Abstract

We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. We initially used a capillary electrophoresis method to gain insight into the role of the VEEV nonstructural protein 3 (nsP3) in viral replication. We identified several regions in nsP3 that are intolerant to small (15 bp) insertions, and thus are presumably functionally important. We also identified nine separate regions in nsP3 that will tolerate small insertions at low temperatures (30°C), but not at higher temperatures (37°C, and 40°C). Because we found this method to be extremely effective at identifying temperature sensitive (ts) mutations, but limited by capillary electrophoresis capacity, we replaced the capillary electrophoresis with massively parallel sequencing and used the improved method to generate a functional map of the entire VEEV genome. We identified several hundred potential ts mutations throughout the genome and we validated several of the mutations in nsP2, nsP3, E3, E2, E1 and capsid using single-cycle growth curve experiments with virus generated through reverse genetics. We further demonstrated that two of the nsP3 ts mutants were attenuated for virulence in mice but could elicit protective immunity against challenge with wild-type VEEV. The recombinant ts mutants will be valuable tools for further studies of VEEV replication and virulence. Moreover, the method that we developed is applicable for generating such tools for any virus with a robust reverse genetics system.

Highlights

  • Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus endemic to regions of South America

  • Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that was first identified in Venezuela in 1938

  • We describe a technique that we have developed that allows for the rapid identification of viral mutants that can be useful for studying the basic biology of viral replication

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Summary

Introduction

Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus endemic to regions of South America. Maintained in a rodent reservoir, VEEV can be transmitted by mosquitoes to horses and humans where it can cause debilitating and potentially fatal encephalitis. There are currently no vaccines for VEE licensed for use in humans. Alphaviruses contain an approximately 11–12 kb single-strand, capped and polyadenylated positive-sense RNA genome. The 59 two-thirds of the genome encode the non-structural proteins; nsP1, nsP2, nsP3, and nsP4, which are involved in genome replication and transcription. The 39 one-third of the genome encodes the structural proteins; capsid, E3, E2, 6K, and E1

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