Calicivirus VP2 forms a portal-like assembly following receptor engagement.

To initiate infection, many viruses enter their host cells by triggering endocytosis following receptor engagement. However, the mechanisms by which non-enveloped viruses escape the endosome are poorly understood. Here we present near-atomic-resolution cryo-electron microscopy structures for feline calicivirus both undecorated and labelled with a soluble fragment of its cellular receptor, feline junctional adhesion molecule A. We show that VP2, a minor capsid protein encoded by all caliciviruses1,2, forms a large portal-like assembly at a unique three-fold axis of symmetry, following receptor engagement. This assembly-which was not detected in undecorated virions-is formed of twelve copies of VP2, arranged with their hydrophobic N termini pointing away from the virion surface. Local rearrangement at the portal site leads to the opening of a pore in the capsid shell. We hypothesize that the portal-like assembly functions as a channel for the delivery of the calicivirus genome, through the endosomal membrane, into the cytoplasm of a host cell, thereby initiating infection. VP2 was previously known to be critical for the production of infectious virus3; our findings provide insights into its structure and function that advance our understanding of the Caliciviridae.

Feline Calicivirus Proteinase-Polymerase Protein Degrades mRNAs To Inhibit Host Gene Expression.

To replicate efficiently and evade the antiviral immune response of the host, some viruses degrade host mRNA to induce host gene shutoff via encoding shutoff factors. In this study, we found that feline calicivirus (FCV) infection promotes the degradation of endogenous and exogenous mRNAs and induces host gene shutoff, which results in global inhibition of host protein synthesis. Screening assays revealed that proteinase-polymerase (PP) is a most effective factor in reducing mRNA expression. Moreover, PP from differently virulent strains of FCV could induce mRNA degradation. Further, we found that the key sites of the PP protein required for its proteinase activity are also essential for its shutoff activity but also required for viral replication. The mechanism analysis showed that PP mainly targets Pol II-transcribed RNA in a ribosome-, 5' cap-, and 3' poly(A) tail-independent manner. Moreover, purified glutathione S-transferase (GST)-PP fusion protein exhibits RNase activity in vitro in assays using green fluorescent protein (GFP) RNA transcribed in vitro as a substrate in the absence of other viral or cellular proteins. Finally, PP-induced shutoff requires host Xrn1 to complete further RNA degradation. This study provides a newly discovered strategy in which FCV PP protein induces host gene shutoff by promoting the degradation of host mRNAs. IMPORTANCE Virus infection-induced shutoff is the result of targeted or global manipulation of cellular gene expression and leads to efficient viral replication and immune evasion. FCV is a highly contagious pathogen that persistently infects cats. It is unknown how FCV blocks the host immune response and persistently exists in cats. In this study, we found that FCV infection promotes the degradation of host mRNAs and induces host gene shutoff via a common strategy. Further, PP protein for different FCV strains is a key factor that enhances mRNA degradation. An in vitro assay showed that the GST-PP fusion protein possesses RNase activity in the absence of other viral or cellular proteins. This study demonstrates that FCV induces host gene shutoff by promoting the degradation of host mRNAs, thereby introducing a potential mechanism by which FCV infection inhibits the immune response.

Strong alkaline electrolyzed water efficiently inactivates SARS-CoV-2, other viruses, and Gram-negative bacteria.

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.

Recovery Optimization and Survival of the Human Norovirus Surrogates Feline Calicivirus and Murine Norovirus on Carpet.

