key: cord-1003128-z3xd9sgx
authors: Anderson, Enyia R.; Hughes, Grant L.; Patterson, Edward I.
title: Inactivation of SARS-CoV-2 on surfaces and in solution with Virusend (TX-10), a novel disinfectant
date: 2020-11-27
journal: bioRxiv
DOI: 10.1101/2020.11.25.394288
sha: ef51c212c304e9690df04188d2e62554ad325a8a
doc_id: 1003128
cord_uid: z3xd9sgx

Until an effective vaccine against SARS-CoV-2 is available on a widespread scale, the control of the COVID-19 pandemic is reliant upon effective pandemic control measures. The ability of SARS-CoV-2 to remain viable on surfaces and in aerosols, means indirect contact transmission can occur and so there is an opportunity to reduce transmission using effective disinfectants in public and communal spaces. Virusend (TX-10), a novel disinfectant, has been developed as a highly effective disinfectant against a range of microbial agents. Here we investigate the ability of Virusend (TX-10) to inactivation SARS-CoV-2. Using surface and solution inactivation assays, we show that Virusend (TX-10) is able to reduce SARS-CoV-2 viral titre by 4log10 PFU/mL within 1 minute of contact. Ensuring disinfectants are highly effective against SARS-CoV-2 is important in eliminating environmental sources of the virus to control the COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is 26 the causative agent of COVID-19 which first emerged in late 2019 [1] . Countries are working 27 to control transmission of SARS-CoV-2 with the ultimate goal of production and large-scale 28 manufacture of an effective vaccine [2] [3] [4] . However, until an effective vaccine is found, control 29 of the virus is limited to implementing measures such as contact tracing, quarantine, 30 enforcing strict social distancing, advising frequent hand hygiene and infection control 31 measures in hospital environments [5] . During the 2002 outbreak of SARS-CoV-1, and the 32 2012 Middle East respiratory syndrome-related (MERS)-CoV outbreak, virus stability 33 facilitated transmission events [6] . Similarly, research has shown that SARS-CoV-2 can remain 34 viable on surfaces, notably plastic and stainless steel for up to 72 hours post inoculation, and in aerosols for at least 3 hours, meaning effective disinfectants can prevent indirect contact 36 transmission [7] . Virusend (TX-10) has been developed to work as a highly effective 37 disinfectant that rapidly inactivates infectious enveloped viruses. As communities begin to 38 reopen and people return to the workplace, effective and quick disinfection of communal 39 areas is paramount to maintaining control of COVID-19. Here we present the evidence that 40

Virusend TX-10 can reduce SAR-CoV-2 virus within one minute both in solution and on 41 surfaces. 42 43

Vero E6 cells (C1008: African green monkey kidney cells), obtained from Public Health 46

England, were maintained in Dulbecco's minimal essential medium (DMEM) containing 10% 47 foetal bovine serum (FBS) and 0.05 mg/ml gentamicin. Cells were kept at 37°C with 5% CO2. 48

Passage 4 or 5 of SARS-CoV-2 isolate (REMRQ0001/Human/2020/Liverpool) from a clinical 49 sample was used to assess inactivation of TX-10. On the fourth and fifth passages the virus 50 was cultured in Vero E6 cells maintained in DMEM with 4% FBS and 0.05mg/mL gentamicin 51 at 37°C and 5% CO2 as previously described [8] . The fifth passage of the virus was harvested 52 48 hours after inoculation and concentrated by passage through a centrifugal column (Amicon 53

Ultra-15 100kDa MWCO). Virus was used immediately after concentrating. 54

Virus Inactivation 56 water for either 30 seconds or 9.5 minutes, after which 900µL of DMEM containing 2% FBS 61 and 0.05 mg/mL gentamicin was added and mixed until dried inoculum was dissolved. The 62 sample was then transferred into a dilution series for virus quantification at exactly 1 minute 63 or 10 minutes after addition of TX-10 to the dried inoculum. Solution inactivation assays used 64 either 8.4log10 or 7.9log10 PFU/mL and were carried out by incubating 25µL of inoculum with 65 100µL of TX-10 or autoclaved water for either 1 minute or 10 minutes. After incubation 10mL 66 of DMEM was added and transferred to a dilution series within 30 seconds of DMEM being 67 added. All experiments were performed in duplicate. 68

