The Viral Evolvment Of HIV-1Drug Resistance Strains In Long-term Antiretroviral Therapy

Antiretroviral Therapy (ART) started since1990s, and now it is widely developed around the world, from single-dose Zidovudine (AZT) to7categories including33kinds of agents. ART has brought hopes to HIV patients, it delayed the development of AIDS, and improved their living quality. China started her ART since2003. With ART, the mortality of HIV patients had dropped from9.5death/100person-year in2003to5.2death/100person-year in2009. Although the ART in China had achieved some success, a number of patients have to use same regimens for long time, because of the lack of antiretroviral drugs. This situation may contribute to the drug-resistance in long-term treatment. This assay focuses on the effect of HIV drug resistance to long-term ART, and hope to provide some useful advice to Chinese ART.The most significant character of HIV virus is the high evolvement rate. The reason is that the reverse transcriptase of HIV has a high rate of mispairing which make it is possible to produce a number of mutant viruses. These mutant viruses exist in patients’plasma as quasispecies.In the research of HIV treatment, drug resistance is a important factor. The high mispairing rate can make some drug resistance mutations that help HIV viruses overcome the pressure of antiretroviral drugs. In long-term ART, the drug resistance is not only for viruses’survival, but also for the viral evolvement.Chinese Center for Disease Control and Prevention, National Center for STD/AIDS Control (NCAIDS) and Prevention and Henan Center for Disease Control and Prevention started a cohort study in a county of Henan province. The blood samples were collected every year. Based on this cohort study, this assay tries to understand the effect of HIV drug resistance mutations to long-term ART. The division of virology and immunology, NCAIDSestablished a standard HIV genotype drug resistance detection, named in-house.We used in-house and SingleGenome Amplification (SGA) to amplified pol gene of HIV protease and part of the transcriptase (2253bp-3553bp,1.3kb). After sequencing, we used Sequencher (4.10.1) and Bioedit(7.0) software to clear and edit the sequence data, and uploaded to Stanford HIV drug resistance database to obtain drug resistance mutations and drug resistance report. We also used the sequence data to build phylogenetic trees to study the viral evolving pathway under drug pressure.In the first part of this assay, we focused on the drug resistance in low viral load (50-1000copies/ml), and the effect to ART treatment. The guideline of WHO HIV drug resistance detection pointed that the lowest limit viral load for drug resistance detection was1000copies/ml, but the lowest limit of latest method of viral load detection can reach50copies/ml. So the drug resistance emerged between viral load50-1000copies/ml had been ignored. Actually, the drug resistance emerged in low viral load can be an early warning for the following virologic failure. This assay studied the drug resistance emerged in low viral load, and the effect to ART treatment. The results shows that Non-nucleotide reverse transcriptase inhibitor related resistance was the most common ones in low viral load samples. In addition, most contributed to resistance to Nevirapine.The second part of this assay lies on the development of drug resistance mutations associated with Nevirapine (NVP), a drug belong to Non-nucleotide reverse transcriptase inhibitor(NNRTI). In China, because of the lack of antiretroviral drugs, a number of patients took same regimen for long time, NVP is usually used in the regimen as NNRTI, patients took NVP even if there were already NVP drug resistance emerged. in nowadays, with the improvement of health care, NVP is considered to be replaced by new NNRTI. In this stage, the research on the NVP associated drug resistance mutations become necessary. This part was also based on the cohort study in Henan province. Plasma samples collection and drug resistance detection were done in four time point. We used standard genotyping and Single Genome Amplification (SGA) to analyze the proportion and changing of K103N,Y181C and G190A. The evaluation of Antiretroviral Therapy (ART) was based on CD4cells counts and HIV-1viral load. The results shows that After receiving first-line antiviral therapy for over72months.the CD4count and viral suppression were still sufficient,78.6%patients could have their CD4count over than200cells/ul. In the SGA sequences, the K103N could decreased and vanished in long-term treatment.Simultaneously,the frequency of Y181C and G190A increased.In the sequences of standard genotyping, the changing of K103N, Y181C and G190A were similar with that in SGA sequences.The last part of this assay was on the development and the effect of compensatory mutations to long-term ART. Although drugresistance mutations can help viruses to overcome the pressure of drugs, they tend to make viruses replicate less efficiently than wild-type viruses, compensatory mutations can solve the problem by provide partial restorations of viral fitness reduction. After long-term ART, HIV viruses can develop a number of drug resistance mutations, and compensatory mutations can also possibly be selected by drug pressure. These compensatory mutations can bring negative effect to ART, and can contribute to AIDS. In this assay, Weuse single genome sequencing (SGA) to get quasispecies POL ge_ne(2253bp-3553bp).Based on the SGA results, we built Markov Chain Monte Carlo (MCMC) phylogenetic trees with molecular clock to find compensatory mutations and try to identify the correlation with primary drug resistance mutations. Besides that, we use Swiss-model to build the structure of whole HIV-1Reverse Transcript (RT) enzyme, and study the hydrogen bond and other molecular bond between the primary drug resistance mutations and the correlated compensatory mutations.Finally, We found two compensatory mutations, they are K43E and L228R, and they may had compensatory effect on M41L and H221Y respectively. The molecular bonds were also support that the compensatory mutations had more possibility to contact the primary drug resistance mutations, and these contacts or bonds may provide some compensatory effect or improvement of regional stability.To the patients receiving ART for long time (over than72months), we should pay attention to the compensatory mutations, while focused on HIV drug resistance surveillance. The compensatory mutations cannot provide direct drug resistance effect, but can possibly associated with primary drug resistance mutations, and may enhance the effect of primary drug resistance mutations.In summary, to fight HIV, we need to focus on the early stage of drug resistance and provide early warning to patients, in order to delay the virologic failure. To the patients taking long-term ART, we need to study the development of drug resistance mutations to improve their treatment effect and living quality.