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LitCovid-PubTator

Id Subject Object Predicate Lexical cue tao:has_database_id
2 26-44 Species denotes bacteriophage Phi6 Tax:10879
3 62-72 Species denotes SARS-CoV-2 Tax:2697049
15 534-544 Species denotes SARS-CoV-2 Tax:2697049
16 581-584 Species denotes MS2 Tax:2710868
17 617-622 Species denotes human Tax:9606
18 1617-1627 Species denotes SARS-CoV-2 Tax:2697049
19 645-654 Chemical denotes SM buffer
20 660-665 Chemical denotes water MESH:D014867
21 753-758 Chemical denotes water MESH:D014867
22 1025-1034 Chemical denotes SM buffer
23 1039-1044 Chemical denotes water MESH:D014867
24 1077-1082 Chemical denotes water MESH:D014867
25 960-970 Disease denotes dry saliva MESH:D015352
33 1675-1680 Species denotes human Tax:9606
34 1830-1840 Species denotes SARS-CoV-2 Tax:2697049
35 2590-2595 Species denotes human Tax:9606
36 2996-3006 Species denotes SARS-CoV-2 Tax:2697049
37 2637-2642 Chemical denotes salts MESH:D012492
38 1720-1728 Disease denotes coughing MESH:D003371
39 2732-2754 Disease denotes dry deposits of saliva MESH:D000079822
42 4663-4665 Gene denotes pH Gene:5053
43 4881-4889 Disease denotes stresses MESH:D000079225
47 5061-5071 Species denotes SARS-CoV-2 Tax:2697049
48 5105-5115 Species denotes SARS-CoV-2 Tax:2697049
49 5281-5291 Species denotes SARS-CoV-2 Tax:2697049
56 6053-6071 Species denotes SARS coronaviruses Tax:694009
57 6098-6108 Species denotes SARS-CoV-2 Tax:2697049
58 6278-6281 Species denotes MS2 Tax:2710868
59 6630-6635 Species denotes human Tax:9606
60 6644-6653 Chemical denotes SM buffer
61 6666-6671 Chemical denotes water MESH:D014867
65 7944-7949 Species denotes human Tax:9606
66 7935-7938 Chemical denotes H2O
67 8201-8215 Chemical denotes saturated salt
74 8586-8591 Species denotes human Tax:9606
75 8573-8575 Chemical denotes SM MESH:D012493
76 8577-8580 Chemical denotes H2O
77 8649-8651 Chemical denotes SM MESH:D012493
78 8660-8663 Chemical denotes H2O
79 8983-8992 Chemical denotes gravitate
87 6972-6975 Species denotes MS2 Tax:2710868
88 7030-7035 Species denotes human Tax:9606
89 7210-7213 Species denotes MS2 Tax:2710868
90 7773-7782 Species denotes bacterial Tax:12333
91 7044-7049 Chemical denotes water MESH:D014867
92 7055-7057 Chemical denotes SM MESH:D012493
93 7436-7444 Disease denotes coughing MESH:D003371
95 10499-10502 Chemical denotes H2O
98 9607-9611 Chemical denotes salt MESH:D012492
99 9539-9549 Disease denotes dry matter MESH:D015352
108 10778-10783 Chemical denotes Water MESH:D014867
109 10870-10875 Chemical denotes water MESH:D014867
110 11099-11104 Chemical denotes water MESH:D014867
111 11185-11187 Chemical denotes SM MESH:D012493
112 11401-11405 Chemical denotes salt MESH:D012492
113 11481-11485 Chemical denotes salt MESH:D012492
114 11581-11585 Chemical denotes NaCl MESH:D012965
115 11607-11609 Chemical denotes SM MESH:D012493
125 13711-13714 Species denotes MS2 Tax:2710868
126 13794-13807 Species denotes Bacteriophage Tax:38018
127 13917-13930 Species denotes bacteriophage Tax:38018
128 13963-13968 Species denotes human Tax:9606
129 14349-14352 Species denotes MS2 Tax:2710868
130 13944-13953 Chemical denotes SM buffer
131 13955-13958 Chemical denotes H2O
132 14356-14358 Chemical denotes SM MESH:D012493
133 14375-14378 Chemical denotes H2O
140 13086-13089 Species denotes MS2 Tax:2710868
141 13463-13466 Species denotes MS2 Tax:2710868
142 11889-11894 Chemical denotes water MESH:D014867
143 11900-11902 Chemical denotes SM MESH:D012493
144 12340-12345 Chemical denotes water MESH:D014867
145 12371-12373 Chemical denotes SM MESH:D012493
151 15355-15358 Species denotes MS2 Tax:2710868
152 14603-14608 Chemical denotes water MESH:D014867
153 15020-15025 Chemical denotes water MESH:D014867
154 15234-15239 Chemical denotes water MESH:D014867
155 15382-15387 Chemical denotes water MESH:D014867
159 15883-15886 Species denotes MS2 Tax:2710868
160 15525-15527 Chemical denotes SM MESH:D012493
161 15890-15892 Chemical denotes SM MESH:D012493
165 17049-17052 Species denotes MS2 Tax:2710868
166 16691-16700 Chemical denotes SM buffer
167 16705-16710 Chemical denotes water MESH:D014867
173 17369-17371 Chemical denotes SM MESH:D012493
174 17384-17387 Chemical denotes H2O
175 17626-17628 Chemical denotes SM MESH:D012493
176 17729-17731 Chemical denotes SM MESH:D012493
177 17820-17825 Chemical denotes water MESH:D014867
184 18415-18420 Species denotes human Tax:9606
185 18650-18655 Species denotes Human Tax:9606
186 18622-18632 Chemical denotes effloresce
187 18909-18914 Chemical denotes salts MESH:D012492
188 19263-19278 Chemical denotes inorganic salts
189 19304-19306 Chemical denotes SM MESH:D012493
193 19829-19831 Gene denotes pH Gene:5053
194 19500-19505 Chemical denotes water MESH:D014867
195 19686-19691 Chemical denotes water MESH:D014867
201 20719-20721 Gene denotes pH Gene:5053
202 20382-20384 Gene denotes pH Gene:5053
203 20293-20297 Chemical denotes salt MESH:D012492
204 20350-20354 Chemical denotes salt MESH:D012492
205 20705-20710 Chemical denotes salts MESH:D012492
210 20884-20886 Gene denotes pH Gene:5053
211 20860-20864 Chemical denotes salt MESH:D012492
