key: cord-0751891-fqv35hvx
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 using a betacoronavirus model
date: 2021-03-03
journal: Biotechnol Bioeng
DOI: 10.1002/bit.27720
sha: 35939098fa40b3419c473a4393c58cfb0cfa147b
doc_id: 751891
cord_uid: fqv35hvx

Heat treatment denatures viral proteins that comprise the virion, making the virus incapable of infecting a host. Coronavirus (CoV) virions contain single‐stranded RNA genomes with a lipid envelope and four proteins, three 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 as low as 75°C with a treatment duration of 15 min can effectively inactivate CoV. The degree of CoV heat inactivation greatly depends on the length of heat treatment time and the temperature applied. With the goal of finding whether sub‐second heat exposure of CoV can sufficiently inactivate CoV, we designed and developed a simple fluidic system that can measure sub‐second heat inactivation of CoV. The system is composed of a stainless‐steel capillary immersed in a temperature‐controlled oil bath followed by an ice bath, through which virus solution can flow at various speeds. Flowing virus solution at different speeds, along with temperature control and monitoring system, allows the virus to be exposed to the desired temperature and treatment durations with high accuracy. Using mouse hepatitis virus, a betacoronavirus, as a model CoV system, we identified that 71.8°C for 0.51 s exposure is sufficient to obtain >5 Log(10) reduction in viral titer (starting titer: 5 × 10(7) PFU/ml), and that when exposed to 83.4°C for 1.03 s, the virus was completely inactivated (>6 Log(10) reduction).

. Compared with other methods, one major advantage of heat treatment is its relatively shorter treatment time and simplistic method, along with the ability to be incorporated into various human-occupied spaces (Batéjat et al., 2021; Kariwa et al., 2006; Yap et al., 2020) . These features allow for such heat treatment methods to be readily implemented into a variety of existing applications or systems that could be retrofitted to add rapid pathogen inactivation functionality, such as to existing 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), as CoV is an enveloped virus that is surrounded by a lipid bilayer with viral spike proteins projecting from the lipid envelope, where both the envelope and the spike protein are susceptible to heat (Schoeman & Fielding, 2019) . Previous studies have shown that at a temperature of 56°C and higher, with heat application time typically longer than 1 min, is needed to efficiently inactivate CoVs such as SARS-CoV and MERS-CoV (>6 Log 10 reduction; Darnell et al., 2004; Pastorino et al., 2020; Yap et al., 2020) . More specifically, at relatively low temperatures (56-65°C), treatment time of 15-60 min were required, while at higher temperatures (70-100°C) a much shorter duration of 1-15 min were needed (Chin et al., 2020; Darnell et al., 2004; Leclercq et al., 2014; Pastorino et al., 2020; Saknimit et al., 1988; Yap et al., 2020) . For example, heat treatment of SARS-CoV-2 at 70°C for 5 min achieved >4.5 Log 10 reduction (Chin et al., 2020) , with another study reported that heat treatment at 92°C for 15 min achieved >6 Log 10 reduction for SARS-CoV-2 (Pastorino et al., 2020) . However, for heat treatment to be utilized for liquid and airborne CoV inactivation in broad ranges of practical settings, such methods need to be applicable at a significantly shorter heat treatment time (even if the temperature itself has to be much higher). Otherwise, there is limited practicality in such heat treatment methods. For example, having to increase the temperature of liquid for minutes would consume a large amount of energy, and having to treat air for minutes is impractical.

Here, we hypothesize that a much shorter heat treatment time may be sufficient to destroy key components of CoVs (e.g., envelope or the spike proteins) to inactivate CoV. Conventional testing methods for the heat treatment of CoVs mostly utilize a simple method of dipping a CoVcontaining tube into a temperature-controlled water bath. Such methods are valid when heat treatment time in the range of minutes is tested but cannot be used for seconds or sub-second testing. In this study, we developed a simple flow-through heating and cooling method utilizing a stainless-steel capillary tube and used the method to investigate the effect of CoV heat treatment at an extremely short heat exposure time of 0.1-1 s (equivalent to actual treatment time of 0.18-2.30 s, obtained through heat transfer simulation) at an actually applied temperature range of 35-100°C. This study provides essential data for the development of sub-second CoV heat inactivation approaches, including methods to efficiently inactivate airborne CoVs indoors. As all CoVs are surrounded by lipid bilayer membranes with similar proteins and have similar physical properties (Schoeman & Fielding, 2019) , we expect that our findings using mouse hepatitis virus (MHV), a betacoronavirus, as a model system that has extensively used as a surrogate coronavirus (Casanova et al., 2009; Hulkower et al., 2011; Körner et al., 2020; Ye et al., 2016) , can be broadly applicable to CoVs in general, including SARS-CoV, SARS-CoV-2, and MERS-CoV, to name a few. One of the potential applications of our findings is to heat inactivate airborne viruses by renovating a ventilation system, and another promising application is to disinfect the sewer system by scaling up the presented system and optimizing the system design. A previous study showed that the time needed for SARS-CoV-2 inactivation can be reduced to 5 min when the treatment temperature is 70°C (Chin et al., 2020) . Taking consideration of our findings, if a filter in an HVAC system can be heated to a high temperature, SARS-CoV-2 in the circulating air can be efficiently killed rapidly. Here, the needed temperature to completely (or mostly) inactivate the viruses can be determined based on our data. Since many other viruses, such as dengue virus, influenza virus, and measles virus, are also enveloped viruses where the envelopes contain surface proteins (Gelderblom, 1996) , we expect that this heat inactivation method can have broad utility in inactivating/disinfecting many other viruses of global consequences.

