Novel assay to quantify recombination in a calicivirus

Area of Science:

• Virology
• Molecular Biology
• Genomics

Background:

• Genomic recombination drives evolution in Caliciviridae viruses, but mechanisms remain unclear.
• Feline calicivirus (FCV) recombination predominantly occurs at a specific hot spot between ORF1 and ORF2.
• Understanding recombination rates is crucial for FCV evolution studies.

Purpose of the Study:

• To develop and validate a quantitative RT-qPCR assay for measuring FCV recombination rates.
• To quantify the rate of recombination between two divergent FCV strains during co-infection in cell culture.
• To confirm the specificity and application of the developed assay for studying FCV recombination.

Main Methods:

• Developed a quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay.
• Utilized virus-specific primers flanking the FCV recombination hot spot (between ORF1 and ORF2).
• Quantified recombinant FCV progeny in co-infected cell cultures and confirmed by sequencing.

Main Results:

• The RT-qPCR assay successfully detected and quantified recombinant FCV progeny.
• Recombination was observed in co-infected cells but not in control experiments.
• The estimated rate of recombination was at least 6.8×10⁻⁶ recombinant genomes per parental genome.

Conclusions:

• The developed RT-qPCR assay is specific and effective for quantifying FCV recombination.
• This method allows for detailed investigation of factors influencing calicivirus recombination.
• Further studies can utilize this assay to explore recombination in vitro and in vivo.