212 20998-21003 Chemical denotes lipid MESH:D008055
213 21035-21040 Chemical denotes water MESH:D014867
219 21605-21615 Species denotes SARS-CoV-2 Tax:2697049
220 21682-21692 Species denotes SARS-CoV-2 Tax:2697049
221 21775-21785 Species denotes SARS-CoV-2 Tax:2697049
222 21555-21563 Disease denotes COVID-19 MESH:C000657245
223 21903-21911 Disease denotes infected MESH:D007239
226 22368-22373 Species denotes human Tax:9606
227 22710-22725 Chemical denotes polysaccharides MESH:D011134
229 22811-22824 Species denotes bacteriophage Tax:38018
243 22890-22893 Species denotes MS2 Tax:2710868
244 23053-23066 Species denotes Bacteriophage Tax:38018
245 23092-23105 Species denotes bacteriophage Tax:38018
246 23816-23829 Species denotes bacteriophage Tax:38018
247 24022-24042 Species denotes Pseudomonas syringae Tax:317
248 24074-24092 Species denotes bacteriophage Phi6 Tax:10879
249 24170-24186 Species denotes Escherichia coli Tax:562
250 24218-24239 Species denotes bacteriophage PhiX174 Tax:10847
251 24318-24334 Species denotes Escherichia coli Tax:562
252 24399-24416 Species denotes bacteriophage MS2 Tax:12022
253 23189-23198 Species denotes bacterial Tax:12333
254 23325-23334 Chemical denotes SM buffer
255 23404-23412 Disease denotes infected MESH:D007239
274 24571-24574 Species denotes MS2 Tax:2710868
275 24775-24780 Species denotes human Tax:9606
276 25038-25041 Species denotes MS2 Tax:2710868
277 24634-24636 Chemical denotes SM MESH:D012493
278 24652-24656 Chemical denotes NaCl MESH:D012965
279 24663-24668 Chemical denotes MgSO4 MESH:D008278
280 24671-24675 Chemical denotes 7H2O
281 24683-24687 Chemical denotes Tris
282 24740-24743 Chemical denotes H2O
283 24846-24854 Chemical denotes melamine MESH:C011907
284 24872-24883 Chemical denotes rhodamine B MESH:C029773
285 26010-26024 Chemical denotes saturated salt
286 26035-26052 Chemical denotes potassium acetate MESH:D019347
287 26077-26096 Chemical denotes potassium carbonate MESH:C037593
288 26119-26133 Chemical denotes sodium bromide MESH:C027938
289 26161-26178 Chemical denotes ammonium chloride MESH:D000643
290 26198-26200 Chemical denotes RH
291 26627-26636 Chemical denotes SM buffer
307 26845-26854 Species denotes bacterial Tax:12333
308 27091-27100 Species denotes bacterial Tax:12333
309 26713-26717 Chemical denotes agar MESH:D000362
310 26755-26757 Chemical denotes LB
311 26770-26774 Chemical denotes agar MESH:D000362
312 26781-26786 Chemical denotes MgSO4 MESH:D008278
313 26797-26802 Chemical denotes CaCl2 MESH:D002122
314 26981-26985 Chemical denotes agar MESH:D000362
315 26994-26996 Chemical denotes LB
316 27010-27014 Chemical denotes agar MESH:D000362
317 27021-27026 Chemical denotes MgSO4 MESH:D008278
318 27037-27042 Chemical denotes CaCl2 MESH:D002122
319 27075-27080 Chemical denotes water MESH:D014867
320 27140-27144 Chemical denotes agar MESH:D000362
321 27269-27273 Chemical denotes agar MESH:D000362
326 27959-27970 Chemical denotes Rhodamine B MESH:C029773
327 27899-27909 Disease denotes SOLA SE II MESH:C537730
328 28132-28138 Disease denotes Chroma
329 28477-28485 Disease denotes SCANplus
331 29017-29021 Gene denotes Supp
333 29208-29212 Gene denotes Supp
335 29512-29529 Disease denotes virus aggregation

LitCovid-PD-HP

Id Subject Object Predicate Lexical cue hp_id
T1 1720-1728 Phenotype denotes coughing http://purl.obolibrary.org/obo/HP_0012735
T2 7364-7369 Phenotype denotes falls http://purl.obolibrary.org/obo/HP_0002527
T3 7436-7444 Phenotype denotes coughing http://purl.obolibrary.org/obo/HP_0012735

LitCovid-sentences

Id Subject Object Predicate Lexical cue
T1 0-136 Sentence denotes Survival of the enveloped bacteriophage Phi6 (a surrogate for SARS-CoV-2) in evaporated saliva microdroplets deposited on glass surfaces
T2 138-146 Sentence denotes Abstract
T3 147-340 Sentence denotes Survival of respiratory viral pathogens in expelled saliva microdroplets is central to their transmission, yet the factors that determine survival in such microdroplets are not well understood.
T4 341-585 Sentence denotes Here we combine microscopy imaging with virus viability assays to study survival of three bacteriophages suggested as good models for respiratory pathogens: the enveloped Phi6 (a surrogate for SARS-CoV-2), and the non-enveloped PhiX174 and MS2.
T5 586-741 Sentence denotes We measured virus viability in human saliva microdroplets, SM buffer, and water following deposition on glass surfaces at various relative humidities (RH).
T6 742-911 Sentence denotes Saliva and water microdroplets dried out rapidly, within minutes, at all tested RH levels (23%, 43%, 57%, and 78%), while SM microdroplets remained hydrated at RH ≥ 57%.
T7 912-1129 Sentence denotes Generally, the survival of all three viruses in dry saliva microdroplets was significantly greater than those in SM buffer and water under all RH (except PhiX174 in water under 57% RH survived the best among 3 media).
T8 1130-1364 Sentence denotes Thus, atmosphere RH and microdroplet hydration state are not sufficient to explain virus survival, indicating that the virus-suspended medium, and association with saliva components in particular, likely play a role in virus survival.
