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Microbiology JoVE (Open Access) Citable · DOI

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

DOI: 10.3791/4145-v
What you'll learn
  • ✓ Perform in vitro RNA transcription and capping of MNV cDNA constructs
  • ✓ Recover infectious murine norovirus using electroporation and lipofection methods
  • ✓ Generate MNV directly from cDNA in cells expressing T7 RNA polymerase
Protocol

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

Difficulty
advanced
Total time
~3–5 days (RNA synthesis, transfection, viral recovery, and titration)
Model organism
Murine norovirus (MNV); Raw 264.7 cells; BSR-T7 cells
Biosafety
BSL-2

Steps

1
Perform RNA transcription and capping for MNV

In vitro transcribe and cap the MNV genome from linearized cDNA template using T7 RNA polymerase and appropriate capping reagents to generate infectious RNA molecules.

▶ 03:16
2
Recover MNV by electroporation into Raw cells

Electroporate in vitro transcribed and capped MNV RNA into Raw 264.7 macrophage cells and monitor for viral recovery and cytopathic effects over several days.

▶ 06:27
3
Recover MNV by lipofection into BSR-T7 cells

Transfect in vitro transcribed MNV RNA into BSR-T7 cells (expressing T7 RNA polymerase) using lipofection reagents to generate infectious virus.

▶ 08:25
4
Directly recover infectious MNV from cDNA template

Transfect linearized MNV cDNA directly into BSR-T7 cells where T7 RNA polymerase transcribes the genome in vivo, enabling direct viral recovery without prior RNA synthesis.

▶ 10:07
5
Analyze and interpret representative results

Review viral titration data, cytopathic effects, and comparative efficiency of the three recovery approaches to validate successful MNV recovery.

▶ 12:20
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