Virus inactivation mechanisms in human urine and fecal sludge

Water, sanitation and hygiene interventions are among the most significant health interventions addressing the worldwide burden of diarrheal disease and environmental enteropathy. Sanitizing human excreta and animal manure (HEAM) is a critical step in reducing the spread of disease and ensuring microbially safe reuse of waste materials. From the perspective of human excreta, it was recently established that despite an increase in global toilet coverage, the proportion of safely managed fecal waste remains low. Therefore, on-site storage and treatment still represent the best opportunities to reduce pathogen load. Among these pathogens, viruses are particularly persistent. However, adequate storage or digestion of HEAM can strongly reduce the number of viruses by creating adverse conditions to their survival. Although temperature, pH and ammonia (NH3) are commonly reported as primary virus inactivation factors, the mechanisms underlying virus reduction remain unclear. This thesis aims to shed more light on these mechanisms and their exploitation for HEAM management. Unlike other living organisms, viruses carry their genetic information under different forms, specifically as single(ss) or double-stranded (ds) RNA or DNA. ssRNA viruses were shown to be the most sensitive virus type during HEAM treatment. Additionally, ssRNA is also the most common genome type among enteric viruses. Therefore, we first investigated the kinetics and mechanisms of inactivation of the ssRNA virus MS2 under temperature, pH and NH3 conditions representative of waste storage. MS2 inactivation was mainly controlled by the activity of NH3 over a pH range of 7.0 to 9.5 and temperatures lower than 40°C. Other bases (e.g., hydroxide, carbonate) additionally contributed to the observed reduction of infective MS2. The loss in MS2 infectivity could be rationalized by a loss in genome integrity, which was attributed to genome cleavage via general base-catalyzed transesterification. The presence of the 2’-hydroxyl group of ribose renders the 3’,5’-phosphodiester linkages of RNA molecules susceptible to base-catalyzed transesterification. The contribution of each base to genome transesterification, and hence inactivation, could be related to the pKa of the conjugated acid of the base by means of a Brønsted relationship. Based on the Brønsted relationship in conjunction with the activity of bases in solution, a model was formulated that enabled an accurate prediction of MS2 inactivation rates in synthetic solutions. Several studies have observed slower inactivation in HEAM among viruses with genome types other than ssRNA. The reasons for this enhanced persistence, however, are not clear. Here, we systematically investigated the inactivation of viruses with different genome type in synthetic solutions with well-controlled temperature, pH and NH3 conditions representative of waste