T9 1365-1628 Sentence denotes Uncovering the exact properties and components that make saliva a favorable environment for the survival of viruses, in particular enveloped ones like Phi6, is thus of great importance for reducing transmission of viral respiratory pathogens including SARS-CoV-2.
T10 1630-1642 Sentence denotes Introduction
T11 1643-1844 Sentence denotes Microdroplets expelled from the human respiratory tract into the air through coughing, talking, and breathing are considered a key source of transmission of respiratory viruses including SARS-CoV-21,2.
T12 1845-2008 Sentence denotes These microdroplets, ranging in size from a few micrometers up to millimeters3–5 travel in the air, and some of them—larger ones in particular—settle on surfaces2.
T13 2009-2085 Sentence denotes Thus, inanimate surfaces are a potential route of virus transmission1,2,6,7.
T14 2086-2197 Sentence denotes The factors that affect virus survival in droplets that settle on surfaces are complex and not well understood.
T15 2198-2350 Sentence denotes Survival is believed to depend upon the physicochemical characteristics and hydration conditions of the immediate microscopic environment of the virion.
T16 2351-2544 Sentence denotes These in turn are determined by several factors, including the composition of the fluid comprising the droplets, the surface properties, the ambient temperature, and the relative humidity (RH).
T17 2545-2687 Sentence denotes A preeminent source for expelled droplets is human saliva, a complex solution that contains salts, a variety of proteins, and surfactants8–11.
T18 2688-2794 Sentence denotes It has been suggested that micrometer-sized dry deposits of saliva droplets on surfaces protect virions12.
T19 2795-2944 Sentence denotes Still, it is not clear how well viruses survive in saliva microdroplets following their deposition on surfaces, and what factors are at play therein.
T20 2945-3131 Sentence denotes The impact of RH on the stability and viability of SARS-CoV-2 and that of other enveloped viruses has been studied mostly in airborne droplets or aerosols13–19 and less on surfaces20–22.
T21 3132-3327 Sentence denotes While survival varies between virus species, increased survival at both low (< 40%) and very high (> 90%) RH is often observed, with decreased survival at intermediate RH levels11,13,14,17,20,23.
T22 3328-3414 Sentence denotes The underlying mechanism of this U-shaped survival as a function of RH is not clear16.
T23 3415-3534 Sentence denotes Only a few studies have attempted to gain mechanistic understanding of how RH affects virus stability in microdroplets.
T24 3535-3745 Sentence denotes Prominent among these are the pioneering studies of the Marr group5,16,18,24, which point to the role of the solvent composition, evaporation dynamics, and RH on virus survival in aerosols and sessile droplets.
T25 3746-3958 Sentence denotes A key factor determining the evaporation rate and equilibrium hydration level of drying droplets on surfaces (and in air as well) is the deliquescence property of solutes, or mostly highly hygroscopic salts25–27.
T26 3959-4184 Sentence denotes Accordingly, although many surfaces in the indoor environment appear dry, they are in fact covered by thin liquid films and micrometer-sized droplets, invisible to the naked eye, known as microscopic surface wetness (MSW) 28.
T27 4185-4334 Sentence denotes MSW can be considered the “envelope” that accommodates microorganisms on surfaces, and as such has profound impact on many aspects of microbial life.
T28 4335-4398 Sentence denotes For example, it can protect microbes from complete desiccation.
T29 4399-4498 Sentence denotes However, MSW is a harsh micro-environment that differs in its properties from those of bulk liquid.
T30 4499-4569 Sentence denotes MSW inherently arises from drying liquids that evaporate on a surface.
T31 4570-4722 Sentence denotes This drying process is accompanied by physicochemical changes such as solute concentrations, pH, reactive oxygenic species, surface tension, and others.
T32 4723-4847 Sentence denotes At the microscale, gradients, local densities, and surface irregularity introduce heterogeneity to MSW environments11,29–31.
T33 4848-4969 Sentence denotes Collectively, MSW imposes severe stresses on microbes therein—including viruses—and affects their survival11,18,20,32,33.
T34 4970-5079 Sentence denotes The current study was motivated by the urgent call for the scientific community to address SARS-CoV-2 spread.
T35 5080-5250 Sentence denotes Like many other viruses, SARS-CoV-2 has been shown to survive well and remain viable on various surfaces, e.g., metal, glass, and plastic, for up to several days19,34–39.
T36 5251-5371 Sentence denotes While not necessarily viable, SARS-CoV-2 RNA has been detected on surfaces in contaminated sites such as hospitals40–43.
T37 5372-5468 Sentence denotes Here, we aim to explore the link between microscopic surface wetness and virus survival therein.
T38 5469-5634 Sentence denotes We focus on two variables that directly affect MSW—solution composition and RH levels—and seek to determine whether and how it is reflected by virus survival trends.
T39 5635-5914 Sentence denotes We use three well-studied bacteriophages previously suggested as good, safe, and easy to work with, as model surrogates for studying surface and air survival of pathogenic viruses44–46: the enveloped Phi6, and two other tailless non-enveloped model bacteriophages as a reference.
T40 5915-6204 Sentence denotes Phi6 is a dsRNA phage of the Cystoviridae family that has been suggested as a good surrogate for studying enveloped RNA viruses including SARS coronaviruses11,14,44,47–49; similar to SARS-CoV-2, it is enveloped by a lipid membrane, has spike proteins, and is of similar size (~ 80–100 nm).
T41 6205-6353 Sentence denotes The other virus strains that we used are the well-studied, non-enveloped MS2 (ssRNA; Leviviridae)45,50–52 and PhiX174 (ssDNA; Microviridae)45,46,53.
T42 6354-6560 Sentence denotes To better understand how RH and the solution composition of microdroplets affect virus survival on surfaces, we combine microscopy imaging to assess MSW state with plaque assays to determine virus survival.
T43 6561-6738 Sentence denotes We compare survival in ‘sprayed’ microdroplets of three suspensions: human saliva, SM buffer, and ‘pure’ water under a range of RH (23–78%) relevant to most indoor environments.
