SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

Antiviral effects of grape seed extract against feline calicivirus, murine norovirus, and hepatitis A virus in model food systems and under gastric conditions.

Grape seed extract (GSE) has antiviral activities against hepatitis A virus (HAV) and human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)). The objectives of this study were to determine (1) time and dose-dependence of GSE against FCV-F9, MNV-1, and HAV at room temperature (RT) and 37 °C over 24 h; and (2) GSE effects in model foods (apple juice (AJ) and 2% milk) and simulated gastric conditions at 37 °C. Viruses at ∼5 log PFU/ml were treated with 0.5-8 mg/ml GSE prepared in water, AJ, milk or gastric juices, or water over 24 h at RT or 37 °C. Infectivity of triplicate treatments was evaluated using plaque assays. GSE effects increased with time and concentration. GSE at 1 mg/ml in AJ reduced MNV-1 to undetectable levels after 1 h and by 1 log in milk after 24 h. GSE at 1 and 2 mg/ml in AJ reduced HAV to undetectable levels after 1 h, while 2 and 4 mg/ml GSE in milk caused ∼1 log reduction after 24 h. GSE at 2 mg/ml in intestinal fluid reduced FCV-F9, MNV-1 and HAV to undetectable levels after 6 h. GSE appears to be a suitable natural option for foodborne viral reduction.

The Inhibitory Effect of Resveratrol from Reynoutria japonica on MNV-1, a Human Norovirus Surrogate.

As a natural nonflavonoid polyphenol compound, resveratrol is the main functional component of Reynoutria japonica and has anti-inflammatory, antioxidant, antiviral, and other physiological activities. In this study, the effect of resveratrol on the viability of RAW264.7 cells was examined, and murine norovirus (MNV-1) was used as a surrogate for human norovirus to evaluate the inhibitory effect of resveratrol. The concentrations of resveratrol resulting in 50% cytotoxicity (CC50) for RAW264.7 cells were 21.32 and 24.97 µg/mL after 24 and 48 h of incubation, respectively, and resveratrol at a concentration lower than the half-effective inhibitory concentration (EC50) could not damage cell DNA. The EC50 of resveratrol on MNV-1 in infected RAW264.7 cells was determined to equal 5.496 µg/mL. After RAW264.7 cells, virus, and a fresh mixture of virus and RAW264.7 cells were treated with resveratrol solution for 1 h (denoted cell pre-treatment, virus pre-treatment, and mixture coprocessing), the RAW264.7 cells obtained after cell pre-treatment exhibited lower virus infection, and MNV-1 obtained after virus pre-treatment and mixture coprocessing showed a decreased infectious capacity. The inhibition ratio of resveratrol on MNV-1 did not significantly differ between the treatments at 4 and 25 °C or among the various pH values except for the lower acidic condition (pH 2). TEM revealed significant changes in the morphology of MNV-1 after treatment with resveratrol, and molecular docking indicated that resveratrol strongly binds to the viral capsid protein of MNV-1. In addition, resveratrol regulated the expression of cytokine that protects against MNV-1 infection. Therefore, at a lower concentration, resveratrol, a natural component from Reynoutria japonica, exerts an inhibitory effect on MNV-1 growth and could be used as a safe additive in food products to improve the nutritional status and control norovirus.

Bioaccumulation Dynamic by Crassostrea gigas Oysters of Viruses That Are Proposed as Surrogates for Enteric Virus Contamination in Environmental Samples.

Oysters are filter-feeders and retain sewage-derived pathogens in their organs or tissues. Since most enteric viruses involved in outbreaks cannot grow in cell culture, studies using viral surrogate models are essential. Some species are proposed as surrogates for enteric viruses in environmental samples, including in bivalve mollusk samples, such as murine norovirus type 1 (MNV-1) and somatic (as φX) or F-specific coliphages (as MS2) bacteriophages. This study evaluated the tissue distribution of viral surrogates for enteric virus contamination after their bioaccumulation by Crassostrea gigas. Oyster tissues were analyzed for the distribution of viral surrogates (MNV-1, φX-174, and MS2) in digestive tissue (DT), gills (GL), and mantle (MT) after 4, 6, and 24 h of experimental bioaccumulation. MNV-1 had higher counts at 6 h in DT (1.2 × 103 PFU/g), followed by GL and MT (9.5 × 102 and 3.8 × 102 PFU/g, respectively). The bacteriophage φX-174 had a higher concentration in the MT at 4 and 6 h (3.0 × 102 PFU/g, in both) and MS2 in the GL after 24 h (2.2 × 102 PFU/g). The bioaccumulation pattern of MNV-1 by oysters was similar to the other enteric viruses (more in DT), while that of phages followed distinct patterns from these. Since the MNV-1 is bioaccumulated by C. gigas and is adapted to grow in cell culture, it is an important tool for bioaccumulation and viral inactivation tests in oysters. Although bacteriophage bioaccumulation was not similar to enteric viruses, they can be indicated for viral bioaccumulation analysis, analyzing MT and GL, since they do not bioaccumulate in DT.

