Enteric Viruses and Free-Living Amoebae: Protozoa as Potential Reservoirs and Transport Vessels for Human Norovirus and Adenovirus

Human norovirus (HNoV) and human adenovirus (HAdV) are major causes of acute viral gastroenteritis globally and environmentally transmitted via the faecal-oral route through contaminated food and water. Recent evidence of these enteric viruses residing within environmental free-living amoebae (FLA)—specifically Vermamoeba vermiformis , Acanthamoeba polyphaga , and Willaertia magna, all abundant in natural and engineered water systems—has significant implications for environmental virology and public health. The incorporation of HNoV into the cytoplasm and vacuoles of Vermamoeba vermiformis and Acanthamoeba polyphaga , as well as the nuclear localization of HAdV within Willaertia magna , was demonstrated using quantitative polymerase chain reaction (qPCR) and fluorescence microscopy. Intact HNoV and HAdV virions persisted inside FLA trophozoites, cysts, and extracellular vesicles for up to 12 days. Moreover, HAdV retained infectivity in buffalo green monkey kidney cells following intracellular persistence, suggesting these viruses can evade amoebal digestion and structural degradation. In the case of HAdV, nuclear incorporation, preservation of capsid integrity, and detection of messenger RNA associated with adenoviral fiber protein synthesis further suggest the possible initiation of virus-related transcriptional activity within the amoeba host. These findings challenge current assumptions about virus removal rates in sewage treatment, food safety protocols, and drinking water production. The enhanced persistence and protection conferred by ubiquitous FLA may also impact microbial risk assessments for recreational water use, particularly in sewage-impacted rivers and lakes, as well as for water reuse applications. Recognition of FLA as reservoirs and transport vessels for enteric viruses necessitates a re-evaluation of existing water and sanitation guidelines to better mitigate environmental transmission pathways. • HNoV and HAdV persist in FLA for 12 days, evading amoebal degradation pathways. • HAdV transcriptional activity was detected within FLA, broadening potential host range for viral activity. • FLA reservoirs may enhance virus persistence, impacting water treatment and reuse. • Updated water guidelines needed to mitigate FLA-mediated virus dispursal and transmission.