Professor Nicholas Ashbolt

The reported persistence of SARS-CoV-2 virions in aquatic environments highlights the need to better understand potential mechanisms that may prolong its dissemination. We evaluated the possibility that amoebae might serve as transport hosts by studying the interaction of the enveloped bacteriophage Phi6, as a potential surrogated along with one of the most common amoebae in engineered aquatic environments, Vermamoeba vermiformis. Using microscopy, imaging flow cytometry and bacteriophage cell culture, our results imply that the SARS-CoV-2 surrogate triggers amoebic mitochondria and induced apoptosis to promote viral persistence in trophozoites. Furthermore, virus-infected amoebae were still infectious after 2 months within FLA cysts. These results suggest that amoebae could contribute to the environmental persistence of SARS-CoV-2, including disinfection processes. In addition, amoebae could be a successful model system for understanding respiratory virus-eukaryotic biology at the cellular and molecular levels.

Intracellular growth of pathogenic
in free-living amoebae (FLA) results in the critical concentrations that are problematic in engineered water systems (EWS). However, being amoeba-resistant bacteria (ARB), how
spp. becomes internalized within FLA is still poorly understood. Using fluorescent microscopy, we investigated in real-time the preferential feeding behavior of three water-related FLA species,
, and
regarding
and two
strains. Although all the studied FLA species supported intracellular growth of
, they avoided this bacterium to a certain degree in the presence of
and mostly fed on it when the preferred bacterial food-sources were limited. Moreover, once
were intracellular, it inhibited digestion of co-occurring
within the same trophozoites. Altogether, based on FLA-bacteria interactions and the shifts in microbial population dynamics, we propose that FLA's feeding preference leads to an initial growth of FLA and depletion of prey bacteria, thus increases the relative abundance of
and creates a "forced-feeding" condition facilitating the internalization of
into FLA to initiate the cycles of intracellular multiplication. These findings imply that monitoring of FLA levels in EWS could be useful in predicting possible imminent high occurrence of
.

Human respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and immunocompromised adults worldwide. Here we report that amoebae-release respirable-sized vesicles containing high concentrations of infectious RSV that persisted for the duration of the experiment. Given the ubiquity of amoebae in moist environments, our results suggest that extracellular amoebal-vesicles could contribute to the environmental persistence of respiratory viruses, including potential resistance to disinfection processes and thereby offering novel pathways for viral dissemination and transmission.

Free-living amoebae (FLA) are ubiquitous protozoa in aquatic/soil habitats and known to resist various disinfection methods commonly used in drinking and wastewater treatment plants. Reoviruses are emerging as useful infectious enteric virus indicators of wastewater treatment efficacy. The possible enhanced protection FLA may provide reoviruses, however, has not been previously described. Using an infectious clinical reovirus isolate in coculture with three FLA, namely, Vermamoeba vermiformis, Acanthamoeba polyphaga, and Willaertia magna, we followed reovirus persistence (by quantitative reverse transcription polymerase chain reaction (RT-qPCR)) and infectivity (TCID50). Virions present in samples persisted over the experimental time period, with most virions remaining infectious. Surprisingly, electron microscopy revealed virions accumulated within the nucleus of amoebae. The current work appears to be the first report of reovirus being internalized within FLA and remaining infectious, providing a previously unreported environmental reservoir and potential mode of dissemination. FLA also appeared to be providing some logs in protection to internalized viruses during UV irradiation, which if not accounted for when determining UV dosage needed for sufficient disinfection may result in unintentional release of pathogens into surrounding water systems.