HIV-1 gp41 genetic diversity and enfuvirtide resistance-associated mutations among enfuvirtide-naïve patients in southern China

Background: Human immunode�ciency virus (HIV) increasing molecular diversity and emergence of drug resistant mutants remain a major concern in Southern China, enfuvirtide (ENF/T-20) is the �rst entry inhibitor used in patients failing highly active antiretroviral therapy (HAART). However, data on HIV-1 gp41genetic diversity and primary ENF resistance-associated mutations among treatment-naïve patients in southern China is limited. The objective was to identify molecular diversity and ENF resistance patterns of HIV-1 in southern China, using envelop (env) gp41 sequences and bioinformatics tools, which may help optimize ART. Methods: From December 2018 to January 2019, 439 blood plasma samples from ENF-naïve HIV-1 patients were collected from Shenzhen, Wuhan and Chongqing, of which, 396 HIV env regions were sequenced and subtyped, and were performed the analysis of drug resistance-associated mutations (DRMs). Results: The main subtypes were circulating recombinant form (CRF) 01_AE (30.6%) and CRF07_BC (48.7%), CRF55_01B had been the fourth subtype in our work, many rare CRFs were observed. Notably, CRF02_AG and CRF_BF strains typically found in Africa and US respectively were identi�ed amongst Chinese HIV-1 patients. Known DRMs were detected in 27.5% (109/396) of ENF treatment-naïve patients. One major DRM (L44M), many secondary DRMs including (N126K, E137K, S138A) and lots of polymorphisms were found in the study, which have been proved to elevate resistance to ENF. Conclusion: HIV-1 molecular diversity among people in southern China observed in the work indicates that HIV-1 variability is becoming increasingly complex in the south of China. A diverse set of primary DRMs discovered in this study described the serious threat to ART, which remind us the urgent need of timely surveillance of HIV-1 viral diversity and drug resistance in China.


Introduction
Human immunode ciency virus type one (HIV-1) presents high genetic diversity, and is suppressed by antiretroviral therapy (ART), but may acquire many drug resistance-associated mutations (DRMs).HIV-1 genetic studies have revealed four groups (M, N, O and P).The M group of HIV-1 is classi ed into nine phylogenetic subtypes (A, B, C, D, F, G, H, J and K) and at least 98 circulating recombinant forms (CRFs) [1,2].Viral diversity analysis has signi cant implication for investigating the global origin and evolution of HIV-1 isolates [3], and future vaccine development [4].Molecular epidemiological studies demonstrated that CRF07_BC, CRF01_AE and CRF08_BC were the dominant subtypes among the population of southern China, with identi cation of drug-resistant strains and novel unique recombinant forms (URFs) [5][6][7].
In 2003, Chinese government made it possible to have free access to ART [8], which not included enfuvirtide (ENF).ENF was the rst HIV fusion inhibitor based on the amino acid sequence of the heptad repeat-2 (HR2) in the glycoprotein 41 (gp41) of HIV-1 envelope (env) protein, which blocks HIV-1 entry and restrict HIV-1 replication, used in combination with other antiretroviral drugs as an alternative to the ordinary ART [9,10].HR1 region substitutions in HIV-1 env gp41, including A30V, L33V, L34M, G36D/S, I37V, V38A, Q39H/R, Q40H, N42T/D, N43D, L44M, L45M, R46M, L54M and et al, have been proved to be associated with ENF resistance in vitro and vivo [11][12][13][14][15][16].The major ENF resistance-associated mutations were identi ed in amino acid position 36-45 of HR1 domain.Other secondary mutations including N126K, E137K, S138A, N140I also were demonstrated to reduce ENF susceptibility [17][18][19][20].ENF resistance-associated mutations have been reported in patients who have accepted highly active antiretroviral therapy (HAART), but less data about the ENF resistance associated mutations among ENFnaïve patients.Hence, before ENF is approved for routine ART by authority in China, the information about ENF primary drug resistance is required.
In this study, we investigated HIV-1 gp41 genetic diversity and the prevalence of antiretroviral drug resistance associated with ENF among treatment-naïve population from southern China.

