key: cord-0983178-lln76cbs
authors: Li, Min; Li, Jiahuan; Yang, Yunlong; Liu, Wenhui; Liang, Zhihui; Ding, Guanyu; Chen, Xiaohe; Song, Qi; Xue, Changying; Sun, Bingbing
title: Investigation of Mouse Hepatitis Virus Strain A59 Inactivation Under Both Ambient and Cold Environments Reveals the Mechanisms of Infectivity Reduction Following UVC Exposure
date: 2022-01-13
journal: J Environ Chem Eng
DOI: 10.1016/j.jece.2022.107206
sha: 7b27539dcf46b8454a588b5414b4e61fb765bbbb
doc_id: 983178
cord_uid: lln76cbs

The surface contamination of SARS-CoV-2 is becoming a potential source of virus transmission during the pandemic of COVID-19. Under the cold environment, the infection incidents would be more severe with the increase of virus survival time. Thus, the disinfection of contaminated surfaces in both ambient and cold environments is a critical measure to restrain the spread of the virus. In our study, it was demonstrated that the 254 nm ultraviolet-C (UVC) is an efficient method to inactivate a coronavirus, mouse hepatitis virus strain A59 (MHV-A59). The inactivation rate to MHV-A59 coronavirus was up to 99.99% when UVC doses were 2.90 and 14.0 mJ/cm(2) at room temperature (23 °C) and in cold environment (-20 °C), respectively. Further mechanistic study demonstrated that UVC could induce spike protein damage to partly impede virus attachment and genome penetration processes, which contributes to 12% loss of viral infectivity. Additionally, it can induce genome damage to significantly interrupt genome replication, protein synthesis, virus assembly and release processes, which takes up 88% contribution to viral inactivation. With these mechanistic understandings, it will greatly contribute to the prevention and control of the current SARS-CoV-2 transmissions in cold chains (low temperature-controlled product supply chains), public area such as airport, school, and warehouse.

The outbreak of COVID-19 has been causing a global pandemic since December 2019. It has spread across 224 countries and territories, and resulted in about 282 million confirmed cases thus far according to WHO Coronavirus (COVID-19) Dashboard [1] . In addition to the airborne transmission of the SARS-CoV-2 viruses [2] , the surface contamination has been becoming a potential issue, and several infection incidents have been reported [3] [4] [5] [6] [7] [8] . Studies have shown that viruses can remain infectious on surfaces for days [3, 9] , and surfaces of common items in daily life, e.g., plastics, glass, cold chain packaging, etc., are vulnerable to contamination by viruses [10, 11] . In cold chain industry, the whole process, i.e., production, packaging and transportation, are generally performed at lower temperatures [12] , this further created a favorable environment for the survival of SARS-CoV-2 on the contaminated surface [13] [14] [15] [16] . When items with contaminated surfaces were transported, it even facilitated the spread of virus domestically or internationally [17, 18] . Since the sampling and screening of the viruses by nucleic acid assay are quite labor intensive, the disinfection of contaminated surfaces is an effective and critical measure to restrain the spread of the virus. J o u r n a l P r e -p r o o f SARS-CoV-2 is single-stranded RNA coronavirus [19] . For disinfection, the use of liquid disinfectants, e.g., isopropyl alcohol (2-propanol) , chlorine dioxide, quaternary ammonium compounds (QACs), and hydrogen peroxide, and heat treatment, have been proved to be quite effective to reduce the infectivity of SARS-CoV-2 [20] [21] [22] [23] [24] . However, above methods have limitations, and they are not effective for the surfaces of large items, e.g., packages in cold chain, warehouse, etc. Ultraviolet-C (UVC), with radiation wavelength in the range of 200-280 nm, has been demonstrated as an efficient method for disinfection of pathogens on surfaces, air and water [25] . It could be used for large area disinfection with the advantages of high efficiency, saving energy and better maintaining the quality of frozen products compared with high-temperature treatment [26] [27] [28] [29] [30] . In addition, it is a safe inactivation without chemical residues compared with using disinfectants [29, 30] . Studies have demonstrated that UVC irradiation can restrain the viable SARS-CoV-2, and even high concentrations of viral stock at 5×10 6 Tissue Culture Infectious Dose 50% (TCID50)/mL were completely inactivated by UVC [31, 32] . However, there is no study on the effectiveness of UVC irradiation on inactivation of viruses residing in ice, and the mechanism of UVC inactivating coronavirus still remains unknown. Therefore, we herein try to investigate the disinfection ability of 254 nm UVC against a mouse coronavirus (MHV-A59), a coronavirus in the same coronavirus genus as SARS-CoV-2 [33, 34] , and further explored its inactivation mechanism. It is expected to provide help to the prevention and J o u r n a l P r e -p r o o f control of the current COVID-19 pandemic or other possible infectious pathogens. 

determination.