Simulation of contamination and elimination of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) from the washing process when handling of potatoes.

Root vegetables, which are in close contact with soil, are particularly vulnerable to soil contamination or decay as they can be contaminated from multiple sources, including primary production and processing. This study investigated effective washing conditions to reduce the microbial contamination of potatoes by using soaking and shaking in the washing process. The reduction of Escherichia coli, Listeria monocytogenes, and Murine norovirus 1 (MNV-1) in four washing processes (soaking only, shaking only, combined soaking-shaking I, and combined soaking-shaking I-shaking II) were compared. The numbers of E. coli and L. monocytogenes decreased by 0.55 and 0.49 log CFU/g after shaking only, 1.96 and 1.80 log CFU/g after soaking, 2.07 and 1.67 log CFU/g after soaking-shaking I, and 2.42 and 1.90 log CFU/g after soaking-shaking I-shaking II, respectively. The combined process reduced the microbial contamination more efficiently than shaking only. The reduction of E. coli in the washing process was higher than that of L. monocytogenes by approximately 0.5 logs. MNV-1 showed a reduction in the soaking and shaking steps by 1.34 and 1.98 log GC/100 g, with no significant reduction observed after the combination process. A combined process of soaking-shaking I-shaking II was effective to eliminate E. coli, L. monocytogenes, and MNV-1 from potatoes during the handling and washing process.

Impact of chlorine dioxide and electron-beam irradiation for the reduction of murine norovirus in low-salted "jogaejeotgal", a traditional Korean salted and fermented clam.

Clam jeotgal, called "jogaejeotgal," is a Korean fermented seafood product with, generally, a high amount of added salt to inhibit the growth of pathogenic microorganisms. This study aimed to evaluate the efficacy of chlorine dioxide (ClO2) and sodium hypochlorite (NaOCl) against murine norovirus 1 (MNV-1), a surrogate for human norovirus, in salt-fermented clam, jogaejeotgal. The sequential effect of ClO2 and electron-beam (e-beam) irradiation on the inactivation of MNV-1 was also investigated. Treatments of up to 300 ppm ClO2 and 1000 ppm NaOCl were used to determine the disinfectant concentrations at which more than 1 log (90%) MNV-1 inactivation occurred. The sequential treatment of ClO2 (50-300 ppm) and e-beam (1-5.5 kGy) was performed after storage at 4 °C for 7 days. There was a 1.9-log reduction of the virus in seasoned clams irradiated at 5.5 kGy after ClO2 treatment at 300 ppm. No significant change (p > 0.05) in physicochemical quality was observed after the combined treatment, suggesting the potential for the use of a combined treatment using ClO2 (300 ppm) and e-beam (5.5 kGy) in the jeotgal manufacturing industry for the reduction of norovirus.

Antiviral Effects of Lindera obtusiloba Leaf Extract on Murine Norovirus-1 (MNV-1), a Human Norovirus Surrogate, and Potential Application to Model Foods.

Noroviruses are the leading cause of acute gastroenteritis and food poisoning worldwide. In this study, we investigated the anti-noroviral activity of Lindera obtusiloba leaf extract (LOLE) using murine norovirus (MNV-1), a surrogate of human norovirus. Preincubation of MNV-1 with LOLE at 4, 8, or 12 mg/mL for 1 h at 25 °C significantly reduced viral infectivity, by 51.8%, 64.1%, and 71.2%, respectively. Among LOLE single compounds, ß-pinene (49.7%), α-phellandrene (26.2%), and (+)-limonene (17.0%) demonstrated significant inhibitory effects on viral infectivity after pretreatment with MNV-1, suggesting that the anti-noroviral effects of LOLE may be due to the synergetic activity of several compounds, with ß-pinene as a key molecule. The inhibitory effect of LOLE was tested on the edible surfaces of lettuce, cabbage, and oysters, as well as on stainless steel. After one hour of incubation at 25°C, LOLE (12 mg/mL) pretreatment significantly reduced MNV-1 plaque formation on lettuce (76.4%), cabbage (60.0%), oyster (38.2%), and stainless-steel (62.8%). These results suggest that LOLE effectively inhibits norovirus on food and metal surfaces. In summary, LOLE, including ß-pinene, may inactivate norovirus and could be used as a natural agent promoting food safety and hygiene.

