Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.

Oncolytic viruses are currently under investigation as anti - cancer therapies due to their innate ability to selectively infect and destroy cancer cells. Major barriers to this anti - tumour effect include inefficient viral spread and immune - mediated neutralization. This study aims to overcome these limitations by taking advantage of the life cycle of the oncolytic clinical candidate known as vaccinia virus (VACV). Naturally, a small proportion (<1%) of VACV progeny are released from infected cells with a cell - derived membrane and become known as extra - cellular enveloped virus (EEV). Due to this additional membrane, EEV can be shielded from many anti -viral immune factors , allowing it to travel further and largely avoid host - mediated neutralization. This form of VACV is important for long range virus dissemination as well as sustained infection. Though the exact mechanism remains to be elucidated, it has been demonstrated that EEV release can be influenced by Abl tyrosine kinase (Abl TK) function. Specific point mutations in viral envelope proteins are known to bring about enhanced viral release, resulting in an elevated proportion of produced EEV. In this study, we investigate the effect of EEV enhancing modifications within various oncolytic VACV strains. Our data reveals that this augmentation of EEV production through the A34R L151E mutation within the Copenhagen (Cop) backbone can enhance the oncolytic potential of VACV in vivo through enhanced spread and immunoevasion.

Equine encephalosis virus (EEV) is the cause of equine encephalosis. The disease is similar to mild forms of African horse sickness (AHS) and the two diseases are easily confused. Laboratory identification and serotyping of EEV is based on viral isolation in BHK-21 cells and a viral plaque inhibition neutralization test (Erasmus <i<et al., 1970). These procedures require long durations to confirm results and it was desirable that a rapid diagnostic assay was developed to distinguish EEV from African horse sickness virus (AHSV). A PCR test developed for AHSV (Quan et al., 2008) formed the basis for development of a similar assay for EEV. The aim was to develop and evaluate a real time PCR assay for the detection of EEV in the blood and organs of horses. FastPCR software was used to design primers to amplify and sequence the EEV S7 (VP7) gene. RNA was extracted from EEV tissue culture isolates, representing all seven serotypes, using a MagMaxTM Express Particle Processor and MagMaxTM-96 Total RNA Isolation kits. A one step reverse transcription PCR (RT-PCR) was carried out to amplify the EEV S7 gene using a GeneAmp Gold RNA PCR core kit. Sequence reactions were carried out using a BigDye terminal v3.1 sequencing kit and analyzed with an ABI 3130xl Genetic Analyzer. After sequences alignment using BioEdit software, conserved regions were identified and Primer Express 3.0 software was used to design EEV primers and TaqMan® MGBTM hydrolysis probes for real-time RT-PCR assay. The EEV real-time RT-PCR assay was specific and did not detect AHSV nor bluetongue virus (BTV). The real-time format was selected because of its convenience, sensitivity and ability to produce results rapidly. Validation of the assay is the next step in establishing it as a routine diagnostic assay. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Veterinary Tropical Diseases / unrestricted