The irradiation system was set as Figure 1A The different UVC irradiation distances, intensities, exposure times and UVC doses used in this study were showed in Table S1 . Because exposure time is a better-controlled condition than UVC intensity, we can change exposure times (from 1 s to 120 s) among intensities to acquire a wide range of UVC doses from 0, 0.105, 0.290, 2.90, 14.0, 83.7 to 107 mJ/cm 2 .

PBS solution was added into a culture dish and frozen overnight at -20 °C.

The thickness of frozen PBS was 6 mm. The frozen PBS was removed from the culture dish and placed on the sensor of the spectrometer. The 254 nm UVC light vertically penetrated the frozen PBS, and the spectrometer sensor was placed right below the light source ( Figure 1A ). The irradiation intensities before and after the penetration of frozen PBS were compared, and the energy loss was shown in Table S2 . assay [12, 35] . Briefly, different UVC-treated virus solutions were diluted 1-10 9 folds with DMEM. In each group, 100 μL of virus solutions with different dilutions were mixed with 100 μL of 6.5×10 4 L929 cells. The CPE on L929 cells was observed and recorded. The CPE results were used to calculate virus titers by Spearman-Karber method [36] . The fractions of infectious MHV-A59 virus after UVC irradiation with different doses were calculated by using virus titers. The raw data of virus titers in different treatment groups were showed in Table S3 .

To determine the infectivity of MHV-A59 before and after UVC irradiation in cold environment, 0.1 mL of MHV-A59 stock solution (2.95×10 10 pfu/mL) was diluted 10 folds with PBS in culture dishes, the diluted virus solutions were frozen at -20 C for 0.5 h. Then, they were immediately irradiated by UVC with different doses (0, 0.105, 0.290, 2.90, 14.0, 83.7 and 107 mJ/cm 2 ) and then J o u r n a l P r e -p r o o f collected in tubes. The raw data were also shown in Table S3 .

The experimental methods to detect genome loss during virus infection process was optimized from the studies of Wigginton et al. [28] and Rattanakul & Oguma [37] . The experimental conditions such as incubation temperature, time and wash buffer used for removing MHV-A59 virus attached to the host cells were optimized, the experimental details were shown as Method S1, Table S4 and Figure S3 .

The specific details on the detection of genome loss in each step determined by pre-optimization experiments were shown as the methods section of supplementary materials (Method S1). The methods were briefly stated here.

Viruses were collected after treated by UVC (0 and 107 mJ/cm 2 ). The 

N 0 and N 1 were the detected gene copies of MHV-A59 virus treated by UVC (0 and 107 mJ/cm 2 ), respectively. The raw data were shown in Table S6 .

MHV-A59 viruses were mixed with L929 cells at a multiplicity of infection Table S4 .

N 0 ' and N 1 ' were the gene copies of MHV-A59 attached to L929 cells after treated by UVC (0 and 107 mJ/cm 2 ), respectively.

MHV lg infectivity = lg' attachment + lg' penetration + lg' replication (5) lg infectivity was total genome reduction measured by infectivity assay (TCID50 assay), it was expressed as:

N 0 ''' and N 1 ''' were the virus titers of untreated and UVC-treated MHV-A59 measured by infectivity assay, respectively (method 2.3), and the result was shown in Table S4 .

Because the loss of replication ability (lg' replication ) consisted of detected genome damage in RT-qPCR assay (lg detected damage ), undetected genome damage and unknown mechanisms during post-replication processes (lg estimated damage ):

lg' replication = lg detected damage + lg estimated damage (7) Thus, the equation 5 was reformed to the following equation:

lg infectivity = lg' attachment + lg' penetration + lg' detected damage + lg' estimated damage (8) Finally, lg estimated damage can be expressed in the following equation:

lg estimated damage = lg infectivity -lg' attachment -lg' penetration -lg' detected damage (9) lg' attachment was the corrected log of genome reduction due to the loss of attachment ability, which could be expressed as:

J o u r n a l P r e -p r o o f lg' attachment = lg attachmentlg detected damage (10) lg' penetration was the corrected log of genome reduction due to the loss of penetration ability, which could be expressed as:

lg' penetration = lg penetrationlg attachment (11) 

The values were expressed as mean ± SD. Two-tailed Student's t-test was used to determined statistical significance for analysis between two groups.

