC (HXB2, 711-5852nt); ⅡB (HXB2, 5852-9513nt); HB030021(Fig S1 and Fig S2). Sub-region tree analysis revealed that parental origin of all CRF01_AE regions of two NFLGs was from cluster 4 linkage, which is more common among MSM in major northern cities. The CRF07_BC segments of two URFs were clustered with 07BC_N in the phylogenetic tree, a newer strain primarily found in MSM in the provinces of the north of China (Fig S3).
In conclusion, this study determined two novel URFs derived from CRF01_AE cluster 4 / CRF07BC_N and CRF07BC_N /B recombinant forms from MSM populations in Hebei Province. Both sub-segments are predominantly popular among MSM in northern Chinese cities. Although CRF07_BC-N appeared later than CRF07_BC-O, the risk of transmission of CRF07_BC-N is higher than that of CRF07_BC-O, both in terms of clustering and interprovincial transmission, especially from Sichuan to the Beijing-Tianjin-Hebei economic zone, and with the promotion of the Beijing-Tianjin-Hebei economic integration policy, the rapid transmission of CRF07_BC-N cluster has been realised in Beijing-Tianjin, Tianjin and Hebei Province. The CRF07_BC-N cluster has become the first HIV subtype in Beijing-Tianjin and the second in Hebei Province. CRF01_AE accelerates the attenuation of CD4 + T cells and disease progression compared to other subtypes, with the CRF01_AE 4 cluster widespread in the MSM population and with a significantly lower level of CD4 + T than that of the CRF01_AE 5 cluster, which is more likely to enter the AIDS stage. Both clusters are predominantly found in northern MSM, who act as a bridge between MSM and HETs. With more and more new URFs combined with CRF_0107 and CRF_07B being developed in China, the more complexly unique CRFs could be prevalent, so it is essential to conduct the HIV-I genetic evolution at the phylogenetic cluster level to focus on vital populations in the subsequent survey, which could reduce the transmission and prevalence of HIV-1 from the high-risk population to general population.
Sequences Data
The gene sequences of HB010119 and HB010132 were deposited in the GenBank with the accession number ON529527 and ON529528, respectively.