Robust Evaluation of Ultraviolet-C Sensitivity for SARS-CoV-2 and Surrogate Coronaviruses.

UV light, more specifically UV-C light at a wavelength of 254 nm, is often used to disinfect surfaces, air, and liquids. In early 2020, at the cusp of the COVID-19 pandemic, UV light was identified as an efficient means of eliminating coronaviruses; however, the variability in published sensitivity data is evidence of the need for experimental rigor to accurately quantify the effectiveness of this technique. In the current study, reliable and reproducible UV techniques have been adopted, including accurate measurement of light intensity, consideration of fluid UV absorbance, and confirmation of uniform dose delivery, including dose verification using an established biological target (T1UV bacteriophage) and a resistant recombinant virus (baculovirus). The experimental results establish the UV sensitivity of SARS-CoV-2, HCoV-229E, HCoV-OC43, and mouse hepatitis virus (MHV) and highlight the potential for surrogate viruses for disinfection studies. All four coronaviruses were found to be easily inactivated by 254 nm irradiation, with UV sensitivities of 1.7, 1.8, 1.7, and 1.2 mJ/cm2/log10 reduction for SARS-CoV-2, HCoV-229E, HCoV-OC43, and MHV, respectively. Similar UV sensitivities for these species demonstrate the capacity for HCoV-OC43, HCoV-229E, and MHV to be considered surrogates for SARS-CoV-2 in UV-inactivation studies, greatly reducing hazards and simplifying procedures for future experimental studies. IMPORTANCE Disinfection of SARS-CoV-2 is of particular importance due to the global COVID-19 pandemic. UV-C irradiation is a compelling disinfection technique because it can be applied to surfaces, air, and water and is commonly used in drinking water and wastewater treatment facilities. UV inactivation depends on the dose received by an organism, regardless of the intensity of the light source or the optical properties of the medium in which it is suspended. The 254 nm irradiation sensitivity was accurately determined using benchmark methodology and a collimated beam apparatus for four coronaviruses (SARS-CoV-2, HCoV-229E, HCoV-OC43, and MHV), a surrogate indicator organism (T1UV), and a resistant recombinant virus (baculovirus vector). Considering the light distribution across the sample surface, the attenuation of light intensity with fluid depth, the optical absorbance of the fluid, and the sample uniformity due to mixing enable accurate measurement of the fundamental inactivation kinetics and UV sensitivity.

UV Inactivation of SARS-CoV-2 across the UVC Spectrum: KrCl* Excimer, Mercury-Vapor, and Light-Emitting-Diode (LED) Sources.

Effective disinfection technology to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can help reduce viral transmission during the ongoing COVID-19 global pandemic and in the future. UV devices emitting UVC irradiation (200 to 280 nm) have proven to be effective for virus disinfection, but limited information is available for SARS-CoV-2 due to the safety requirements of testing, which is limited to biosafety level 3 (BSL3) laboratories. In this study, inactivation of SARS-CoV-2 in thin-film buffered aqueous solution (pH 7.4) was determined across UVC irradiation wavelengths of 222 to 282 nm from krypton chloride (KrCl*) excimers, a low-pressure mercury-vapor lamp, and two UVC light-emitting diodes. Our results show that all tested UVC devices can effectively inactivate SARS-CoV-2, among which the KrCl* excimer had the best disinfection performance (i.e., highest inactivation rate). The inactivation rate constants of SARS-CoV-2 across wavelengths are similar to those for murine hepatitis virus (MHV) from our previous investigation, suggesting that MHV can serve as a reliable surrogate of SARS-CoV-2 with a lower BSL requirement (BSL2) during UV disinfection tests. This study provides fundamental information on UVC's action on SARS-CoV-2 and guidance for achieving reliable disinfection performance with UVC devices. IMPORTANCE UV light is an effective tool to help stem the spread of respiratory viruses and protect public health in commercial, public, transportation, and health care settings. For effective use of UV, there is a need to determine the efficiency of different UV wavelengths in killing pathogens, specifically SARS-CoV-2, to support efforts to control the ongoing COVID-19 global pandemic and future coronavirus-caused respiratory virus pandemics. We found that SARS-CoV-2 can be inactivated effectively using a broad range of UVC wavelengths, and 222 nm provided the best disinfection performance. Interestingly, 222-nm irradiation has been found to be safe for human exposure up to thresholds that are beyond those effective for inactivating viruses. Therefore, applying UV light from KrCl* excimers in public spaces can effectively help reduce viral aerosol or surface-based transmissions.