Study samples
From December 2018 to January 2019, 439 blood plasma samples from ENF treatment-naïve HIV-1 population were collected from the Third People's Hospital of Shenzhen (228 samples), Wuhan Jinyintan Hospital (44 samples) and Chongqing Centers for Disease Control and Prevention (CDC) (167 samples), and quantitated by HIV-1 nucleic acid detection kit (Zhuhai Livzon Diagnostics Inc., Zhuhai, China).Among these samples, 396 HIV env regions were successfully sequenced.

HIV-1 genotype and phylogenetic analysis
We edited and aligned the sequences using Geneious 9.1.2,compared with gp41 reference sequences.Env sequences were submitted to the Los Alamos HIV BLAST tool for initial HIV-1 subtyping (https://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html).All sequences were aligned and compared with HIV-1 reference sequences (Accession numbers were shown in supplementary materials S-Table 1) derived from the Los Alamos HIV sequence database (https://www.hiv.lanl.gov)and then the nucleotide alignments were subjected to phylogenetic inference for further HIV-1 subtyping through the neighbor-joining method and Kimura 2-parameter model in 1000 bootstrap replicates implemented in the MEGA 7.0.2(https://www.megasoftware.net).For nalization of phylogenetic trees, we used FigTree v1.4.3 (University of Edinburgh, UK) to prepare gures as previously described [21].Intra-genomic breakpoints and boot-scanning analyses were performed on sequences with possibly unidenti ed strains through jpHMM program (http://jphmm.gobics.de/submission_hiv.html)[22].The sequences did not match established CRFs were identi ed as URFs.Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) was used to generate the genome maps of URFs.
Drug resistance mutation analysis DRM analyses in env regions were performed in the HIV rd program (https://www.hivrd.ics.ufba.br/).HIV rd uses HIV-1 HXB2 env gp41 protein sequence (nucleotide: 7863-7892, amino acids: 36-45) as a reference sequence to align the user query sequences [23].The mutation in this region can confer HIV-1 resistance to ENF [24], and polymorphisms are other amino acid differences amongst the gp41 region in the absence of ENF [25].

Statistical analysis
Polymorphisms were analyzed using VESPA program (https://www.hiv.lanl.gov/content/sequence/VESPA/vespa.html).The VESPA program detects signature patterns (atypical amino acid or nucleotide residues) in a set of query sequences relative to a set of background sequences [26].Chin-square in SPSS 21.0 software was utilized for statistical analyses.

Results
HIV-1 gp41 subtyping analysis Among 439 samples, a total of 396 samples were successfully sequenced and included in this study, of which 198, 156, 42 were collected from Shenzhen, Chongqing and Wuhan respectively.Gp41 of HIV-1 variant were sequenced and subtyped through jpHMM program and HIV-1 BLAST tool, further con rmed by phylogenetic analysis (Fig. 1).As is shown in Table 1, CRF07_BC (48.7%, 193/396) and CRF01_AE (30.6%, 121/396) were the two main subtypes.A diverse set of genotypes were also identi ed amongst gp41 regions, including CRF08_BC, CRF55_01B, B, CRF67_01B, CRF_BF, CRF02_AG, CRF52_01B, CRF59_01B, CRF79_0107 and CRF85_BC.Few URFs (0.5%, 2/396) were found in the study (Fig. 2).The distribution of HIV-1 subtypes among the three cities in the study demonstrated that the HIV-1 isolates in Shenzhen were more variable than Chongqing and Wuhan (P < 0.01), and the proportions of CRF07_BC and CRF08_BC in Chongqing were higher than the other two cities (P < 0.01).CRF55_01B outnumbered subtype B to become the fourth HIV-1subtype.

