Functional characterization of coxsackievirus A9- eGFP vector

Abstract

Picornaviruses are small, single-stranded, positive-sense, and non-enveloped RNA viruses, that cause various diseases in humans and mammals. More than 300 human picornavirus types are identified as responsible for diseases from respiratory illness to life-threatening CNS disease, flaccid paralysis, and meningitis. Even though there are identified picornavirus types, there are no direct antiviral drugs against this virus, and barely any vaccines are available. RNA viruses like picornaviruses are prone to mutations that alter the virus tropism and pathogenesis, which complicate the development of therapeutic strategies. For picornavirus research, viral cDNA clones or viral vectors research is necessary, and the standard methods used for developing cDNA clones however are old-fashioned and complex. In cancer therapy, picornaviruses show promise as vectors for delivering therapeutic materials to affected cells to destroy the cells completely. The project aims to characterize the functionality of the CVA9-eGFP virus vector in cell culture by measuring its infection efficiency, replication dynamics, the expression of the eGFP marker gene, and the potentiality of these vectors for cancer therapy. The T7-promoter-based system was used for the in vivo rescue of CVA9 and CVA9-eGFP virus particles from the viral cDNA or T7-promoter-tagged PCR amplicons. An optimized long-PCR protocol utilized for successful amplification of the full-length CVA9 viral genome with T7-promoter for virus rescue using the T7 RNA polymerase (T7RNAP) system. Moreover, different promoters, such as T7 and CMV promoters’ efficiency for virus rescue and viral replication were evaluated. The experiment involved several steps from transforming the E. coli with plasmid vectors, isolating and purifying different CVA9 and CVA9-eGFP DNA, verification through restriction digestion and agarose gel electrophoresis, generating T7-promoter-tagged PCR amplicons, transfecting mammalian cells, and eventually observing the infected cells using an EVOS FL AUTO fluorescence microscope. CVA9-eGFP PCR amplicons successfully transfect the BHK-21 and T7-BSR cells which generate infectious virus particles indicating that the CVA9-eGFP vectors function properly within mammalian cells. Addressing the fundamental research questions and testing the hypothesis, this research gives us valuable insights into the functionality and behaviour of the CVA9-eGFP vector. Using the information from this research in molecular virology fields offers a foundation for developing novel therapeutic properties against picornavirus diseases and cancer.