An Improved Reverse Genetics System for Mammalian Orthoreoviruses

Virology. 2010 Mar 15;398(2):194-200. doi: 10.1016/j.virol.2009.11.037. Epub 2009 Dec 29.

Abstract

Mammalian orthoreoviruses (reoviruses) are highly useful models for studies of double-stranded RNA virus replication and pathogenesis. We previously developed a strategy to recover prototype reovirus strain T3D from cloned cDNAs transfected into murine L929 fibroblast cells. Here, we report the development of a second-generation reovirus reverse genetics system featuring several major improvements: (1) the capacity to rescue prototype reovirus strain T1L, (2) reduction of required plasmids from 10 to 4, and (3) isolation of recombinant viruses following transfection of baby hamster kidney cells engineered to express bacteriophage T7 RNA polymerase. The efficiency of virus rescue using the 4-plasmid strategy was substantially increased in comparison to the original 10-plasmid system. We observed full compatibility of T1L and T3D rescue vectors when intermixed to produce a panel of T1LxT3D monoreassortant viruses. Improvements to the reovirus reverse genetics system enhance its applicability for studies of reovirus biology and clinical use.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cricetinae
  • DNA, Recombinant / genetics
  • DNA, Viral / genetics
  • Electrophoresis, Polyacrylamide Gel
  • Fluorescent Antibody Technique
  • Genetic Vectors / genetics*
  • Orthoreovirus, Mammalian / genetics*
  • Plasmids / genetics
  • Reoviridae Infections / virology*
  • Transfection

Substances

  • DNA, Recombinant
  • DNA, Viral