Synergistic Effects of Combined Chlorine and Vitamin B1 on the Reduction of Murine Norovirus-1 on the Oyster (Crassostrea gigas) Surface.

This study investigated the synergistic effects of combined chlorine (200, 500, 700, and 1000 ppm) and vitamin B1 (1000, 2000, and 3000 ppm) on the murine norovirus-1 (MNV-1), a human norovirus (NoV) surrogate, on oyster surface. Vitamin B1 slightly reduced MNV-1 (0.04-0.3 log-reduction), whereas chlorine significantly reduced MNV-1 (0.4-1.0 log-reduction). The combined chlorine and vitamin B1 resulted in a 0.52-1.97 log-reduction of MNV-1. The synergistic reduction in the MNV titer was not dependent on the concentrations of chlorine and vitamin B1, and it ranged between 0.08 and 1.03 log10 PFU/mL. The largest synergistic reduction observed was for the combined 700 ppm chlorine and 1000 ppm vitamin B1. The pH and mechanical texture of the oysters were not significantly changed by the combined 0-1000 ppm chlorine and 3000 ppm vitamin B1. The overall sensory acceptability were significantly (P < 0.05) reduced in oysters treated with 1000 ppm chlorine and 3000 ppm vitamin B1 than in those treated with 0-700 ppm chlorine and 3000 ppm vitamin B1. This study suggests that the combined 700 ppm chlorine and 3000 ppm vitamin B1 could potentially be used to reduce NoV on oyster surface without causing concomitant changes in the mechanical texture, pH, or sensory qualities of the oysters.

Survivin Overexpression Has a Negative Effect on Feline Calicivirus Infection.

It is known that levels of the anti-apoptotic protein survivin are reduced during Murine norovirus MNV-1 and Feline calicivirus (FCV) infection as part of the apoptosis establishment required for virus release and propagation in the host. Recently, our group has reported that overexpression of survivin causes a reduction of FCV protein synthesis and viral progeny production, suggesting that survivin may affect early steps of the replicative cycle. Using immunofluorescence assays, we observed that overexpression of survivin, resulted in the reduction of FCV infection not only in transfected but also in the neighboring nontransfected CrFK cells, thus suggesting autocrine and paracrine protective effects. Cells treated with the supernatants collected from CrFK cells overexpressing survivin showed a reduction in FCV but not MNV-1 protein production and viral yield, suggesting that FCV binding and/or entry were specifically altered. The reduced ability of FCV to bind to the surface of the cells overexpressing survivin, or treated with the supernatants collected from these cells, correlate with the reduction in the cell surface of the FCV receptor, the feline junctional adhesion molecule (fJAM) 1, while no effect was observed in the cells transfected with the pAm-Cyan vector or in cells treated with the corresponding supernatants. Moreover, the overexpression of survivin affects neither Vaccinia virus (VACV) production in CrFK cells nor MNV-1 virus production in RAW 267.4 cells, indicating that the effect is specific for FCV. All of these results taken together indicate that cells that overexpress survivin, or cell treatment with the conditioned medium from these cells, results in the reduction of the fJAM-1 molecule and, therefore, a specific reduction in FCV entry and infection.

Differences in the viability of murine norovirus in different aquatic locations.

Norovirus is detected from shellfish and environmental water more frequently in winter than in other seasons. However, there is no report regarding its viability in actual seawater in situ. We investigated the viability of murine norovirus strain 1 (MNV-1), a surrogate for human norovirus, in 2 types of aquatic locations, a seawater pool carrying oceanic water and inner bay carrying brackish water. Sterilized seawater was inoculated with MNV-1 and enclosed in dialysis tubes, which were placed at the 2 locations. MNV-1 exhibited higher level of viability in brackish than in oceanic water. Factors that influenced the viability of MNV-1 included salt concentration as well as temperature of the seawater. Therefore, based on our findings, coastal brackish water that is routinely used for harvesting or cleaning seafood at fishing ports may promote the viability of norovirus.

