key: cord-313795-jr3n3uo9 authors: McAuley, Julie L.; Deerain, Joshua M.; Hammersla, William; Aktepe, Turgut E.; Purcell, Damian J.F.; Mackenzie, Jason M. title: Liquid chalk is an antiseptic against SARS-CoV-2 and influenza A respiratory viruses date: 2020-11-02 journal: bioRxiv DOI: 10.1101/2020.11.02.364661 sha: doc_id: 313795 cord_uid: jr3n3uo9 The COVID-19 pandemic has impacted and enforced significant restrictions within our societies, including the attendance of the public and professional athletes in gyms. Liquid chalk is a commonly used accessories in gyms and is comprised of magnesium carbonate and alcohol that quickly evaporates on the hands to leave a layer of dry chalk. We investigated whether liquid chalk is an antiseptic against highly pathogenic human viruses including, SARS-CoV-2, influenza virus and noroviruses. Chalk was applied before or after virus inoculum and recovery of infectious virus was determined to mimic the use in the gym. We observed that addition of chalk before or after virus contact lead to a significant reduction on recovery of infectious SARS-CoV-2 and influenza but had little impact on norovirus. These observations suggest that the use and application of liquid chalk can be an effective and suitable antiseptic for major sporting events, such as the Olympic Games. 86 was aseptically smeared onto 2-4 discrete areas covering approximately 2cm round surface 87 (approx. 50ul) on a sterile tissue culture dish and allowed to dry. 50uL virus inoculum was then 88 applied. Where the inoculum did not absorb into the dry chalk, a slurry of chalk:virus mixture 89 was created using a sterile tip and mixing. 15min later, 500uL infection media (identical to 90 culture media but without the presence of sera) was added then mixed with the chalk:virus 91 sample and collected. Excess chalk was pelleted at 400 g for 3min, then a 50% tissue culture 92 infectious dose (TCID 50 ) assay performed on the supernatant as previously described [6, 7] . 93 For the virus first assay, 50uL virus inoculum was added to 2-4 discrete areas on a sterile tissue 94 culture dish, then chalk added and spread to cover an approximate 2cm round surface on a 95 sterile tissue culture dish. After 15min incubation at room temperature, 500uL infection media 132 whereas Chalk #4 also had a significant impact, but some residual virus could be recovered. 154 To further our study, we also tested the antiviral effect of Liquid Chalk against another 155 highly infectious and pathogenic respiratory viral pathogen IAV. The experiments were 156 performed exactly as described above and the virus TCID 50 /mL was titrated on MDCK cells. 157 As can be observed in Figure 2 , all four Liquid Chalk products were effective in restricting the 158 recovery of IAV compared to SARS-CoV-2. However, for IAV the effect was greater when transmission of SARS-CoV-2 (the 52 causative agent of the COVID-19 pandemic), influenza A virus (H1N1) (IAV) and norovirus, 53 using the surrogate model of mouse norovirus Vero cells (American Type Culture Collection [ATCC]) were maintained in Minimal Essential 58 Media (MEM) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 10 μM 59 HEPES, 2 mM glutamine and antibiotics Madin-Darby canine kidney (MDCK) cells were grown in Roswell Park 61 Memorial Institute (RPMI) media supplemented with 10% FBS, 2 mM glutamine and 62 antibiotics. RAW 264.7 cells were maintained in Dulbecco's Modified Eagle's Medium 63 (DMEM) with 10% FBS and 1% GlutaMAX. Cell cultures were maintained at 37 o C in a 5% 64 CO 2 incubator. SARS-CoV-2 isolate hCoV-19/Australia/VIC01 Cytotoxicity assay 96® Non-Radioactive Cytotoxicity Assay 50ul of various 71 liquid chalk samples were aseptically air-dried, resuspended in 500ul of cell culture media and 72 centrifuged at 400xg for 3 mins to remove excess chalk particles (this sample is depicted as 73 "neat"). Neat supernatant was 10-fold serially diluted in respective tissue culture media, added 74 to the 96-well plate containing cells and incubated at 37ᴼC for 24 hours. Following the 75 incubation period, 10ul of 10x Lysis Solution was added for 45 mins to the control wells to 8 170 applied first and dried, the recovery of infectious influenza A virus was significantly reduced 171 compared to the chalk control, but markedly more infectious virus