Effect of elevated temperature on SARS-CoV-2 viability

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide disruption of global health putting healthcare workers at high risk. To reduce the transmission of SARS-CoV-2, in particular during treating the patients, our team aims to develop an optimized isolation chamber. The present study was conducted to evaluate the role of temperature elevation against SARS-CoV-2 viability, where the information would be used to build the isolation chamber. 0.6 mL of the Indonesian isolate of SARS-CoV-2 strain 20201012747 (approximately 10 13 PFU/mL) was incubated for one hour with a variation of temperatures: 25, 30, 35, 40, 45, 50, 55, 60, and 65°C in digital block heater as well as at room temperature (21-23°C) before used to infect Vero E6 cells. The viability was determined using a plaque assay. Our data found a significant reduction of the viral viability from 10 13 PFU/mL to 10 9 PFU/mL after the room temperature was increase to 40°C. Further elevation revealed that 55°C and above resulted in the total elimination of the viral viability. Increasing the temperature 40°C to reduce the SARS-CoV-2 survival could create mild hyperthermia conditions in a patient which could act as a thermotherapy. In addition, according to our findings, thermal sterilization of the vacant isolation chamber could be conducted by increasing the temperature to 55°C. In conclusion, elevating the temperature of the isolation chamber could be one of the main variables for developing an optimized isolation chamber for COVID-19 patients.


Introduction
Coronavirus disease 2019 (COVID- 19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has inflicted disruptions in many aspects of health systems globally.SARS-CoV-2 is an enveloped, non-segmented, positive sense, single-stranded RNA virus with a genome of approximately 30 kilobases. 1,2The virus is mainly transmitted by nasopharyngeal droplets of an infected person; however, it could also be transmitted through aerosol. 3The viability of the virus in the environment is influenced by several factors, including climatic parameters such as humidity and temperature. 4Temperature has been proposed as a factor that affects SARS-CoV-2 transmission. 57][8] A study in State of Rio de Janeiro, Brazil found that the maximum and average of temperature were correlated negatively with COVID-19 infection. 6Data from 117 countries also found a negative association between temperature and COVID-19 transmissibility in which an increase of 1°C could decrease COVID-19 prevalence by approximately 5.4%. 7Several other investigations, however, have shown no evidence of a substantial influence of temperature on SARS-CoV-2 transmission. 9,10vertheless data reveals that SARS-CoV-2 is highly susceptible to heat. 11A recent study has shown that the virus could survive for at least 14 days at 4°C while only two days at 37°C and five minutes at 70°C. 11Another study suggested that, at 40°C SARS-CoV-2-infected epithelial cells have reduced viral transcription and replication. 12Although studies on the effect of elevated temperature on SARS-CoV-2 have been carried out, the temperature ranges used are limited. 13In addition, the effect of temperature on viruses originating from Indonesia has not yet been published.As part of our project to optimize the isolation chamber for COVID-19 patients, we determined the effect of temperature on the resistance of SARS-CoV-2 originating from Indonesia by evaluating the viral viability with a range of temperatures from room temperature (21-23°C) to 65°C.Understanding viral survivability is critical for developing a temperature optimized isolation chamber that could minimize the risk of infection to healthcare workers and optimize energy consumption while ensuring comfort for patients.In addition, the information of this study might important for those who are living in the tropics.

SARS-CoV-2
SARS-CoV-2 strain 20201012747 isolated from Jakarta, Indonesia was used in this study.The virus originated from a patient with severe COVID-19 manifestation.The virus was kindly supplied by the Eijkman Institute for Molecular Biology.Before used in this study, the virus has been passaged twice on Vero E6 cells (ECACC, Vero C1008) (RRID: CVCL_0574).The virus was classified as an ancestral SARS-CoV-2 strain and isolated on 12 October 2020.

Exposure of temperatures
To expose to different temperatures, 0.6 mL of SARS-CoV-2 stock in 1.5 mL sterile tubes were incubated at room temperature RT (21-23°C), 25, 30, 35, 40, 45, 50, 55, 60, or 65°C for 1 hour in a digital block heater (Cat.no.5382000031, Eppendorf, Germany).A separated experiment was conducted for each temperature; three replicates were used for each temperature and each replicate was repeated three times.