The coronavirus used in this study is mouse hepatitis virus strain A59 (MHV-A59) and has been described previously (Bond et al., 1979; Wippold et al., 2020) . Mouse L2 cells, which are susceptible to MHV infection, were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4 mM glutamine and 10% defined calf serum (Hyclone) at 37°C 5% CO 2 environment (Sturman & Takemoto, 1972) .

Plaque assays were conducted as described previously, and viral titer quantified at 2 days postinfection after removing the agarose overlay and staining the cells with crystal violet (Leibowitz et al., 2011) .

Samples were assayed in triplicate and the number of plaques was counted. Infectivity was determined and expressed as plaqueforming units (PFUs) per ml. was bent and immersed in an oil bath and then in an ice bath sequentially. A relatively small inner diameter tubing was selected to minimize the volume of the solution so that the virus solution can be rapidly heated and cooled down. SS was utilized to maximize heat conduction from the exterior to the interior of the tubing. Since a short pulse of the high-temperature application was desired to accurately assess the impact of the temperature on viral infectivity, an ice bath was utilized to rapidly cool down the heated viral solution. A temperature-controlled oil bath (Instatherm® Economy Bath/Controller Kit, Ace Glass, Inc., VWR) equipped with a Type J thermocouple temperature sensor was employed to control the heat treatment temperature. Vegetable cooking oil (Member's mark, Sam's Club) was used in the oil bath. A mixture of ice and water was used as the ice bath to cool down the treated virus solution immediately after treatment. A syringe pump (Fusion 200, Chemyx) was employed to control the flow rate of the injected virus solution.

Silicone tubing was used to connect the syringe to the inlet of the SS tubing and also to collect the heat-treated samples from the outlet of the SS tubing. Low-thermal-conducting silicone tubing was utilized to further limit the temperature exposure to only a short section of the overall tubing. The collected virus samples were stored at −80°C until plaque assays were conducted to measure the infectivity of the heat-treated viruses.

Stock MHV viruses of 2.7 × 10 9 PFU/ml were diluted by DMEM media with 10% FBS to 5 × 10 7 PFU/ml, a high-enough titer to ac- Table S1 . A viral solution flowing through the tubing while the oil bath temperature was set to room temperature (22°C) was used as a control to account for any potential viral titer reduction due to virus adhering to the tubing surfaces and other potential losses.

The COMSOL Multiphysics 5.5a software (COMSOL Inc.) was used for the temperature profile simulation of the viral solution.

To perform this finite element analysis, a 3D geometry was chosen, where the geometry was created using the following initial and boundary conditions (Zhang et al., 2019 (Zhang et al., , 2020 : no-slip conditions on the tubing surfaces; the fluid is Newtonian and the flow within the channel is incompressible; no viscous stress and convective flux on the tubing outlet; convective heat flux is considered as the source of heat influx from the oil to the tubing.

The simulation was performed using non-isothermal flow (nitf) multi-physical interfaces laminar flow (spf) coupled with heat transfer in solids and fluids (ht) under the stationary study model.

The initial inlet flow temperature and ambient air temperature were set to 22°C. The material physical properties of viral solution were set to be identical to those of water. The material properties of the SS tubing were set to: density = 7850 kg/m 3 ; thermal conductivity = 16.2 W/(m K); heat capacity at constant pressure = 500 J/(kg K). Here, we assume the overall heat loss by the oil bath is equal to the overall heat gain of water flowing by (the heat loss to ambient air is deemed negligible). Then, the temperaturedependent heat transfer coefficient (H) was calculated based on Equation (1) (Bergman et al., 2011; Shashi Menon, 2015) and the real-time temperature measurement (see Section 2.5 for more details). The calculated H value is 473.6 W/(m 2 K) under the thermal treatment condition of 125°C (oil bath temperature) for 0.5 s (based on the length of merged SS tubing and set flow rate), while the H value increased to 675.5 W/(m 2 K) when the treatment condition is 170°C for 0.1 s. The H values obtained in this study are close to previously published results (Kobasko et al., 2010) .

