key: cord-0982830-9m7qbk71
authors: Rockey, N.; Arts, P. J.; Li, L.; Harrison, K. R.; Langenfeld, K.; Fitzsimmons, W. J.; Lauring, A. S.; Love, N. G.; Kaye, K. S.; Raskin, L.; Roberts, W. W.; Hegarty, B.; Wigginton, K. R.
title: Humidity and deposition solution play a critical role in virus inactivation by heat treatment on N95 respirators
date: 2020-06-23
journal: nan
DOI: 10.1101/2020.06.22.20137448
sha: f236d525744527069eaace8f04d6d769479d3d42
doc_id: 982830
cord_uid: 9m7qbk71

Supply shortages of N95 respirators during the coronavirus disease 2019 (COVID-19) pandemic have motivated institutions to develop feasible and effective N95 respirator reuse strategies. In particular, heat decontamination is a treatment method that scales well and can be implemented in settings with variable or limited resources. Prior studies using multiple inactivation methods, however, have often focused on a single virus under narrowly defined conditions, making it difficult to develop guiding principles for inactivating emerging or difficult-to-culture viruses. We systematically explored how temperature, humidity, and virus deposition solutions impact the inactivation of viruses deposited and dried on N95 respirator coupons. We exposed four virus surrogates across a range of structures and phylogenies, including two bacteriophages (MS2 and phi6), a mouse coronavirus (murine hepatitis virus, MHV), and a recombinant human influenza A virus subtype H3N2 (IAV), to heat treatment for 30 minutes in multiple deposition solutions across several temperatures and relative humidities (RH). We observed that elevated RH was essential for effective heat inactivation of all four viruses tested. For heat treatments between 72{degrees}C and 82{degrees}C, RH greater than 50% resulted in > 6-log10 inactivation of bacteriophages and RH greater than 25% resulted in > 3.5-log10 inactivation of MHV and IAV. Furthermore, deposition of viruses in host cell culture media greatly enhanced virus inactivation by heat and humidity compared to other deposition solutions such as phosphate buffered saline, phosphate buffered saline with bovine serum albumin, and human saliva. Past and future heat treatment methods or technologies must therefore explicitly account for deposition solutions as a factor that will strongly influence observed virus inactivation rates. Overall, our data set can inform the design and validation of effective heat-based decontamination strategies for N95 respirators and other porous surfaces, especially for emerging or low-titer viruses that may be of immediate public health concern such as SARS-CoV-2.

Effective decontamination of medical equipment is critical for controlling infectious 64 diseases in clinical settings. This is heightened during pandemics, when shortages of personal 65 protective equipment (PPE), such as N95 respirators, lead to occupational risks for healthcare 66 workers. During the coronavirus disease 2019 (COVID-19) pandemic, high demand for N95 67 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. 

Arguably the simplest and most accessible approach to N95 respirator decontamination is 77 to harness the biocidal activity of heat and moisture. Treating medical equipment with 78 pressurized saturated steam in autoclaves for one hour, for example, leads to high levels of virus 79 inactivation (6-9); however, the high temperatures and pressures in autoclave sterilizers affect 80 N95 respirator integrity (10, 11). In contrast, N95 respirator treatments at lower temperatures, up 81 to 80-90°C for an hour or longer, do not affect filter performance and fit (12) (13) (14) . To date, studies 82 assessing virus inactivation on N95 respirators at elevated temperatures below 100 °C have 83 included limited viruses and conditions. Influenza viruses heated to 65°C with 85% relative 84 humidity (RH) for 20-30 minutes resulted in greater than 3-log10 inactivation (15, 16) . Dry heat at 85 70°C for 1 hour led to > 3-log10 inactivation of severe acute respiratory syndrome coronavirus 2 86 (SARS-CoV-2) (2). In cases where heat treatment is not a feasible decontamination approach, the 87 CDC recommends limited reuse of N95 respirators after incubation in paper bags at room 88 temperature for an excess of five days (17) . The justification for this recommendation is based on 89 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.22.20137448 doi: medRxiv preprint 5 experiments evaluating stability of SARS-CoV-2 on surfaces (18); however, the efficacy of this 90 practice on N95 respirators has yet to be validated. 91 Previous virus inactivation studies that focus on other types of surfaces can inform N95 92 respirator decontamination strategies. Such studies suggest that both temperature and humidity 93 can affect virus inactivation in droplets dried on porous or nonporous surfaces (19 CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 23, 2020. is in the same genus as SARS-CoV-2 and is thus expected to exhibit a similar fate outside of its 134 host compared to SARS-CoV-2.

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 23, 2020. (Tables S6 to S9 ). For all four viruses, inactivation was lowest at 1% RH at 147 both 72°C and 82°C (Figure 1 ). For treatments with RHs above 25% for 72°C and above 13% 148 for 82°C, inactivation was beyond the detection limits of all viruses. Consequently, we were 149 unable to observe the real inactivation trends at elevated RHs for any of the viruses tested. The 150 dynamic ranges for inactivation varied greatly among the four viruses. Specifically, the dynamic 151 ranges for inactivation were between 5.5-log10 and 8.1-log10 for the bacteriophages, ~4.0-log10 152 for IAV, and ~3.7-log10 for MHV.