REVISED Amendments from Version 1
In this new version, we have added the information that this research is useful for people living in the tropics in the Introduction section.We have added more important information in the Methods section such as: (a) the description for virus propagation; and (b) information related to the SARS-CoV-2 used in this study such as the origin and the lineage.In Discussion section we have removed the hypothesis that lifting the ambient temperature around an infected patient for one hour to 40°C might reduce the risk of infection to health care workers.We have added more limitations of our study including: (a) we did not assess the role of other factors that might influence the SARS-CoV-2 transmission such as humidity; and (b) the sensitivity of the virus to temperature was tested in a liquid state only and we did not assess the aerosol state.
Any further responses from the reviewers can be found at the end of the article

Plaque assay
After the incubation at different temperatures, the viruses were diluted with a 10-fold serial dilution with 2% MEM (10 -1 to 10 -12 ) and 0.1 mL was inoculated onto 95-100% confluent monolayers of Vero E6 cells for 1 hour at 37°C with 5% CO 2 with manual gentle shaking every 20 mins.After 1 hour of incubation, each well was then covered with 1 mL of 2% carboxymethyl cellulose (Cat.no.17854-1KG, Merck, Germany) containing MEM, 2% FBS (Cat.no.26140095), 3.5 mM Na 2 CO 3 (Cat.no.25080094), 25 mM HEPES (Cat.no.DMEM 15630080), 1% non-essential amino acid (Cat.no.11140050), and 1% penicillin-streptomycin-amphotericin B (Cat. no.15240062); All from Gibco (Thermo Fisher Scientific, MA, USA).The plates were incubated in cell incubator for 72 hours at 37°C and 5% CO 2 .The cells were then fixed with 4% paraformaldehyde for 4 hours at room temperature, and stained with 1% crystal violet.The plaques were counted manually.All works with infectious SARS-CoV-2 were conducted in the Biosafety Level 3 Laboratory at the Eijkman Institute for Molecular Biology in Jakarta.The plaque forming unit (PFU/mL) was calculated by dividing the number of plaques by dilution factor.

Results
The virus was observed to remain stable from RT to 35°C with an average plaque count of 10 13 PFU/mL. 23A reduction in the viral count was observed in the 40°C treatment group at 10 9 PFU/mL.Increasing the temperature to 45°C resulted in a further reduction of the viral viability resulting in 10 6 PFU/mL.The last temperature with visible plaque was in the 50°C treatment group with a result of 10 2 PFU/mL.The reduction of SARS-CoV-2 viability had a temperature-dependent trend within the temperature range of 35 to 50°C (Figure 1).No plaques were visible in the 55, 60 and 65°C treatment groups.

Discussion
Nosocomial transmission of SARS-CoV-2 has been identified to occur via multiple routes in healthcare facilities 14 indicating that uncomplicated measures like wearing personal protective equipment along with surface cleaning and decontamination 15 could be used effectively to reduce the transmission of infection.Other than that, modification to facilities such as, including isolation chambers with temperature control could help minimize the transmission. 11,16As noted in a previous study, 11 temperature affects the stability of the virus in aerosol or on a surface.When the temperature of the room is not elevated, SARS-CoV-2 could remain stable for up to 72 hours on a surface such as plastic and stainless steel and three hours in aerosols. 16Based on the findings of this present study, to reduce the viral viability significantly, a higher room temperature might be important.However, according to a previous report, the average maximum temperature for Indonesian people to still feel comfortable falls between 24 and 29°C 17 and to increase the room temperature would probably cause discomfort to the patient.
The administration of heat to the isolation chamber could be conducted prior to patient handling to reduce the likelihood of SARS-CoV-2 transmission.The other possibility is using heat in the isolation chamber as a means of thermotherapy.Thermotherapy is where mild-temperature elevation or hyperthermia (39-42°C) is used as a treatment against SARS-CoV-2 infection. 180][21] Following the increase in temperature, heat-shock proteins (HSPs) are released which downregulates the progression of sepsis-induced acute lung injury. 22However, HSPs could become hosts to several viruses (such as human papillomavirus, adenovirus, and dengue virus) promoting their infectivity. 23In the case of SARS-CoV-2, its infectivity is more likely to be degraded than promoted by the HSPs. 18Therefore, heat administration to the isolation chamber should not be performed on COVID-19 patients with human papillomavirus, adenovirus, or dengue virus co-infections.In addition, it should not be attempted on patients with severe-to-critical COVID-19 as they would be more likely to have an increased risk of mortality following the thermotherapy or heat administration. 24e of the limitations of this study was we did not assess the role of other factors that might influence the SARS-CoV-2 transmission such as humidity.Therefore, the results of our study should be incorporated with data of the other studies assessing the effects of humidity on SARS-CoV-2 viability.In addition, we assessed the effect of the temperature on SARS-CoV-2 in a liquid state only and this might have influenced the results.Assessing the effect of the temperature on different states might could provide better understanding.

