For this study, preliminary testing with Escherichia coli MS-2 (MS-2) and Escherichia coli T4 (T4) bacteriophages was conducted to develop virucidal methodology for verifying the inactivation after treatment with the test formulations following AOAC germicidal methodologies. After the determination of various experimental parameters (i.e. exposure, concentration) of the formulations, final testing was conducted on BCV. All experiments were conducted with various organic challenges (horse serum, bovine feces, compost) for results that more accurately represent field use condition.
The MS-2 and T4 were slightly more resistant than BCV and required a 2 minutes exposure while BCV was completely inactivated after a 1 minute exposure. These results were also consistent for the testing conducted in the presence of the various organic challenges indicating that the test formulations are highly effective for real world application.
In general, virucidal activity and mechanism of action of various disinfectants is largely understudied. Standards for virucidal testing are being developed, however none are mandated at this time for verifying virus inactivation. It is largely accepted and recommended to follow Association of Official Analytical Chemists (AOAC) germicidal methods for virucidal efficacy testing. The major structural targets of disinfectants against viruses include the viral envelope (predominantly lipid), the capsid (predominantly protein), and the genome (nucleic acid material). According to the Klein-DeForest Scheme, mechanism of action of various chemical disinfectants against viruses is largely due to the presence or absence of a viral envelope, with the latter typically being more resistant.
Viruses are typically categorized into:
-Groups A (lipophilic with envelope) viruses,
-Groups B (hydrophilic, non-enveloped) viruses, or
-Groups C (intermediate solubility, nonenveloped) viruses
The structural target on the virus is dependent on the type of disinfectant used. Because coronaviruses are an enveloped virion, they fall into the Klein-DeForest susceptibility groupA. Group A is susceptible to compounds including halogens, aldehydes, Quaternary Ammonium Compounds (QAC), phenolics, alcohols, peroxide, proteases, and detergents. It is important to note that the susceptibility is referring to the ability of the disinfectant to disrupt surface properties typically important in attachment to cellular receptors, and does not always result in complete degradation and loss of replication functionality of the nucleic acid.
In this research, BCV was used as a surrogate for the virus that causes SARS to study inactivation. Since there are few published protocols for determining virucidal activity of disinfectants, it was important to develop methodology in which exposure duration could be controlled by neutralizing, or quenching, the antiviral effects. Due to the necessity of an in vitro cell culture for propagation of BCV, the subsequent removal of the toxicity due to the nature of the chemicals present in the test formulations was necessary to conduct the virucidal efficacy tests.
Disinfectant activity is often reduced in the presence of organic materials due to cross reactivity with compounds in the organic load effectiveness.
In order to be representative of environmental conditions, the test formulations were also challenged in the presence of organic material including horse serum (for MS-2 and T4) or bovine feces and compost (for BCV).
Sandia National Laboratories test decontamination formulations to inactivate SARS coronavirus by using a close relative, BCV that would react similarly. The results indicate that the test formulation (DF-200) were all highly effect in complete BCV inactivation in contact times as low as 1 minute even in the presence of various organic challenges. It is important to note that the term complete inactivation indicates the failure to recover the test virus, due to an inability to detect the last infective virus particle.
The loss of infectivity of the treated viruses is a result of the disruption of important structures on the surface (namely the viral receptor cites) that physically prevent successful attachment and subsequent penetration intothe host cell. Because standard and official methods or protocols do not currently exist for testing disinfectants as virucides, factors including time of contact, organic load, tissue culture system, sterility, and temperature were all carefully controlled and monitored in this research.
Refer by Rapid Inactivation of SARS-like Coronaviruses Report, Sandia National Laboratories, operated for the United States Department of Energy by Sandia Corporation.