Mouse Hepatitis Virus Research Articles

Efficacy of detergent-based cleaning methods against coronavirus MHV-A59 on porous and non-porous surfaces

This study evaluated the efficacy of detergent-based surface cleaning methods against Murine Hepatitis Virus A59 (MHV) as a surrogate coronavirus for SARS-CoV-2. MHV (5% soil load in culture medium or simulated saliva) was inoculated onto four different high-touch materials [stainless steel (SS), Acrylonitrile Butadiene Styrene plastic (ABS), Formica, seat fabric (SF)]. Immediately and 2-hr post-inoculation, coupons were cleaned (damp wipe wiping) with and without pretreatment with detergent solution or 375 ppm hard water. Results identified that physical removal (no pretreatment) removed >2.3 log10 MHV on ABS, SS, and Formica when surfaces were cleaned immediately. Pretreatment with detergent or hard water increased effectiveness over wet wiping 2-hr post-inoculation; pretreatment with detergent significantly increased (p ≤ 0.05) removal of MHV in simulated saliva, but not in culture media, over hard water pretreatment (Formica and ABS). Detergent and hard water cleaning methods were ineffective on SF under all conditions. Overall, efficacy of cleaning methods against coronaviruses are material- and matrix-dependent; pre-wetting surfaces with detergent solutions increased efficacy against coronavirus suspended in simulated saliva. This study provides data highlighting the importance of incorporating a pre-wetting step prior to detergent cleaning and can inform cleaning strategies to reducing coronavirus surface transmission.

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in murine β-coronavirus-induced neuroinflammation

Mouse hepatitis virus (MHV; m-β-CoV) serves as a useful model for studying the cellular factors involved in neuroinflammation. To understand the role of matrix metalloproteinases (MMPs) in neuroinflammation, brain tissues from m-β-CoV-infected mice were harvested at different days post-infection (d.p.i) and investigated for Mmp expression by RT-qPCR. Mmp-2, -3, -8, -12 showed significant mRNA upregulation peaking with viral replication between 5 and 6 d.p.i. Elevated levels of MMP regulator TIMP-1 are suggestive of a TIMP-1 mediated host antiviral response. Biological network assessment suggested a direct involvement of MMP-3, -8, -14 in facilitating peripheral leukocyte infiltrations. Flow cytometry confirmed the increased presence of NK cells, CD4+ and CD8+ T cells, neutrophils, and MHCII expressing cells in the m-β-CoV infected mice brain. Our study revealed that m-β-CoV upregulated Park7, RelA, Nrf2, and Hmox1 transcripts involved in ROS production and antioxidant pathways, describing the possible nexus between oxidative pathways, MMPs, and TIMP in m-β-CoV-induced neuroinflammation.

Inhibiting ACSL1-Related Ferroptosis Restrains Murine Coronavirus Infection.

Murine hepatitis virus strain A59 (MHV-A59) was shown to induce pyroptosis, apoptosis, and necroptosis of infected cells, especially in the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59 is unknown. We utilized murine macrophages and a C57BL/6 mice intranasal infection model to address this. In primary macrophages, the ferroptosis inhibitor inhibited viral propagation, inflammatory cytokines released, and cell syncytia formed after MHV-A59 infection. In the mouse model, we found that in vivo administration of liproxstatin-1 ameliorated lung inflammation and tissue injuries caused by MHV-A59 infection. To find how MHV-A59 infection influenced the expression of ferroptosis-related genes, we performed RNA-seq in primary macrophages and found that MHV-A59 infection upregulates the expression of the acyl-CoA synthetase long-chain family member 1 (ACSL1), a novel ferroptosis inducer. Using ferroptosis inhibitors and a TLR4 inhibitor, we showed that MHV-A59 resulted in the NF-kB-dependent, TLR4-independent ACSL1 upregulation. Accordingly, ACSL1 inhibitor Triacsin C suppressed MHV-A59-infection-induced syncytia formation and viral propagation in primary macrophages. Collectively, our study indicates that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serve as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.