Carpets have been implicated in prolonged and reoccurring outbreaks of human noroviruses (HuNoV), the leading cause of acute gastroenteritis worldwide. Viral recovery from environmental surfaces, such as carpet, remains undeveloped. Our aim was to determine survival of HuNoV surrogates on an understudied environmental surface, carpet. First, we measured the zeta potential and absorption capacity of wool and nylon carpet fibers, we then developed a minispin column elution (MSC) method, and lastly we characterized the survival of HuNoV surrogates, feline calicivirus (FCV) and murine norovirus (MNV), over 60 days under 30 and 70% relative humidity (RH) on two types of carpet and one glass surface. Carpet surface charge was negative between relevant pH values (i.e., pH 7 to 9). In addition, wool could absorb approximately two times more liquid than nylon. The percent recovery efficiency obtained by the MSC method ranged from 4.34 to 20.89% and from 30.71 to 54.14% for FCV and MNV on carpet fibers, respectively, after desiccation. Overall, elution buffer type did not significantly affect recovery. Infectious FCV or MNV survived between <1 and 15 or between 3 and 15 days, respectively. However, MNV survived longer under some conditions and at significantly (P < 0.05) higher titers compared to FCV. Albeit, surrogates followed similar survival trends, i.e., both survived longest on wool then nylon and glass, while 30% RH provided a more hospitable environment compared to 70% RH. Reverse transcription-quantitative PCR signals for both surrogates were detectable for the entire study, but FCV genomic copies experienced significantly higher reductions (<3.80 log10 copies) on all surfaces compared to MNV (<1.10 log10 copies).IMPORTANCE Human noroviruses (HuNoV) are the leading cause of acute gastroenteritis worldwide. Classical symptoms of illness include vomiting and diarrhea which could lead to severe dehydration and death. HuNoV are transmitted by the fecal-oral or vomitus-oral route via person-to-person contact, food, water, and/or environmental surfaces. Published laboratory-controlled studies have documented the environmental stability of HuNoV on hard surfaces, but there is limited laboratory-based evidence available about survival on soft surfaces, e.g., carpet and upholstered furniture. Several epidemiological reports have suggested soft surfaces may be HuNoV fomites illustrating the importance of conducting a survival study. The three objectives of our research were to demonstrate techniques to characterize soft surfaces, develop a viral elution method for carpet, and characterize the survival of HuNoV surrogates on carpet. These results can be used to improve microbial risk assessments, the development of much-needed soft surface disinfectant, and standardizing protocols for future soft surface studies.

Blueberry proanthocyanidins against human norovirus surrogates in model foods and under simulated gastric conditions.

Blueberry proanthocyanidins (B-PAC) are known to decrease titers of human norovirus surrogates in vitro. The application of B-PAC as therapeutic or preventive options against foodborne viral illness needs to be determined using model foods and simulated gastric conditions in vitro. The objective of this study was to evaluate the antiviral effect of B-PAC in model foods (apple juice (AJ) and 2% reduced fat milk) and simulated gastrointestinal fluids against cultivable human norovirus surrogates (feline calicivirus; FCV-F9 and murine norovirus; MNV-1) over 24 h at 37 °C. Equal amounts of each virus (5 log PFU/ml) was mixed with B-PAC (1, 2 and 5 mg/ml) prepared either in AJ, or 2% milk, or simulated gastric fluids and incubated over 24 h at 37 °C. Controls included phosphate buffered saline, malic acid (pH 7.2), AJ, 2% milk or simulated gastric and intestinal fluids incubated with virus over 24 h at 37 °C. The tested viruses were reduced to undetectable levels within 15 min with B-PAC (1, 2 and 5 mg/ml) in AJ (pH 3.6). However, antiviral activity of B-PAC was reduced in milk. FCV-F9 was reduced by 0.4 and 1.09 log PFU/ml with 2 and 5 mg/ml B-PAC in milk, respectively and MNV-1 titers were reduced by 0.81 log PFU/ml with 5 mg/ml B-PAC in milk after 24 h. B-PAC at 5 mg/ml in simulated intestinal fluid reduced titers of the tested viruses to undetectable levels within 30 min. Overall, these results show the potential of B-PAC as preventive and therapeutic options for foodborne viral illnesses.

Antiviral effects of black raspberry (Rubus coreanus) seed extract and its polyphenolic compounds on norovirus surrogates.

Black raspberry seeds, a byproduct of wine and juice production, contain large quantities of polyphenolic compounds. The antiviral effects of black raspberry seed extract (RCS) and its fraction with molecular weight less than 1 kDa (RCS-F1) were examined against food-borne viral surrogates, murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9). The maximal antiviral effect was achieved when RCS or RCS-F1 was added simultaneously to cells with MNV-1 or FCV-F9, reaching complete inhibition at 0.1-1 mg/mL. Transmission electron microscopy (TEM) images showed enlarged viral capsids or disruption (from 35 nm to up to 100 nm) by RCS-F1. Our results thus suggest that RCS-F1 can interfere with the attachment of viral surface protein to host cells. Further, two polyphenolic compounds derived from RCS-F1, cyanidin-3-glucoside (C3G) and gallic acid, identified by liquid chromatography-tandem mass spectrometry, showed inhibitory effects against the viruses. C3G was suggested to bind to MNV-1 RNA polymerase and to enlarge viral capsids using differential scanning fluorimetry and TEM, respectively.

