Icariin, Formononetin and Caffeic Acid Phenethyl Ester Inhibit Feline Calicivirus Replication In Vitro.

Cats infected with feline calicivirus (FCV) often display oral ulcers and inflammation of the upper respiratory tract, which can lead to death in severe cases. Antiviral therapy is one of the most effective ways to control FCV infection. Natural compounds in Chinese herbal medicines and medicinal plants provide abundant resources for research on antiviral drugs. In this study, we found that icariin (ICA), formononetin (FMN) and caffeic acid phenethyl ester (CPAE) show low cytotoxicity towards F81 cells, that the three natural compounds have apparent antiviral effects on FCV in vitro, and that they can inhibit different FCV strains. Then, we found that ICA and FMN mainly function in the early stage of FCV infection, while CAPE can function in both the early and late stages of FCV infection. Finally, we found that ICA has an antagonistic effect on FMN and CAPE in FCV infection, and FMN has a synergistic effect with CAPE against FCV infection. Our results showed that ICA, FMN and CAPE may be potential drug candidates for FCV-induced diseases.

Magnolia officinalis and Its Honokiol and Magnolol Constituents Inhibit Human Norovirus Surrogates.

Norovirus is a major cause of foodborne disease and nonbacterial gastroenteritis globally. This study evaluated the antiviral effects of Magnolia officinalis extract and its honokiol and magnolol constituents against human norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV) in vitro, and in model food systems. Pretreatment or cotreatment of M. officinalis extract at 1 mg/mL reduced MNV and FCV titers by 0.6-1.8 log. Honokiol and magnolol, which are the major polyphenols in the extract, showed significant antiviral effects against MNV and FCV. The virus-infected cells that were treated with M. officinalis extract exhibited significantly increased glutathione levels (p < 0.05). The extract, honokiol, and magnolol revealed ferric ion-reducing and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities in a dose-dependent manner. Furthermore, MNV and FCV titers were reduced by >1.6 log or to undetectable levels in apple, orange, and plum juices and by 0.9 and 1.6 log in milk, respectively, when they were treated with the extract at 5 mg/mL. Therefore, the present study suggests that M. officinalis extract can be used as an antiviral food material to control norovirus foodborne diseases.

Saxifraga spinulosa-Derived Components Rapidly Inactivate Multiple Viruses Including SARS-CoV-2.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and norovirus (NV) are highly contagious pathogens that threaten human health. Here we focused on the antiviral potential of the medicinal herb, Saxifraga spinulosa (SS). Water-soluble extracts of SS were prepared, and their virus-inactivating activity was evaluated against the human virus pathogens SARS-CoV-2 and IAV; we also examined virucidal activity against feline calicivirus and murine norovirus, which are surrogates for human NV. Among our findings, we found that SS-derived gallocatechin gallate compounds were capable of inactivating all viruses tested. Interestingly, a pyrogallol-enriched fraction (Fr 1C) inactivated all viruses more rapidly and effectively than did any of the component compounds used alone. We found that 25 µg/mL of Fr 1C inactivated >99.6% of SARS-CoV-2 within 10 s (reduction of ≥2.33 log10 TCID50/mL). Fr 1C resulted in the disruption of viral genomes and proteins as determined by gel electrophoresis, electron microscopy, and reverse transcription-PCR. Taken together, our results reveal the potential of Fr 1C for development as a novel antiviral disinfectant.

In-Depth Characterization of Bioactive Extracts from Posidonia oceanica Waste Biomass.

Posidonia oceanica waste biomass has been valorised to produce extracts by means of different methodologies and their bioactive properties have been evaluated. Water-based extracts were produced using ultrasound-assisted and hot water methods and classified according to their ethanol-affinity (E1: ethanol soluble; E2: non-soluble). Moreover, a conventional protocol with organic solvents was applied, yielding E3 extracts. Compositional and structural characterization confirmed that while E1 and E3 extracts were mainly composed of minerals and lipids, respectively, E2 extracts were a mixture of minerals, proteins and carbohydrates. All the extracts showed remarkably high antioxidant capacity, which was not only related to phenolic compounds but also to the presence of proteins and polysaccharides. All E2 and E3 extracts inhibited the growth of several foodborne fungi, while only E3 extracts decreased substantially the infectivity of feline calicivirus and murine norovirus. These results show the potential of P. oceanica waste biomass for the production of bioactive extracts.

In vitro efficacy of povidone iodine and hydroxyethyl cellulose, alone and in combination, against common feline ocular pathogens.

Infectious ocular disease, such as conjunctivitis, is common in cats and can be caused by several viruses and bacteria, either as a single infection or as co-infections. In this study, povidone-iodine (PVP-I), alone or compounded with hydroxyethyl cellulose (HEC), was investigated for its efficacy against these pathogens in vitro. Whilst PVP-I alone was effective at inhibiting feline herpesvirus type 1 (FHV-1), Chlamydia felis, and Mycoplasma felis, PVP-I with HEC exerted a synergistic inhibitory effect against FHV-1 and C. felis. In contrast, only minimal inhibition of feline calicivirus was observed. These results demonstrate that PVP-I, alone and in combination with HEC, is effective against some feline ocular pathogens when tested in cell lines in vitro. In vivo studies investigating the systemic safety, ocular tolerance, and clinical efficacy of this combination in cats would be necessary before it could be recommended as a therapy in affected cats.