The N-Terminal Region of Middle East Respiratory Syndrome Coronavirus Accessory Protein 8b Is Essential for Enhanced Virulence of an Attenuated Murine Coronavirus.

ABSTRACTMiddle East respiratory syndrome coronavirus (MERS-CoV) is a beta coronavirus that emerged in 2012, causing severe pneumonia and renal failure. MERS-CoV encodes five accessory proteins. Some of them have been shown to interfere with host antiviral immune response. However, the roles of protein 8b in innate immunity and viral virulence was rarely studied. Here, we introduced individual MERS-CoV accessory protein genes into the genome of an attenuated murine coronavirus (Mouse hepatitis virus, MHV), respectively, and found accessory protein 8b could enhance viral replication in vivo and in vitro and increase the lethality of infected mice. RNA-seq analysis revealed that protein 8b could significantly inhibit type I interferon production (IFN-I) and innate immune response in mice infected with MHV expressing protein 8b. We also found that MERS-CoV protein 8b could initiate from multiple internal methionine sites and at least three protein variants were identified. Residues 1-23 of protein 8b was demonstrated to be responsible for increased virulence in vivo. In addition, the inhibitory effect on IFN-I of protein 8b might not contribute to its virulence enhancement as aa1-23 deletion did not affect IFN-I production in vitro and in vivo. Next, we also found that protein 8b was localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, which was disrupted by C-terminal region aa 88-112 deletion. This study will provide new insight into the pathogenesis of MERS-CoV infection.IMPORTANCE Multiple coronaviruses (CoV) cause severe respiratory infections and become global public health threats such as SARS-CoV, MERS-CoV, and SARS-CoV-2. Each coronavirus contains different numbers of accessory proteins which show high variability among different CoVs. Accessory proteins are demonstrated to play essential roles in pathogenesis of CoVs. MERS-CoV contains 5 accessory proteins (protein 3, 4a, 4b, 5, 8b), and deletion of all four accessory proteins (protein 3, 4a, 4b, 5), significantly affects MERS-CoV replication and pathogenesis. However, whether ORF8b also regulates MERS-CoV infection is unknown. Here, we constructed mouse hepatitis virus (MHV) recombinant virus expressing MERS-CoV protein 8b and demonstrated protein 8b could significantly enhance the virulence of MHV, which is mediated by N-terminal domain of protein 8b. This study will shed light on the understanding of pathogenesis of MERS-CoV infection.

Phosphatidylserine receptors enhance SARS-CoV-2 infection.

Phosphatidylserine (PS) receptors enhance infection of many enveloped viruses through virion-associated PS binding that is termed apoptotic mimicry. Here we show that this broadly shared uptake mechanism is utilized by SARS-CoV-2 in cells that express low surface levels of ACE2. Expression of members of the TIM (TIM-1 and TIM-4) and TAM (AXL) families of PS receptors enhance SARS-CoV-2 binding to cells, facilitate internalization of fluorescently-labeled virions and increase ACE2-dependent infection of SARS-CoV-2; however, PS receptors alone did not mediate infection. We were unable to detect direct interactions of the PS receptor AXL with purified SARS-CoV-2 spike, contrary to a previous report. Instead, our studies indicate that the PS receptors interact with PS on the surface of SARS-CoV-2 virions. In support of this, we demonstrate that: 1) significant quantities of PS are located on the outer leaflet of SARS-CoV-2 virions, 2) PS liposomes, but not phosphatidylcholine liposomes, reduced entry of VSV/Spike pseudovirions and 3) an established mutant of TIM-1 which does not bind to PS is unable to facilitate entry of SARS-CoV-2. As AXL is an abundant PS receptor on a number of airway lines, we evaluated small molecule inhibitors of AXL signaling such as bemcentinib for their ability to inhibit SARS-CoV-2 infection. Bemcentinib robustly inhibited virus infection of Vero E6 cells as well as multiple human lung cell lines that expressed AXL. This inhibition correlated well with inhibitors that block endosomal acidification and cathepsin activity, consistent with AXL-mediated uptake of SARS-CoV-2 into the endosomal compartment. We extended our observations to the related betacoronavirus mouse hepatitis virus (MHV), showing that inhibition or ablation of AXL reduces MHV infection of murine cells. In total, our findings provide evidence that PS receptors facilitate infection of the pandemic coronavirus SARS-CoV-2 and suggest that inhibition of the PS receptor AXL has therapeutic potential against SARS-CoV-2.