Interactions between Human Norovirus Surrogates and Acanthamoeba spp.

Human noroviruses (HuNoVs) are the most common cause of food-borne disease outbreaks, as well as virus-related waterborne disease outbreaks in the United States. Here, we hypothesize that common free-living amoebae (FLA)-ubiquitous in the environment, known to interact with pathogens, and frequently isolated from water and fresh produce-could potentially act as reservoirs of HuNoV and facilitate the environmental transmission of HuNoVs. To investigate FLA as reservoirs for HuNoV, the interactions between two Acanthamoeba species, A. castellanii and A. polyphaga, as well as two HuNoV surrogates, murine norovirus type 1 (MNV-1) and feline calicivirus (FCV), were evaluated. The results showed that after 1 h of amoeba-virus incubation at 25°C, 490 and 337 PFU of MNV-1/ml were recovered from A. castellanii and A. polyphaga, respectively, while only few or no FCVs were detected. In addition, prolonged interaction of MNV-1 with amoebae was investigated for a period of 8 days, and MNV-1 was demonstrated to remain stable at around 200 PFU/ml from day 2 to day 8 after virus inoculation in A. castellanii. Moreover, after a complete amoeba life cycle (i.e., encystment and excystment), infectious viruses could still be detected. To determine the location of virus associated with amoebae, immunofluorescence experiments were performed and showed MNV-1 transitioning from the amoeba surface to inside the amoeba over a 24-h period. These results are significant to the understanding of how HuNoVs may interact with other microorganisms in the environment in order to aid in its persistence and survival, as well as potential transmission in water and to vulnerable food products such as fresh produce.

A comparison of the thermal inactivation kinetics of human norovirus surrogates and hepatitis A virus in buffered cell culture medium.

Human noroviruses and hepatitis A virus (HAV) are considered as epidemiologically significant causes of foodborne disease. Therefore, studies are needed to bridge existing data gaps and determine appropriate parameters for thermal inactivation of human noroviruses and HAV. The objectives of this research were to compare the thermal inactivation kinetics of human norovirus surrogates (murine norovirus (MNV-1), and feline calicivirus (FCV-F9)) and HAV in buffered medium (2-ml vials), compare first-order and Weibull models to describe the data, calculate Arrhenius activation energy for each model, and evaluate model efficiency using selected statistical criteria. The D-values calculated from the first-order model (50-72 °C) ranged from 0.21-19.75 min for FCV-F9, 0.25-36.28 min for MNV-1, and 0.88-56.22 min for HAV. Using the Weibull model, the tD = 1 (time to destroy 1 log) for FCV-F9, MNV-1 and HAV at the same temperatures ranged from 0.10-13.27, 0.09-26.78, and 1.03-39.91 min, respectively. The z-values for FCV-F9, MNV-1, and HAV were 9.66 °C, 9.16 °C, and 14.50 °C, respectively, using the Weibull model. For the first order model, z-values were 9.36 °C, 9.32 °C, and 12.49 °C for FCV-F9, MNV-1, and HAV, respectively. For the Weibull model, estimated activation energies for FCV-F9, MNV-1, and HAV were 225, 278, and 182 kJ/mol, respectively, while the calculated activation energies for the first order model were 195, 202, and 171 kJ/mol, respectively. Knowledge of the thermal inactivation kinetics of norovirus surrogates and HAV will allow the development of processes that produce safer food products and improve consumer safety.

High hydrostatic pressure activity on the disinfection of clams artificially contaminated with feline calicivirus.

