key: cord-0817736-p1yzzcy2
authors: Jiang, Yuqian; Zhang, Han; Wippold, Jose A.; Gupta, Jyotsana; Dai, Jing; de Figueiredo, Paul; Leibowitz, Julian L.; Han, Arum
title: Sub-second heat inactivation of coronavirus
date: 2020-10-06
journal: bioRxiv
DOI: 10.1101/2020.10.05.327528
sha: e4cf1196bfaca2dc4e9f77571b9943fa38bca474
doc_id: 817736
cord_uid: p1yzzcy2

Heat treatment denatures viral proteins that comprise the virion, making virus incapable of infecting a host. Coronavirus (CoV) virions contain single-stranded RNA genomes with a lipid envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a temperature of as low as 75 °C and treatment duration of 15 min can effectively inactivate CoV. The applicability of a CoV heat inactivation method greatly depends on the length of time of a heat treatment and the temperature needed to inactivate the virus. With the goal of finding conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple system that can measure sub-second heat inactivation of CoV. The system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil bath followed by an ice bath, through which virus solution can be flowed at various speeds. Flowing virus solution at different speeds, along with a real-time temperature monitoring system, allows the virus to be accurately exposed to a desired temperature for various durations of time. Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 85.2 °C for 0.48 s exposure is sufficient to obtain > 5 Log10 reduction in viral titer (starting titer: 5 × 107 PFU/mL), and that when exposed to 83.4 °C for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log10 reduction). IMPORTANCE Three coronaviruses (CoVs) have now caused global outbreaks within the past 20 years, with the COVID19 pandemic caused by SARS-CoV-2 still ongoing. Methods that can rapidly inactivate viruses, especially CoVs, can play critical roles in ensuring public safety and safeguarding personal health. Heat treatment of viruses to inactive them can be an efficient and inexpensive method, with the potential to be incorporated into various human-occupied spaces. In this work, a simple system that can heat-treat viruses for extremely short period was developed and utilized to show that sub-second exposure of CoV to heat is sufficient to inactivate CoV. This opens up the possibility of developing instruments and methods of disinfecting CoV in diverse settings, including rapid liquid disinfection and airborne virus disinfection. The developed method can also be broadly utilized to assess heat sensitivity of viruses other viral pathogens of interest and develop sub-second rapid heat inactivation approaches.

envelope and 4 proteins, 3 of which are associated with the lipid envelope and thus are thought to 23 be easily denatured by heat or surfactant-type chemicals. Prior studies have shown that a 24 temperature of as low as 75 ℃ and treatment duration of 15 min can effectively inactivate CoV. 25 The applicability of a CoV heat inactivation method greatly depends on the length of time of a 26 heat treatment and the temperature needed to inactivate the virus. With the goal of finding 27 conditions where sub-second heat exposure of CoV can sufficiently inactivate CoV, we designed 28 and developed a simple system that can measure sub-second heat inactivation of CoV. The 29 system is composed of capillary stainless-steel tubing immersed in a temperature-controlled oil 30 bath followed by an ice bath, through which virus solution can be flowed at various speeds.

Flowing virus solution at different speeds, along with a real-time temperature monitoring system, 32 allows the virus to be accurately exposed to a desired temperature for various durations of time. 33 Using mouse hepatitis virus (MHV), a beta-coronavirus, as a model system, we identified that 34 85.2 ℃ for 0.48 s exposure is sufficient to obtain > 5 Log 10 reduction in viral titer (starting titer: 35 5 × 10 7 PFU/mL), and that when exposed to 83.4 ℃ for 0.95 s, the virus was completely 36 inactivated (zero titer, > 6 Log 10 reduction).

Three coronaviruses (CoVs) have now caused global outbreaks within the past 20 years, with the 39 COVID19 pandemic caused by SARS-CoV-2 still ongoing. Methods that can rapidly inactivate 40 viruses, especially CoVs, can play critical roles in ensuring public safety and safeguarding 41 personal health. Heat treatment of viruses to inactive them can be an efficient and inexpensive method, with the potential to be incorporated into various human-occupied spaces. In this work, 43 a simple system that can heat-treat viruses for extremely short period was developed and utilized 44 to show that sub-second exposure of CoV to heat is sufficient to inactivate CoV. This opens up 45 the possibility of developing instruments and methods of disinfecting CoV in diverse settings, 46 including rapid liquid disinfection and airborne virus disinfection. The developed method can 47 also be broadly utilized to assess heat sensitivity of viruses other viral pathogens of interest and 48 develop sub-second rapid heat inactivation approaches. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the 55 currently ongoing global pandemic, the coronavirus disease of 2019 (COVID-19) (1-3). The 56 main transmission routes of SARS-CoV-2 include direct or indirect contact with objects or 57 contaminated surfaces, short-range person-to-person transmission via droplets from coughing or 58 sneezing, and long-range airborne transmission via aerosols (4). Therefore, environmental 59 sterilization and virus inactivation are of great importance to prevent and control the spread of 60 the virus. Currently, the most commonly used methods to sterilize or inactivate viruses include 61 treatment using chemical agents, UV irradiation exposure, and heat treatment, which have all 62 been intensively assessed and reported (5-10). Compared with other methods, one major 63 advantage of heat treatment is its relatively shorter treatment time and simplistic method, along 64 with the ability to be incorporated into various human-occupied space (11-13), which allows for 65 the technique to be readily implemented into a variety of existing applications or systems that 66 could be readily retrofitted to add rapid pathogen inactivation functionality, such as existing 67 heating, ventilation, and air conditioning (HVAC) systems as well as sewer systems.

