Targeting viral replication

Methods

Summary

We will clone the genome of vaccinia virus strain MVA into a BAC vector and establish this within E. coli.  We will modify the viral genome using new molecular tools that we have developed within our lab for genome engineering. Using the piggyBAC system we will refactor the expression and introduce viral transcriptional genes into the chromosome of the host strain. We will then test the capacity to recover infectious MVA from the transfected vBAC using the fowlpox helper virus and without it. If we have the complete gene set required to "kick start" the vBAC we should see viral progeny via a GFP reporter expression. Further  phenotypic characterisation will be done to confirm the essentiality of the genes identified.

Challenges

The risks are minimal. The techniques and proof of principal have already been published.

Pre Analysis Plan

Our phenotypic analysis will include comparisons with the wild type virus. We will monitor viral titre, rate of spread of infection by intermittently quantifying the number of viral foci using a GFP reporter encoded upon the viral genome. All of the experiments will be repeated a significant number of times to obtain accurate measures. We will use qPCR to quantitate expression of specific viral genes to track progression of the population through the infection life cycle.