84. Enhanced Detection Method for Retrovirus in Vaccine

Abstract

Detecting any presence of retroviruses in biological material is important to ensure safety and prevent retroviral infection, which has been associated with various disorders including malignancies, immunodeficiencies, and neurological disorders. Highly specific and sensitive PCR techniques are used to detect specifically the presence of a known retrovirus. In contrast, the presence of an unknown retrovirus can only be examined by non-specific methods such as transmission electron microscopy and growth of virus on susceptible cells. However, detection methodologies involving cell culture techniques may be ineffective because certain retroviruses are not cytopathic in vitro. Retroviruses possess RNA-dependent DNA polymerase known as reverse transcriptase (RT), which is inherent in all kinds of retrovirus. Taking advantage of its ubiquitous nature in retroviruses, enzymatic activity of RT may be measured to detect retroviral contamination in biological products. On the basis of the polymerase enhanced reverse transcriptase (PERT) assay, two different assay systems, PCR-based RT assay (PERT-PCR) and ELISA-based RT assay (PERT-ELISA), were developed. Respective optimum conditions were determined. Sensitivity, linearity and reproducibility of the methods were studied on purified RT. Both PERT-PCR and PERT-ELISA could detect as little as 10-10 unit/ml of RT. Nonetheless, no additional increase in sensitivity was observed by performing ELISA after PCR amplification. Treatment with RNase after cDNA synthesis increased sensitivity over 100 times, and the addition of synthetic DNA competitors, which suppress RT-like activities of cellular DNA-dependent DNA polymerases, greatly increased specificity of the assay. A modified form of PERT-PCR assay was used and subsequently RT activities were detected in vaccines and culture supernatant of retrovirus-producing cells. This enhanced form of PERT-PCR assay is easy to perform and is able to detect up to 10-10 unit/ml of purified RT, as well as RT in vaccines and cell culture supernatant. Thus, this study presents an enhanced assay, applicable to screening of all replication-competent retrovirus, quantification of the viral load, drug susceptibility testing of RT, and control of virus inactivation in biological products.