Detection And Analysis Of HIV-1 Phenotypic And Genotypic Drug Resistance

The emergence and transmission of drug resistant human immunodeficiency virus-1 (HIV-1) compromise antiretroviral treatment for HIV-1. Thus, detection for drug resistance and understanding the prevalence of HIV-1 drug resistance is significantly important for the prevention and control of HIV. In present, there are some detecting and diagnostic methods for HIV-1 drug resistance. Among them, HIV-1 phenotypic susceptibility tests based on the recombinant pesudoviruses showed obviously advantages. However, these tests still have some disadvantages. Therefore, our study focuses on developing and optimizing a recombinant pesudovirus test assay for drug resistant HIV-1. And further to analyze the prevalence of HIV-1 drug resistance in some regions of China.Firstly, based on a recombinant HIV-1 pesudovirus system, an efficient phenotypic assay for detecting drug resistant HIV-1 was developed and validated in many ways. The HIV-1 envelope gene deficient vector pSG3Δenv modified with inserting two restriction endonuclease sites by site direct mutation was used to construct the reference pesudovirus. The 1.5kb fragment harboring PR and RT drug resistant genes was cloned into the modified back bone vector pSG3Δenv-m to construct a recombinant vector. After the recombinant vector and HIV-1 envelope expressed plasmid pcDNA3.1-env were co-transfected into the 293FT cells, single infected pesudovirus was released from the cells. Sixteen pesudoviruses of HIV-1 were constructed in our study. TZM-b1 cells were used as target cells of the pesudoviruses. The phenotypic susceptibility of reference and recombinant pesudoviruses were detected by the Lucifer’s reporter gene system. We validated the assays on the repeatability, accuracy and effectiveness. Comparing the results of phenotype and genotype, we validated the assays effectiveness and explained the drug resistant characteristics of the pesudoviruses. The phenotypic susceptible fold- changes of 16 recombinant pesudoviruses were detected involved in 12 antiretroviral drugs. The 192 reactions of phenotypic susceptible assays were carried, and 42 reactions (21.4%) were at the high level of drug resistance with FC>10, and 17 reactions (8.9%) with FC>100. The phenotypic and genotypic results were compared. the two were basically consistent.In the comparison, we found that there were some results inconsistent. We had analyzed those results. There were 3 pesudoviruses which contained 3-4 TAMs and mutations E44 and V118I. The emergence of those mutations might have the relationship with increasing resistance to 3TC and FTC. And one of those 3 pesudoviruses had contained the mutation T69D, the resistance to ZAT of which was significantly increasing. This mutation might have the some synergism to NNRTIs resistance. The mutation H221Y emerged in 3 pesudoviruses. The phenotype results showed that, the mutation H221Y might be associated with the resistance to NVP. These mutations synergism were not very clear so far and further studies are needed. The pesoduvirus phenotypic susceptibility detecting system constructed in this study combined the advantages of phenotype and the genotype detection. Compare with other detecting systems, this system is able to specifically and comprehensively explain the resistance characteristics. Because the HIV-lbackbone vector plasmid does not contain the Lucifer’s reporter genes it could avoid internal signal interference of the system. The results of this system could be more accurately reflect the virus drug resistance conditions. These detected systems also have great application prospects.We also investigated the HIV-1 drug resistance prevalence of Guangxi Zhuang autonomous region. Sixty one untreated HIV-1 samples were detected by genotype assays with the international common primers and PCR methods. The 61 HIV strains belonged to 4 genotypes respectively, CRF01_AE, CRF07_BC, CRF08_BC and B. There were 8 samples (13.1%) associated with the different level of drug resistance to PIs and RTIs, including 3 subtype B,3 subtype CRF01_AE and 2 subtype CRF08_BC. Among them, sample GX54 and GX48 have the most seriously drug resistance levels to PIs and RTIs. GX54 contained major PIs mutation L90M,9 TAMs and 3 NNRTIs mutations. GX48 contained 6 TAMs and 1 NNRTIs mutation. The other 6 samples only have low levels resistance to RTIs, and Each of them only contained one or two drug resistant mutations. In addition, the amino acid diversity of protease (1-99 amino acids) and reverse transcriptase (1-225 amino acids) of 61 samples was analyzed by the gene software. The results showed that 13 amino acid sites of protease and 15 amino acid sites of reverse transcriptase had over 10% amino acids substituted frequency. The PR and RT contained two and seven most activity amino acid sites respectively and the substituted frequency of the amino acids was over 40%. The results showed that most of the mutations were the TAMs. RT site 69 and 215 exist the most amino acids substitutions. These results indicated that there are some activity amino acid sites in the PR and RT genes, which may be associated with the emergences and evolutions of drug resistance HIV-1.In conclusion, we constructed and validated a pesudovirus phenotypic susceptibility system for HIV drug resistance detection. Sixteen recombinant pesudoviruses were obtained and the phenotypic susceptibilities were detected and evaluated. In addition, we also investigated the HIV-1 drug resistance prevalence of Guangxi Zhuang autonomous region and the drug resistance characteristics of this region were analyzed. Some amino acid sites more likely associated with the drug resistance were found and further studies are needed.