Cytotoxicity for surface inactivation was determined by inoculating stainless-steel discs with 71 50µL of DMEM containing 2% FBS and 0.05 mg/mL gentamicin and allowed to air dry at room 72 temperature for 1 hour. Dried inoculum was incubated with 100µl of TX-10 or autoclaved 73 water for 5 minutes, after which 900µL of DMEM containing 2% FBS and 0.05 mg/mL 74 gentamicin was added and mixed until dried inoculum was dissolved. The sample was then 75 transferred into a dilution series and a standard plaque assay performed. Cytotoxicity for 76 solution assays were performed by incubating 25 µL of DMEM containing 2% FBS and 0.05 77 mg/mL gentamicin with 100 µL of TX-10 for 5 minutes, after which 10mL of DMEM was added 78 and sample transferred to a dilution series for standard plaque assays. The cytotoxicity assays 79 were performed in duplicate. 80 81

Suppression for solution inactivation was assayed by adding 25µL of inoculum to 100µL of TX-83 transferred into a dilution series and a standard plaque assay preformed. The suppression 85 assay was performed in duplicate. 86 87

Samples from each condition were serial diluted 10-fold for quantification by standard plaque 89 assay using Vero E6 cells. Cells were incubated for 72 hours at 37°C and 5% CO2, then fixed 90 with 10% formalin and stained with 0.05% crystal violet solution. Plaques were counted to 91 calculate virus titre. All samples were performed in technical duplicates. 92 93

For inactivation assays, Virusend TX-10 was directly placed on SARS-CoV-2 inoculum, for an 95 incubation period of either 1 minute or 10 minutes. On the hard surface, contact time of 1 96 minute with Virusend TX-10 reduced SARS-CoV-2 titres to below the limit of detection for 97 both high and low titre inoculum (Fig 1) . A titre of 7.3log10 PFU/mL was recovered from the 98 high titre, hard surface control samples. Similarly, incubation with Virusend TX-10 for 10 99 minutes reduced the virus titre to below the limit of detection, compared with 7.0log10 100 PFU/mL recovered from the high titre control. With a low titre inoculum, Virusend TX-10 also 101 reduced SARS-CoV-2 titres to below the limit of detection after contact times of 1 and 10 102 minutes on hard surfaces. Titres of 5.3log10 PFU/mL and 5.9log10 PFU/mL were recovered from 103 the 1-and 10-minute control samples, respectively. Cytotoxicity assays with Virusend TX-10 104 in the absence of virus were used to determine the limit of detection, the point at which Vero reduction of at least 4.0log10 PFU/mL of infectious SARS-CoV-2 with high titre inoculum and a 108 reduction of at least 2.3log10 PFU/mL with low titre inoculum (Fig 1) . For inactivation assays in solution, Virusend TX-10 was placed directly into solution with SARSreduced the high titre inoculum from 6.00log10 PFU/mL, in the water control, to below the 120 limit of detection (Fig 2A) . A 10-minute incubation with Virusend TX-10 also reduced viral titre from 6.0log10 PFU/mL to below the limit of detection (Fig 2B) . With the low titre inoculum, 122 the addition of Virusend TX-10 reduced SARS-CoV-2 to below the limit of detection at both 1 123 minute and 10 minute incubation times (Fig 2) . Titres of 5.6log10 PFU/mL were recovered from 124 control samples at 1 minute and 10 minutes. A suppression assay for solution inactivation 125 assays was used to demonstrate that dilution with 10mL of DMEM suppressed Virusend TX-126 10 inactivation of SARS-CoV-2 upon the completion of the assay. The addition of Virusend TX-127 10 to virus inoculum in 10mL of DMEM recovered a virus titre of 5.7log10 PFU/mL with high 128 titre inoculum and 5.6log10 PFU/mL with low titre inoculum. Cytotoxicity assays for solution 129 inactivation assays showed the limit of detection for these assays was 2.0log10 PFU/mL. incubated with high titre (HT) virus inoculum for 1 minute and 10 minutes. When low titre 133 (LT) inoculum was used, both incubation periods reduced the titre by at least 3.6log10 PFU/mL, 134

