key: cord-268034-7id7sfsu
authors: Auerswald, Heidi; Yann, Sokhoun; Dul, Sokha; In, Saraden; Dussart, Philippe; Martin, Nicholas J.; Karlsson, Erik A.; Garcia-Rivera, Jose A.
title: Assessment of Inactivation Procedures for SARS-CoV-2
date: 2020-05-28
journal: bioRxiv
DOI: 10.1101/2020.05.28.120444
sha: 
doc_id: 268034
cord_uid: 7id7sfsu

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus disease 2019 (COVID-19), presents a challenge to laboratorians and healthcare workers around the world. Handling of biological samples from individuals infected with the SARS-CoV-2 virus requires strict biosafety and biosecurity measures. Within the laboratory, non-propagative work with samples containing the virus requires, at minimum, Biosafety Level-2 (BSL-2) techniques and facilities. Therefore, handling of SARS-CoV-2 samples remains a major concern in areas and conditions where biosafety and biosecurity for specimen handling is difficult to maintain, such as in rural laboratories or austere field testing sites. Inactivation through physical or chemical means can reduce the risk of handling live virus and increase testing ability worldwide. Herein we assess several chemical and physical inactivation techniques employed against SARS-CoV-2 isolates from Cambodian COVID-19 patients. This data demonstrates that all chemical (AVL, inactivating sample buffer and formaldehyde) and heat treatment (56°C and 98°C) methods tested completely inactivated viral loads of up to 5 log10.

To determine if any viable virus remained post inactivation, 50% Polyethylene glycol 8000 133 (Sigma-Aldrich, St. Louis, USA) in PBS was added (1/5 of total sample volume) to an aliquot 134 from each sample condition and incubated overnight at 4°C. Following incubation, virus was 135 recovered by centrifugation at 1,500 rpm for 1h. Precipitates were washed twice with sterile PBS, 136 re-constituted with infection medium, and used for infecting the TCID50 on Vero E6 cells and 137 recovery cultures on Vero cells. Negative controls were treated the same way to examine 138 cytotoxicity of possible remaining traces of inactivation solutions. 139 140 SARS-CoV-2 real-time RT-PCR 141

Following inactivation, RNA from one aliquot per condition per virus isolate and negative control 142 was immediately extracted with the QIAamp Viral RNA Mini Kit (Qiagen) and stored at -80°C 143 until further processing. Real-time RT-PCR assays for SARS-CoV-2 RNA detection were 144 performed in duplicate using the Charité Virologie algorithm (Berlin, Germany) to detect both E 145 and RdRp genes [9] . In brief, real-time RT-PCR was performed using the SuperScript™ III One-146

Step RT-PCR System with Platinum™ Taq Inc., La Jolla, CA,, USA). Analysis of variance was performed comparing mean Ct values for each 157 inactivation method. Difference between standard (AVL) and each specific inactivation method 158 was determined using Dunnett's test for many-to-one comparison. A p-value of less than 0.05 was 159 considered to indicate statistical significance. Agreement, including bias and 95% confidence interval, between Ct values following inactivation by AVL and other methods was assessed using 

All chemical and thermal inactivation methods resulted in the reduction of viable SARS-CoV-2 to 186 undetectable levels. Untreated virus isolates had a concentration of viable virus up to 6.67 x 10 5 187 (isolate 2310) before treatment (Table 1) Previous studies have been conducted on the effectiveness of chemical inactivation 220 techniques on SARS-CoV-2 [11, 12] , the majority of these based on infectious agents of concern 221 such as Ebola [13] and SARS and MERS coronaviruses [14] . As with other viruses, the primary 222 step in the molecular detection of SARS-CoV-2 is viral lysis to begin the extraction of nucleic 223 acids. The buffers used in this lysis step yield varying results [11, 13, 15, 16] ; however, unlike 224 previous studies [11] , this study found that AVL buffer alone was successfully able to fully 225 inactivate up to 5 log10 of virus from three different primary isolates of SARS-CoV-2. Apart from 226 differences in isolates utilized and a slight reduction in titer, it is unclear as to the reasons why 227 AVL buffer fully inactivated in this study versus others, but further work is warranted to determine 228 the exact effectiveness of this step alone.

Inactivating sample transport media, either made in-house or commercially available, also 230 presents an attractive way to inactivate samples at the point of sampling to ensure safe handling 231 along the transport chain and within the laboratory. These inactivating transport media include the 232 key components of many viral lysis buffers including chaotropic agents (GITC), detergents (Triton 233 X-100) and buffering agents (EDTA, Tris-HCL) to inactivate a preserve viral RNA. Previous 234 studies have shown that GITC-lysis buffers are able to inactivate SARS-CoV-2 samples [11, 12] ; 235 however, the addition of Triton-X may be necessary for complete inactivation [11] . In line with 236 these studies, commercial sample transport media containing both GITC and Triton-X was 237 successfully able to inactivate up to 5 log10 of virus with no loss of molecular diagnostic sensitivity. 238

Apart from sample media and buffers utilized for diagnostic testing, various disinfectant 239 and inactivating chemicals are available for sample treatment. Formaldehyde has a long history of 240 use for inactivation against a number of viruses and in a number of fixation techniques, including 241 vaccine preparations [17, 18] . Formaldehyde has been shown to successfully inactivate both SARS 242 and MERS [14, 19, 20] and has been suggested to be a viable alternative for disinfection and 243 inactivation of 20] . Formaldehyde treatment did successfully inactive up to 5 244 log10 of virus; however, this treatment severely impacted viral detection in subsequent molecular 245 testing. This decreased detection is not unexpected as formaldehyde treatment results in RNA 246 degradation and modification [21] . Therefore, formaldehyde treatment does not appear to be a 247 solution for increased molecular SARS-CoV-2 testing; however, it does remain a viable alternative 248 for sample inactivation or disinfection. 249

Perhaps the most studied technique thus far regarding SARS-CoV-2 has been thermal 250 inactivation at various times and temperatures [11, [22] [23] [24] . Several previous studies have shown 251 heat to be an effective inactivation technique against other coronaviruses, including SARS, MERS, and human seasonal strains [14, 23, 25] . Similar to previous studies, 56 o C heat treatment for 30 or 253 60 minutes was fully able to inactivate up to 5 log10 of SARS-CoV-2 from three different isolates 254 [11, 22] . Interestingly, while other studies utilized 95 o C for 5 to 10 minutes for inactivation, heat 255 treatment at 98 o C for only 2 minutes was also able to completely inactivate up to 5 log10 of virus. 256

These results are very promising as high heat treatment is extremely rapid and may be a vital 257 addition to the testing arsenal, as RT-PCR can possibly be performed directly from these samples 258 without the need for nucleic acid extraction [26, 27] . Interestingly, the shortened time period of 259 high heat treatment may mitigate some of the reduction in detection seen in previous studies and 260 make this technique more employable [11] . 261

Overall, the agreement and retained sensitivity amongst RT-PCR results, combined with 262 the fact that all methods resulted in 100% virus inactivation up to a viral load of 5 log10, suggests 263 that any of the tested methods, except formaldehyde, are useful to inactivate SARS-CoV-2 264 samples. Given the WHO recommendation to "test, test, test," these data can help to optimize 265 sample inactivation for austere or remote areas. Indeed, it may be possible to use basic tools such 266 as a stopwatch and boiling water to achieve 100% virus inactivation without compromising sample 267 integrity, significantly decreasing possible exposure during sample transportation and handling, 268 allowing for dissemination of testing to labs with decreased biosafety and biosecurity capacity, 

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