ABSTRACT 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. SIMILAR CONTENT BEING VIEWED BY OTHERS STABILITY OF FELINE CORONAVIRUS IN AEROSOLS AND DRIED IN

ubiquitous organisms in natural and engineered environments1,2,3. They live at interfaces (water-soil, water-animal, water-plants and water-air), adherent on various surfaces and feed on

microorganisms3. While relatively few amoebae species are pathogens in their own right4, they are known natural environmental reservoirs for a range of amoeba-resisting bacterial pathogens,

such as _Legionella pneumophila_, a water-based bacterium responsible for Legionnaires’ disease that results in major community health burden5,6,7,8. More recently, amoebae have been

identified as potential reservoirs for non-enveloped respiratory and enteric viruses such as adenoviruses, coxsackieviruses, reovirus and the giant amoeba virus _Mimivirus_9,10,11,12.

Several highly transmissible respiratory enveloped viruses with epidemic potential have emerged in last two decades, with the ongoing COVID-19 pandemic being the most significant to date13,

yet their potential interaction with (sewage/faecal-borne) amoebae is unreported. Human respiratory syncytial virus (RSV) is a large (120–300 nm diameter) pleomorphic enveloped virus with a

non-segmented, negative-sense, single-stranded RNA that belongs to the Pneumoviridae family and is recognised as one of the most common causes of acute respiratory tract infections in

children, older people, and immunocompromised adults14,15,16. Despite the enormous burden of RSV disease, there is currently no efficacious vaccine nor antiviral drug therapy available17.

RSV is a highly contagious pathogen and transmission is thought to be primarily by large droplets and fomites, but is yet to be fully resolved18. However, clinical and epidemiological

studies of patients infected with RSV raised the possibility of faecal–oral transmission as described for other respiratory viruses19,20,21. Herein we used RSV as a model for potential

interactions of enveloped respiratory viruses with amoebae to ascertain their possible role as an environmental reservoir and vehicle for dissemination and transmission. Within two hours of

introducing GFP-RSV to an active culture of _Willaertia magna_ (co-culture) the virus was observed within trophozoites and expelled vesicles (Fig. 1a). In a separate experiment, and after 72

 h post introduction, fluorescence microscopy showed expelled respirable-sized amoebal-vesicles filled with GFP-RSV (Fig. 1b). Transmission electron microscopy (TEM) revealed pleomorphic RSV

particles from different cross-sections within _W. magna_ phagosomes (Fig. 2a). Further to this, the presence of RSV inside purified extracellular amoebal-vesicles was confirmed by TEM

(Fig. 2b). Using the ImageJ software package22, the virions measurements (Table 1) were consistent with previous conventional EM studies23,24,25. 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 infectious26,27.

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 TCID50 analysis, demonstrated that RSV_-_EAVs were indeed infectious with titres peaking at ~104 TCID50 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 phase-contrast 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 vesicles28,29,30,31. 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 antibodies32. 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 vesicles11.

Overall, virus-laden vesicles would increase the (dose) likelihood to infect susceptible host cells33, as well as the virus’ infectivity, as demonstrated for enteroviruses with equivalent

numbers of virions free versus within vesicles29,34,35. 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 hosts39. 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 inhalation40,41. Taken together these interesting observations provide evidence to suggest that amoebae

may contribute to the environmental persistence and transmission of respiratory viruses associated with natural aquatic environments and engineered water systems. Notably, extracellular

amoebal-vesicles could enable non-enveloped 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, biocides42 and antiviral host factors43,44. 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

epidemic45, and associated with toilets and COVID-19 cases in hospitals46. 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. METHODS 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% CO2 in vented 75 cm2 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% CO2 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 described37. 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% (TCID50) using the Reed–Muench formula48. 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% CO2. 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

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endpoints. _Am. J. Trop. Med. Hyg._ 27, 493–497 (1938). Google Scholar  Download references ACKNOWLEDGEMENTS These studies were supported by Alberta Innovates (grant # 201300490), Alberta,

Canada. We would like to thank Dr. David Marchant (University of Alberta; Canada) for supplying the green fluorescent protein-expressing RSV (GFP-RSV) strain used in this study and Arlene

Oatway for help with transmission electron microscopy (Microscopy Facility Biological Sciences, University of Alberta). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Public Health,

University of Alberta, Edmonton, AB, Canada Rafik Dey & Nicholas J. Ashbolt * Dept. Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada Rafik Dey, Melanie

A. Folkins & Nicholas J. Ashbolt * School of Environment, Science and Engineering, Southern Cross University, Lismore, NSW, Australia Nicholas J. Ashbolt Authors * Rafik Dey View author

publications You can also search for this author inPubMed Google Scholar * Melanie A. Folkins View author publications You can also search for this author inPubMed Google Scholar * Nicholas

J. Ashbolt View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS R.D. conceived the work. Sample preparations and assays were carried out by M.F.

and R.D. R.D. and N.J.A wrote the paper. CORRESPONDING AUTHOR Correspondence to Rafik Dey. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

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ARTICLE Dey, R., Folkins, M.A. & Ashbolt, N.J. Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses. _npj Biofilms Microbiomes_ 7, 25 (2021).

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