T44 6739-6862 Sentence denotes The link between the microscopic environment of viruses, including hydration conditions, and virus viability, is discussed.
T45 6864-6871 Sentence denotes Results
T46 6872-7116 Sentence denotes To study virus survival in microdroplets deposited on a smooth, inanimate surface, we sprayed Phi6, MS2, and PhiX174 viruses suspended in three aqueous media—human saliva, water, and SM buffer—on glass-bottom 12-well plates (Fig. 1, “Methods”).
T47 7117-7263 Sentence denotes Fluorescent beads (2 µm in diameter) in an equivalent concentration to those of the Phi6 and MS2 viruses (~ 106/mL), were added to the suspension.
T48 7264-7537 Sentence denotes Sprayed microdroplet size ranged between tens to hundreds of microns in diameter (Fig. 2A,B), which falls within the range of respiratory fluid microdroplets exhaled while coughing, speaking, and breathing4,5 and gravitating toward surfaces (i.e., not the < 5 µm aerosols).
T49 7538-7842 Sentence denotes The sprayed well plates were placed under constant temperature and RH conditions (23%, 43%, 57%, 78%) for 14 h, and subsequently microscopy images were taken and virus survival was estimated by the plaque assay using the corresponding bacterial host as a reporter for virus viability (Fig. 1, “Methods”).
T50 7843-7904 Sentence denotes Figure 1 Schematic illustration of the experimental workflow.
T51 7905-8109 Sentence denotes Phage suspended in SM buffer, H2O, and human saliva were applied onto a glass-bottom 12-well plate by a spraying device, resulting in microdroplets ranging in size from ~ 10 microns to ~ 1 mm in diameter.
T52 8110-8325 Sentence denotes Plates were transferred to sealed containers that maintained specific RH conditions (using saturated salt solutions), and the containers were placed in an incubator to maintain constant temperature (25 °C) for 14 h.
T53 8326-8519 Sentence denotes At the end of the incubation period, the surface of the plates was imaged by microscopy, and subsequently the wells were re-suspended and phage concentration was determined by the plaque assay.
T54 8520-8812 Sentence denotes Figure 2 Representative microscopy images of sprayed SM, H2O, and human saliva on a glass surface. (A) typical shape and size of SM buffer, H2O, and saliva immediately after spray application onto a glass surface. (B) Droplet size distribution calculated based on the images shown in Panel A.
T55 8813-9093 Sentence denotes Typical droplet size is between a few tens of microns to hundreds of microns in diameter, which is within the range of expelled saliva microdroplets that are expected to gravitate more rapidly toward surfaces (as opposed to aerosols, which remain suspended in the air for longer).
T56 9094-9244 Sentence denotes To better understand the microscale hydration state that viruses experience, we first examined the surfaces under the microscope 14 h post deposition.
T57 9245-9314 Sentence denotes Representative images of the surface at t = 14 h are shown in Fig. 3.
T58 9315-9384 Sentence denotes The saliva microdroplets dried out rapidly, within minutes (see Supp.
T59 9385-9441 Sentence denotes Video 1), and appeared completely dry at all tested RHs.
T60 9442-9678 Sentence denotes As can be seen in Fig. 3, saliva microdroplets left a thin layer deposit (~ 3–5 µm thickness) of dry matter with aggregated substances (some of these aggregates are salt crystals, but some of them are not, as observed by Vejerano et al.
T61 9679-9683 Sentence denotes 11).
T62 9684-9799 Sentence denotes Beads were dispersed fairly uniformly within these microdroplet deposits, as previously demonstrated for virions11.
T63 9800-10058 Sentence denotes While the beads are at least an order of magnitude larger than individual virions, their visualization can help us grasp the concentration of virions in droplets, and possibly their spatial distribution (assuming lack of virion aggregation—see “Discussion”).
T64 10059-10187 Sentence denotes We estimated ~ 10 virions in a 100-µm droplet (see “Methods”), which is consistent with the observed bead distribution (Fig. 3).
T65 10188-10320 Sentence denotes Figure 3 Microscopy images of microdroplets deposited on the glass-bottom well plates after 14 h incubation at a range of RH levels.
T66 10321-10498 Sentence denotes SM buffer droplets remained hydrated at 57% RH and above, partially hydrated at 43% RH, and completely dry at 23% RH (medium salt crystallization can be seen in dried droplets).
T67 10499-10573 Sentence denotes H2O and saliva droplets appeared to be dry under all tested RH conditions.
T68 10574-10777 Sentence denotes Fluorescent micro-beads (2 µm) (red) were added at a concentration similar to the virus concentration, and thus may help to visualize the expected distribution of viruses within evaporated microdroplets.
T69 10778-10839 Sentence denotes Water microdroplets dried out rapidly at all RH levels (Supp.
T70 10840-10959 Sentence denotes Video 2), as expected, as the water suspension contained very low concentrations of deliquescent solutes (e.g., salts).
T71 10960-11087 Sentence denotes At RH = 78%, some tiny microdroplets (~ few µms in diameter) were observed around single beads or small bead clusters (Fig. 3).
T72 11088-11171 Sentence denotes These tiny water microdroplets were likely retained due to strong capillary forces.
T73 11172-11252 Sentence denotes In contrast, SM buffer microdroplets were hydrated at 57% RH and above (Fig. 3).
T74 11253-11332 Sentence denotes Stable microdroplets of tens of µms in diameter could be clearly seen (Fig. 3).
T75 11333-11429 Sentence denotes At RH = 43%, some of the microdroplets, but not all, dried out, and salt crystals were observed.
T76 11430-11514 Sentence denotes At RH = 23%, all microdroplets dried out, and only salt crystals were seen (Fig. 3).
T77 11515-11633 Sentence denotes These observations can be explained by the efflorescence point of NaCl, the major solute in SM buffer: ~ 45–48% RH 54.
T78 11634-11696 Sentence denotes Next, we estimated virus survival under all tested conditions.
T79 11697-11843 Sentence denotes The content of each well was suspended, and viral viability in the suspensions were evaluated (PFU/mL) by the plaque assay (Fig. 4 and “Methods”).
