Virucidal Activity against SARS-CoV-2 Flashcards
What four disinfectants were tested in this study for activity against SARS-CoV-2?
QAC (MCP) 5%
QAC (FWD) 5%
W30 (amphoteric surfactant) 1%
Medical EtOH 95%
Vero cells were cultured at 37dc with 5% CO2 in DMEM (medium) supplemented with 10% fetal bovine serum (FBS)
Explain the use of FBS
- interfering substance
How was cytotoxicity of the disinfectants assessed?
- Vero cells in medium with disinfectant but without virus (ensure that diluted disinfectants was not cytotoxic to the cell cultures)
- Test pdt serially diluted, incubated, dilutions discarded and cells overlaid with fresh medium => observed for cytotoxic effect
Inactivation assay was performed using ____
describe the steps involved
quantitative suspension test
- diluted virus-disinfectant mixture was taken out to inoculate and infect the host cells (Vero cells)
- incubate
- extract nucleic acids of viruses (viral RNA) and use qPCR to quantify infectious virus titre after virus inactivation
How was virus titre determined?
Viral RNA isolated from serially diluted virus stock was amplified using qPCR, used to draw a standard curve to calculate corresponding virus titre
What is the plaque assay?
think this is the positive control?
Virus samples serially diluted 10 fold in medium and layered on the cells cultured for 12-24h in triplicates
1h incubation at 37dc, cells cultured in CMC medium for 72-96h at 37dc to allow plaque formation
treat with 4% paraformaldehyde to inactivate infectious virus
plaques stained with crystal voilet, visualized and counted
What reduction factor was considered as evidence of sufficient virucidal activity?
RF >= 4
determined by difference of log titre of virus control minus log titre of test virus
Describe the QAC activity against SARS-CoV-2
MCP and FWD highly effective within 15s of contact time, at very low concentrations
FWD at 0.06% ineffective
MCP at 0.06% can inactivate virus with more than 8min contact time
After keeping QAC dilutions at room temp for 1 week,
virucidal activity decreased a little but still effective at longer contact time
Results suggest that FWD can be used as an alternative for MCP
Describe the activity of W30 against SARS-CoV-2
W30 not as effective as QAC, require longer contact time of 2 minutes
W30 at lower conc., require longer contact time
However, it has lower toxicity to human (since it is a surfactant)
- advantage: non-toxic, less irritating, non-flammable
Though it is not as effective as QAC, it can be used in many household cleaning products including household products, soap, skin hygiene
Describe activity of EtOH against SARS-CoV-2
Generally, alcohol optimum conc range is from 60-90%
Generally, alcohol ineffective below 50%
However, ethanol and isopropanol shown to be effective >30%, within 30s
38% ethanol was sufficient to completely inactivate virus within 15s
Which of the 4 disinfectants showed similar dose-dependent inactivating SARS-CoV-2 pattern?
QAC (MCP and FWP) and W30
- better inactivation effect at higher conc., and longer exposure time
Why might MCP be harmful to the environment?
Contain nonylphenol ethoxylates (NPE) - highly toxic to aquatic environment
*FWD does not contain NPE
What was a potential disadvantage to the integrated cell culture-qPCR method?
Did not use gold standard method of CPE and TCID50 assay/plaque assay
when initial virus titre used was low, RF >4 could not be reached or longer incubation time was required
How was it mitigated?
- pretreatment of virus with disinfectant and quantify the virus titres using qPCR
- only infectious virus can be amplified in the host cell genome
- cells incubated for optimised period to amplify the virus (decrease limit of quantification, improve sensitivity of detection)
What was the role of ethanol in the study?
38% ethanol was sufficient to completely inactivate virus
- this proved the method used in the study was feasible (since ethanol alr evaluated)