BACKGROUND: The contamination of shellfish with gastroenteric viruses may cause outbreaks because they are often eaten raw or under-cooked. High-hydrostatic pressure treatments have already proven to be effective in reducing high viral load in shellfish samples. The objectives are the assessment of the viral load reduction of contaminated clams using HHP treatments at different pressures and times and the study of the changes caused by these treatments in some food physical parameters. METHODS: Clams were contaminated with a solution containing Feline Calicivirus; they were closed in envelopes and treated with 300, 400, 500, 600 MPa for 1, 3, 5, 7 min for every pressure value. After the treatment the residual viral titre was calculated. The texture parameters were obtained after treating clams samples at the same pressure values but only for 3 and 7 min and analysing them with a TPA test. RESULTS: HHP treatments of 500 and 600 MPa were sufficient to cause a total inactivation at every timelength considered while with 300 and 400 MPa after 1 min, concentrations of 1.13 and 0.55 respectively were found. In general hardness and gumminess tend to increase after the treatment whereas springiness and cohesiveness decrease a bit. CONCLUSIONS: HHP treatments showed good sterilization ability against FCV but it's necessary to consider that FCV has a lower resistance to disinfection than Human norovirus. Texture changes are in line with what is reported in literature.

Survival and inactivation of human norovirus surrogates in blueberry juice by high-pressure homogenization.

Human noroviruses (HNoV) have been implicated in gastrointestinal outbreaks associated with fresh produce, juices, and ready-to-eat foods. In order to determine the risk of HNoV transmission by contaminated blueberry juice, survival characteristics of cultivable HNoV surrogates (murine norovirus, MNV-1; feline calicivirus, FCV-F9; and bacteriophage MS2) in blueberry juice (pH = 2.77) after 0, 1, 2, 7, 14, and 21 days at refrigeration temperatures (4°C) were studied. High-pressure homogenization (HPH) was studied as a novel processing method for noroviral surrogate inactivation in blueberry juice. Blueberry juice or phosphate-buffered saline (PBS; pH 7.2 as control) was inoculated with each virus, stored over 21 days at 4°C or subjected to HPH, and plaque assayed. FCV-F9 (∼5 log(10) PFU/mL) was undetectable after 1 day in blueberry juice at 4°C. MNV-1 (∼4 log(10) PFU/ml) showed minimal reduction (1 log(10) PFU/mL) after 14 days, with greater reduction (1.95 log(10) PFU/mL; p < 0.05) after 21 days in blueberry juice at 4°C. Bacteriophage MS2 (∼6 log(10) PFU/mL) showed significant reduction (1.93 log(10) PFU/mL; p < 0.05) after 2 days and was undetectable after 7 days in blueberry juice at 4°C. FCV-F9 remained viable in PBS for up to 21 days (2.28 log(10) PFU/mL reduction), while MNV-1 and MS2 survived after 21 days (1.08 and 0.56 log(10) PFU/mL reduction, respectively). Intriguingly, FCV-F9 and bacteriophage MS2 showed reduction after minimal homogenization pressures in blueberry juice (pH = 2.77), possibly due to the combination of juice pH, juice components, and mechanical effects. MNV-1 in blueberry juice was only slightly reduced at 250 (0.33 log(10) PFU/mL) and 300 MPa (0.71 log(10) PFU/mL). Virus surrogate survival in blueberry juice at 4°C correlates well with the ease of HNoV transmission via juices. HPH for viral inactivation in juices is dependent on virus type, and higher homogenization pressures may be needed for MNV-1 inactivation.

Antiviral effects of black raspberry (Rubus coreanus) juice on foodborne viral surrogates.

Abstract Human noroviruses (HuNoVs) are the most frequent cause of foodborne viral gastroenteritis, causing approximately 90% of non-bacterial epidemic outbreaks around the world. Rubus coreanus is a species of black raspberry, rich in polyphenols, and known to exert anti-inflammatory, antibacterial, and antiviral activities. In the present study, the antiviral effects of R. coreanus juice (black raspberry [BRB] juice) on foodborne viral surrogates, murine norovirus-1 (MNV-1) and feline calicivirus-F9 (FCV-F9), were compared with those of cranberry juice, grape juice, and orange juice by plaque assays. Among the four juices tested, BRB juice was the most effective in reducing plaques formation of these viruses. Time-of-addition experiments were designed to determine the mechanism of action of BRB juice on MNV-1 and FCV-F9. The maximal antiviral effect of BRB juice against MNV-1 was observed when it was added to RAW 264.7 cells (mouse leukemic monocyte macrophage cell line) simultaneously with the virus. Pre-treatment of either Crandell Reese Feline Kidney cells or FCV-F9 with BRB juice exhibited significant antiviral activity. The inhibition of viral infection by BRB juice on MNV-1 and FCV-F9 probably occurs at the internalization of virions into the cell or the attachment of the viral surface protein to the cellular receptor. The polyphenol components in BRB (i.e., gallic acid and quercetin), however, did not show any activity against these viruses. Our data provide great promise for the utilization of BRB in the prevention of foodborne viral outbreaks.

