Application of viability PCR to investigate the capsid integrity of human enteric viruses in drinking water sources and the persistence of those viruses in surface waters.

Here, we investigated the occurrence of indigenous human enteric viruses with intact capsids (i.e., those that are potentially infectious) in drinking water sources by applying viability PCR using improved propidium monoazide with an enhancer (PMAxx-Enhancer-PCR). A comparison of the results obtained with PCR alone and PMAxx-Enhancer-PCR suggested that adenovirus, norovirus GI, and rotavirus present in drinking water sources tended to have damaged capsids and be inactivated by damage to the capsid. In contrast, astrovirus, norovirus GII, and enteroviruses tended to have intact capsids and be potentially infectious. To elucidate the main cause of damage to the viral capsid, we next investigated the effects of environmental stresses (water temperature, particulate matter, enzymatic activity, and sunlight) on viral persistence in surface waters used as drinking water sources through virus-spiking incubation experiments by using infectivity assays, PCR alone, and PMAxx-Enhancer-PCR. The pseudo-first-order decay rate constants of the viruses obtained with PMAxx-Enhancer-PCR were comparable to those obtained with PCR alone when only water temperature, particulate matter, and enzymatic activity were stresses. In contrast, when sunlight was present in addition to the other three stresses, PMAxx-Enhancer-PCR showed larger decay rate constants than PCR alone. Thus, sunlight appears to be an environmental stress that determines the capsid integrity of indigenous human enteric viruses in drinking water sources. Indigenous pepper mild mottle virus (PMMoV) was present at >100 times higher concentrations in drinking water sources than human enteric viruses in both cases of using PCR alone and PMAxx-Enhancer-PCR, supporting the suitability of PMMoV as a surrogate for human enteric viruses for evaluating virus removal in actual drinking water treatment plants. The incubation experiments suggested that PMMoV is more resistant to environmental stresses than human enteric viruses, partly due to its more robust capsid structure. This may contribute to the high concentration of PMMoV in drinking water sources.