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Related Experiment Videos

Efficient bunyavirus rescue from cloned cDNA.

Anice C Lowen1, Carol Noonan, Angela McLees

  • 1Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 5JR, Scotland, UK.

Virology
|November 30, 2004
PubMed
Summary

Researchers improved Bunyamwera virus recovery using a T7 RNA polymerase cell line. This simplified method efficiently generates infectious Bunyaviridae viruses from cloned cDNA for research.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Bunyaviruses are trisegmented, negative-sense RNA viruses.
  • Previous methods for Bunyamwera virus recovery involved recombinant vaccinia virus expressing T7 RNA polymerase.
  • Improving the efficiency and reproducibility of Bunyavirus rescue systems is crucial for research.

Purpose of the Study:

  • To enhance the efficiency of Bunyamwera virus recovery from cloned cDNA.
  • To compare different methods of providing T7 RNA polymerase for virus rescue.
  • To simplify the Bunyavirus rescue procedure.

Main Methods:

  • Utilized a BHK-derived cell line (BSR-T7/5) constitutively expressing T7 RNA polymerase.
  • Transfected cells with three plasmids encoding full-length antigenome viral RNAs.

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  • Quantified virus recovery by plaque-forming units (pfu).

    • Main Results:

    • The BSR-T7/5 cell line supported efficient and reproducible Bunyamwera virus recovery.
    • Routine generation of >10(7) pfu per rescue experiment was achieved.
    • Simplified procedure using only three plasmids demonstrated effective virus recovery.

    Conclusions:

    • Constitutive expression of T7 RNA polymerase in BSR-T7/5 cells significantly improves Bunyavirus rescue efficiency.
    • A simplified three-plasmid system enables efficient recovery of Bunyamwera virus.
    • The improved rescue system is potentially applicable to other Bunyaviridae genera and arenaviruses.