Inactivation of human norovirus surrogates by benzalkonium chloride, potassium peroxymonosulfate, tannic acid, and gallic acid.

Novel methods to effectively disinfect contact surfaces and prevent human norovirus transmission are essential. The effect of benzalkonium chloride (BAC), potassium peroxymonosulfate (KPMS), tannic acid (TA), and gallic acid (GA) on enteric virus surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), and bacteriophage MS2 was studied. Viruses at high (∼7 log10 PFU/mL) or low (∼5 log10 PFU/mL) titers were mixed with equal volumes of BAC (0.2, 0.5, and 1 mg/mL), KPMS (5, 10, and 20 mg/mL), TA (0.02 and 0.2 mg/mL), GA (0.2, 0.4, and 0.8 mg/mL), or water and incubated for 2 h at room temperature. Viral infectivity after triplicate treatments was evaluated using plaque assays in duplicate. Low titers of FCV-F9 and MNV-1 were completely reduced, while low-titer MS2 was reduced by 1.7-1.8 log10 PFU/mL with BAC at all three concentrations. High-titer FCV-F9 was reduced by 2.87, 3.08, and 3.25 log10 PFU/mL, and high-titer MNV-1 was reduced by 1.55, 2.32, and 2.75 log10 PFU/mL with BAC at 0.1, 0.25, and 0.5 mg/mL, respectively. High-titer MS2 was reduced by ∼2 log10 PFU/mL with BAC at all three concentrations. KPMS at all three concentrations reduced high and low titers of FCV-F9 and MS2 and low-titer MNV-1 to undetectable levels, while high-titer MNV-1 was reduced by 0.92 and 3.44 log10 PFU/mL with KMPS at 2.5 and 5 mg/mL, respectively. TA at 0.2 mg/mL only reduced high-titer FCV-F9 by 0.98 log10 PFU/mL and low-titer FCV-F9 by 1.95 log10 PFU/mL. GA at 0.1, 0.2, and 0.4 mg/mL reduced low-titer FCV-F9 by 2.50, 2.36, and 0.86 log10 PFU/mL, respectively with negligible effects against high-titer FCV-F9. BAC and KPMS show promise to be used as broad-spectrum contact surface disinfectants for prevention of noroviral surrogate contamination.

Simultaneous comparison of murine norovirus, feline calicivirus, coliphage MS2, and GII.4 norovirus to evaluate the efficacy of sodium hypochlorite against human norovirus on a fecally soiled stainless steel surface.

Free chlorine as hypochlorite is recommended to decontaminate fecally contaminated surfaces to control human norovirus (NoV). We evaluated the efficacy of sodium hypochlorite to decontaminate GII.4 NoV and three surrogates of human NoVs, feline calicivirus (FCV), murine norovirus (MNV), and coliphage MS2, on a fecally soiled stainless steel surface. Reduction of infectivity of FCV, MNV, and MS2 was measured by plaque assay and the decline of genomic copy numbers of GII.4 NoV by reverse transcriptase-polymerase chain reaction. Sodium hypochlorite solution at 5000 ppm could inactivate FCV by 3 log(10) plaque forming units after approximately 1.9 minutes of contact time, but required longer exposure times of 3.2 and 4.5 minutes to reduce MNV and MS2 by 3 log(10), respectively. However, detection of viral RNA by reverse transcriptase-polymerase chain reaction assay may not be reliable to estimate the effectiveness of sodium hypochlorite against human NoV. Of three NoV surrogates, FCV is not the most resistant of the virus tested for inactivation by hypochlorite and thus is not the worst-case model for estimating NoV inactivation. Although the use of 5000 ppm of hypochlorite for fecally soiled surfaces is effective, it may require longer exposure times of ≥3 minutes to control NoVs. Surface precleaning before hypochlorite disinfection is recommended to initially reduce the fecal organic load for better virus inactivation and should be a part of the environmental hygiene response measures during an NoV outbreak or where NoV fecal contamination of environmental surfaces is likely or suspected to be present.