Efficacy of an Automated Multiple Emitter Whole-Room Ultraviolet-C Disinfection System Against Coronaviruses MHV and MERS-CoV.

Efficient and automated methods of disinfecting surfaces contaminated with the Middle Eastern respiratory syndrome coronavirus (MERS-CoV) may prevent the spread of the virus. Here we report the efficacy and use of an automated triple-emitter whole room UV-C disinfection system to inactivate mouse hepatitis virus, strain A59 (MHV-A59) and MERS-CoV viruses on surfaces with a >5 log10 reduction.

Receptor-independent infection by mutant viruses newly isolated from the neuropathogenic mouse hepatitis virus srr7 detected through a combination of spinoculation and ultraviolet radiation.

The mouse hepatitis virus (MHV) has a high mutation rate, leading to various neuropathologies after infection. The srr7 mutant was isolated from the MHV strain cl-2, which induces characteristic spongiform degeneration in the brain. To investigate outcomes of srr7 infection, we re-cloned srr7(H2) from the viral stock srr7(Mix). During this re-cloning, we obtained the mutant viruses, Mu-1, Mu-2, and Br-1 which was isolated from the mice brain infected with srr7(Mix). We examined mutant viruses for infectivity independent of the major MHV receptor (MHVR), because these mutants exhibited high virulence similar to cl-2, which is MHVR-independent. To confirm MHVR-independence in vitro, we used a combination of spinoculation and ultraviolet radiation to detect distinct plaque formation (SpinoPlaque(UV+)) afrer infection of BHK cells, which do not express MHVR. Using this technique, we found that the unique neuropathologies caused by infection with the mutant viruses result from infecting neurons, which do not express MHVR. Infection with the mutant viruses was 100% correlated with SpinoPlaque(UV+) formation. This is in contrast to infection with srr7, which does not from SpinoPlaque(UV+).

Equine arteritis virus subgenomic RNA transcription: UV inactivation and translation inhibition studies.

The expression of the genetic information of equine arteritis virus (EAV), an arterivirus, involves the synthesis of six subgenomic (sg) mRNAs. These are 5' and 3' coterminal since they are composed of a leader and a body sequence, which are identical to the 5' and 3' ends of the genome, respectively. Previously, it has been suggested that cis-splicing of a genome-length precursor RNA is involved in their synthesis. This was reevaluated in a comparative analysis of the sg RNA synthesis of EAV, the coronavirus mouse hepatitis virus (MHV), and the alphavirus Sindbis virus. UV transcription mapping showed that the majority of the EAV sg RNAs made at later stages of infection is not derived from a genome-length precursor. However, complete independence of sg RNA synthesis from that of genomic RNA was never observed during the course of infection. The possibility that this resulted from UV irradiation-induced effects on the synthesis of the viral replicase was investigated by inhibiting translation using cycloheximide. For EAV, ongoing protein synthesis was found to be more important for the synthesis of sg RNA than for that of genomic RNA. In general, MHV transcription was extremely sensitive to translation inhibition, whereas EAV genomic RNA synthesis became independent of de novo protein synthesis late in infection.

Tumor necrosis factor expression during mouse hepatitis virus-induced demyelinating encephalomyelitis.

Neutralizing anti-tumor necrosis factor alpha (TNF-alpha) antibody treatment of mice infected with the neurotropic JHMV strain of mouse hepatitis virus showed no reduction of either virus-induced encephalomyelitis or central nervous system demyelination. TNF-alpha-positive cells were present in the central nervous system during infection; however, TNF-alpha could not be colocalized with JHMV-infected cells. In vitro, TNF-alpha mRNA rapidly accumulated following JHMV infection; however, no TNF-alpha was secreted because of inhibition of translation. Both live and UV-inactivated virus inhibited TNF-alpha secretion induced by lipopolysaccharide. These data show that TNF-alpha is not secreted from infected cells and indicate that if contributes to either JHMV-induced acute encephalomyelitis nor primary demyelination.

