Virus purification highlights the high susceptibility of SARS-CoV-2 to a chlorine-based disinfectant, chlorous acid.

Chlorous acid water (HClO2) is known for its antimicrobial activity. In this study, we attempted to accurately assess the ability of chlorous acid water to inactivate SARS-CoV-2. When using cell culture supernatants of infected cells as the test virus, the 99% inactivation concentration (IC99) for the SARS-CoV-2 D614G variant, as well as the Delta and Omicron variants, was approximately 10ppm of free chlorine concentration with a reaction time of 10 minutes. On the other hand, in experiments using a more purified virus, the IC99 of chlorous acid water was 0.41-0.74ppm with a reaction time of 1 minute, showing a strong inactivation capacity over 200 times. With sodium hypochlorite water, the IC99 was 0.54ppm, confirming that these chlorine compounds have a potent inactivation effect against SARS-CoV-2. However, it became clear that when using cell culture supernatants of infected cells as the test virus, the effect is masked by impurities such as amino acids contained therein. Also, when proteins (0.5% polypeptone, or 0.3% BSA + 0.3% sheep red blood cells, or 5% FBS) were added to the purified virus, the IC99 values became high, ranging from 5.3 to 76ppm with a reaction time of 10 minutes, significantly reducing the effect. However, considering that the usual usage concentration is 200ppm, it was shown that chlorous acid water can still exert sufficient disinfection effects even in the presence of proteins. Further research is needed to confirm the practical applications and effects of chlorous acid water, but it has the potential to be an important tool for preventing the spread of SARS-CoV-2.

Comparative evaluation of chlorous acid and sodium hypochlorite activity against SARS-CoV-2.

A novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), suddenly emerged in China in 2019, spread globally and caused the present COVID-19 pandemic. Therefore, to mitigate SARS-CoV-2 infection effective measures are essential. Chlorous acid (HClO2) has been shown to be an effective antimicrobial agent. However, at present there is no experimental evidence showing that HClO2 can inactivate SARS-CoV-2. Therefore, in this study, we examined the potential of HClO2 to inactivate SARS-CoV-2 in presence or absence of organic matter and the results were compared with that of sodium hypochlorite (NaClO), another potent antimicrobial agent. When concentrated SARS-CoV-2 was incubated with 10 ppm HClO2 for 10 s, viral titre was decreased by 5 log of 50% tissue culture infective dose per mL (TCID50 ml-1). However, the same concentration of NaClO could not inactivate SARS-CoV-2 as effectively as HClO2 did even after incubation for 3 min. Furthermore, 10 ppm HClO2 also inactivated more than 4.0 log of TCID50 within 10 s in the presence of 5 % fetal bovine serum used as mixed organic matters. Our results obtained with HClO2 are more effective against SARS-CoV-2 as compared to NaClO that can be used for disinfectant against SARS-CoV-2 .

Identifying the chloroperoxyl radical in acidified sodium chlorite solution.

The present study identified the active radical species in acidic sodium chlorite and investigated the feasibility of quantifying these species with the diethylphenylenediamine (DPD) method. Electron spin resonance (ESR) spectroscopy was used to identify the active species generated in solutions containing sodium chlorite (NaClO2). The ESR signal was directly observed in an acidified sodium chlorite (ASC) aqueous solution at room temperature. This ESR signal was very long-lived, indicating that the radical was thermodynamically stable. The ESR parameters of this signal did not coincide with previously reported values of the chlorine radical (Cl●) or chlorine dioxide radical (O = Cl●-O and O = Cl-O●). We refer to this signal as being from the chloroperoxyl radical (Cl-O-O●). Quantum chemical calculations revealed that the optimal structure of the chloroperoxyl radical is much more thermodynamically stable than that of the chlorine dioxide radical. The UV-visible spectrum of the chloroperoxyl radical showed maximum absorbance at 354 nm. This absorbance had a linear relationship with the chloroperoxyl radical ESR signal intensity. Quantifying the free chlorine concentration by the DPD method also revealed a linear relationship with the maximum absorbance at 354 nm, which in turn showed a linear relationship with the chloroperoxyl radical ESR signal intensity. These linear relationships suggest that the DPD method can quantify chloroperoxyl radicals, which this study considers to be the active species in ASC aqueous solution.

Microbicidal effects of weakly acidified chlorous acid water against feline calicivirus and Clostridium difficile spores under protein-rich conditions.

Sanitation of environmental surfaces with chlorine based-disinfectants is a principal measure to control outbreaks of norovirus or Clostridium difficile. The microbicidal activity of chlorine-based disinfectants depends on the free available chlorine (FAC), but their oxidative potential is rapidly eliminated by organic matter. In this study, the microbicidal activities of weakly acidified chlorous acid water (WACAW) and sodium hypochlorite solution (NaClO) against feline calcivirus (FCV) and C. difficile spores were compared in protein-rich conditions. WACAW inactivated FCV and C. difficile spores better than NaClO under all experimental conditions used in this study. WACAW above 100 ppm FAC decreased FCV >4 log10 within 30 sec in the presence of 0.5% each of bovine serum albumin (BSA), polypeptone or meat extract. Even in the presence of 5% BSA, WACAW at 600 ppm FAC reduced FCV >4 log10 within 30 sec. Polypeptone inhibited the virucidal activity of WACAW against FCV more so than BSA or meat extract. WACAW at 200 ppm FAC decreased C. difficile spores >3 log10 within 1 min in the presence of 0.5% polypeptone. The microbicidal activity of NaClO was extensively diminished in the presence of organic matter. WACAW recovered its FAC to the initial level after partial neutralization by sodium thiosulfate, while no restoration of the FAC was observed in NaClO. These results indicate that WACAW is relatively stable under organic matter-rich conditions and therefore may be useful for treating environmental surfaces contaminated by human excretions.

Antimicrobial activity and stability of weakly acidified chlorous acid water.

The antimicrobial activity of weakly acidified chlorous acid water (WACAW) against Staphylococcus aureus, non-pathogenic Escherichia coli, enterohemorrhagic E. coli (EHEC O157:H7), Candida albicans, and spore-forming Bacillus and Paenibacillus species was evaluated in vitro. The antiviral activity was also examined using feline calicivirus (FCV). Diluted WACAW (>100 ppm) effectively reduced the number of non-spore-forming bacteria (>4 log10 CFU reductions) within 5 min. Treatment with this sanitizer at 400 ppm for 30 min achieved>5 log10 CFU reductions in spore-forming Bacillus and Paenibacillus species while an equivalent concentration of sodium hypochlorite (NaClO) resulted in only a 0.98 and 2.72 log10 CFU reduction, respectively. The effect of this sanitizer against FCV was equivalent to that of NaClO. Immersion in WACAW (400 ppm) achieved >4 and 2.26 log10 CFU reductions in Campylobacter jejuni and EHEC, respectively, on artificially contaminated broiler carcass pieces. Finally, theantimicrobial activity of this sanitizer was shown to be maintained for at least 28 d when in contact with nonwoven fabric (100% cotton). This study showed that pH control of chlorous acid is expected to modify its antimicrobial activity and stability. WACAW is expected to have applications in various settings such as the food processing and healthcare industries.