Interaction of Free-living protozoa with water-borne human pathogenic viruses and protection from disinfection

Acanthamoeba causes Granulomatous Amoebic Encephalitis (GAE) and Amoebic Keratitis (AK) in humans and in its cystic form is resistant to extreme environmental conditions. Both human pathogenic water-borne viruses and free-living protozoa share the same aquatic environment. This study set out to test the ability of both Acanthamoeba and Tetrahymena to internalise and protect enteric viruses; coxsackievirus (B3, B5), poliovirus (PV) and rotavirus (RV) following co-culture. Viral uptake was assessed by infection of cultured mammalian cells, by indirect immunofluorescence (IF), and by reverse transcriptase-polymerase chain reaction (RT-PCR). The results showed that none of the free suspended viruses were internalised in Acanthamoeba or Tetrahymena. However, both coxsackievirus B3N and rotavirus Wa could be detected within Acanthamoeba by IF and confirmed by RT-PCR when the amoebae were co-cultured (fed) with virally infected mammalian cells. The co-cultured amoeba was allowed to encyst but following this procedure no viruses were detected either by cell culture or RT-PCR. In a second series of experiments, the efficacy of solar disinfection (SODIS) against viruses either alone or when co-cultured with Acanthamoeba was assessed. SODIS reduced the viral infectivity by over 3log10 after 1 h for CVB3N and over 2log10 for PV after 2 h. Repeating these experiments in the presence of riboflavin, a 6log10 reduction was observed for CVB3N after 1 h of light exposure and 6log10 after 6 h for all other viruses tested. The results suggest that Acanthamoeba does not internalise or protect viruses in suspension. However, if a virus is located with an infected mammalian cell then it may be internalised; a new potential mechanism for virus dissemination in the environment. Secondly, solar disinfection is an effective treatment method for water contaminated with viruses which is further enhanced by the addition of riboflavin. This study provides a practical example of low technology methods which could be utilised to provide safe drinking water in various circumstances.