Responses of mice to murine coronavirus immunization.

Oral and/or intranasal inoculation of susceptible mouse genotypes with the JHM strain of mouse hepatitis virus (MHV-JHM) consistently results in T cell dysfunction as reflected by in vitro proliferative responses to mitogens or allogeneic cells. One approach to examining the mechanism responsible for the observed functional T cell suppression is to determine whether virus replication is required for its induction. To this end, mice were inoculated oronasally with MHV-JHM that was inactivated with short-wave ultraviolet light, beta-propiolactone or psoralen. Mice were also inoculated with live MHV-JHM after recovery from homotypic or heterotypic MHV infection. Spleen cells from BALB mice inoculated oronasally with inactivated MHV-JHM yielded extremely variable in vitro proliferative responses after concanavalin A stimulation. MHV-susceptible mice exposed oronasally or intraperitoneally to virus inactivated by any of the minimum effective treatments failed to seroconvert. Immunization with psoralen-treated virus intraperitoneally in Freund's complete adjuvant or oronasally failed to protect from live virus challenge, but survivors had elevated virus-specific serum IgG antibody titers compared to mock-immunized controls at two weeks post-challenge. Spleen cells from mice that were challenged after recovery from homotypic live virus infection did not exhibit the profound in vitro T cell suppression normally observed during the acute stage of primary infection. In contrast, MHV-JHM challenge of mice vaccinated with heterotypic live MHV-S resulted in significantly depressed in vitro T cell function. The combined data suggest that either virus replication or exposure to more concentrated antigen may be required for induction of the dramatic T cell dysfunction that occurs as a consequence of MHV-JHM infection as well as for a detectable MHV-specific humoral response.

Effects of regional spinal X-irradiation on demyelinating disease caused by Theiler's virus, mouse hepatitis virus or experimental allergic encephalomyelitis.

The effects of X-irradiation on the course of chronic demyelinating disease were examined in mice with experimental allergic encephalitis (EAE), mouse hepatitis virus (MHV) or Theiler's virus (DAV) infection. One month after the induction of EAE or 2-16 months after inoculation of DAV, exposure of the cervical spinal cord to 20 Gy X-rays caused local exacerbation of disease activity but spinal irradiation did not affect MHV-induced demyelination. In EAE, there was a significant increase in the number of inflammatory cells in the irradiated part of the cord. Mice infected with DAV showed locally increased demyelination and axonal degeneration but no change in the titer of infectious virus within the cord. Thus in DAV infection, as in EAE, the exacerbation of disease seemed to be due to vascular or immunological factors rather than viral reactivation.

Physico-chemical properties of mouse hepatitis virus (MHV-2) grown on DBT cell culture.

Some properties of a strain of mouse hepatitis virus, MHV-2, grown on DBT cells were determined using a plaque assay on the cells. Viral growth was not inhibited by the presence of actinomycin D or 5-iodo-2-deoxyuridine. MHV-2 was completely inactivated by ether, chloroform, sodium deoxycholate or beta-propiolactone, but showed a moderate resistance to trypsin. Heating at 56 C for 30 min did not completely abolish the virus infectivity. The virus was stable after heating at 50 C for 15 min in 1M-MgCl2 or 1M-MgSO4 as well as at 37 C for 60 min at pH 3.0 to 9.0. Infectivity was decreased to 1/100 and 1/400 after storing at 4 C for 30 days and 37 C for 24 hr, respectively. The virus passed through a 200-nm but not a 50-nm Sartorius membrane filter. The buoyant density of MHV-2 was 1.183 g/cm3 in sucrose gradient, and the fraction contained coronavirus-like particles measuring 70 to 130 nm in diameter. Survival rate was 10% after exposure to ultraviolet at 150 ergs/mm2. Freezing and thawing or sonication at 20 kc for 3 min did not affect the virus titer. No hemagglutinin was demonstrable with red blood cells of the chicken, Japanese quail, mouse, rat, hamster, guinea pig, sheep, bovine or human.