T80 11844-12100 Sentence denotes Virus viability in sprayed droplets (saliva, water, and SM buffer) was compared with a control made from the same preparation that was kept in sealed tubes throughout the duration of the experiment, and then sprayed after 14 h and immediately re-suspended.
T81 12101-12261 Sentence denotes Strikingly, the highest survival of all tested viruses was found in evaporated saliva microdroplet deposits, compared to the other media at any given RH levels.
T82 12262-12404 Sentence denotes The exception was PhiX174, which had similar levels of survival in saliva and water at 57% RH and saliva and SM buffer at 78% RH respectively.
T83 12405-12537 Sentence denotes Survival in saliva of all three viruses was relatively high, with less than 1.5 log reduction in viability, compared to the control.
T84 12538-12747 Sentence denotes Phi6 survival ranged between a minimum of ≈ 3 × 102 PFU/mL (RH = 57%) to a maximum of ≈ 2 × 104 PFU/mL (RH = 23%) after 14 h in evaporated saliva droplets, compared to ≈ 1 × 104 PFU/mL in the control (Fig. 4).
T85 12748-12869 Sentence denotes The higher survival at RH = 23% might be explained by lower loss in the resuspension after spraying, compared to control.
T86 12870-13085 Sentence denotes Similar to Phi6, PhiX174 survival in saliva droplets was lowest at RH = 57% with ≈ 6 × 102 PFU/mL and highest at RH = 23% (> 1 × 104 PFU/mL), again with higher survival than in the suspended medium (4 × 103 PFU/mL).
T87 13086-13252 Sentence denotes MS2 survival in saliva microdroplets increased with RH, between ≈ 2 × 104 PFU/mL at RH = 23% to ≈ 105 PFU/mL at RH = 78%, compared to 5 × 105 PFU/mL in bulk (Fig. 4).
T88 13253-13360 Sentence denotes The correlation between reduction in viability in saliva (in log10 values) varied between the phages (Supp.
T89 13361-13366 Sentence denotes Figs.
T90 13367-13375 Sentence denotes S1, S2).
T91 13376-13536 Sentence denotes While Phi6 had negative correlation with weak regression correlation (with R2 = 0.52), MS2 showed positive correlation with weak linear correlation (R2 = 0.31).
T92 13537-13670 Sentence denotes PhiX174 showed a non-monotonous U-shaped survival as a function of RH, with close to zero correlation and low R2 values (R2 = 0.008).
T93 13671-13781 Sentence denotes Figure 4 Survival of Phi6, PhiX174, and MS2 respectively as a function of RH and medium type on glass surface.
T94 13782-13793 Sentence denotes Left panel:
T95 13794-13897 Sentence denotes Bacteriophage titer concentration (PFU/mL) after 14 h incubation at 23%, 43%, 57%, and 78% RH at 25 °C.
T96 13898-14019 Sentence denotes A 105–106 titer of bacteriophage suspended in SM buffer, H2O, or human saliva was applied by spraying on a glass surface.
T97 14020-14121 Sentence denotes A control sample was left suspended in the spraying device for the duration of the incubation period.
T98 14122-14277 Sentence denotes Following the incubation period, phage concentration was determined by plaque assay (mean ± SD of four replicates; points represent individual replicates).
T99 14278-14290 Sentence denotes Right panel:
T100 14291-14462 Sentence denotes Relative log reduction of viability of Phi6, PhiX174, and MS2 in SM buffer (green), H2O (red) and saliva (blue) (mean ± SD of four replicates) in evaporated microdroplets.
T101 14463-14584 Sentence denotes Each data point is derived by subtracting the log value of the inoculum from the log value of the virus titer at each RH.
T102 14585-14811 Sentence denotes The fact that the water microdroplets were dry at all RH levels, appears to have a large impact on the enveloped virus Phi6, which showed more than 4 log reduction in viability (Fig. 4), with zero observed plaques at RH ≥ 43%.
T103 14812-14982 Sentence denotes In contrast, the two non-enveloped viruses exhibited lower log reduction in viability of about 1–3 (> few hundreds PFU/mL compared to ≈104 PFU/mL in the control, Fig. 4).
T104 14983-15084 Sentence denotes While PhiX174 survival in evaporated water droplets was linearly correlated with RH (R2 = 0.87, Supp.
T105 15085-15090 Sentence denotes Figs.
T106 15091-15165 Sentence denotes S1, S2), hydration conditions did not show any noticeable change (Fig. 3).
T107 15166-15344 Sentence denotes Overall, PhiX174 showed significantly lower viability in evaporated water microdroplets in comparison to saliva microdroplets, except comparable survival at 57% RH (Fig. 4, Supp.
T108 15345-15354 Sentence denotes Fig. S3).
T109 15355-15452 Sentence denotes MS2 survival in evaporated water microdroplets was significantly lower than that in saliva (Supp.
T110 15453-15507 Sentence denotes Fig. S3), with a U-shaped RH dependence (Fig. 4, Supp.
T111 15508-15517 Sentence denotes Fig. S1).
T112 15518-15744 Sentence denotes In the SM buffer, while microdroplets remained hydrated at 57% and 78% RH (Fig. 3), survival of all three viruses was significantly lower than in saliva under the corresponding RH levels (t-test, P < 0.05, See “Methods”, Supp.
T113 15745-15754 Sentence denotes Fig. S3).
T114 15755-15854 Sentence denotes The only exception was PhiX174, which exhibited similar survival levels in SM and saliva at 78% RH.
T115 15855-16093 Sentence denotes Overall, Phi6, PhiX174, and MS2 in SM buffer deposits showed 3–4 log reduction in viability (reflected by no more than tens to hundreds of PFU/mL) across the tested RH range, exhibiting the familiar U-shaped trend (showing R2 < 0.5, Supp.
T116 16094-16103 Sentence denotes Fig. S1).
T117 16105-16115 Sentence denotes Discussion
T118 16116-16354 Sentence denotes Aiming to obtain some mechanistic insights into factors that determine virus survival in microdroplets deposited on surfaces, we explored a wide range of RH, three types of solutions, and three model viruses (enveloped and non-enveloped).
T119 16355-16563 Sentence denotes Combining surface microscopy imaging and the virus viability assays, we were able to explore the link between the tested variables as manifested with respect to microscopic hydration state and virus survival.
