Quantitative Distribution of Infectious F-Specific RNA Phage Genotypes in Surface Waters

F-specific RNA phages (FRNAPHs) are considered potential viral indicators of water pollution due to their occurrence and stability in water environments. However, their suitability as viral indicators is not fully elucidated because the characteristics of FRNAPHs are variable depending on the genotype. In this study, for the characterization of infectious FRNAPH genotypes, integrated culture reverse transcription-PCR coupled with the most probable number approach was applied to surface water samples. Further, to recover low concentrations of FRNAPH genotypes, an FRNAPH recovery method was developed. The novel FRNAPH recovery method using a noncharged microfiltration membrane could effectively recover FRNAPH strains without inactivation, while a method using an electronegative microfiltration membrane resulted in the inactivation of some strains. Infectious FRNAPH genotypes in surface water samples were successfully quantified with an efficiency comparable to that of the conventional plaque assay. Genotype I (GI) and GII FRNAPHs tended to be predominant at locations impacted by treated and untreated municipal wastewater, respectively. The numbers and proportions of infectious FRNAPHs tended to be higher during the winter season when water temperature decreased.

Properties of FRNAPHs are highly variable depending on their genotypes. Previous typing methods for FRNAPHs are not quantitative and/or are based on molecular assays, which cannot differentiate infective strains from inactive strains. Due to the reasons mentioned above, the utility of FRNAPHs as viral indicators of water pollution has not been fully validated. In this study, a quantitative genotyping method for infectious FRNAPHs was developed and applied to surface water samples. The method enabled characterization of infectious FRNAPH genotypes in terms of their occurrence and seasonality. Moreover, comparison of the method to a conventional molecular assay (reverse transcription-quantitative PCR) enabled characterization of their stability. Our approach can provide novel findings for further validation of FRNAPHs as viral indicators of water pollution.