The mouse hepatitis virus, MHV-A59, was selected as a surrogate mouse virus model for SARS-CoV-2 disinfection [38] [39] [40] [41] . Both MHV-A59 virus and SARS-CoV-2 belong to beta-coronavirus, and their structure and behavior in environment was similar [33, 34] . A 254 nm UVC lamp was chosen as light source because of its high virus inactivation capability [42, 43] , the UV spectrum and irradiation system setup were shown as Figure S1A and Figure 1 , respectively. TCID50 assay was used to determine the infectivity of MHV-A59 before and after UVC treatment. The selected model virus concentration was J o u r n a l P r e -p r o o f beyond 5×10 4 gene copies/cm 2 on the surface of culture dish, which was calculated from RT-qPCR assay (Table S6 ). This titer was much higher than the possible virus load on contaminated surfaces, which was estimated to be less than 2×10 2 gene copies/cm 2 [44] [46, 47] . However, it was inhibited in cold temperature [48, 49] . Calculated by Eq S1, the UVC susceptibility constant for the MHV-A59 was 0.454 m 2 /J at room temperature, and it was 0.159 m 2 /J at -20 °C ( Figure S2 ). The former was three times as much as the UVC susceptibility constant for SARS-CoV-2, which was calculated to be about 0.135 -0.162 m 2 /J (MOI = 5) [27] . Besides, UVC dose for 90% inactivation (D90 value) of MHV-A59 and SARS-CoV-2 were 21.0 and 21.5 J/m 2 , respectively [50] . These results suggested that the UVC sensitivity for MHV-A59 is highly similar to SARS-CoV-2. All together, these results demonstrated that UVC could efficiently inactivate MHV-A59 virus at room temperature and even in cold environment, which may be supposed to be applicable for SARS-CoV-2 because the two viruses belong to beta-coronavirus and are similar in structure and UVC susceptibility.

Based on the fact that UVC could inactivate MHV-A59 coronavirus, the inactivation mechanism was further explored. It has been demonstrated that coronaviruses could infect host cells through the following steps [38, 51] Table S4 ). Two conversed gene segments (GS) were chosen for RT-qPCR assay, which were GS1 from membrane (M) protein and GS2 from nucleocapsid (N) protein, respectively. The details of gene information for RT-qPCR were shown as Table S4 . The result from GS1

showed that the lg detected damage was 1.17 ± 0.10 ( Figure 3A , calculated from Table S6 by Eq 2), meaning the viral gene number of the untreated group was 10 1.17 (14.8) folds higher than that of the UVC-treated group.

Then, UVC-treated or untreated MHV-A59 virus was mixed with L929 cells (MOI = 1). The gene loss in each key step during the virus infection could be determined by modified experiments from previous studies [28, 37] . The experimental optimization was shown as method S1 and the results were shown in the Table S4 and Figure S3 . In brief, as for attachment assay, the J o u r n a l P r e -p r o o f mixture of MHV-A59 virus and L929 cells was incubated in 1 mL of cold PBS at 4 °C for 1.5 h to ensure the attachment of viruses to the host cells. The incubation conditions were optimized to inhibit the coronaviral fusion to host cells [37, 52] . The attachment assay result was expressed by the logarithm of genome reduction (lg attachment ) (Eq 3 and Table S6 ). The lg attachment was 1.50 ± 0.01 and higher than lg attachment ( Figure 3A ). It suggested that MHV-A59 attachment was influenced by UVC treatment, and the viral gene number of the untreated group was 10 1.50 (31.6) folds higher than that of the UVC-treated group. Finally, the viruses and L929 cells were mixed in same condition as the attachment assay, but incubated at 37 °C for another 0.5 h to allow the genome penetration [52] . The viruses attached to membrane but not entering into cells were removed by washing the mixture with PBS containing 0.02% SDS, it was determined in this study (Method S1, Table S4 and Figure S3 ).