T120 16564-16753 Sentence denotes In summary, we observed significantly higher survival of all three viruses in evaporated saliva microdroplets in comparison to SM buffer and water, 14 h after deposition on a glass surface.
T121 16754-16895 Sentence denotes The only exceptions were for Phix174 at the higher range of tested RH levels, where survival in saliva was comparable to that of other media.
T122 16896-17015 Sentence denotes Phage survival in saliva microdroplets as a function of RH showed different trends (at the studied range of RH levels):
T123 17016-17132 Sentence denotes Phi6 survival decreased with RH, MS2 survival increased with RH, and PhiX174 showed a U-shaped survival-RH relation.
T124 17133-17271 Sentence denotes Our results indicate that RH and hydration conditions are not sufficient to explain virus survival in microdroplets deposited on surfaces.
T125 17272-17410 Sentence denotes For example, the log reduction of Phi6 at 78% RH was ≈ 1, ≈ 3, and ≈ 5 when suspended in saliva, SM buffer, and H2O respectively (Fig. 4).
T126 17411-17552 Sentence denotes This implies that the physicochemical changes that characterize a microdroplet’s drying process have pivotal implications for virus survival.
T127 17553-17615 Sentence denotes Thus, the effect of RH on virus survival is context dependent.
T128 17616-17698 Sentence denotes Likewise, SM droplets were hydrated at 78% RH and completely desiccated at 23% RH.
T129 17699-17802 Sentence denotes Nonetheless, Phi6 survival in SM droplets under these widely differing hydration states was comparable.
T130 17803-17914 Sentence denotes We conclude that water availability and hydration status of a droplet cannot explain virus survival on its own.
T131 17915-18334 Sentence denotes The observation that at a given RH, the microscopic hydration conditions of deposited droplets of various media can differ so widely (see along the rows of Fig. 3) suggests that RH does not directly affect virus stability and viability in drying microdroplets deposited on surfaces, but rather RH indirectly affects survival through its effect on physicochemical conditions at the scale that matters for viruses (~ µm).
T132 18335-18499 Sentence denotes Remarkably, both enveloped and non-enveloped viruses survive well in evaporated human saliva microdroplets deposited on glass surfaces at a wide range of RH levels.
T133 18500-18649 Sentence denotes The observation that saliva droplets were completely dry even at 78% RH was somewhat surprising, as we expected saliva to effloresce at 40–50% RH 54.
T134 18650-18750 Sentence denotes Human saliva is a complex fluid that somehow protects viruses in drying droplets and aerosols 11,12.
T135 18751-18890 Sentence denotes A rough approximation estimates that in our experiments, droplets of ~ 100 microns in diameter contained around 10 virions (see “Methods”).
T136 18891-19029 Sentence denotes Thus, the mass of salts, proteins, and surfactants in these droplets are a few orders of magnitude higher than is the total virion mass11.
T137 19030-19228 Sentence denotes As suggested by Marr et al., association between virion and saliva proteins (or other components) can protect the virions for prolonged periods (even days) in such dried saliva droplets on surfaces.
T138 19229-19401 Sentence denotes In addition, the lower content of inorganic salts in saliva55 than that in SM buffer may explain part of the large differences in virion survival between the two solutions.
T139 19402-19538 Sentence denotes A second remarkable result is the very low survival (> 4 log reduction) of Phi6 in the evaporated water microdroplets, regardless of RH.
T140 19539-19652 Sentence denotes This result may indicate damage associated with the lipid membrane of the enveloped phage under these conditions.
T141 19653-19775 Sentence denotes We note that in the control bulk water solution, survival of Phi6 was high and comparable to that in SM buffer and saliva.
T142 19776-19900 Sentence denotes A possible explanation therefor may be attributed to pH changes that occur during evaporation and drying of microdroplets11.
T143 19901-20013 Sentence denotes The absence of protecting components such as salt crystals or saliva proteins may explain the low survival rate.
T144 20014-20219 Sentence denotes This indicates that the physicochemical properties, experienced by the virions in the SM droplets from time of deposition through evaporation until reaching equilibrium (typically within minutes, see Supp.
T145 20220-20267 Sentence denotes Video 3), affect virus stability and viability.
T146 20268-20419 Sentence denotes The dramatic increase in salt concentrations, and/or the time of exposure to high salt concentrations and reduced pH, likely cause damage to the virus.
T147 20420-20603 Sentence denotes This in turn, suggests that the evaporation kinetics of suspended droplets on surfaces may affect the survival of viruses, in a similar manner as that suggested in aerosol droplets16.
T148 20604-20726 Sentence denotes Thus, complete dryness could in fact protect the virions from those high concentrations of dissolved salts and low pH5,12.
T149 20727-20783 Sentence denotes Another less-understood issue is viral aggregation56,57.
T150 20784-20974 Sentence denotes Spontaneous aggregation of virions, for example as a response to changes in salt concentrations and pH50,56,58, may occur in drying microdroplets, and thus may play a role in virus survival.
T151 20975-21135 Sentence denotes We speculate that with lipid-enveloped viruses suspended in water, aggregation of viruses imposed by hydrophobicity or other mechanisms might play a vital role.
T152 21136-21337 Sentence denotes If significant viral aggregation occurs, it has implications for interpretation of PFU/mL survival estimates (true for any virus survival study), and for the distribution of virions in drying droplets.
T153 21338-21490 Sentence denotes Large virion aggregates (> tens of virions) might also affect microdroplets’ drying dynamics due to capillary pinning, as we have shown for bacteria 28.
T154 21491-21564 Sentence denotes The results of this study provide important insights concerning COVID-19.
T155 21565-21766 Sentence denotes Although performed with a surrogate for SARS-CoV-2, it indicates, as have other studies, that enveloped viruses like SARS-CoV-2 survive well in evaporated saliva droplets on inanimate surfaces19,34–39.
T156 21767-21990 Sentence denotes Because SARS-CoV-2 transmission and disease severity appear to depend upon viral load59, it is likely that in indoor environments where infected individuals stay for long periods, viable viruses persist on fomites for days.