Physics-controlled mesh with the element size of "finer" was applied for the simulation. A mesh refinement testing using the typical condition of 125°C and 0.5 s exposure time was conducted ( Figure S1 ). The mesh convergence test result (see Figure S1 for more details) indicates the mesh is refined enough to obtain a solution that can be trusted (the relative error is 2%-3% when compared to the "finer" mesh result with the threshold value, where the convergent value at "extremely fine" mesh condition was used as threshold value). where A: surface area where the heat transfer takes place;

T oil : temperature of the surrounding oil;

T ss : temperature of the solid surface;

C w : heat capacity of water;

Q w : flow rate of water;

ρ: density of water;

T in : water temperature at inlet; and

T out : water temperature at outlet. Figure S2b ), which were then used as the actual temperatures virus solution was exposed to.

The In this laminar flow regime, we anticipate a temperature gradient along the radial direction of the pipe. At steady state, the temperature gradient does not change with time, but is a function of the location along the axial direction. Figure 2a shows the temperature change of flowing virus solution within the SS tubing when the oil bath temperature was set to 125°C and a moderate exposure time of 0.5 s (flow rate = 92,300 µl/h) was used. Here, the SS tubing temperature increases rapidly as it enters the oil bath (pink zone), then gradually drops when the tubing is exposed to air (yellow zone), and then rapidly drops as it enters the ice bath (blue zone). The reason that the SS temperature inside the oil bath (pink zone) is not the same as the oil bath temperature is due to the cooling effect of room temperature virus solution continuously flowing into the tubing, especially since the length of the tubing immersed inside the oil bath was relatively short (5 cm). Using a much longer tubing inside the oil bath would have eventually made the SS tubing temperature the same as the oil bath temperature, but that makes it impossible to apply a short heat exposure, the reason why a relatively short tubing length was utilized.

The virus solution temperature follows the SS tubing temperature relatively closely. The highest solution temperature of 85.9°C was achieved right as it comes out from the oil bath (this highest temperature is referred to as the simulated temperature (Max) in Table S1 ) and maintains its temperature relatively stable (temperature drop of only 6.5°C over a 10 cm length), until arriving at the segment of the SS tubing immersed in the ice bath. Therefore, the effective heat treatment region was designated from the temperature point within the oil bath (pink zone) where the temperature rises above that of the pre-ice bath temperature (the ending temperature of the yellow zone) to the pre-ice bath temperature. The "actual exposure time" was calculated based on this "effective heat treatment region" (the length is slightly longer than 10.0 cm). For example, in the case of 125°C 0.5 s heat treatment condition, this "actual exposure time" is around two times longer than the set exposure time, which in this case was 1.03 s. 

An average viral load of SARS-CoV-2 is 7 × 10 6 per ml (Stadnytskyi et al., 2020) , therefore we chose 5 × 10 7 PFU/ml of MHV, which is a slightly higher concentration and would also provide a rigorous test for the rapid heat inactivation system presented here. Our study investigated the effect of rapid heat treatment (<1 s) on coronavirus inactivation by flowing the MHV virus solution through a stainless tubing immersed in an oil bath, which temperature was controlled from 55 to 170°C. We found that the coronavirus was inactivated F I G U R E 2 The simulated temperature distribution of the entire heat inactivation testing system when using the oil bath to apply heat. The top color bar represents the axial cross-sectional SS tubing, and traverse sectional views of positions i-iv are displayed in the right circles. However, when the set exposure time was reduced to 0.1 s, MHV could not be sufficiently inactivated even when the oil bath set temperature was increased to 170°C (Figure 3a) .

These results were then re-plotted based on the actual treatment temperature and exposure time, as shown in Figure 3b . As discussed in Section 3.2, the actual exposure time calculated was around two times longer than the set exposure time. For visualization purposes, the actual exposure times were rounded to 2, 1, 0.5, and 0.2 s (Table S1 , the temperature ranges for the rounding were When these results were plotted into a 2D format ( Figure S3b ), the correlation between the viral inactivation and actual exposure temperature showed that heat treatment temperature as high as around 70°C is required to effectively provide sub-second heat inactivation (>5 Log 10 reduction) regardless of the actual treatment time. 

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