Trisodium phosphate for foodborne virus reduction on produce.

Human noroviruses (NoVs) are recognized as the major cause of acute nonbacterial foodborne gastroenteritis outbreaks in both developed and developing countries. They are resistant to most chemical inactivation processes, and can survive in the environment for long periods. The aim of this research was to apply trisodium phosphate (TSP) on spiked produce (lettuce and peppers) for the reduction of foodborne NoV surrogates, feline calicivirus (FCV-F9), and murine norovirus (MNV-1). Washed and dried lettuce (3 × 3 cm²) and Jalapeno peppers (25-30 g/pepper) were spiked with FCV-F9 and MNV-1 at titers of ∼7 log10 plaque forming unit (PFU)/mL or ∼5 log10 PFU/mL and dried aseptically in a biosafety hood for 5 min. Samples were treated with 2% TSP, 5% TSP, 200 mg/L sodium hypochlorite, or water for 15 or 30 sec. Treatments were immediately neutralized with cell culture media containing 10% fetal bovine serum, and viruses were recovered and evaluated using standardized plaque assays. No significant differences between the two contact times on viral reduction was observed (p > 0.05). All three chemicals reduced FCV-F9 titers at ∼5 log10 PFU/mL to undetectable levels, but MNV-1 at ∼5 log10 PFU/mL was decreased by ∼2-3 log10 PFU/mL with 200 mg/L sodium hypochlorite and 2% TSP, and to undetectable levels by 5% TSP. FCV-F9 at ∼7 log10 PFU/mL was reduced by >5 log10 PFU/mL with 2% TSP, in comparison to 200 mg/L sodium hypochlorite that showed ≤ 1.4 log10 PFU/mL reduction. MNV-1 at ∼7 log10 PFU/mL was decreased by ∼2-3.4 log10 PFU/mL with 2% TSP; and by <1.3 log10 PFU/mL with 200 mg/L sodium hypochlorite. FCV-F9 and MNV-1 at ∼7 log10 PFU/mL were reduced to undetectable levels by 5% TSP. Treatments by 5% TSP for 30 sec did not result in any statistically significant color changes of the tested produce. TSP at 5% appears suitable as an alternative treatment to chlorine washes for NoV reduction on produce, without any noticeable visual quality changes.

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods.

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

Characterization of a continuous feline mammary epithelial cell line susceptible to feline epitheliotropic viruses.

Mucosal epithelial cells are the primary targets for many common viral pathogens of cats. Viral infection of epithelia can damage or disrupt the epithelial barrier that protects underlying tissues. In vitro cell culture systems are an effective means to study how viruses infect and disrupt epithelial barriers, however no true continuous or immortalized feline epithelial cell culture lines are available. A continuous cell culture of feline mammary epithelial cells (FMEC UCD-04-2) that forms tight junctions with high transepithelial electrical resistance (>2000Omegacm(-1)) 3-4 days after reaching confluence was characterized. In addition, it was shown that FMECs are susceptible to infection with feline calicivirus (FCV), feline herpesvirus (FHV-1), feline coronavirus (FeCoV), and feline panleukopenia virus (FPV). These cells will be useful for studies of feline viral disease and for in vitro studies of feline epithelia.

Temperature inactivation of Feline calicivirus vaccine strain FCV F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative real-time polymerase chain reaction-A novel method for predicting virus infectivity.

A one-step reverse transcription quantitative real-time polymerase chain reaction (RT-QPCR) method in combination with RNase treatment and low copy number samples was developed in order to examine the effect of temperature on the ability of virus capsids to protect their RNA content. The method was applied to a non-cultivable virus (GII.4 norovirus) and Feline calicivirus vaccine strain F-9 (FCV) which is often used as a norovirus surrogate. Results demonstrated that FCV RNA is exposed maximally after 2min at 63.3 degrees C and this correlated with a greater than 4.5log reduction in infectivity as assessed by plaque assay. In contrast human GII.4 norovirus RNA present in diluted clinical specimens was not exposed maximally until 76.6 degrees C, at least 13.3 degrees C greater than that for FCV. These data suggest that norovirus possesses greater thermostability than this commonly used surrogate. Further, these studies indicate that current food processing regimes for pasteurisation are insufficient to achieve inactivation of GII.4 NoVs. The method provides a novel molecular method for predicting virus infectivity.

