key: cord-0776418-7vj5x1nv
authors: Welch, Stephen R.; Davies, Katherine A.; Buczkowski, Hubert; Hettiarachchi, Nipunadi; Green, Nicole; Arnold, Ulrike; Jones, Matthew; Hannah, Matthew J.; Evans, Reah; Burton, Christopher; Burton, Jane E.; Guiver, Malcolm; Cane, Patricia A.; Woodford, Neil; Bruce, Christine B.; Roberts, Allen D. G.; Killip, Marian J.
title: Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives
date: 2020-07-10
journal: bioRxiv
DOI: 10.1101/2020.07.08.194613
sha: fc0dd268287d891e6edc915886d2ec574249c81b
doc_id: 776418
cord_uid: 7vj5x1nv

The COVID-19 pandemic has necessitated a rapid multi-faceted response by the scientific community, bringing researchers, health officials and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.

Infection with the novel human betacoronavirus SARS-CoV-2 can cause a severe or fatal 44 respiratory disease, termed COVID-19 (1-3). As the COVID-19 pandemic has developed, 45 millions of clinical samples have been collected for diagnostic evaluation. SARS-CoV-2 has 46 been classified as a Hazard Group 3 pathogen in the UK, and as such, deliberate work with the virus must be carried out in high containment laboratories (containment level 3 (CL3) in the UK) × g for two mins. For Amicon Ultra filters, 500µl of sample was added, centrifuged at 14,000 × g 150 for 10 mins, followed by three washes with 500µl PBS. Sample was then collected by 151 resuspending contents of the filtration device with 500µl PBS. To assess remaining cytotoxicity, instructions. Normalized values of absorbance (relative to untreated cells) were used to fit a 4-For commercial products, virus preparations (tissue culture fluid, titers ranging from 1 × 163 10 6 to 1 × 10 8 PFU/ml) were treated in triplicate with reagents at concentrations and for contact 164 times recommended in the manufacturers' instructions for use, where available, or for 165 concentrations and times specifically requested by testing laboratories. Where a range of 166 concentrations was given by the manufacturer, the lowest ratio of product to sample was tested 167 (i.e. lowest recommended concentration of test product). Specimen transport tube reagents were 168 tested using a ratio of one volume of tissue culture fluid to ten volumes of reagent, unless a 169 volume ratio of sample fluid to reagent was specified by the manufacturer. Detergents, fixatives 170 and solvents were tested at the indicated concentrations for the indicated times. For testing of 171 alternative sample types, virus was spiked into the indicated sample matrix at a ratio of in Ct across the course of a passage were observed. Table 1 shows the dilution 239 factor of reagent-treated sample required to achieve the CC20 after filtration, with <1 indicating 240 complete removal of cytotoxicity. These data were used to determine the relative cytotoxicity 241 removed by one filtration step for each combination of reagent and matrix ( Figure 1A) .

All unfiltered reagents tested here were cytotoxic, but the degree of cytotoxicity varied 243 considerably as did the optimal filtration matrix for each reagent. remaining cytotoxicity would be removed by first or second (10 -1 -10 -2 ) dilutions in the TCID50 257 assay allowing evaluation of titer reduction using these reagents with the caveat that the effective 258 assay limit of detection (LOD) would be higher. Passing treated samples through more than one 259 column, or increasing the depth of the resin/bead bed within the spin column can also improve 260 cytotoxicity removal for some reagents (unpublished data).

In addition to cytotoxicity removal, a successful filtration method must also purify virus 262 without adversely affecting titer or integrity. We therefore assessed SARS-CoV-2 recovery after 263 each filtration method. Using an input titer of 1.35 × 10 6 TCID50/mL, triplicate purifications of 264 virus through Sephadex LH-20 or Pierce detergent removal spin columns resulted in recovery of 265 100% of input virus ( Figure 1B ). In contrast, the recoverable titer after one filtration through 266 Amicon Ultra filters was 2.13 × 10 5 TCID50/mL, an 85% reduction from input. Purification with 267 S400HR and Bio-Beads SM2 matrices resulted in recoverable titers of 1.08 × 10 6 TCID50/mL 268 and 8.99 × 10 5 TCID50/mL, a loss of 30% and 35% of input virus, respectively. passage. 285 We also sought to inform sample processing by examining inactivation by molecular 286 extraction lysis buffers used in several manual and automated extraction protocols within SARS-

CoV-2 diagnostic and research laboratories. We could demonstrate a ≥4 log10 reduction in 288 TCID50 titer for all but two molecular extraction reagents when evaluated using tissue culture 289 fluid ( show that at both 0.1% and 0.5% v/v concentration, virus titers in tissue culture fluid were 319 reduced by ≥4.9 log10 TCID50s, even with less than 2 min contact time (Table 4) . Furthermore, 320 we were unable to recover infectious virus from samples treated with 0.5% Triton X-100 for 10 321 mins or longer. We also saw effective inactivation of SARS-CoV-2 by SDS, NP40 and Triton X-322 100 in spiked NP and OP swab specimen fluid, but, importantly, we were not able to replicate 323 this in spiked serum; 1% Triton X-100 only reduced titers in human serum by a maximum of 2 324 log10 TCID50s with contact times of up to two hours.

In addition to evaluating inactivation efficacy by detergents, we assessed the effects of 326 treatment on RNA integrity to determine their suitability for inactivation prior to nucleic acid 327 testing. Extracted RNA from treated samples was tested using a SARS-CoV-2-specific qRT-328 PCR, and the Ct difference between detergent-treated samples and mock-treated controls 329 determined (Table 4) TCID50s at the highest concentration tested (2%).

Samples containing infectious SARS-CoV-2 require an initial inactivation step before 353 downstream processing; given the rapid emergence of SARS-CoV-2, these inactivation protocols inactivation studies for all viruses (known and novel), not only SARS-CoV-2. We have applied 406 these methods to obtain SARS-CoV-2 inactivation data for a wide range of reagents in use (or 407 proposed for use) in SARS-CoV-2 diagnostic and research laboratories. In addition to guiding 408 laboratory risk assessments, this information enables laboratories to assess alternative reagents 

Molecular Extraction Reagents Buffer

Other Formaldehyde