The logarithm of genome reduction in genome penetration assay (lg penetration ) was 1.70 ± 0.02 (Eq 4 and Table S6 ), and it was higher than lg attachment . It suggested that viral genome penetration process was also affected by UVC treatment, and the viral gene number of the untreated group was 10 1.70 (50.1) folds higher than that of the UVC-treated group.

The total genome reduction (lg infectivity ) was denoted by virus titer reduction of UVC-treated MHV-A59 in the infectivity assay (Eq 6 and Table S6 ), and it was 4.50 ± 0.12 ( Figure 3A ). In addition, the undetected viral genome reduction during genome replication to virus release processes (lg estimated damage ) J o u r n a l P r e -p r o o f could be estimated to be about 2.80 (Eq 7-9). Furthermore, the detected results from GS2 in each assay were similar to that from GS1 ( Figure 3A and Table S6 ).

In order to quantify how UVC treatment could affect the MHV-A59 virus infection, the loss of infectivity in each infection process was determined based on the viral genome reduction ( Figure 3B ), the calculation methods were shown as method 2.4.4 [37] . The total infectivity assay (lg infectivity ) was regarded as 100% loss of infectivity. As for the attachment process detected with GS1, the genome reduction (lg' attachment ) was the difference between attachment assay (lg attachment ) and the former RT-qPCR assay (lg detected damage ) (Eq 10), thus the lg' attachment contributed to 7.4% loss of infectivity ( Figure 3B ). As for the genome penetration, the genome reduction (lg' penetration ) was the difference between genome penetration assay (lg penetration ) and attachment assay (lg attachment ) (Eq 11). The lg' penetration contributed to 4.4% loss of infectivity ( Figure 3B ). It suggested that spike (S) proteins, which were responsible for attachment and genome penetration of coronavirus [53, 54] , were damaged by UVC. It agreed with previous study that proteins could be denatured following 254 nm UVC treatment [55, 56] . Therefore, it could be expected that S protein denaturation would be induced by UVC irradiation to inhibit viral attachment and genome penetration processes. The loss of infectivity during genome replication to virus release (lg' replication ) could be associated with detected genome damage (lg detected damage ), undetected genome damage during J o u r n a l P r e -p r o o f post-replication processes (lg estimated damage ) (Eq 7). The former (lg detected damage ) and the latter (lg estimated damage ) contributed to 26.0% and 62.2% loss of infectivity, respectively. Furthermore, the detected results from GS2 showed highly similar percentages in each assay with GS1. The sensitivity of the RT-qPCR method depends on the location and size of designed oligonucleotide sequences for genome damage [37] . A long-range PCR would be more appropriate to assess UV-induced genome damage [57] . When a short target genome segment was used, the detected genome damage may be underestimated, as shown in this study. It could be an important reason for the value of estimated genome damage contributed the higher loss of infectivity.

Based on the above data, a potential mechanism of UVC inactivation was proposed in Figure 3C . It was shown that the virus attachment and the genome penetration ability was decreased, which was probably caused by viral S protein denaturation. Besides, genome replication, protein synthesis, virus assembly and release were significantly interrupted by genome damage. The genome damage was a more important factor to induce the inactivation of MHV-A59, which agreed with previous studies that UVC inactivated virus through damaging virus genes [46] .

In our study, we demonstrated that the 254 nm UVC is an efficient method to inactivate MHV-A59 (99.99%) at room temperature ( Table S1 .

J o u r n a l P r e -p r o o f 

WHO coronavirus disease (COVID-19) dashboard

The Lancet Respiratory, COVID-19 transmission-up in the air

Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1

COVID-19 Outbreak Caused by Contaminated Packaging of Imported Cold-Chain Products

Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care

Investigating SARS-CoV-2 surface and air contamination in an acute healthcare setting during the peak of the COVID-19 pandemic in London

Role of the Healthcare Surface Environment in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Transmission and Potential Control Measures

The evidence of indirect transmission of SARS-CoV-2 reported in Guangzhou, China

Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents

Evidence of Foodborne Transmission of the Coronavirus (COVID-19) through the Animal Products Food Supply Chain

Potential role of inanimate surfaces for the spread of coronaviruses and their inactivation with disinfectant agents. Infection Prevention in Practice

Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV

Impact of meteorological factors on the COVID-19 transmission: A multi-city study in China

Impact of temperature on the dynamics of the COVID-19 outbreak in China

Virology, pathogenesis, diagnosis and in-line treatment of COVID-19

The Long-Term Presence of SARS-CoV-2 on Cold-Chain Food Packaging Surfaces Indicates a New COVID-19 Winter Outbreak: A Mini Review. Frontiers in Public Health

Frozen food: is it safe to eat during COVID-19 pandemic? Public Health

Cold-chain food contamination as the possible origin of COVID-19 resurgence in Beijing

Molecular Architecture of the SARS-CoV-2 Virus

Inactivation of Severe Acute Respiratory Coronavirus Virus 2 (SARS-CoV-2) and Diverse RNA and DNA Viruses on Three-Dimensionally Printed Surgical Mask Materials

Inactivation of coronaviruses by heat

Ethanol and isopropanol inactivation of human coronavirus on hard surfaces

Are Quaternary Ammonium Compounds, the Workhorse Disinfectants, Effective against Severe Acute Respiratory Syndrome-Coronavirus-2?

Virucidal Activity of Chlorine Dioxide Gas for Reduction of Coronavirus on Surfaces and PPE. Occupational Diseases and Environmental Medicine

Ultraviolet irradiation doses for coronavirus inactivation -review and analysis of coronavirus photoinactivation studies. GMS Hygiene and Infection Control

Ultraviolet Germicidal Irradiation Handbook: UVGI for Air and Surface Disinfection

UV-C irradiation is highly effective in inactivating SARS-CoV-2 replication

Virus inactivation mechanisms: impact of disinfectants on virus function and structural integrity

Low Cost Homemade System to Disinfect Food Items from SARS-CoV-2

Advantages and Limitations on Processing Foods by UV Light

Susceptibility of SARS-CoV-2 to UV irradiation

Effectiveness of 222-nm ultraviolet light on disinfecting SARS-CoV-2 surface contamination

Evolutionary Trajectory for the Emergence of Novel Coronavirus SARS-CoV-2. Pathogens

Molecular Evolution of Human Coronavirus Genomes

Determination of 50% endpoint titer using a simple formula

Analysis of Hydroxyl Radicals and Inactivation Mechanisms of Bacteriophage MS2 in Response to a Simultaneous Application of UV and Chlorine

Antiviral and Anti-Inflammatory Treatment with Multifunctional Alveolar Macrophage-Like Nanoparticles in a Surrogate Mouse Model of COVID-19

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology

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

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater

Predicted inactivation of viruses of relevance to biodefense by solar radiation

The use of action spectrua to determine the physical state of DNA in vivo

Longitudinal monitoring of SARS-CoV-2 RNA on high-touch surfaces in a community setting

A review of epidemic investigation on cold-chain food-mediated SARS-CoV-2 transmission and food safety consideration during COVID-19 pandemic

RNA under attack: cellular handling of RNA damage

Ultraviolet shadowing of RNA can cause significant chemical damage in seconds

Ultraviolet Radiation for Microorganism Inactivation in Wastewater

Effect of Temperature on Photoinduction

Genomic Modeling as an Approach to Identify Surrogates for Use in Experimental Validation of SARS-CoV-2 and HuNoV Inactivation by UV-C Treatment

Coronavirus infection of the central nervous system: host-virus stand-off

The avian coronavirus infectious bronchitis virus undergoes direct low-pH-dependent fusion activation during entry into host cells

Coronaviruses: an overview of their replication and pathogenesis

Cell entry mechanisms of SARS-CoV-2

Photochemical Reactions of Natural Macromolecules. Photoreactions of proteins

Proteomic analysis of UVC irradiation-induced damage of plasma proteins: Serum amyloid P component as a major target of photolysis

Long-range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Investigation and Resources. Guanyu Ding, Xiaohe Chen, Changying Xue and Qi Song: Software and Validation

Writing -Original Draft. Bingbing Sun, Changying Xue and Qi Song: Writing -Review & Editing; Bingbing Sun: Supervision and Funding acquisition

 UVC inactivation occurs either at room temperature or in cold environment

 Spike protein damage impedes virus attachment and genome penetration processes.  Genome damage interrupts further virus replication processes

 It has potentials to be applied in SARS-CoV-2 inactivation in cold chain packaging

This work was supported by the National Natural Science Foundation of 

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J o u r n a l P r e -p r o o f