Lack of influence by endosymbiont Wolbachia on virus titer in the common bed bug, Cimex lectularius.

BACKGROUND: The common bed bug, Cimex lectularius, is an obligatory blood-feeding ectoparasite that requires a blood meal to molt and produce eggs. Their frequent biting to obtain blood meals and intimate association with humans increase the potential for disease transmission. However, despite more than 100 years of inquiry into bed bugs as potential disease vectors, they still have not been conclusively linked to any pathogen or disease. This ecological niche is extraordinarily rare, given that nearly every other blood-feeding arthropod is associated with some type of human or zoonotic disease. Bed bugs rely on the bacteria Wolbachia as an obligate endosymbiont to biosynthesize B vitamins, since they acquire a nutritionally deficient diet, but it is unknown if Wolbachia confers additional benefits to its bed bug host. In some insects, Wolbachia induces resistance to viruses such as Dengue, Chikungunya, West Nile, Drosophila C and Zika, and primes the insect immune system in other blood-feeding insects. Wolbachia might have evolved a similar role in its mutualistic association with the bed bug. In this study, we evaluated the influence of Wolbachia on virus replication within C. lectularius. METHODS: We used feline calicivirus as a model pathogen. We fed 40 bed bugs from an established line of Wolbachia-cured and a line of Wolbachia-positive C. lectularius a virus-laden blood meal, and quantified the amount of virus over five time intervals post-feeding. The antibiotic rifampicin was used to cure bed bugs of Wolbachia. RESULTS: There was a significant effect of time post-feeding, as the amount of virus declined by ~90% over 10 days in both groups, but no significant difference in virus titer was observed between the Wolbachia-positive and Wolbachia-cured groups. CONCLUSIONS: These findings suggest that other mechanisms are involved in virus suppression within bed bugs, independent of the influence of Wolbachia, and our conclusions underscore the need for future research.

Annexin A2 associates to feline calicivirus RNA in the replication complexes from infected cells and participates in an efficient viral replication.

Cellular proteins have been identified to participate in calicivirus replication in association with viral proteins and/or viral RNAs. By mass spectrometry from pull-down assays, we identified several cellular proteins bound to the feline calicivirus (FCV) genomic RNA; among them the lipid raft-associated scaffold protein Annexin (Anx) A2. AnxA2 colocalizes with FCV NS6/7 protein and with the dsRNA in infected cells; moreover, it was found associated with the viral RNA in the membrane fraction corresponding to the replication complexes (RCs), suggesting its role during FCV replication. AnxA2-knockdown from CrFK cells prior to infection with FCV caused a delay in the cytopathic effect, a strong reduction of viral non-structural proteins and dsRNA production, and a decrease of FCV yield in both cell-associated and supernatant fractions. Taken together, these results indicate that AnxA2 associates to the genomic RNA of FCV and is required for an efficient FCV replication.

Evaluation of murine norovirus, feline calicivirus, poliovirus, and MS2 as surrogates for human norovirus in a model of viral persistence in surface water and groundwater.

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25 degrees C and 4 degrees C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25 degrees C (0.18 and 0.09 log(10)/day for FCV, 0.13 and 0.10 log(10)/day for PV, 0.12 and 0.06 log(10)/day for MS2, and 0.09 and 0.05 log(10)/day for MNV) but not significant at 4 degrees C. According to a multiple linear regression model, the NV NA reduction rates (0.04 +/- 0.01 log(10)/day) were not significantly different from the NA reduction rates of MS2 (0.05 +/- 0.03 log(10)/day) and MNV (0.04 +/- 0.03 log(10)/day) and were significantly different from those of FCV (0.08 +/- 0.03 log(10)/day) and PV (0.09 +/- 0.03 log(10)/day) at 25 degrees C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.