An increase in treatment temperature from 72°C to 82°C resulted in a 1.3-log10 and 2.0-154 log10 average increase in inactivation for MS2 and phi6, respectively, across different RHs 155 ( Figure 1 ). This trend of greater inactivation at 82°C compared to 72°C was consistent across 156 nearly all RH conditions for both viruses, though increases were not statistically significant for 157 all settings (Table S10) . Likewise, for IAV and MHV at 1% RH, the average inactivation 158 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.22.20137448 doi: medRxiv preprint 8 increase was 1.1-log10 as temperature increased from 72°C to 82°C (Figure 1 ). The influence of 159 temperature on inactivation was not calculated for IAV and MHV above 1% RH as assay respectively, across all temperatures within assay limits (Table S11 ). MS2, for example, 166 underwent an average of 1.4-log10 inactivation at 1% RH, and a 4.9-log10 inactivation at 13%.

MHV and IAV inactivation increased by 1.2-log10 and 1.3-log10, respectively, from 1% to 13% 168 RH at 72°C.

No consistent trend in inactivation was observed between mammalian viruses and the 170 bacteriophages for treatment conditions within assay limits. For example, the four viruses 171 demonstrated similar inactivation levels at 1% RH at both 72°C and 82°C, but IAV and MHV 172 were inactivated less than the bacteriophages at 72°C and 13% RH (Table S12 ). The small 173 dynamic range of IAV and MHV limited our ability to determine strong inactivation trends for 174 these viruses.

The MHV and IAV media used in the above experiments consisted of Dulbecco's 176 modified Eagle's medium (DMEM) with different supplementary ingredients (Table S4) .

Control experiments with phi6 and MS2 deposited in both of these media types and treated at 178 72°C and 13% RH demonstrated that similar inactivation was experienced in either DMEM 179 medium composition ( Figure S2 ).

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The copyright holder for this preprint this version posted June 23, 2020. (Table S14 ). The difference in 197 inactivation between MS2 and phi6 was less pronounced when viruses were deposited in IAV 198 medium. There, phi6 was inactivated, on average, 0.6-log10 more than MS2. We note that 199 differences between phi6 and MS2 inactivation were not due to differences in their respective 200 stock solutions, because the two viruses were deposited in a single mixed stock solution.

To determine if protein content in IAV contributed to the observed differences in virus 202 inactivation between IAV medium and PBS, we tested MS2 and phi6 inactivation with a PBS 203 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.22.20137448 doi: medRxiv preprint deposition solution that was supplemented with the same amount of BSA as was present in the 204 IAV medium. Bacteriophage inactivation in PBS + BSA deposition solution was significantly 205 less than in IAV medium for both MS2 and phi6 across nearly all conditions tested (Figure 3 , 206 Table S13), with average reductions of 3.0-log10 and 3.7-log10 for MS2 and phi6, respectively.

With respect to the specific effect of BSA in the deposition solution, MS2 was inactivated 1.3-208 log10 more when the PBS deposition solution contained BSA. Two out of four of the 209 temperature/RH conditions exhibited statistically significant differences (Table S13 ). For phi6, (Table S13) . Overall, these results showed that virus 213 inactivation in PBS + BSA was more similar to inactivation in PBS than in IAV medium for phi6 214 and MS2.

To better represent virus-containing droplets present on N95 respirators, we tested MS2 216 and phi6 inactivation in freshly collected human saliva sterilized using UV treatment. Phage 217 inactivation in saliva was within 0.7 log10 of inactivation in PBS, on average ( Figure 3 ).

Inactivation of the two viruses deposited in IAV medium was significantly greater than 219 inactivation when deposited in saliva across nearly all treatments (3.6-log10 and 3.5-log10 greater 220 for MS2 and phi6, respectively; Table S13 ). On average, inactivation levels of MS2 deposited in 221 saliva were 0.5-log10 larger than inactivation levels in PBS ( Figure 3 ). For phi6, inactivation in 222 saliva was 0.8-log10 less than in PBS ( Figure 3 ). These results indicate that deposition in saliva is 223 more similar to deposition in PBS than in IAV medium.