T157 21991-22208 Sentence denotes Thus, as long as not proved otherwise, indirect transmission through inanimate surfaces—in particular those with prolonged and high contact like mobile phones and touch screens—is not unlikely, and must be considered.
T158 22209-22356 Sentence denotes This study provides further evidence that viruses can survive well in evaporated saliva microdroplets for hours after their deposition on surfaces.
T159 22357-22483 Sentence denotes What makes human saliva a favorable environment for virus survival is not well understood and thus calls for further research.
T160 22484-22787 Sentence denotes Interdisciplinary research is required to uncover how the physicochemical microenvironment surrounding the virion affects its survival in saliva microdroplets, and may pinpoint specific components of saliva, such as proteins, polysaccharides, salts or surfactants, that are essential for virus survival.
T161 22789-22796 Sentence denotes Methods
T162 22798-22832 Sentence denotes Bacteria and bacteriophage strains
T163 22833-22989 Sentence denotes Bacteriophages Phi6 (DSM-21518), PhiX174 (DSM-4497), and MS2 (DSM-13767) were purchased from the German Collection of Microorganisms and Cell Cultures DSMZ.
T164 22990-23052 Sentence denotes Vacuum-dried phages were revitalized as per DSMZ instructions.
T165 23053-23079 Sentence denotes Bacteriophage propagation:
T166 23080-23178 Sentence denotes A two-cycle bacteriophage propagation protocol was used to obtain ~ 10 mL titers of > 1010 PFU/mL.
T167 23179-23279 Sentence denotes Overnight bacterial host cultures were diluted at 1:50 into 1 mL fresh media and grown to OD600 0.3.
T168 23280-23399 Sentence denotes A single plaque isolate, suspended in 100 µL SM buffer, was used to infect the fresh culture (first propagation cycle).
T169 23400-23529 Sentence denotes The infected culture was shake incubated at the appropriate temperature for the host strain for 3 h, or until lysis was observed.
T170 23530-23631 Sentence denotes Meanwhile, the overnight host cultures were diluted again in 9 mL fresh media and grown to OD600 0.3.
T171 23632-23804 Sentence denotes 1 mL of the first propagation sample was used to infect 9 mL of fresh host cultures (second propagation cycle) that was shake incubated for 3 h or until lysis was observed.
T172 23805-23927 Sentence denotes The second bacteriophage propagation was centrifuged (4,200RCF, 10 min) and filtered through a 0.22 µm filter (Millex GV).
T173 23928-24021 Sentence denotes Phages were stored at 4 °C and their concentrations were determined by the drop plaque assay.
T174 24022-24169 Sentence denotes Pseudomonas syringae (DSM-21482), a host strain for bacteriophage Phi6, was purchased from DSMZ and cultivated in TSB (Tryptic Soy Broth) at 28 °C.
T175 24170-24317 Sentence denotes Escherichia coli (DSM-13127), a host strain for bacteriophage PhiX174, was purchased from DSMZ and cultivated in LB (Luria–Bertani broth) at 37 °C.
T176 24318-24448 Sentence denotes Escherichia coli (ATCC 15,597) (kindly provided by Adi Stern), a host strain for bacteriophage MS2, was cultivated in LB at 37 °C.
T177 24450-24523 Sentence denotes Viability of bacteriophages in microdroplets deposited on a glass surface
T178 24524-24820 Sentence denotes Phage stock solution (108 PFU/mL for Phi6; and MS2 and 107 PFU/mL for PhiX174) were diluted tenfold with: (1) SM Buffer (100 mM NaCl, 8 mM MgSO4 × 7H2O, 50 mM Tris–Cl pH 7.5, 0.01% w/v gelatin) (2) filter-sterilized H2O (W4502, sigma) and (3) natural human saliva (donated by one of the authors).
T179 24821-24958 Sentence denotes Fluorescent beads (2 µm, melamine resin labeled by rhodamine B, 94,009 Fluka) were added to the suspension with a 5 × 102 final dilution.
T180 24959-25061 Sentence denotes We aimed to have an equivalent concentration of beads to those of the Phi6 and MS2 viruses (~ 106/mL).
T181 25062-25351 Sentence denotes The beads were added for two purposes: (i) to mimic micrometer-sized particulates (e.g., bacteria cells) that are spread on real-world surfaces and have been shown to affect the formation of microscopic surface wetness28; and to (ii) help interpret the virion distribution within droplets.
T182 25352-25540 Sentence denotes Solutions were loaded into 5 mL refillable spray bottles (purchased at a local cosmetics store) and a portion of the load was sprayed on a 12-well glass bottom plate (P12-1.5H-N, Cellvis).
T183 25541-25637 Sentence denotes Each well was sprayed by pressing the spray nozzle twice (delivering a total volume of ~ 50 µL).
T184 25638-25746 Sentence denotes Spray was applied to the well through a 50 mL falcon tube from which we chopped 1.5 cm from its conical end.
T185 25747-25820 Sentence denotes The plates were placed without the cover lid inside a sealed plastic box.
T186 25821-26000 Sentence denotes As this study’s focus is virus survival in the indoor environment, we chose to work at a temperature of 25 °C and a range of RH (23% to 78%), which spans most indoor environments.
T187 26001-26295 Sentence denotes A 100 mL saturated salt solution (potassium acetate 268.6 g/100 mL, 23% RH; potassium carbonate 111 g/100 mL, 43% RH; sodium bromide 94.6 g/100 mL, 57% RH; and ammonium chloride 39.5 g/100 mL, 78% RH) was placed in the bottom of each box to maintain relative humidity of 23%, 43%, 57%, and 78%.
T188 26296-26356 Sentence denotes The boxes were placed in an incubator set at 25 °C for 14 h.
T189 26357-26528 Sentence denotes Control: spray bottles with the remaining (unsprayed) suspensions were left in incubation for 14 h and sprayed onto the 12-well plates at the end of the incubation period.
T190 26529-26663 Sentence denotes All plates were imaged at the end of the 14-h incubation period and then suspended with 500 µL of SM buffer for the drop plaque assay.
T191 26665-26683 Sentence denotes Virus plaque assay
T192 26684-26804 Sentence denotes Plates containing the bottom agar layer were poured in advance (TSB or LB with 15 g/L agar, 5 mM MgSO4, and 5 mM CaCl2).