Inhibition of Murine Norovirus and Feline Calicivirus by Edible Herbal Extracts.

Human noroviruses (HuNoVs) cause foodborne and waterborne viral gastroenteritis worldwide. Because HuNoV culture systems have not been developed thus far, no available medicines or vaccines preventing infection with HuNoVs exist. Some herbal extracts were considered as phytomedicines because of their bioactive components. In this study, the inhibitory effects of 29 edible herbal extracts against the norovirus surrogates murine norovirus (MNV) and feline calicivirus (FCV) were examined. FCV was significantly inhibited to 86.89 ± 2.01 and 48.71 ± 7.38% by 100 µg/mL of Camellia sinensis and Ficus carica, respectively. Similarly, ribavirin at a concentration of 100 µM significantly reduced the titer of FCV by 77.69 ± 10.40%. Pleuropterus multiflorus (20 µg/mL) showed antiviral activity of 53.33 ± 5.77, and 50.00 ± 16.67% inhibition was observed after treatment with 20 µg/mL of Alnus japonica. MNV was inhibited with ribavirin by 59.22 ± 16.28% at a concentration of 100 µM. Interestingly, MNV was significantly inhibited with 150 µg/mL Inonotus obliquus and 50 µg/mL Crataegus pinnatifida by 91.67 ± 5.05 and 57.66 ± 3.36%, respectively. Treatment with 20 µg/mL Coriandrum sativum slightly reduced MNV by 45.24 ± 4.12%. The seven herbal extracts of C. sinensis, F. carica, P. multiflorus, A. japonica, I. obliquus, C. pinnatifida, and C. sativum may have the potential to control noroviruses without cytotoxicity.

Antiviral effect of theaflavins against caliciviruses.

Caliciviruses are contagious pathogens of humans and various animals. They are the most common cause of viral gastroenteritis in humans, and can cause lethal diseases in domestic animals such as cats, rabbits and immunocompromised mice. In this study, we conducted cytopathic effect-based screening of 2080 selected compounds from our in-house library to find antiviral compounds against three culturable caliciviruses: feline calicivirus, murine norovirus (MNV) and porcine sapovirus (PoSaV). We identified active six compounds, of which two compounds, both related to theaflavins, showed broad antiviral activities against all three caliciviruses; three compounds (abamectin, a mixture of avermectin B1a and B1b; avermectin B1a; and (-)-epigallocatechin gallate hydrate) were effective against PoSaV only; and a heterocyclic carboxamide derivative (BFTC) specifically inhibited MNV infectivity in cell cultures. Further studies of the antiviral mechanism and structure-activity relationship of theaflavins suggested the following: (1) theaflavins worked before the viral entry step; (2) the effect of theaflavins was time- and concentration-dependent; and (3) the hydroxyl groups of the benzocycloheptenone ring were probably important for the anti-calicivirus activity of theaflavins. Theaflavins could be used for the calicivirus research, and as potential disinfectants and antiviral reagents to prevent and control calicivirus infections in animals and humans.

Curcumin-Mediated Photodynamic Inactivation of Norovirus Surrogates.

Photodynamic inactivation (PDI) is extensively used to inactivate different type of pathogens through the use of photosensitizers (PS). Curcumin has been identified as an excellent natural photosensitizer with some potential applications in the food industry. The aim of this study was to assess the antiviral activity of photoactivated curcumin on norovirus surrogates, feline calicivirus (FCV), and murine norovirus (MNV). Initially, different concentrations of curcumin (13.5-1358 µM) were individually mixed with each virus at titers of ca. 6-7 log TCID50/ml and photoactivated by LED blue light with light dose of 3 J/cm2. Results showed that photoactivated curcumin at 50 µg/mL reduced FCV titers by almost 5 log after incubation at 37 °C for 30 min. Lower antiviral activity (0.73 log TCID50/mL reduction) was reported for MNV. At room temperature, curcumin at 5 µg/mL reduced FCV titers by 1.75 log TCID50/mL. These results represent a step forward in improving food safety using photoactivated curcumin as an alternative natural additive to reduce viral contamination.

In vitro antiviral effect of germacrone on feline calicivirus.