Innate Immune Training with Bacterial Extracts Enhances Lung Macrophage Recruitment to Protect from Betacoronavirus Infection

Training of the innate immune system with orally ingested bacterial extracts was demonstrated to have beneficial effects on infection clearance and disease outcome. The aim of our study was to identify cellular and molecular processes responsible for these immunological benefits. We used a murine coronavirus (MCoV) A59 mouse model treated with the immune activating bacterial extract Broncho-Vaxom (BV) OM-85. Tissue samples were analysed with qPCR, RNA sequencing, histology, and flow cytometry. After BV OM-85 treatment, interstitial macrophages accumulated in lung tissue leading to a faster response of type I interferon (IFN) signalling after MCoV infection resulting in overall lung tissue protection. Moreover, RNA sequencing showed that lung tissue from mice receiving BV OM-85 resembled an intermediate stage between healthy and viral infected lung tissue at day 4, indicating a faster return to normal tissue homoeostasis. The pharmacologic effect was mimicked by adoptively transferring naive lung macrophages into lungs from recipient mice before virus infection. The beneficial effect of BV OM-85 was abolished when inhibiting initial type I IFN signalling. Overall, our data suggest that BV OM-85 enhances lung macrophages allowing for a faster IFN response towards a viral challenge as part of the oral-induced innate immune system training.

Microbiological survey of Korean mouse facilities from 2014 to 2019.

We surveyed mouse microbiological contamination rates by testing rates for common contaminants using serological, culture, and parasitological methods. A total of 21,292 experimentally housed mice from 206 animal facilities, including hospitals, universities, companies, and research institutes, were tested over a 6-year period from 2014 to 2019. The most commonly found contaminants were various species of nonpathogenic protozoa (47.2%). The most common pathogenic bacteria were Staphylococcus aureus (21.2%), Pasteurella pneumotropica (12.5%), and Pseudomonas aeruginosa (5.8%). Mouse hepatitis virus (6.1%) was detected, but no other viral or bacterial pathogens were found. These results establish that the main pathogens that currently contaminate mouse facilities in Korea are opportunistic pathogens and that contamination with important pathogens, such as those in Categories B or C, has decreased.

A Novel Potentially Recombinant Rodent Coronavirus with a Polybasic Cleavage Site in the Spike Protein.

ABSTRACTThe emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reignited global interest in animal coronaviruses and their potential for human transmission. While bats are thought to be the wildlife reservoir of SARS-CoV and SARS-CoV-2, the widespread human coronavirus OC43 is thought to have originated in rodents. Here, we sampled 297 rodents and shrews, representing eight species, from three municipalities of southern China. We report coronavirus prevalences of 23.3% and 0.7% in Guangzhou and Guilin, respectively, with samples from urban areas having significantly higher coronavirus prevalences than those from rural areas. We obtained three coronavirus genome sequences from Rattus norvegicus, including a Betacoronavirus (rat coronavirus [RCoV] GCCDC3), an Alphacoronavirus (RCoV-GCCDC5), and a novel Betacoronavirus (RCoV-GCCDC4). Recombination analysis suggests that there was a potential recombination event involving RCoV-GCCDC4, murine hepatitis virus (MHV), and Longquan Rl rat coronavirus (LRLV). Furthermore, we uncovered a polybasic cleavage site, RARR, in the spike (S) protein of RCoV-GCCDC4, which is dominant in RCoV. These findings provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the value of a One Health approach to virus discovery.IMPORTANCE Surveillance of viruses among rodents in rural and urban areas of South China identified three rodent coronaviruses, RCoV-GCCDC3, RCoV-GCCDC4, and RCoV-GCCDC5, one of which was identified as a novel potentially recombinant coronavirus with a polybasic cleavage site in the spike (S) protein. Through reverse transcription-PCR (RT-PCR) screening of coronaviruses, we found that coronavirus prevalence in urban areas is much higher than that in rural areas. Subsequently, we obtained three coronavirus genome sequences by deep sequencing. After different method-based analyses, we found that RCoV-GCCDC4 was a novel potentially recombinant coronavirus with a polybasic cleavage site in the S protein, dominant in RCoV. This newly identified coronavirus RCoV-GCCDC4 with its potentially recombinant genome and polybasic cleavage site provides a new insight into the evolution of coronaviruses. Furthermore, our results provide further information on the potential for interspecies transmission of coronaviruses and demonstrate the necessity of a One Health approach for zoonotic disease surveillance.