Conclusions
This present study also has proven that increasing indoor temperature to 55°C is sufficient to terminate the virus.Further increment to the temperature would not be necessary and only results in higher energy consumption.Similarly, a previous study also reported the inactivation of 90% of SARS-CoV-2 achieved at 54.5°C after 36 minutes. 13However, for use in treatment 55°C, might be too high for patients to tolerate.In that case, we only suggest the use of such temperature to thermally sterilize the isolation chamber prior to its use.
reduce the risk of transmission to the healthcare worker.
On one hand, patient continuously shed virus to the environment.Provided that respiratory droplets and aerosols are the major routes of transmission, pre-heating the chamber for one hour before the healthcare worker coming in will have minimal effect on the continuous droplet and aerosol generation.If the purpose is to reduce the risk of fomite transmission, simple surface disinfection will be more efficient and safer.The authors provided evidence by other groups that report negative association between air temperature and COVID-19 transmission in different countries.However, these findings are controversial.Many other studies found no evidence on higher temperature will have negative impact on COVID-19 transmission and some even found the reverse.
On the other hand, it may not be desirable to place a patient in an undesirable environment at 40 degree C, not to mention the increased risk of heat related illness, like heat exhaust or heat stroke, which can be lethal.I think this needs to be considered very carefully.In addition, it is mentioned in the abstract that increasing the environmental temperature to 40 degree C will create mild hyperthermia condition in patient and reduce SARS-CoV-2 survival.However, there is no evidence to support this claim.The viability of virus in culture medium without present of any cell may not be the same as in the respiratory track covered with epithelial cells at the same temperature.
There are other flaws in this manuscript including the experimental design and data presentation.The viability of the virus was only studied in medium.Although the authors mentioned this as a limitation of the study, assessing of the viral stability in droplet / aerosol exposed to hot air would be more relevant and essential for coming to the current conclusion.Plaque assay is fine for determining the viral titre of infectious virus.However, the information on limit of detection of the assay is missing.No plaque observed in the assay does not necessarily mean that the viral titre is zero.The input control without one hour incubation was also missing, so one has no idea about the initial titre.Statistical analysis was also missing.

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility?No source data required

Are the conclusions drawn adequately supported by the results? No
The methods were adequate to answer the research question.Choosing the plaque assay for determination of infective virus particles is sufficient.Using PCR would add no further information.Any description for virus propagation is missing.As the process and purity might have an influence on the results, I would add that information.Temperature steps were chosen in 5 °C intervals.I wondered why more physiological temperature were not also chosen, such as 31°C, 34°C…(as representatives in tracheal temperature when breathing e.g.20°C warm air).This was recently addressed by https://doi.org/10.1093/infdis/jiac264.The sensitivity of the virus to temperature was only tested in a liquid state.The question about the stability as virus aerosol remained unanswered.It is a pity that the authors did not compare different virus strains.They focused only on SARS-CoV-2 strain 20201012747 isolated from Jakarta, Indonesia.I found no information about the Statistics used.I also missed some recent literature found by literature search.
In general, I was not really convinced by the hypothesis that lifting the ambient temperature around an infected patient for one hour to 40 degree would substantially reduce the risk of infection to health care workers.As infection progresses, any exhalation of patient' breath would increase the virus load in the air again.It is also necessary to understand what breathing of 40 °C warm air means for the air temperature in the trachea and in the alveoli as it could be reduced to core body temperature again -meaning that it remains without any effect if we think of using that for treatment of patients.Other methods for lifting body temperature might be more effective.
It is hard to say whether the information has great importance for others as the information provided is not substantially new.Other researchers might be interested in reading the study, in particular researchers focusing on transmission of SARS-CoV-2.Overall, this is a very small publication showing little results.These results are not outstanding, in terms of the content of the results per se and not in terms of the methods/techniques used and also not in terms of the effort involved, which might also justify indexing.Reviewer Expertise: Expertise in in vitro / ex vivo infection research, focus on respiratory diseases.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Introduction
Add that this research is useful for people living in the tropics.

Discussion
Temperature is not the sole cause of disease breading, so in this section it is necessary to add research limitations, such as humidity factor which are not included in this research study.

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It is advisable to add research related to indoor temperature.

Conclusions
"This present study also has proven that increasing temperature" -> Indoor temperature

References 1 .
Minor point: M&M part: please change from two times to twice ○ Stauft CB, Sangare K, Wang TT: Differences in New Variant of Concern Replication at Physiological Temperatures In Vitro.J Infect Dis.2022.PubMed Abstract | Publisher Full TextIs the work clearly and accurately presented and does it cite the current literature?NoIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?No source data requiredAre the conclusions drawn adequately supported by the results?NoCompeting Interests: No competing interests were disclosed.

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Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes