Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses

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.

It is important to note, that amoebae trophozoites were visibly unaffected by the presence of internalised RSV virus.
Based on the GFP expression, it appeared that the RSV within amoebal-vesicles could still be infectious 26,27 . Therefore, it was of interest to assess the infectivity of freshly isolated RSV-EAVs (Fig.  3a). The EAVs containing RSV were collected 24 h post infection and viral titres, as measured by traditional TCID 50 analysis, demonstrated that RSV-EAVs were indeed infectious with titres peaking at~10 4 TCID 50 mL −1 (Fig. 3c), at a similar infectivity to RSV-only controls. Minor losses could be explained by the supernatant washing steps. On closer observation using phasecontrast microscopy there was also clear cytopathic effect induced by infectious RSV-EAVs in Hela cells, preventing the formation of the cells monolayer and affecting their appearance after 5 days of infection (Fig. 3d).
Recently, multiple independent studies have revealed that different viruses may exploit the secretory autophagy pathway to exit cells via released vesicles [28][29][30][31] . These amoebal-released packaged viruses could prolong their environmental infectivity (via fomites/aerosols/water system), as well when internalised by avoiding immune systems detection, such as evading recognition by neutralising antibodies 32 . Also, in a previous study utilising infectious Coxsackievirus B virions (i.e. a non-enveloped, enteric virus) we reported virions localised in Vermamoeba vermiformis trophozoites and expelled vesicles 11 . Overall, virus-laden vesicles would increase the (dose) likelihood to infect susceptible host cells 33 , as well as the virus' infectivity, as demonstrated for enteroviruses with equivalent numbers of virions free versus within vesicles 29,34,35 . Extracellular vesicles containing enteric viruses are naturally shed in human and animal faeces (and amoebae grow in sewage/animal excreta, including bat guano) [36][37][38] , which could be ingested and transmit to other hosts 39 . Interestingly, as evident in Figs 1 and 2, the released amoebal-vesicles are 2-3 μm in diameter, the size range expected to penetrate to the lower respiratory tract via mouth or nose inhalation 40,41 .
Taken together these interesting observations provide evidence to suggest that amoebae may contribute to the environmental persistence and transmission of respiratory viruses associated with 1 natural aquatic environments and engineered water systems. Notably, extracellular amoebal-vesicles could enable nonenveloped and enveloped virion dissemination and aid in the transmission of respiratory viruses. Amoeba-packaged viruses (in trophozoites, cysts and vesicles) may also protect virions from inactivation via sunlight, biocides 42 and antiviral host factors 43,44 . Hence, we recommend further study of the persistence and transmission of respiratory viruses in faecal droplets and aerosols to assess this newly proposed risk pathway; noting that sewage droplets/aerosols were shown to be important during the first SARS epidemic 45 , and associated with toilets and COVID-19 cases in hospitals 46 . Understanding how enveloped viruses persists in our environmental systems and interact with amoebae will contribute to our understanding of the epidemiology and   microbial ecology of respiratory viruses and potentially permit the development of methods to further aid in their management.

Strains and culture conditions
The virus used in this study was green fluorescent protein-expressing RSV (GFP-RSV) containing the viral glycoproteins (S, G and F) 47 . The RSV was propagated on 80-90% confluent HeLa cells (ATCC CCL-2) in DMEM medium containing 10% FBS, and 1% penicillin-streptomycin at 37°C and 5% CO 2 in vented 75 cm 2 cell-culture flasks.
The amoebae used in this study was Willaertia magna (ATCC 50035), a member of the Vahlkampfiidae family that was isolated from bovine faeces. Amoebae were grown in tissue culture flasks in SCGYEM (Serum-Casein-Glucose-Yeast-Extract-Medium: ATCC medium 1021) at 25°C in a 5% CO 2 incubator. The trophozoites were maintained in exponential growth phase by sub-culturing every 3-4 days in fresh SCGYEM. Amoebae were harvested by tapping the flasks to dislodge surface-adhered cells and subsequent centrifugation in a 15 mL screw-cap tube (FALCON, Fischer Scientific, Edmonton, Canada 3033) at 2000 × g for 10 min. Cells were washed three times with sterile distilled water to remove carried-over nutrients in the supernatants.

Imaging flow cytometry analysis
ImageStream ® cytometry analysis and the instrument gating strategy for amoebae was performed as previously described 37 . Briefly, W. magna trophozoites were infected for 2 h with GFP-RSV at MOI of 100, washed and re-suspended in PBS prior to processing through the ImageStream ® X Mark II (Millipore Sigma). Cells were examined at 60× magnification. Analysis was performed using the IDEAS software (Amnis, Seattle) and cells (fluorescent viruses and amoebae) were identified on the basis of bright field morphology, size and GFP signal.

Isolation of extracellular amoebal-vesicles (EAVs) containing RSV
W. magna and RSV were co-cultured at a ratio of 1:100 in conical Falcon tubes containing 3 mL of SCGYEM medium, vortexed to favour virus interaction with amoebae and then transferred to 6-well culture plates (Fisher Scientific 130185). After overnight incubation at 30°C, samples were analysed using a phase-contrast microscope (Leica CTR 4000) to detect the presence of EAVs in the supernatant while amoebal trophozoites remain attached to the surface of the well plates. To isolate and separate the EAVs containing RSV from the attached trophozoites, supernatants were removed and transferred into new well plates several times. In brief, supernatants were gently removed with care taken not to disturb the attached amoebae on well plate surfaces, and transferred to new well plates for 10-20 min to allow any amoebal trophozoites to attach to surfaces (Fig. 3b). The isolated EAVs containing RSV were collected and washed twice with PBS by centrifugation at 4000 × g for 5 min to remove uninternalized viruses. The purified EAVs were then used for infectivity assays and microscopy.

RSV infectivity assays
RSV was released from amoebal vesicles by three consecutive freeze−thaw cycles. RSV infectivity (EAVs containing RSV and RSV-only control) was measured by infecting confluent HeLa cells in quadruplicate using 48-well plates and serial dilution of the virus in HeLa cells maintenance medium. Cells were observed daily for cytopathic effects for seven days and CPE was measured by the tissue culture infectious dose 50% (TCID 50 ) using the Reed-Muench formula 48 .

Transmission electron microscopy
Axenic cultures of W. magna were co-cultured with RSV at a MOI of 100 on Thermonax ® cover slips (Thermo Fisher 174985). After decanting the medium, amoebae were fixed at room temperature with 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences 15960). The samples were submitted for processing at the imaging core at University of Alberta, faculty of biological sciences. Sectioned and carbon-coated samples were observed with a Hitachi H-7650 transmission electron microscope.

Fluorescence microscopy
Co-cultures of W. magna-GFP-RSV were carried in 12-well tissue culture plates overlaid with microscopy cover slips (Fisher Scientific 12-5461) and incubated at 25°C with 5% CO 2 . After 72 h of infection, the medium was removed and cells fixed with 4% paraformaldehyde for 5 min at room temperature and then washed with phosphate-buffered saline three times. Images were taken with an EVOS FL fluorescent cell imaging system (ThermoFisher Scientific).

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.

DATA AVAILABILITY
The data sets generated during and/or analysed during the current study are either shown in the manuscript or available from the corresponding author on reasonable request.