Viral Inactivation with Emphasis on SARS-CoV-2 Using Physical and Chemical Disinfectants.

Background Recently, an outbreak of a novel human coronavirus SARS-CoV-2 has become a world health concern leading to severe respiratory tract infections in humans. Virus transmission occurs through person-to-person contact, respiratory droplets, and contaminated hands or surfaces. Accordingly, we aim at reviewing the literature on all information available about the persistence of coronaviruses, including human and animal coronaviruses, on inanimate surfaces and inactivation strategies with biocides employed for chemical and physical disinfection. Method A comprehensive search was systematically conducted in main databases from 1998 to 2020 to identify various viral disinfectants associated with HCoV and methods for control and prevention of this newly emerged virus. Results The analysis of 62 studies shows that human coronaviruses such as severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS) coronavirus or endemic human coronaviruses (HCoV), canine coronavirus (CCV), transmissible gastroenteritis virus (TGEV), and mouse hepatitis virus (MHV) can be efficiently inactivated by physical and chemical disinfectants at different concentrations (70, 80, 85, and 95%) of 2-propanol (70 and 80%) in less than or equal to 60 s and 0.5% hydrogen peroxide or 0.1% sodium hypochlorite within 1 minute. Additionally, glutaraldehyde (0.5–2%), formaldehyde (0.7–1%), and povidone-iodine (0.1–0.75%) could readily inactivate coronaviruses. Moreover, dry heat at 56°C, ultraviolet light dose of 0.2 to 140 J/cm2, and gamma irradiation could effectively inactivate coronavirus. The WHO recommends the use of 0.1% sodium hypochlorite solution or an ethanol-based disinfectant with an ethanol concentration between 62% and 71%. Conclusion The results of the present study can help researchers, policymakers, health decision makers, and people perceive and take the correct measures to control and prevent further transmission of COVID-19. Prevention and decontamination will be the main ways to stop the ongoing outbreak of COVID-19.

Tea–Essential Oil–Metal Hybrid Nanocoatings for Bacterial and Viral Inactivation

Natural plant-derived antimicrobial nanocoatings have been synthesized by mixing brewed tea with cinnamaldehyde oil. Concurrent addition of copper or silver salts produces hybrid tea-cinnamaldehyde-copper or tea-cinnamaldehyde-silver nanocoatings, respectively. Tea-cinnamaldehyde, tea-cinnamaldehyde-copper, and tea-cinnamaldehyde-silver coatings are all found to display strong antibacterial efficacy against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus (Log10 Reduction = 8.44 and 7.90, respectively). Tea-cinnamaldehyde-copper and tea-cinnamaldehyde-silver hybrid nanocoatings deposited onto nonwoven polypropylene provide 98.6 and 99.8% deactivation, respectively, toward murine coronavirus MHV-A59 (a potential surrogate for COVID-19 global pandemic coronavirus SARS-CoV-2). Key advantages of this approach are that the coating fabrication involves just a single step, utilizes cheap reagents (which are widely available over the counter to the general public), does not require any equipment apart from a container, and the coatings spontaneously adhere to a variety of substrate materials (including silicon, glass, polyester, nonwoven polypropylene, poly(tetrafluoroethylene), and cotton). Tea is one of the most ubiquitous beverages in the world, meaning that these antimicrobial coatings could be produced locally almost anywhere and by anyone without the need for any specialized technical training or expertize (for example, at remote field hospitals during humanitarian crises and in low-income countries). © 2021 The Authors. Published by American Chemical Society.

Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) invitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

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

ABSTRACTUV 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.

ORF3a of SARS-CoV-2 promotes lysosomal exocytosis-mediated viral egress.

Viral entry and egress are important determinants of virus infectivity and pathogenicity. β-coronaviruses, including the COVID-19 virus SARS-CoV-2 and mouse hepatitis virus (MHV), exploit the lysosomal exocytosis pathway for egress. Here, we show that SARS-CoV-2 ORF3a, but not SARS-CoV ORF3a, promotes lysosomal exocytosis. SARS-CoV-2 ORF3a facilitates lysosomal targeting of the BORC-ARL8b complex, which mediates trafficking of lysosomes to the vicinity of the plasma membrane, and exocytosis-related SNARE proteins. The Ca2+ channel TRPML3 is required for SARS-CoV-2 ORF3a-mediated lysosomal exocytosis. Expression of SARS-CoV-2 ORF3a greatly elevates extracellular viral release in cells infected with the coronavirus MHV-A59, which itself lacks ORF3a. In SARS-CoV-2 ORF3a, Ser171 and Trp193 are critical for promoting lysosomal exocytosis and blocking autophagy. When these residues are introduced into SARS-CoV ORF3a, it acquires the ability to promote lysosomal exocytosis and inhibit autophagy. Our results reveal a mechanism by which SARS-CoV-2 interacts with host factors to promote its extracellular egress.

Comparison of five polyethylene glycol precipitation procedures for the RT-qPCR based recovery of murine hepatitis virus, bacteriophage phi6, and pepper mild mottle virus as a surrogate for SARS-CoV-2 from wastewater

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery efficiency of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery efficiency of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery efficiency of MHV (0.070%–2.6%) and phi6 (0.071%–0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (8.2 × 108 to 5.6 × 109) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation (r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

Enigmatic Neural Pathways: Potentiating Viral Neuroinvasion Into the CNS.

Enigmatic Neural Pathways: Potentiating Viral Neuroinvasion Into the CNS.

Selection of a SARS-CoV-2 Surrogate for Use in Surface Disinfection Efficacy Studies with Chlorine and Antimicrobial Surfaces.

Initial recommendations for surface disinfection to prevent SARS-CoV-2 transmission were developed using previous evidence from potential surrogates. To the best of our knowledge, no appropriate surrogate for SARS-CoV-2 has been identified or confirmed for chlorine and antimicrobial surface disinfection. We completed a study to evaluate the efficacy of two hypothesized antimicrobial surfaces, and four chlorine solutions on nonporous and porous surfaces, against SARS-CoV-2 and three potential SARS-CoV-2 surrogates [coronavirus mouse hepatitis virus (MHV) and bacteriophages Phi6 and MS2], to identify a BSL-1 or BSL-2 virus to use in future studies. We found SARS-CoV-2 can be reduced >4 log10 on porous and nonporous surfaces within 30 s upon exposure to 0.5% NaOCl. The results indicate coronavirus MHV-GFP is inactivated faster than SARS-CoV-2 (MHV-GFP ≥ 6.08 log10; SARS-CoV-2 = 0.66 log10 at 30 s with 0.05% NaOCl on steel) and MS2 is inactivated more slowly. Phi6 is inactivated like SARS-CoV-2, and we propose Phi6 as a slightly conservative surrogate for SARS-CoV-2 chlorine disinfection. Additionally, disinfection of bacteriophages on wood was challenging, and exposure to antimicrobial surfaces had no disinfection efficacy as tested. We recommend using 0.5% chlorine on surfaces for a minimum of 30 s of contact to disinfect SARS-CoV-2 and recommend additional research on Phi6 disinfection with varied surfaces and conditions.

In Vitro Evaluation of the Virucidal Activity of Different Povidone-Iodine Formulations Against Murine and Human Coronaviruses.

