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Digital archive of theses discussed at the University of Pisa


Thesis etd-12112010-155240

Thesis type
Tesi di dottorato di ricerca
Thesis title
Studio delle possibili interazioni tra virus di derivazione umana e protisti in ambiente idrico
Academic discipline
Course of study
tutor Prof.ssa Carducci, Annalaura
  • Protozoi
  • Virus
Graduation session start date
The ability of protists to host some bacterial pathogens such as Legionella, which have developed resistance mechanisms to escape phagocytosis and/or digestion by protists, it is well known, while some studies suggest that protists may have a possible role as occasional hosts for viruses. In waste water treatment plants this mechanism could be a possible cause of the virus resistance to disinfection.
The aim of this study was to investigate, in both experimental and field conditions, a possible interaction in the environment between human viruses and protozoa. The research was carried out in different phases: the first step has been the research of enteric viruses in the aquatic environment, and their resistance to disinfection treatments, by monitoring raw sewages, treated effluents and shellfishes with biomolecular and cultural tests. Results evidenced that adenoviruses (HAdv) was the more present virus in environment and more resistant to treatments. Adenoviruses was always present in sewages at the entrance and at the exit of the plant with an abatement rate of about 2 Logs, besides was also found in depurated shellfish, so it was chosen for the infection experimental phases of the study.
In a second preliminary phase protist strains isolated from marine and fresh waters, besides algae used as food were analyzed with biomolecular tests for HAdv, enterovirus, rotavirus, hepatitis A virus, norovirus GGI and GGII presence. Results showed the absence of human viruses in the tested strains, except HAdv whose genome was found in 2 strains (RoM8 and CoMa6) and in the algae. None of the tested samples showed cytopathic effect, but the PCR performed on cell lysates resulted positive, indicating the virus infectivity.
Before the start of the experimental tests two possible methods to be used to detect the possible presence of enteric viruses in protists were tested: PCR and immunofluorescence (IF). The use of PCR has proved to be failure because of difficult to completely eliminate the virus from the culture medium and then proceed to the analysis of the cell, immunofluorescence instead gave good results and was therefore chosen for further investigations.
Successively it was carried out the experimental infection phases: a strain of the protozoan ciliate Euplotes octocarinatus testing negative for HAdv was chosen for experimental infection with Adenovirus Type 2 and maintained in contact for variable times (5, 15, 30, 45 min, 1, 3, 6, 24, 48, 72 h and 7, 10, 45, 105 days). After 4 washes followed by centrifugation, the samples were then analyzed by direct immunofluorescence (IF) in more replicates. Subsequently to evaluate if virus can survive inside the cells, after 1 h of incubation, Euplotes cells were centrifuged and pellets were washed four times in order to remove unadsorbed virus, suspended in mineral water and maintained at room temperature for various lengths of time (24, 72 h and 7, 8, 11, 14, 16 days) before IF analysis. The microscopic observation showed that viruses enter the protozoa after a contact of 15 minutes and was then visible in most cells during the subsequent contact times. The fluorescence within protozoa appears localize mostly in the cytoplasm below the macronucleus presumably within the digestive vacuoles, because the cells contain pockets of fluorescence that had the approximate size, shape and localization of food vacuoles. Besides the results of the survival of HAdv in Euplotes cultures showed that, the viruses remained inside the cells until at least 35 days postinfection with a gradual reduction in the number of fluorescent cells, in particular from 21 days.
Finally, to study the interactions between protozoa and HAdv in field conditions, wild protists were isolated from active sludges and submitted to IF for HAdv detection. These protists showed a diffuse IF, probably due to the surface absorption of HAdv confirming the interaction between protists and adenoviruses.