Feline calicivirus (FCV) often causes respiratory tract and oral disease in cats and is a highly contagious virus. Widespread vaccination does not prevent the spread of FCV. Furthermore, the low fidelity of the RNA-dependent RNA polymerase of FCV leads to the emergence of new variants, some of which show increased virulence. Currently, few effective anti-FCV drugs are available. Here, we found that germacrone, one of the main constituents of volatile oil from rhizoma curcuma, was able to effectively reduce the growth of FCV strain F9 in vitro. This compound exhibited a strong anti-FCV effect mainly in the early phase of the viral life cycle. The antiviral effect depended on the concentration of the drug. In addition, germacrone treatment had a significant inhibitory effect against two other reference strains, 2280 and Bolin, and resulted in a significant reduction in the replication of strains WZ-1 and HRB-SS, which were recently isolated in China. This is the first report of antiviral effects of germacrone against a calicivirus, and extensive in vivo research is needed to evaluate this drug as an antiviral therapeutic agent for FCV.

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.

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.

Expression of antiviral cytokines in Crandell-Reese feline kidney cells pretreated with Korean red ginseng extract or ginsenosides.

The antiviral activity and protective mechanism of Korean red ginseng (KRG) is not well understood. The aim of this study was to investigate the protective mechanism of KRG extract and ginsenosides against feline calicivirus (FCV), a human norovirus surrogate. CRFK cells that were pretreated for 48h with 10µg/mL of KRG extract or purified ginsenoside Rb1 or Rg1, were inoculated with FCV. RNA extracted from each treated group was examined for the expression of antiviral cytokines, including interferon-α (IFN-α), interferon-ß (IFN-ß), interferon-ω (IFN-ω), Mx, and zinc finger antiviral protein shorter isoform (ZAPS), by relative real-time reverse transcription-polymerase chain reaction. mRNA expression of IFN-α, IFN-ß, IFN-ω, Mx, and ZAPS was significantly induced in the FCV-challenged group pretreated with the KRG extract or ginsenosides, and it was higher than the group treated with FCV alone. Mx protein expression was confirmed by western blotting of CRFK cells pretreated with the ginsenoside Rb1 or with Rg1. Induction of antiviral cytokines contributes to the reduction of the viral titer in CRFK cells pretreated with the KRG extract and purified ginsenosides. In future studies, the antiviral protective mechanism of KRG should be demonstrated using other viruses such as human norovirus.

Enzymatic synthesis of novel phenol acid rutinosides using rutinase and their antiviral activity in vitro.

Novel rutinosides of vanillic acid, sinapic acid, ferulic acid, and caffeic acid were prepared via a rutinase-catalyzed transglycosylation reaction. Reaction mixtures containing rutin, phenolic acid, and rutinase derived from tartary buckwheat ( Fagopyrum tataricum ) seeds were incubated in 20 mM acetate buffer (pH 5.0) at 40 °C. The resulting rutinoside of each phenolic acid was purified by HPLC, and the structure was determined by NMR and FAB-MS analysis. Antiviral activity was determined using feline calicivirus (FCV) strain F9, which is a typical norovirus surrogate. It was found that rutinosylation of the phenolic acids increased their antiviral activity against FCV, with the sinapic acid rutinoside being the most effective. These results will contribute to the development of antiviral agents against noroviruses.

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.

Ozone inactivation of norovirus surrogates on fresh produce.

Preharvest contamination of produce by foodborne viruses can occur through a variety of agents, including animal feces/manures, soil, irrigation water, animals, and human handling. Problems of contamination are magnified by potential countrywide distribution. Postharvest processing of produce can involve spraying, washing, or immersion into water with disinfectants; however, disinfectants, including chlorine, have varying effects on viruses and harmful by-products pose a concern. The use of ozone as a disinfectant in produce washes has shown great promise for bacterial pathogens, but limited research exists on its efficacy on viruses. This study compares ozone inactivation of human norovirus surrogates (feline calicivirus [FCV] and murine norovirus [MNV]) on produce (green onions and lettuce) and in sterile water. Green onions and lettuce inoculated with FCV or MNV were treated with ozone (6.25 ppm) for 0.5- to 10-min time intervals. Infectivity was determined by 50% tissue culture infectious dose (TCID(50)) and plaque assay for FCV and MNV, respectively. After 5 min of ozone treatment, >6 log TCID(50)/ml of FCV was inactivated in water and ∼2-log TCID(50)/ml on lettuce and green onions. MNV inoculated onto green onions and lettuce showed a >2-log reduction after 1 min of ozone treatment. The food matrix played the largest role in protection against ozone inactivation. These results indicate that ozone is an alternative method to reduce viral contamination on the surface of fresh produce.

Virucidal efficacy of four new disinfectants.

Virucidal efficacy was evaluated for four recently available disinfectants: chlorine dioxide, potassium peroxymonosulfate, a quaternary ammonium compound, and citricidal (grapefruit extract). Sodium hypochlorite (3%) and tap water were used as positive and negative controls respectively. Feline herpesvirus, feline calicivirus, and feline parvovirus were exposed to the manufacturers' recommended dilutions of the evaluated disinfectants. Both chlorine dioxide and potassium peroxymonosulfate completely inactivated the three viruses used in this study. These disinfectants can aid in controlling nosocomial transmission of viruses with less of the deleterious effects of sodium hypochlorite. The quaternary ammonium compound evaluated in this study and citricidal were not effective against feline calicivirus and feline parvovirus.