IntroductionPolyvinylpyrrolidone–iodine (PVP-I) demonstrates broad-spectrum anti-infective activity and is available in different formulations for oral rinse and topical use in medical and personal care settings. The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has underscored the need to supplement available preventive strategies.MethodsWe assessed virucidal activity of PVP-I formulations, including 0.5% (w/v) solution, 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution, versus placebos when challenged with coronaviruses in two in vitro studies. Murine coronavirus strain A59 (American Type Culture Collection [ATCC]® VR-764™), human coronavirus strain OC43 (ZeptoMetrix Corp. #0810024CF), human coronavirus strain NL63 (ZeptoMetrix Corp. #0810228CF), and human coronavirus strain 229E (ATCC® VR-740™) were used as surrogates for SARS-CoV-2. Both studies used the American Society for Testing and Materials in vitro time-kill method.ResultsAll active PVP-I formulations in study 1 demonstrated virucidal activity at 15 s, with mean log10 reduction of greater than 4.56 or greater than 99.99% inactivation; a cytotoxic effect against the National Collection of Type Cultures clone 1469 host cells was observed with 5.0% (w/v) solution, 7.5% (w/v) scrub, and 10.0% (w/v) solution. Active PVP-I formulations in study 2 demonstrated effective virucidal activity against coronaviruses in less than 15 s; log10 reduction in viral titer for each coronavirus strain was consistently higher for 10.0% (w/v) solution and 0.5% (w/v) solution versus 7.5% (w/v) scrub.ConclusionBoth studies demonstrated in vitro virucidal activity of PVP-I formulations when challenged with SARS-CoV-2 surrogate coronaviruses. Although promising, further investigations are needed to evaluate SARS-CoV-2 inactivation.

Nanostructured Copper Surface Kills ESKAPE Pathogens and Viruses in Minutes.

In the search for a fast contact-killing antimicrobial surface to break the transmission pathway of lethal pathogens, nanostructured copper surfaces were found to exhibit the desired antimicrobial properties. Compared with plain copper, these nanostructured copper surfaces with Cu(OH)2 nano-sword or CuO nano-foam were found to completely eliminate pathogens at a fast rate, including clinically isolated drug resistant species. Additionally these nanostructured copper surfaces demonstrated potential antiviral properties when assessed against bacteriophages, as a viral surrogate, and murine hepatitis virus, a surrogate for SARS-CoV-2. The multiple modes of killing, physical killing and copper ion mediated killing contribute to the superior and fast kinetics of antimicrobial action against common microbes, and ESKAPE pathogens. Prototypes for air and water cleaning with current nanostructured copper surface have also been demonstrated.

Ozone Treatment Is Insufficient to Inactivate SARS-CoV-2 Surrogate under Field Conditions.

COVID-19 caused by SARS-CoV-2 caused a worldwide crisis, highlighting the importance of preventive measures in infectious diseases control. SARS-CoV-2 can remain infectious on surfaces for up to several weeks; therefore, proper disinfection is required to mitigate the risk of indirect virus spreading. Gaseous ozone treatment has received particular attention as an easily accessible disinfection tool. In this study, we evaluated the virucidal effectiveness of gaseous ozone treatment (>7.3 ppm, 2 h) on murine hepatitis virus (MHV)-contaminated stainless-steel surface and PBS-suspended virus under field conditions at ambient (21.8%) and high (49.8–54.2%) relative humidity. Surficial virus was soiled with 0.3 g/L of BSA. Parallelly, a half-hour vaporization with 7.3% hydrogen peroxide was performed on contaminated carriers. The obtained results showed that gaseous ozone, whilst quite effective against suspended virus, was insufficient in sanitizing coronavirus contaminated surfaces, especially under low RH. Increased humidity created more favorable conditions for MHV inactivation, resulting in 2.1 log titre reduction. Vaporization with 7.3% hydrogen peroxide presented much better virucidal performance than ozonation in a similar experimental setup, indicating that its application may be more advantageous regarding gaseous disinfection of surfaces contaminated with other coronaviruses, including SARS-CoV-2.

SARS-CoV-2 causes senescence in human cells and exacerbates the senescence-associated secretory phenotype through TLR-3.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse β-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.