to below the limit of detection. Diagonal pattern represents cytopathic effect caused by TX-135 10 and solid black represents the titre of infectious virus following each treatment. Limit of 136 detection (LOD) (2.0log10 PFU/mL) is shown across the graph with a dotted red line. solutions of 75% ethanol and 10% sodium hypochlorite are able to reduce SARS-CoV-2 titre 148 by at least 2.0log10 PFU/mL and 3.25log10 PFU/mL, respectively, within 5 minutes [9] . 149 However, the WHO has recommended diluting household bleach 1:100 to reduce irritation to 150 the user and contact times of 10 to 60 minutes to disinfect surfaces and when immersing 151 items [12] . Rapid household disinfectants could reduce transmission in private residence and 152 public spaces, such as offices. Here we have shown that Virusend TX-10 is able to reduce 153 SARS-CoV-2 virus titre by at least 4.0log10 PFU/mL in 1 minute of contact time making it and 154 effective disinfectant for households and public spaces. 155

An initial obstruction to the work presented here, was the need for a high virus titre to show 157 a 4.0log10 PFU/mL reduction due to the cytotoxicity of Virusend TX-10 to Vero E6 cells. The 158 limit of detection indicated the point at which cytopathic effect in Vero E6 cells is caused by 159

Virusend TX-10 and not the virus. Therefore, to achieve a 4.0log10 PFU/mL reduction, SARS-CoV-2 had to be concentrated after harvesting to give stock titres of 8.4log10 and 9.8log10 161 PFU/mL. When a lower stock virus titre of 7.9log10 PFU/mL was used, a 4.0log10 PFU/mL 162 reduction could not be demonstrated and would not meet the strict requirements of 163 European Standard testing. However, these assays still showed a similar trend of inactivation. 164

The use of higher viral titre in these assays indicates the effectiveness of Virusend TX-10, 165 which may be necessary to inactivate SARS-CoV-2 in environments that are contaminated 166 CoV-2 specifically, is important to minimise community transmission of SARS-CoV-2. 185 186 Current advice focuses on increasing public engagement in essential control measures, such 187 as high levels of hygiene in the home [17] . Virusend TX-10 can reduce the strain of demand 188 on current hygiene product resources, to be used within private residences, communal public 189 areas such as offices and hospital environments [18] [19] [20] . It can reduce viral titres on surfaces 190 and in solution by at least 4.0log10 PFU/mL within 1 minute of contact making it highly suitable 191 for rapid disinfection of private households and public spaces such as hospitals and offices. 192

The development of disinfectants such as Virusend TX-10 and others is important as we 193 continue efforts to reduce transmission of SARS-CoV-2. 

A new coronavirus 210 associated with human respiratory disease in China

The early landscape of coronavirus disease 2019 vaccine development in the UK 215 and rest of the world

Ensuring 219 global access to COVID-19 vaccines

The COVID-19 vaccine development landscape

The role 227 of community-wide wearing of face mask for control of coronavirus disease 2019 228 (COVID-19) epidemic due to SARS-CoV-2

Role of fomites in SARS transmission during 232 the largest hospital outbreak in Hong Kong

Aerosol and Surface Stability of SARS-CoV-2 as Compared with 237

Methods of Inactivation of SARS-CoV-2 for Downstream Biological 242

Factors 246 affecting stability and infectivity of SARS-CoV-2

Feasibility of controlling COVID-19 outbreaks by isolation of cases and contacts

-252 living survival and environmental transmission

Respiratory Infections in Health Care. WHO Guidelines Approved by the Guidelines 261 Review Committee

CoV-2 RNA detection in the air and on surfaces in the COVID-19 ward of a hospital in 264

Recombinant 268 mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers 269 in vitro and is fully pathogenic in vivo

Critical review of norovirus surrogates in food safety research: 273 rationale for considering volunteer studies

hepatitis C virus grown in cell culture

Preparing for a challenging winter

Potential methanol toxicity and the importance of 282 using a standardised alcohol-based hand rub formulation in the era of COVID-19

The energy and environmental footprints of 287

Energy (Oxf)

Production of Hand Sanitizer for Public Health Protection: The UK and US Academic 292