HIV-1Drug Resistance Testing And Screening Novel Resistance Associated Mutations In Fujian Province

Objective This study aimed to assess the fake virus phenotypicresistance detection system of self-built human immunodeficiency virus type1(HIV-1) which would used in clinical drug-resistance testing.Thus understoodthe prevalence of HIV-1drug-resistance in Fujian Province and looked for newresistance-associated mutation sites.Methods1. Genotypic and phenotypic resistance testing were both detectedfor clinical samples which collected from antiretroviral therapy (ART) failurepatients in Fujian province from2005to2011. A small part of untreatedsamples were also detected.The results of these two kinds of sample werecompared with each other,and analsis the prevalence of HIV-1drug-resistancestrains in Fujian Province.2. Using the chi-square test(or Fisher exact) to compare mutation sites ofthe protease region(1to99amino acids) and reverse transcriptase region(1to300amino acids) in antiviral treatment and no treatment sequence of CRF01_AE subtypes one by one. CRF01_AE subtypes were collected from HIV-1pandemic strain in Fujian Province. Variation of mutations between the ARTfailure and the na ve were compared. The novel mutations with higherfrequencices in the ART failure than in the na ve were screened. After the abovework was finished, a suspicious namely S68G drug-resistant mutations werefurther identified basing on site-directed mutagenesis and the phenotypicresistance testing system.Results1. Compared the phenotypic drug-resistance test results of29fake viruswith genotypic drug-resistance interpretation results.It turned out that thephenotypic and genotypic assays were basically consistent, except for twodemonstrating resistance to AZT in genotypic analysis but susceptible to AZTin phenotypic analysis and one susceptible to3TC in genotypic analysis but resistance to3TC in phenotypic analysis.2. Results of genotypic and phenotypic resistance testing showed that drugresistance rate was low in untreated patients,but the overall resistance rate washigher among the patients who failed in ART,especially for the nucleosidereverse transcriptase inhibitors (NRTIs) and non-nucleoside reversetranscriptase inhibitors (NNRTIs).3. Ten new suspicious drug-resisitance mutation sites were found inCRF01_AE subtype sequences in Fujian Province. These mutation sites hadsignificant higher occurrence in antiviral treatment failure population untreatedpopulation. they were K70R mutation of the protease and mutations I5V, K20R,I31L, K43Q, S68G, I142V, V111I, K173L,T200A of the reverse transcriptaseregion.4. The S68G mutation and reversion mutation virus clones wereintroduced by site-directed mutagenesis and the50%inhibitory concentration(IC50)change folds of each mutation/reversion mutated virus weremeasured.The results showed there were not any difference in the IC50value forthe S68G mutation and reversion mutation virus clones analyed of AZT, D4Tand3TC, respectively.Conclusion1. The HIV-1pesudovirus phenotypic resistance testing system possess astrong clinical drug testing practicality. It’s also a promising research tool tocarry out further basic studies including new drug resistance asscoiatedmutations of HIV-1.2. In Fujian province, the overall incidence of drug resistance was loweramong the patients who has not yet receiving ART, however, the patients withvirological suppressing failure was higher, especially for NRTIs and NNRTIs.3. Ten suspicious antiviral therapy or resistance-associated novelmutations were screened for subtype CRF01_AE in Fujian province, and S68Gmutation site (in RT) was not signficant effects on the susceptibility of AZT,D4T and3TC,respectively.