Discussion
To date, this is the rst study on the prevalence of ENF resistance-associated polymorphisms and mutations in HIV-1 isolates among treatment-naïve patients in southern China.What's more, we investigated the env gp41 diversity of HIV-1 in these patients.
Molecular characterization of the HIV-1 isolates circulating within the HIV-1 patients in southern China revealed the most prevalent subtypes were CRF01_AE and CRF07_BC, while CRF08_BC and subtype B showed the lower prevalence in comparison to the national report in China in 2006 [27].The prevalence of subtype B mainly transmitted by blood transfusion has signi cantly decreased, due to the prohibition of paid blood donation, which is demonstrated by the low percentage of subtype B in the present study.The absence of HIV-1 patients from Guizhou, Yunnan and Sichuan provinces in this study may account for the observed low proportion of CRF08_BC, since CRF08_BC predominates in these areas [27].The geographic subtype distribution (Numbers of samples ≥ 10) suggested that CRF07_BC and CRF01_AE were dominant subtypes in three areas, which was consistent with previous reports among high-risk populations [28][29][30].Notably, high prevalence of CRF55_01B was observed in our study, compared with the previous reports [27,29,31].CRF55_01B rstly identi ed in Shenyang composed of subtype B and CRF01_AE, with 4 intra-genomic breakpoints in the polymerase (pol) gene [32].Evidence showed that CRF55_01B originated and outbroke from men who have sex with men (MSM) in Shenzhen [33], and CRF55_01B have spread throughout China, along with population migration [34].Furthermore, several rare CRFs including CRF52_01B, CRF59_1B, CRF67_01B, CRF79_0107 and CRF85 were isolated from HIV-1 patients, describing that HIV-1 recombination between different genotypes is becoming increasingly complex in China.
Signi cantly, CRF_BF and CRF02_AG strains typically found in South American countries and Africa respectively, were identi ed among Chinese HIV-1 patients [35][36][37][38].To our knowledge, CRF_BF was rstly reported in China.More importantly, the two uncommon strains were both found in Shenzhen, which was an international metropolis with progressively international travel and immigration.Therefore, the identi cation of imported CRF_BF and CRF02_AG in Chinese indicates the newly emerging migration modes in the global HIV-1 pandemic.Furthermore, Few URFs were available in our study, HIV recombination can be presented in different regions (gag, pol and env) between different subtypes, therefore, more gene regions should be included in the further study for comprehensive and accurate HIV-1 classi cation.
In this study, we evaluated the distributions of env gp41 polymorphisms and ENF resistance-associated mutations among HIV-1 strains from 396 ENF-naïve HIV-1 infected population in Shenzhen, Chongqing and Wuhan in the period 2018-2019.In total, 27.5% HIV-1 isolates had primary ENF resistanceassociated mutations and polymorphisms, higher than a previous study in Hong Kong (20.8%),China [39].Only one major ENF resistance-associated mutation (amino acid positions: 36-45), L44M, was discovered in the study, which has associated with 1.8 fold resistance to enfuvirtide in vitro [40].In The subtype distribution of three dominant mutations indicated R46K/M/Q and S138A were predominant in subtype CRF07_BC, while E137K was mainly found in subtype B. The prevalence of resistanceassociated mutations and polymorphisms may have relationship with HIV-1 subtypes.However, DRM determinations were analyzed by HIV rd Program, which is based on subtype B and has biased the results for non-B strains [23], further phenotypic resistance analysis should be performed to con rm the susceptibility to ENF among non-B subtypes.

Conclusion
In summary, our study demonstrated increasing HIV-1 genetic diversity and high prevalence of primary ENF resistance-associated mutations and polymorphisms in the south Chinese population.The identi cation of imported CRF02_AG and CRF_BF strains in Chinese individuals, especially the rst emergence of US-derived CRF_BF isolate, suggest the newly emerging migration modes of the global HIV-1 pandemic.Additionally, though few major DRM was observed, a large amount of secondary and minor DRMs were isolated from ENF treatment-naïve patients, which can be a serious threat to ART after the failure of the rst-line ART.The complex HIV-1 evolution and high prevalence of DRMs to ENF remind us the urgent need for continued molecular surveillance to monitor the HIV-1 diversity and primary DRMs in Chinese population.
Phylogenetic tree analysis of env gp-41 sequences among HIV-1 isolates in the southern China.
Sequences from HIV-1 infected patients and references are respectively in blank and red in the trees and boxes.