Assays for the specific growth rate and cell-binding ability of rotavirus

Syun Suke Kadoya, Daisuke Sano

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 °C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Original languageEnglish
Article numbere58821
JournalJournal of Visualized Experiments
Volume2019
Issue number143
DOIs
Publication statusPublished - 2019 Jan

Keywords

  • Cell-binding ability
  • Immunology and infection
  • Issue 143
  • MA104 cell line
  • Plaque assay
  • RT-qPCR
  • Rotavirus
  • Specific growth rate

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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