Murine norovirus (MNV-1) exposure in vitro to the purine nucleoside analog Ribavirin increases quasispecies diversity.

Ribavirin is a pharmaceutical antiviral used for the treatment of RNA virus infections including norovirus, hepatitis C virus, hepatitis E virus, Lassa virus, respiratory syncytial virus, and rhinovirus. Despite the drug's history and documented efficacy, the antiviral mechanism of Ribavirin remains unclear. Mechanisms proposed include depletion of the intracellular GTP pool, immunomodulatory effects, induction of error catastrophe, inhibition of viral polymerase activity, and/or inhibition of viral capping. In the present study, we leveraged deep sequencing data to demonstrate that Ribavirin increases murine norovirus (MNV-1) viral diversity. By serial passaging MNV-1 in RAW 264.7 cells for twenty generations in the presence of Ribavirin, we demonstrated statistically significant increases in both the number of unique haplotypes and the average pairwise difference (APD). Based on statistically significant differences in the probability of nucleotide mutations based on Roche 454 sequencing, we also demonstrated that single nucleotide substitutions are increased in the presence of Ribavirin. Finally, we demonstrated Ribavirin's impact on statistically significantly reducing the relative proportion of the dominant sequence within the quasispecies.

Virucidal efficacy of treatment with photodynamically activated curcumin on murine norovirus bio-accumulated in oysters.

Norovirus (NoV) is one of the most important seafood- and water-borne viruses, and is a major cause of acute gastroenteritis outbreaks. In the present study we investigated the effect of curcumin as a sensitizer to photodynamic treatment both in buffer and in oysters against murine norovirus 1 (MNV-1), a surrogate of NoV. MNV-1 cultured in buffer and MNV-1 bio-accumulated in oysters were irradiated with a novel LED light source with a wavelength of 470nm and an energy of 3.6J/cm(2). Inactivation of MNV-1 was investigated by plaque assays. After virus was extracted from the gut of oysters treated over a range of curcumin concentrations, the ultrastructural morphology of the virus was observed using electron microscopy, and the integrity of viral nucleic acids and stability of viral capsid proteins were also determined. Results showed that the infectivity of MNV-1 was significantly inhibited by 1-3logPFU/ml, with significant damage to viral nucleic acids in a curcumin dose-dependent manner after photodynamic activation. Virus morphology was altered after the photodynamic treatment with curcumin, presumably due to the change of the viral capsid protein structures. The data suggest that treatment of oysters with photodynamic activation of curcumin is a potentially efficacious and cost-effective method to inactivate food-borne NoV. Further studies are necessary to evaluate the toxicology of this approach in detail and perform sensory evaluation of the treated product.

Evaluation of the swab sampling method to recover viruses from fomites.

The monitoring of virus contamination on fomites, especially at hospitals has been used for a more effective evaluation of the microbiological quality of surfaces. Swab sampling is the method used currently, although the use of an internal control process (ICP) has not yet been assessed. The aim of this study is to determine the recovery rate of murine norovirus 1 (MNV-1) and bacteriophage PP7 on different surfaces in order to assess their potential use as an ICP. For this purpose both viruses were spiked experimentally both on porous and non-porous formic as well as on rubberized surfaces. Quantitative PCR (qPCR) showed a variable efficiency with a percentage recovery ranging from 0.6 to 77% according to viruses and surfaces. A global analysis suggested that MNV-1 could be used as a potential ICP for the swab sampling method.

Solar water disinfection (SODIS): Impact on hepatitis A virus and on a human Norovirus surrogate under natural solar conditions.

