A Study Of Genetic Diversity And Drug Resistance Among HIV/AIDS Individuals In Yunnan, China

BackgroundsAIDS, i.e. Acquired Immune Deficiency Syndrome, is a serious human lifethreatening infectious disease caused by human immunodeficiency virus type 1(HIV-1). The etiological agent of AIDS is HIV-1, which belongs to the genus Lentivirus of the family Retroviridae. Pol, one of three structural proteins of HIV-1, is essential during the life cycle of the virus and plays a key role in viral attachment to susceptible cells.To date AIDS remains incurable. Highly active antiretroviral therapy(HAART) is the only treatment method that can effectively inhibit HIV replication, reconstruct the immune function, relieve clinical symptoms and prolong the life of patients. But it is not clear that the HIV virus, for both treated and untreated AIDS patients, can produce drug resistance and intercelluar viral transmission. Therefore, it is of great significance to look for an effective anti-HIV strategy.In this work, we study for the first time the HIV/AIDS epidemic in Yunnan Province, China in 2014, specifically Honghe and Lincang, both badly affected regions of HIV genetic diversity and primary drug resistance; simulataneously we study HAART failure as well as HIV gene mutation and secondary drug resistance among patients in Yunnan Province, China in 2013. The findings would help better understand HIV genetic evolution /diversity, disease monitoring, interference in transmission, and thus to assure optimal prevention and therapeutic strategy of HIV infection and design of vaccines.MethodsThe in-house method was used for the first time to amplify the pol gene of HIV-1 structural proteins of 254 untreated HIV/AIDS individuals in Honghe and Lincang prefectures in 2014 and 1354 treated HIV/AIDS individuals of Yunnan province in 2013. The sequenced results of PCR amplification were assembled and checked by ContigExpress and BioEdit software. The online tool HIV BLAST was used to determine roughly the genotype of the pol gene. The recombination in HIV-1 pol gene was identified by online tool RIP and jphMM in Los Alamos HIV Database. The Simplot software localized recombination breakpoint in the sequences. The drug resistance mutations were evaluated by the Stanford University Algorithm. Based on subtypte distribution of HIV-1 in Yunnan, MEGA6.0 software was used to construct the phylogenetic tree of B/C subtype in order to compare it with the reference strains of B and C subtypes in different regions and estimate the genetic relationship.Results1. The prevalence of primary drug resistance in Honghe and Lincang in 2014 showed 11.8%. The drug resistance rate in Honghe was 12.0% and 11.4% in Lincang. RPV was most prone to drug resistance, and the frequency of drug resistance was 9.1%. The most common points E138 A, V179 D and T69 Ins of drug Resistance variants were detected in RPV. The prevalence of HIV-1 drug resistance in 2013 was 1.5% in Yunnan. The frequency of acquired drug resistance in HAART-failure individuals in Yunnan province was 46.2%. The prevalence of resistance to any drugs was 21.8% including NRTIs, NNRTIs or PIs. 23.6% and 0.8% individuals showed resistance to dual or triple drugs. According to the analysis results in 16 prefectures of Yunnan province, the hardest hit area of acquired HIV-1 drug resistance was Dehong. Honghe, Kunming and Wenshan also exhibited more HIV-1 drug resistant variants than other prefectures. Of NRTIs, the most common resistant drugs were 3TC and FTC, and the frequencies were both 23.9%. M184V/I(23.0%) were the most common mutations occurring in HIV-1 NRTI-related resistance variants. Of NNRTIs, EFV and NVP showed higher resistance and the resistance rates were both 41.7%. Secondary drug resistance was ETR(19.7%). K103N/S, G190A/S/E and Y181C/I/V were the top three mutations showing in NNRTI-related mutations and the frequencies were 21.1%, 11.4% and 10.0% respectively. Of PIs, the most common points of drug Resistance were NFV(2.1%) and L33 F, M46 I and I54 V in the PI. The TDF and LPV/r were used as second-line antiretroviral drugs successfully, and the Resistance frequencies were 7.5% and 0.4%, respectively.2. To assess the difference of mutations in the RT region of pol gene among CRF01_AE, CRF07/08_BC, we conducted a statistical analysis in the RT region. The results showed that the known drug resistance mutations including A62, T69, V179 and E138 exhibited difference in the three subtypes. We also identified 27 novel mutations including V35、T39、K43、S48、V60、K122 and I135, of which several mutations were known to be related with decreasing antiretroviral drug susceptibility.3. To evaluate the timing of drug resistance in HAART failure individuals we screened 997 patients with first episodes of virologic failure in 