Virus inactivation at ambient conditions. To assess whether virus inactivation at 225 ambient conditions was also affected by deposition solution, we tested bacteriophage inactivation 226 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.22.20137448 doi: medRxiv preprint at room temperature (20°C) and 36% RH using different deposition matrices. This RH and 227 temperature were selected at the lower end of standard thermohygrometric ranges for healthcare 228 facilities (37). After 24 hours, we observed relatively low levels of inactivation (< 2 log10 on 229 average) for both MS2 and phi6 in all deposition matrices ( Figure 4 ). MS2 inactivation in IAV 230 medium was significantly higher than in either PBS (p = 0.0061) or saliva (p = 0.029). Although 231 the average inactivation was higher for phi6 deposited in IAV medium than phi6 in PBS and 232 saliva, the differences were not statistically significant. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. Figure S3 ). In other words, the improved inactivation observed for 82°C compared to 72°C was 261 driven more by the added water content than by the temperature. In several studies that have 262 taken a modeling approach, absolute humidity was deemed a better predictor of IAV inactivation 263 than temperature and RH at both elevated temperatures (26) and ambient temperature ranges ( CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. several studies where diverse viruses have been dried on surfaces and exposed to 4-40°C, 275 increasing RH from 20-30% to 50-80% also increased inactivation (19, 22, 25) . Other studies 276 have observed the opposite trend when viruses were dried on surfaces (25) MS2 under nearly all conditions tested. Previous research on viruses dried on surfaces and 314 exposed to room temperature also found increased persistence of non-enveloped viruses 315 compared to enveloped viruses (48, 49). As viruses dry on surfaces, it has been suggested that the 316 air water interface may damage the lipid membranes of enveloped viruses (19, 30) and the 317 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.22.20137448 doi: medRxiv preprint increased salt concentration can cause the lipid membrane to become rigid (50). Moreover, some 318 studies observed that increased salt concentrations can protect non-enveloped viruses (30, 32).

We did not observe that the enveloped mammalian IAV and MHV viruses were 320 consistently more susceptible to inactivation than the non-enveloped MS2 (Figure 1 ). In fact,

MHV was less susceptible than both bacteriophages to heat treatment at 72C and 13% RH when 322 deposited in culture media (Figure 1 ). We note that our dynamic ranges for MHV and IAV were when deposited in their culture media relative to PBS (Figures 2 and 3) . At room temperature, 340 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. . https://doi.org/10.1101/2020.06.22.20137448 doi: medRxiv preprint only MS2 was inactivated to a greater extent when deposited in its culture medium (Figure 4) . 341 There has been limited prior work comparing virus survival in PBS versus culture media. In 342 contrast to our results, Yang et al observed that IAV deposited in culture media supplemented 343 with fetal calf serum exhibited less inactivation compared to viruses deposited in PBS when 344 exposed to room temperature and RH from 20 to 60% (30). This discrepancy may be due to the 345 fact that the deposition solution was allowed to dry before heat treatment in our study, whereas (Tables S2 and S3) . 354 Proteins in the deposition solution could have a protective effect on viruses (29, 30). Our results, 355 however, show that inactivation with viruses deposited in PBS + BSA was significantly less than 356 when applied in IAV medium, which contains BSA at the same levels ( Figure 4) . Therefore, 357 protein content also does not appear to explain the observed differences. Another possible 358 explanation is that the L-glutamine present in IAV medium degrades into glutamate and 359 ammonia (52, 53). Ammonia is known to cause virus inactivation in solution (54), although 360 further work is needed to test this hypothesis in dried droplets and at elevated temperatures. 

Although the deposition solution was not explicitly stated in this study, inactivation of SARS-

CoV-2 could have been significantly overestimated if a culture medium was used for deposition.

This is of critical importance for clinical settings, because these results may lead healthcare 385 workers to disinfect respirators at 70°C for 60 minutes in an oven without controlled humidity 386 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. (58) . The MS2 and phi6 experimental stocks were combined, 432 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 23, 2020. (Table S2) , each used in a subset of experiments, included 1X PBS (Table S3) , IAV 469 medium, with the exception that no trypsin was added (Table S4) , MHV medium (Table S4) , 470 PBS with 0.1875% BSA (PBS + BSA), or human saliva. For each saliva experiment, fresh saliva 471 was collected from a volunteer and UV treated to sterilize (59, 60). Volunteers did not eat within 472 2 hours prior to collection and rinsed their mouth with water ten minutes before collection (61, 473 62). Saliva was collected in two wells of a 12-well plate in a thin layer. The saliva was 474 immediately treated for five minutes using a custom-built collimated beam equipped with 0.16 475 mW cm -2 UV254 lamps (model G15T8, Philips). Lamp intensity was measured using chemical 476 actinometry (63, 64) . MHV (~10 6 PFU/ml) in MHV media (Table S4 ) and IAV (~10 7 TCID50/ml) 477 in IAV media (Table S4) were used for droplet deposition.

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The copyright holder for this preprint this version posted June 23, 2020. immobilized on a coated metal test tube rack with metal binder clips ( Figure S1 ). The metal rack 497 with coupons was then transferred to the oven at the predetermined temperature and humidity 498 settings. Treatment times were started when the oven RH was within 1% of the target RH for 13 499 -71% RH and within 5% of target RH for 1% and 89% RH. The oven reached these conditions 500 within five minutes. Heat decontamination experiments with phi6 and MS2 deposited in PBS 501 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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