This study evaluates the effectiveness of solar water disinfection (SODIS) in the reduction and inactivation of hepatitis A virus (HAV) and of the human Norovirus surrogate, murine Norovirus (MNV-1), under natural solar conditions. Experiments were performed in 330 ml polyethylene terephthalate (PET) bottles containing HAV or MNV-1 contaminated waters (10(3) PFU/ml) that were exposed to natural sunlight for 2 to 8 h. Parallel experiments under controlled temperature and/or in darkness conditions were also included. Samples were concentrated by electropositive charged filters and analysed by RT-real time PCR (RT-qPCR) and infectivity assays. Temperature reached in bottles throughout the exposure period ranged from 22 to 40ºC. After 8 h of solar exposure (cumulative UV dose of ~828 kJ/m2 and UV irradiance of ~20 kJ/l), the results showed significant (P<0.05) reductions from 4.0 (+/-0.56)x10(4) to 3.15 (+/-0.69)x10(3) RNA copies/100ml (92.1%, 1.1 log) for HAV and from 5.91 (+/-0.59)x10(4) to 9.24 (+/-3.91)x10(3) RNA copies/100 ml (84.4%, 0.81 log) for MNV-1. SODIS conditions induced a loss of infectivity between 33.4% and 83.4% after 4 to 8 h in HAV trials, and between 33.4% and 66.7% after 6 h to 8 h in MNV-1 trials. The results obtained indicated a greater importance of sunlight radiation over the temperature as the main factor for viral reduction.

Aqueous Extracts of Hibiscus sabdariffa Calyces Decrease Hepatitis A Virus and Human Norovirus Surrogate Titers.

Hibiscus sabdariffa extract is known to have antioxidant, anti-diabetic, and antimicrobial properties. However, their effects against foodborne viruses are currently unknown. The objective of this study was to determine the antiviral effects of aqueous extracts of H. sabdariffa against human norovirus surrogates (feline calicivirus (FCV-F9) and murine norovirus (MNV-1)) and hepatitis A virus (HAV) at 37 °C over 24 h. Individual viruses (~5 log PFU/ml) were incubated with 40 or 100 mg/ml of aqueous hibiscus extract (HE; pH 3.6), protocatechuic acid (PCA; 3 or 6 mg/ml, pH 3.6), ferulic acid (FA; 0.5 or 1 mg/ml; pH 4.0), malic acid (10 mM; pH 3.0), or phosphate buffered saline (pH 7.2 as control) at 37 °C over 24 h. Each treatment was replicated thrice and plaque assayed in duplicate. FCV-F9 titers were reduced to undetectable levels after 15 min with both 40 and 100 mg/ml HE. MNV-1 was reduced by 1.77 ± 0.10 and 1.88 ± 0.12 log PFU/ml after 6 h with 40 and 100 mg/ml HE, respectively, and to undetectable levels after 24 h by both concentrations. HAV was reduced to undetectable levels by both HE concentrations after 24 h. PCA at 3 mg/ml reduced FCV-F9 titers to undetectable levels after 6 h, MNV-1 by 0.53 ± 0.01 log PFU/ml after 6 h, and caused no significant change in HAV titers. FA reduced FCV-F9 to undetectable levels after 3 h and MNV-1 and HAV after 24 h. Transmission electron microscopy showed no conclusive results. The findings suggest that H. sabdariffa extracts have potential to prevent foodborne viral transmission.

Transfer of noroviruses between fingers and fomites and food products.

Human norovirus (NoV) contaminated hands are important routes for transmission. Quantitative data on transfer during contact with surfaces and food are scarce but necessary for a quantitative risk assessment. Therefore, transfer of MNV1 and human NoVs GI.4 and GII.4 was studied by artificially contaminating human finger pads, followed by pressing on stainless steel and Trespa® surfaces and also on whole tomatoes and cucumber slices. In addition, clean finger pads were pressed on artificially contaminated stainless steel and Trespa® surfaces. The transfers were performed at a pressure of 0.8-1.9 kg/cm(2) for approximately 2s up to 7 sequential transfers either to carriers or to food products. MNV1 infectivity transfer from finger pads to stainless steel ranged from 13 ± 16% on the first to 0.003 ± 0.009% on the sixth transfer on immediate transfer. After 10 min of drying, transfer was reduced to 0.1 ± 0.2% on the first transfer to 0.013 ± 0.023% on the fifth transfer. MNV1 infectivity transfer from stainless steel and Trespa® to finger pads after 40 min of drying was 2.0 ± 2.0% and 4.0 ± 5.0% respectively. MNV1 infectivity was transferred 7 ± 8% to cucumber slices and 0.3 ± 0.5% to tomatoes after 10 min of drying, where the higher transfer to cucumber was probably due to the higher moisture content of the cucumber slices. Similar results were found for NoVs GI.4 and GII.4 transfers measured in PCR units. The results indicate that transfer of the virus is possible even after the virus is dried on the surface of hands or carriers. Furthermore, the role of fingers in transmission of NoVs was quantified and these data can be useful in risk assessment models and to establish target levels for efficacy of transmission intervention methods.