2013. We found that when the HAART time was more than 18 months, the prevalence of drug resistance in virologic failure individuals exceeded 50%, which is obviously higher than <18 months. During HAART, HIV-1 virus exhibited resistance to NVP and EFV initially, and the median duration time of drug resistance that emerged was about 33 months. The variants showed intermediate/high-level resistance to EFV and high-level to NVP. ABC, 3TC and FTC were the drugs to which resistance occurred earliest in NRTIs and the median genesis of drug resistance was 47 months approximately. The Resistance level of ABC was mainly low-level and those of 3TC and FTC were high-level.4. The prevalence of acquired drug resistance in Honghe prefecture from 2010 to 2013 was similar to that of Yunnan in 2013. Of NRTIs, the frequency of DDI(21.0%) was higher than other drugs and resistance peaked in 2010. Besides M184V/I mutation, A62 V mutation increased obviously in this prefecture and the frequency was 12.6%. Of NNRTIs, EFV and NVP were also showing high resistance and K103N/S, G190A/S/E, Y181C/I/V mutations were the most common mutations in HIV-1 variants. It is worth noticing that HIV/AIDS individuals in Honghe exhibited V179D/E/F mutation and the frequency was higher in comparison with Yunnan as a whole.5. The genotyping results in primary and acquired drug resistance showed that the major epidemic variants in Yunnan were CRF08_BC, CRF07_BC and CRF01_AE. Recombination forms, including B’/C recombinant and subtype C, were found to be the most common. Additionally, CRF01_AE and B’ recombination were also found. Based on subtype distribution of HIV-1 in Yunnan, we found that of the 10 B subtype sequences, five showed greater affinity with B subtypes in Guizhou(B.CN.07. GZ070030. JF932486), and only 2 showed close affinity with strains in Yunnan; among the 22 C subtype sequences, 13 were found to distantly related to those Yunnan and India, 5 were found to be closely related to C subtype in India, and 4 were found to be closely related to C subtype in Yunnan.Conclusions1. The prevalence of primary drug resistance in Honghe and Lincang in 2014 showed 11.8%, especially in Honghe was 12.0%. The prevalence of HIV-1 drug resistance in 2013 was 1.5% in Yunnan. The frequency of acquired drug resistance in HAART-failure individuals in Yunnan province was 46.2%. Of NRTIs, the most common resistant drugs were 3TC and FTC, M184V/I was the most common mutation occurring in HIV-1 NRTI-related resistance variants. Of NNRTIs, EFV and NVP showed higher resistance and secondary drug resistance was ETR. K103N/S, G190A/S/E and Y181C/I/V were the top three mutations showing in NNRTI-related mutations. Of PIs, the most common points of drug resistantce were NFV and L33 F, M46 I and I54 V in the PI.2. To assess the difference of mutations in the RT region of pol gene among CRF01_AE, CRF07/08_BC, we conducted a statistical analysis in the RT region. The results showed that the known drug resistance mutations including A62, T69, V179 and E138 exhibited difference in the three subtypes. We also identified 27 novel mutations of which several mutations were known to be related with decreasing antiretroviral drug susceptibility.3. To evaluate the timing of drug resistance in HAART failure individuals we screened 997 patients with first episodes of virologic failure in 2013. We found that when the HAART time was more than 18 months, the prevalence of drug resistance in virologic failure individuals exceeded 50%, which is obviously higher than <18 months. During HAART, HIV-1 virus exhibited resistance to NVP and EFV initially, and the median duration time of drug resistance that emerged was about 33 months. The variants showed intermediate/ high-level resistance to EFV and high-level to NVP. ABC, 3TC and FTC were the drugs to which resistance occurred earliest in NRTIs and the median genesis of drug resistance was 47 months approximately. The resistance level of ABC was mainly low-level and those of 3TC and FTC were high-level.4. The genotyping results in primary and acquired drug resistance showed that the major epidemic variants in Yunnan were CRF08_BC, CRF07_BC and CRF01_AE. Recombination forms, including B’/C recombinant and subtype C, were found to be the most common. Additionally, CRF01_AE and B’ recombination were also found. Based on subtype distribution of HIV-1 in Yunnan, we found that B subtype showed greater affinity with B subtypes in Guizhou. C subtypes were found to distantly relate to those Yunnan and India.These results suggested that study of genetic diversity and drug resistance among HIV/AIDS individuals in Yunnan, China could provide scientific evidence for disease monitoring, interference in transmission, improvement of antiretroviral therapeutic strategy and design of vaccines.