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Establishment of a superinfection exclusion method for pestivirus titration using a recombinant reporter pestiviruses

  • 0Laboratory of Microbiology, Department of Disease Control, Faculty of Veterinary Medicine, Hokkaido University, Hokkaido, Japan.
The Journal of Veterinary Medical Science +

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February 14, 2024

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February 14, 2024

View abstract on PubMed

Summary

This summary is machine-generated.

A new luciferase-based method simplifies pestivirus titration. This technique uses recombinant reporter viruses and measures luminescence, offering a faster alternative to traditional methods for detecting viruses like classical swine fever virus (CSFV).

Area Of Science

  • Virology
  • Molecular Biology
  • Biotechnology

Background

  • Pestiviruses exhibit two biotypes based on cytopathogenicity.
  • Titration of non-cytopathogenic pestiviruses typically requires indirect methods like immunostaining or interference assays.
  • Current titration methods are time-consuming and labor-intensive due to the need for microscopic observation.

Purpose Of The Study

  • To develop a novel, efficient, and potentially automatable method for pestivirus titration.
  • To utilize the superinfection exclusion phenomenon and a luciferase reporter system for virus quantification.

Main Methods

  • Development of recombinant reporter pestiviruses incorporating a NanoLuc luciferase (HiBiT) subunit.
  • Application of the superinfection exclusion phenomenon: cells infected with classical swine fever virus (CSFV) were superinfected with a reporter CSFV (vGPE<sup>-</sup>/HiBiT).
  • Quantification of virus growth via luminescence measurement in culture fluid, correlating with virus titer.

Main Results

  • The novel luciferase-based method provided pestivirus titers comparable to traditional immunoperoxidase staining.
  • The method demonstrated applicability for titrating both classical swine fever virus (CSFV) and border disease virus (BDV).
  • Successful heterologous superinfection using reporter CSFV for BDV titration was achieved.

Conclusions

  • A novel luciferase-based titration method using superinfection exclusion offers a simple and efficient alternative for pestivirus quantification.
  • This technique facilitates automated virus detection, significantly reducing time and labor compared to conventional methods.
  • The method's versatility extends to different pestiviruses, including CSFV and BDV, highlighting its broad potential.

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