VP2 mediates the release of the feline calicivirus RNA genome by puncturing the endosome membrane of infected cells.

Feline calicivirus (FCV) is one of the few members of the Caliciviridae family that grows well in cell lines and, therefore, serves as a surrogate to study the biology of other viruses in the family. Conley et al. (14) demonstrated that upon the receptor engagement to the capsid, FCV VP2 forms a portal-like assembly, which might provide a channel for RNA release. However, the process of calicivirus RNA release is not yet fully understood. Our findings suggest that the separation of the FCV capsid from its genome RNA (gRNA) occurs rapidly in the early endosomes of infected cells. Using a liposome model decorated with the FCV cell receptor fJAM-A, we demonstrate that FCV releases its gRNA into the liposomes by penetrating membranes under low pH conditions. Furthermore, we found that VP2, which is rich in hydrophobic residues at its N-terminus, functions as the pore-forming protein. When we substituted the VP2 N-terminal hydrophobic residues, the gRNA release efficacy of the FCV mutants decreased. In conclusion, our results suggest that in the acidic environment of early endosomes, FCV VP2 functions as the pore-forming protein to mediate gRNA release into the cytoplasm of infected cells. This provides insight into the mechanism of calicivirus genome release.IMPORTANCEResearch on the biology and pathogenicity of certain caliciviruses, such as Norovirus and Sapovirus, is hindered by the lack of easy-to-use cell culture system. Feline calicivirus (FCV), which grows effectively in cell lines, is used as a substitute. At present, there is limited understanding of the genome release mechanism in caliciviruses. Our findings suggest that FCV uses VP2 to pierce the endosome membrane for genome release and provide new insights into the calicivirus gRNA release mechanism.

Feline Calicivirus P39 Inhibits Innate Immune Responses by Autophagic Degradation of Retinoic Acid Inducible Gene I.

Feline calicivirus (FCV) is a feline pathogen that can cause severe upper respiratory tract disease in cats, thus posing a major threat to their health. The exact pathogenic mechanism of FCV is still unclear, although it has been identified as having the ability to induce immune depression. In this study, we discovered that FCV infection triggers autophagy and that its non-structural proteins, P30, P32, and P39, are responsible for initiating this process. Additionally, we observed that altering autophagy levels via chemical modulation resulted in different influences on FCV replication. Moreover, our findings indicate that autophagy can modify the innate immunity induced by FCV infection, with increased autophagy further suppressing FCV-induced RIG-I signal transduction. This research provides insights into the mechanism of FCV replication and has the potential to aid in the development of autophagy-targeted drugs to inhibit or prevent FCV infection.

Radiosensitivity increase in FCV-F9 virus using combined treatments with natural antimicrobials and γ-irradiation.

AIMS: The objective was to evaluate the possible synergistic effect of cranberry juice (CJ) and commercial citrus extract (BS) against FCV-F9 viral titre in vitro in combination with γ-irradiation and to determinate the D10 values and radiosensitivity increase. METHODS AND RESULTS: Virus samples were treated with a formulation containing a mixture of BS or CJ. Results showed a D10 of 0·05, 0·42% and 1·34 kGy for the virus treated with the BS, the CJ and the irradiation alone respectively. Concentrations needed to reduce 6 log TCID50  ml-1 of viral titre were BS-0·3%, CJ-2·52% and 8·04 kGy. Irradiation combined with BS-0·01% and CJ-0·1% against FCV-F9 virus showed D10 values of 0·74 and 0·72 kGy, respectively, resulting in a viral radiosensitization of 1·28 and 1·50 for respective treatments. CONCLUSION: The higher viral radiosensitization observed after combining γ-irradiation with BS-0·01% and CJ-0·1% indicates that CJ and BS could be used as antiviral agents alone or in combination with γ-irradiation to prevent NoV outbreaks. SIGNIFICANCE AND IMPACT OF THE STUDY: Cranberry juice and BS could be used in hurdle approaches in combined treatment with γ-irradiation to assure food safety without a detrimental effect on nutritional value and maintain low processing cost.

Role of phosphate groups on antiviral activity of casein phosphopeptide against feline calicivirus as a surrogate for norovirus.

BACKGROUND: Current research on the gastrointestinal digestion of milk-casein strongly suggests the existence of novel bioactive peptides with antiviral activities that are attributable to their immunostimulatory effects. In the present study, we investigated the antiviral activity of casein peptides rich in phosphate groups, such as casein phosphopeptide (CPP-III). RESULTS: We prepared two types of CPP with different phosphorylation levels to clarify the role of the phosphate group. