Researchers developed a method using DEAE-cellulose column chromatography to select and quantify rare revertants (S+ variants) in populations of small-plaque mutants (S-) of fowl plague virus. The method exploits biochemical differences in how these viral variants bind to and elute from chromatography columns. S- mutants that respond to polycations elute at higher salt concentrations (0.4-0.6 M NaCI) compared to the original large-plaque strain (0.2-0.4 M NaCI), allowing enrichment of revertants over 2000-fold. The authors validated this approach using artificial virus mixtures and successfully determined both spontaneous and chemically-induced reversion frequencies across multiple mutants. Chemical mutagenesis with O-methylhydroxylamine substantially increased reversion rates compared to spontaneous reversion. The method enables detection of reversion frequencies as low as 10^-8 and can be applied to studying mutagenic specificity and genetic stability of viral stocks.

Key findings

• DEAE-cellulose column chromatography selectively separates large-plaque S+ variants from small-plaque S- mutants based on differential elution at different salt concentrations.
• The method achieves over 2000-fold enrichment of rare S+ revertants, enabling detection of reversion frequencies as low as 10^-8.
• Chemical mutagenesis with O-methylhydroxylamine increases reversion frequency substantially (up to 8.5 × 10^-4) compared to spontaneous reversion rates.
• Experimental reversion frequency values obtained by this method closely approximate actual values when tested with artificial viral mixtures at known ratios.
• This chromatographic approach can be applied to studying mutagenic specificity, genetic stability, and detecting rare recombinants in viral populations.