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Updated: May 31, 2026
Plaque Assay for Murine Norovirus
Published on: August 22, 2012
Plaque formation assay for human parainfluenza virus type 1
Keijo Fukushima1, Tadanobu Takahashi, Masahiro Takaguchi
1Department of Biochemistry, School of Pharmaceutical Sciences and Global COE Program for Innovation in Human Health Sciences, University of Shizuoka, 52–1 Yada, Shizuoka 422–8526, Japan.
Abstract:
Human parainfluenza virus type 1 (hPIV1) generally does not show visible plaques in common cell lines, including Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells, by plaque formation assays for human parainfluenza virus type 3 (hPIV3) and Sendai virus. In several conditions of the plaque formation assay, complete elimination of serum proteins in the overlay medium was necessary for visualization of hPIV1-induced plaque formation in LLC-MK(2) cells. We developed a plaque formation assay for hPIV1 isolation and titration in LLC-MK(2) cells using an initial overlay medium of bovine serum albumin-free Eagle's minimum essential medium containing agarose and acetylated trypsin for 4-6 d followed by a second overlay staining medium containing agarose and neutral red. The assay allowed both laboratory and clinical hPIV1 strains to form large plaques. The plaque reduction assay was also performed with rabbit anti-hPIV1 antibody as a general evaluation model of viral inhibitors to decrease both the plaque number and size. The results indicate that the plaque formation assay is useful for hPIV1 isolation, titration, evaluation of antiviral reagents and epidemiologic research.
Insights
A new plaque formation assay enables visualization of human parainfluenza virus type 1 (hPIV1) plaques in LLC-MK(2) cells. This method is crucial for hPIV1 isolation, titration, and antiviral research.
Area of Science:
- Virology
- Cell Biology
Background:
- Human parainfluenza virus type 1 (hPIV1) typically does not form visible plaques in standard cell lines using existing plaque assays.
- Serum proteins in overlay media often inhibit hPIV1 plaque visualization in Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells.
Purpose of the Study:
- To develop a reliable plaque formation assay for the isolation and titration of hPIV1.
- To establish a method for evaluating antiviral reagents and conducting epidemiologic research on hPIV1.
Main Methods:
- Developed a modified plaque assay using serum-free Eagle's minimum essential medium with agarose and acetylated trypsin for initial overlay, followed by neutral red staining.
- Utilized Lewis lung carcinoma-monkey kidney (LLC-MK(2)) cells for hPIV1 plaque formation.
- Employed a plaque reduction assay with anti-hPIV1 antibody to assess viral inhibitors.
Main Results:
- The new assay successfully induced large plaque formation for both laboratory and clinical hPIV1 strains in LLC-MK(2) cells.
- Complete elimination of serum proteins was essential for plaque visualization.
- The plaque reduction assay demonstrated efficacy in evaluating antiviral activity.
Conclusions:
- The developed plaque formation assay is effective for hPIV1 isolation and titration.
- This assay serves as a valuable tool for assessing antiviral efficacy and for hPIV1-related epidemiologic studies.
