Optimization of human immunodeficiency virus type one replication assay using single cycle replicable virions

HIV virions with replication capacity are needed for HIV researches, like investigating for new anti HIV agents. Here HIV-1 replication assay was optimized with HIV-1 single cycle replicable (SCR) virions to improve biological safety condition. pSPAX2, pmzNL4-3 and pMD2G plasmids were co-transfected to the HEK293T by using polyfect reagent to produce the SCR HIV-1 virions. Virions were quantified using capture ELISA P24. Different MOI of SCR virions were used for infecting of Target cells (HEK) and the load of the supernatant P24 was monitored days after infection. Single cycle replication assay (SCRA) was developed using kinetic studies data. The P24 load of the infected cells supernatant has linear relation to the beginning infectious MOI. 24 hours post infection with HIV-1 SCR virions the viral particle production was detectable. The highest load of P24 in infected cells supernatant was detected 48 hours after infection. Using this developed method, the 50 and 95 percent inhibitory concentration of (IC95 and IC50) Indinavir and Nevirapine were calculated as 25nM and 50nM. In this study, using SCR HIV-1 virions the SCRA was developed. SCR HIV-1 virions are replicable only for one cycle and this improves the safety of developed assays. The accuracy of assay was examined by quantifying the anti HIV-1 potential of two commercial anti-AIDS drugs and the calculated activity for test agents was equal to previously known amounts.