remained in the samples 172 treated with chalk 1 and chalk 3 compared to the virus first samples that underwent the same 173 treatment (data not significant) As a comparator, we also investigated the ability of Liquid Chalk to inactivate another 178 highly infectious viral pathogen, norovirus. As human norovirus is difficult to cultivate in 179 laboratory conditions, we utilised the widely appreciated surrogate murine norovirus (MNV) 180 for our studies Again, the experiments were identical to those described above except the 181 viral TCID 50 /mL was performed on RAW264.7 (murine macrophage) cells We observed that Chalks #1-3 had very little impact on virus recovery, with a 0.5 log 184 reduction the best that we observed. However, in contrast to SARS-CoV-2 we observed an 185 approximate 1 log reduction upon application of Chalk #4. This is interesting as the major 186 difference between MNV and SARS-CoV-2 and IAV is that MNV is a non-enveloped virus, 187 whereas the other two contain a host-derived lipid membrane as their outer most layer. Thus Norovirus remains infectious when exposed to Liquid Chalk. Norovirus was not 192 rendered non-infectious when treated with gym chalk, regardless of whether the virus was 193 added to dry chalk, or chalk was added to virus inoculum As different alcohols are the major constituents of liquid chalk, we additionally 198 evaluated the impact of alcohol alone on the recovery of infectious virus. As can be observed 199 in Table 1, exposure of SARS-CoV-2 and IAV to both ethanol and isopropanol is detrimental 200 to the infectiousness of these viruses up to a dilution of 40% v/v for IAV and 20% v/v for 201 SARS-CoV2. Given proprietary information regarding the alcohol content of chalks There have recently been two press releases, one using seasonal CoV and not SARS Neither 208 public release investigated the efficiency against other highly infectious viral pathogens. To 209 our knowledge this study is the first to demonstrate antiviral activity of a range of commercially 210 available and commonly used Liquid Chalks. Given the uncertainty of re-opening gyms due to 211 contact transmission from potentially contaminated equipment, our findings that Liquid Chalks 212 have anti-viral activity against SARS-CoV-2 may aid in decision making for re-opening gyms 213 in the future Victorian Infectious Diseases Reference Laboratory at the Doherty Institute, in providing our 219 laboratory with isolated SARS-CoV-2 material Author Contributions TEA and JMD performed then experiments themselves; WH provided the liquid chalk collated and analysed the data and wrote the manuscript 223 Funding: The University of Melbourne acknowledges the support of a grant administered by 224 the State Government of Victoria to JMM 225 Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in 226 the design of the study Hand sanitizers: A review of ingredients, mechanisms 231 of action, modes of delivery, and efficacy against coronaviruses Efficacy of ethanol against viruses in hand disinfection Inactivation of 237 Severe Acute Respiratory Syndrome Coronavirus 2 by WHO-Recommended Hand Rub 238 Formulations and Alcohols. Emerging Infectious Disease journal Inactivation of SARS-CoV-2 by commercially 241 available alcohol-based hand sanitizers Isolation and rapid 244 sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with 11 COVID-19 in Australia Validation of 249 a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay 250 for rapid detection of SARS-CoV-2 RNA Expression of the 1918 influenza A virus PB1-F2 enhances the pathogenesis of viral and 254 secondary bacterial pneumonia STAT1-Dependent Innate 258 Immunity to a Norwalk-Like Virus Mouse 260 norovirus replication is associated with virus-induced vesicle clusters originating from 261 membranes derived from the secretory pathway Murine norovirus: a model system to study 265 norovirus biology and pathogenesis Liquid Chalk Proven in CU Labs to Kill Coronavirus, Potentially Helping collated and analysed the data and wrote the manuscript 302 Competing interests: All authors declare they have no competing interests with this study 303 Data sharing: All results gained during this study will be made available via journal access 304 after acceptance and publication of the article on open access university website