Ammonia as an In Situ Sanitizer: Influence of Virus Genome Type on Inactivation

ABSTRACT

Treatment of human excreta and animal manure (HEAM) is key in controlling the spread of persistent enteric pathogens, such as viruses. The extent of virus inactivation during HEAM storage and treatment appears to vary with virus genome type, although the reasons for this variability are not clear. Here, we investigated the inactivation of viruses of different genome types under conditions representative of HEAM storage or mesophilic digestion. The goals were to characterize the influence of HEAM solution conditions on inactivation and to determine the potential mechanisms involved. Specifically, eight viruses representing the four viral genome types (single-stranded RNA [ssRNA], double-stranded RNA [dsRNA], single-stranded DNA [ssDNA], and double-stranded DNA [dsDNA]) were exposed to synthetic solutions with well-controlled temperature (20 to 35°C), pH (8 to 9), and ammonia (NH₃) concentrations (0 to 40 mmol liter⁻¹). DNA and dsRNA viruses were considerably more resistant than ssRNA viruses, resulting in up to 1,000-fold-longer treatment times to reach a 4-log inactivation. The apparently slower inactivation of DNA viruses was rationalized by the higher stability of DNA than that of ssRNA in HEAM. Pushing the system toward harsher pH (>9) and temperature (>35°C) conditions, such as those encountered in thermophilic digestion and alkaline treatments, led to more consistent inactivation kinetics among ssRNA and other viruses. This suggests that the dependence of inactivation on genome type disappeared in favor of protein-mediated inactivation mechanisms common to all viruses. Finally, we recommend the use of MS2 as a conservative indicator to assess the inactivation of ssRNA viruses and the stable ΦX174 or dsDNA phages as indicators for persistent viruses.

IMPORTANCE Viruses are among the most environmentally persistent pathogens. They can be present in high concentrations in human excreta and animal manure (HEAM). Therefore, appropriate treatment of HEAM is important prior to its reuse or discharge into the environment. Here, we investigated the factors that determine the persistence of viruses in HEAM, and we determined the main mechanisms that lead to their inactivation. Unlike other organisms, viruses can have four different genome types (double- or single-stranded RNA or DNA), and the viruses studied herein represent all four types. Genome type appeared to be the major determinant for persistence. Single-stranded RNA viruses are the most labile, because this genome type is susceptible to degradation in HEAM. In contrast, the other genome types are more stable; therefore, inactivation is slower and mainly driven by the degradation of viral proteins. Overall, this study allows us to better understand the behavior of viruses in HEAM.