Low-cost and high efficiency DNA-based Reverse Genetics Platforms for manufacturing RNA virus products.

Abstract
The majority of full-length, infection-capable RNA viruses and derivative products available in the marketplace
are produced by serially passaging and amplifying existing virus stocks in cell culture. Furthermore, new virus
strains and variants are generated for distribution by similar cell culture adaptation and amplification of field
isolates. Unfortunately, the very high mutation rates of RNA virus polymerases coupled with the presence of
strong selection pressures during cell culture amplification of virus stocks often results in the selection of mutants
with dramatically altered in vitro and/or in vivo phenotypes as compared to naturally circulating wild-type strains.
The unpredictable nature of cell-adaptive mutation often causes serial passage-acquired mutations to be
different in different laboratories, even when virus stocks are amplified on the same cell type. This creates
unwanted phenotypic variability, potentially rendering experiments non-reproducible and experimental data
unreliable. Additionally, the rapid generation, validation and dissemination of new strains/variants of emerging
viruses is also hindered by these limitations. Currently, all stocks available from organizations such as the
American Type Culture Collection (ATCC) are passaged during the isolation and development process.
Furthermore, products for sale by organizations such as ATCC are provided in small volumes at high costs and
end users are compelled to re-amplify these stocks for experimental use. We established Advanced Virology
specifically to address these critical issues impacting the natural fidelity and uniformity of commercial virus stocks
and to provide stocks in experiment-ready volumes such that no end user amplification or stock validation is
required. Our guiding principle is to use sequencing of field isolates of RNA viruses, when possible, to preserve
the genotypes of naturally circulating strains and then use DNA-based reverse genetics systems to rescue wild
type viruses in vitro. DNA-based reverse genetics systems have been developed by academic laboratories for
producing RNA viruses in the absence of serial passaging. Use of these systems minimizes the occurrence of
unplanned mutation while increasing efficiency and control of the production process. However, current reverse
genetics systems are confined to academic laboratories skilled in the art and not optimized for large-scale
production of virus stocks due to both the low efficiency and very high costs of some intermediate steps (e.g., in
vitro RNA synthesis, RNA electroporation) in their production protocols. In this project, we propose to reduce
production costs and increase efficiency of production by developing DNA-only systems that involve direct use
of DNA vectors in all steps of the virus production process either by transfection of DNA into cells or creation of
stable cell lines inducibly expressing virus proteins. We will use alphaviruses and Influenza virus as prototypical
positive- and negative-sense RNA viruses to develop scalable, low-cost and efficient production platforms.
Overall, we aim to facilitate the broader use of high-quality virus stocks in order to increase the reliability, quality,
efficiency and pace of virology research.

Grant Number: 1R43IP001282-01
NIH Institute/Center: ALLCDC
Principal Investigator: Nishank Bhalla