Heat inactivation is a relatively easy, safe, and efficient method to disinfect coronavirus (CoV), 69 as CoV is an enveloped virus that is surrounded by a lipid bilayer with viral spike proteins 70 projecting from the lipid envelope, where both the envelope and the spike protein are susceptible 71 to heat (14) . Previous studies have shown that at a temperature of 56 ℃ and higher, with heat 72 application time typically longer than 1 min, is needed to efficiently inactivate CoVs such as 73 SARS-CoV and MERS-CoV (> 6 Log 10 reduction) (5, 6, 13). More specifically, at relatively low 74 treatment temperatures (56 -65 ℃), treatment time of 15 -60 min was required, while at higher 75 treatment temperatures (70 -100 ℃) a much shorter duration of 1 to 15 min was needed (5, 6, 13, (15) (16) (17) . For example, heat treatment of SARS-CoV-2 at 70 ℃ for 5 min achieved > 4.5 Log 10 77 reduction (15), with another study reported that heat treatment at 92 ℃ for 15 min achieved > 6 78 Log 10 reduction for SARS-CoV-2 (5). However, for heat treatment to be effectively utilized for 79 liquid and airborne CoV inactivation in broad ranges of practical settings, such methods need to 80 be effective at a significantly shorter heat treatment time (even if the temperature itself has to be 81 higher), otherwise there is limited practicality in such heat treatment methods. For example, 82 having to increase the temperature of liquid for minutes would consume large amount of energy, 83 and having to treat air for minutes becomes impractical.

Here, we hypothesize that a much shorter heat treatment time may be sufficient to destroy key 85 components of CoVs (e.g., envelope or the spike proteins) to completely inactivate CoV.

Conventional heat treatment testing method mostly utilizes a simple method of dipping a CoV-87 containing tube into a temperature-controlled water bath. Such methods are valid when heat immersed in an oil bath and then in an ice bath sequentially. A relatively small inner diameter 120 tubing was selected to minimize the volume of solution so that the virus solution can be rapidly heated and cooled down, and SS was utilized to maximize heat conduction from the exterior to 122 the interior of the tubing. Since a short pulse of high-temperature application was desired to 123 accurately assess the impact of the temperature on viral infectivity, an ice bath was utilized to 124 rapidly cool down the heated viral solution. A temperature-controlled oil bath (Instatherm ®

Economy Bath/Controller Kit, Ace Glass, Inc., VWR, USA) equipped with a type J 126 thermocouple temperature sensor was employed to control the heat treatment temperature. Table S1 . A viral solution flowing through the tubing while the oil bath 145 temperature was set to room temperature (22 ℃) was used as a control to account for any 146 potential viral titer reduction due to virus adhering to the tubing surfaces and other potential 147 losses. The simulation was performed using non-isothermal flow (nitf) multi-physical interfaces speeds and its temperature was measured. This measured temperature inside the SS tubing (2 cm 199 away from the heated water bath) was then compared to that of the bulk solution temperature. 

As reported, an average viral load of SARS-CoV-2 is 7 × 10 6 per milliliter (26) could not be sufficiently inactivated even when the oil bath temperature was increased to 170 ℃.

Therefore, the remaining infectivity after heat treatments comparing different conditions was re-268 plotted by the actual thermal treatment temperature and duration, and is shown in Fig. 3 . Among We studied the thermal treatment conditions to efficiently inactivate a coronavirus and developed 292 a system and protocol that can inactivate infectibility of MHV. We successfully developed an 293 the temperature applied to the CoV solution can be monitored in real time. Through experimental 295 measurement and computational thermal simulation, we validated the real temperature that the 296 CoV solutions are exposed to. Using this setup, we identified that 85.2 ℃ for 0.48 s exposure is 297 sufficient to obtain > 5 Log 10 reduction in viral titer (starting titer: 5 × 10 7 PFU/mL), and that 298 when exposed to 83.4 ℃ for 0.95 s, the virus was completely inactivated (zero titer, > 6 Log 10 299 reduction). This is the first experimental result that shows that sub-second exposure to high 300 temperature is sufficient to inactive the infectibility of CoV. Since heat treatment is a simple, 301 inexpensive, and efficient approach to inactivate coronaviruses, our method can be used to study 302 the thermal sensitivity of viruses, as well as providing critical data that can be used to develop 303 efficient CoV heat inactivation methods that can be broadly applied to real-world settings. 

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