T193 26805-26961 Sentence denotes On the day of the experiment, overnight bacterial host cultures were diluted at 1:50 into 1 mL fresh media and shake incubated until they reached OD600 0.3.
T194 26962-27086 Sentence denotes Meanwhile, the top agar (TSB or LB with 7.5 g/L agar, 5 mM MgSO4, and 5 mM CaCl2) was melted and kept in a 55 °C water bath.
T195 27087-27203 Sentence denotes The bacterial host culture was combined with the top agar at a ratio of 1:40, and poured on top of the bottom layer.
T196 27204-27436 Sentence denotes The phages were serially diluted in SM buffer, and after the top agar solidified, either 10 µL, 100 µL, or 1000 µL were pipetted and spread out onto either one quarter, one half, or a full plate (10 × 10 cm petri dish) respectively.
T197 27437-27566 Sentence denotes Plates were left open until dry, and incubated at the appropriate temperature for the host strain (as described above) overnight.
T198 27568-27578 Sentence denotes Microscopy
T199 27579-27682 Sentence denotes 12-well plates were mounted on a stage top without warming (room temperature) during image acquisition.
T200 27683-27877 Sentence denotes Images were collected using an Eclipse Ti-E inverted epi-fluorescence microscope (Nikon) equipped with a Plan Apo 20x/0.75 NA air objective and the Perfect Focus System for maintenance of focus.
T201 27878-27958 Sentence denotes An LED light source (SOLA SE II, Lumencor) was used for fluorescence excitation.
T202 27959-28140 Sentence denotes Rhodamine B-marked fluorescent beads were excited through a 560/40 filter, and emission was collected with a T585lpxr dichroic mirror and 630/75 filter (filter cube #49008, Chroma).
T203 28141-28289 Sentence denotes Images were acquired with a SCMOS camera (ZYLA 4.2PLUS, Andor Technology, Ltd.) controlled with NIS Elements 5.02 software (Nikon Instruments Inc.).
T204 28290-28394 Sentence denotes 10 × 10 adjacent fields of views (covering a total area of 2.82 × 2.82 mm) were monitored per each well.
T205 28395-28510 Sentence denotes Multiple stage positions were collected using a motorized encoded scanning stage (SCANplus IM 130 × 85, Märzhäuse).
T206 28512-28549 Sentence denotes Statistical analyses and calculations
T207 28551-28571 Sentence denotes Statistical analyses
T208 28572-28800 Sentence denotes To investigate the relationships between the dependent variable of virus viability (in log10 transformed values) and the independent variable of RH, we fitted statistical models in MATALB using ordinary least-squares regression.
T209 28801-28929 Sentence denotes We evaluated linear correlations based on R2, F-tests, and P-values for statistical significance of the linear models (see Supp.
T210 28930-29022 Sentence denotes Fig S1), and Correlation Coefficients based on Pearson correlation coefficients r (see Supp.
T211 29023-29031 Sentence denotes Fig S2).
T212 29032-29213 Sentence denotes Statistical significance of the differences in viability reductions between saliva and the other two aqueous media, under equal RH levels, was assessed using Welch t-test (see Supp.
T213 29214-29222 Sentence denotes Fig S3).
T214 29223-29269 Sentence denotes Statistical analyses were performed in MATLAB.
T215 29271-29323 Sentence denotes Estimation of the number of viruses per microdroplet
T216 29324-29378 Sentence denotes The initial concentration of viruses was ~ 106 per mL.
T217 29379-29478 Sentence denotes The original volume of a droplet that left a 100-µm diameter deposition was estimated at ~ 0.01 nL.
T218 29479-29639 Sentence denotes Assuming there is no significant virus aggregation and that viruses are uniformly distributed, we obtain ~ 106 per mL × 10–5 = 10 virions per such microdroplet.
T219 29641-29666 Sentence denotes Supplementary Information
T220 29668-29690 Sentence denotes Supplementary Video 1.
T221 29691-29713 Sentence denotes Supplementary Video 2.
T222 29714-29736 Sentence denotes Supplementary Video 3.
T223 29737-29765 Sentence denotes Supplementary Information 1.
T224 29767-29783 Sentence denotes Publisher's note
T225 29784-29902 Sentence denotes Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
T226 29904-29929 Sentence denotes Supplementary Information
T227 29930-30021 Sentence denotes The online version contains supplementary material available at 10.1038/s41598-020-79625-z.
T228 30023-30039 Sentence denotes Acknowledgements
T229 30040-30111 Sentence denotes We thank Adi Stern for kindly providing the host E. coli strain of MS2.
T230 30112-30114 Sentence denotes N.
T231 30115-30167 Sentence denotes K. is supported by research grants from the James S.
T232 30168-30302 Sentence denotes McDonnell Foundation (Studying Complex Systems Scholar Award, Grant #220020475) and from the Israel Science Foundation (ISF #1396/19).
T233 30303-30383 Sentence denotes This study was partially supported by the Edmond de Rothschild Foundation (to N.
T234 30384-30388 Sentence denotes K.).
T235 30390-30410 Sentence denotes Author contributions
T236 30411-30452 Sentence denotes A.F., T.O., and N.K. conceived the study.
T237 30453-30488 Sentence denotes A.F and T.O. performed experiments.
T238 30489-30530 Sentence denotes M.G. and T.O. performed image processing.
T239 30531-30576 Sentence denotes A.F., T.O., and M.G. performed data analyses.
T240 30577-30651 Sentence denotes All authors discussed the results and contributed to the final manuscript.
T241 30652-30680 Sentence denotes N.K. supervised the project.
T242 30681-30723 Sentence denotes A.F., T.O., and N.K. wrote the manuscript.
T243 30725-30742 Sentence denotes Data availability
T244 30743-30979 Sentence denotes The datasets generated during and/or analyzed during the current study are available in the Figshare repository at https://figshare.com/articles/Virus_survival_in_evaporated_saliva_microdroplets_deposited_on_inanimate_surfaces/12546563.
T245 30981-31000 Sentence denotes Competing interests
T246 31001-31044 Sentence denotes The authors declare no competing interests.