To date, this is the first 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 . The prevalence of subtype B mainly transmitted by blood transfusion has significantly 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 . 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 [28–30]. Notably, high prevalence of CRF55_01B was observed in our study, compared with the previous reports [27, 29, 31]. CRF55_01B firstly identified in Shenyang composed of subtype B and CRF01_AE, with 4 intra-genomic breakpoints in the polymerase (pol) gene . Evidence showed that CRF55_01B originated and outbroke from men who have sex with men (MSM) in Shenzhen , and CRF55_01B have spread throughout China, along with population migration . 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.
Significantly, CRF_BF and CRF02_AG strains typically found in South American countries and Africa respectively, were identified among Chinese HIV-1 patients [35–38]. To our knowledge, CRF_BF was firstly 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 identification 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 classification.
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 resistance-associated mutations and polymorphisms, higher than a previous study in Hong Kong (20.8%), China . 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 . In China, ENF has been applied as salvage therapy for patients failing ordinary ART since 2015, but not been included in the Free AIDS Antiretroviral Therapy Manual , the low usage of ENF in China may contribute to the observed low prevalence of major ENF resistance mutations in our study (0.25%, 1/396), compared to the reports in Brazil (3.8%, 3/80) , Spain (3.0%, 6/200)  and US (16.7%, 2/12)  among ENF-untreated patients. However, several mutations (N126K, E137K, S138A) in the HR2 region presented in the study can lead to increased viral fusion activity and reduced susceptibility to ENF . Notably, these mutations in HR2 domain may dramatically reduce ENF susceptibility about 100-1000-fold, in combination with substitutions in HR1 domain (amino acid positions: 36–45) [43, 44]. In addition, a lot of minor mutations and polymorphisms (A30V, Q32L, L33V, L34M, S35A/F/T, Q39H, R46K/M/Q, E49D/K/Q, Q52H, H53N/Q/R, Q56K/R, I62V) associated with potential resistance ENF were discovered in our study have reported in previous studies [16, 25, 45, 46].
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 resistance-associated mutations and polymorphisms may have relationship with HIV-1 subtypes. However, DRM determinations were analyzed by HIVfird Program, which is based on subtype B and has biased the results for non-B strains , further phenotypic resistance analysis should be performed to confirm the susceptibility to ENF among non-B subtypes.