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Related Experiment Videos

Generating West Nile Virus from an Infectious Clone.

Rianna Vandergaast1, Brenda L Fredericksen2

  • 1Imanis Life Sciences, Rochester, MN, 55902, USA. vandergaast.rianna@imanislife.com.

Methods in Molecular Biology (Clifton, N.J.)
|May 19, 2016
PubMed
Summary

Generating infectious West Nile virus (WNV) clones is challenging due to genome instability. This study presents a streamlined two-plasmid protocol for efficient WNV recovery, aiding WNV biology research.

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Area of Science:

  • Virology
  • Molecular Biology
  • Genetics

Background:

  • Infectious clones are essential for studying West Nile virus (WNV) biology.
  • Flaviviral genome instability historically limited the generation of WNV infectious clones.
  • Recent advances in cloning and two-plasmid systems have improved WNV clone generation.

Purpose of the Study:

  • To describe a protocol for recovering WNV from a two-plasmid infectious clone system.
  • To facilitate the generation of infectious WNV for research purposes.

Main Methods:

  • Utilized a two-plasmid WNV infectious clone system.
  • Digested constructs with restriction enzymes to create complementary restriction sites.
  • Annealed DNA fragments to form a linear template for in vitro transcription.
Keywords:
ElectroporationIn vitro transcriptionInfectious RNAInfectious cloneTwo-plasmid systemWest Nile virus

Related Experiment Videos

  • Synthesized infectious RNA, transfected cells, and recovered WNV from supernatant.

    • Main Results:

    • Successfully recovered WNV from the two-plasmid system.
    • The protocol enables the generation of infectious RNA for WNV recovery.
    • This method is applicable to various WNV strains and chimeric viruses.

    Conclusions:

    • The described protocol offers an efficient method for recovering WNV from two-plasmid infectious clones.
    • This technique simplifies the generation of WNV, supporting further research into WNV biology and pathogenesis.
    • The protocol's versatility allows for the creation of diverse WNV constructs, including pathogenic and chimeric strains.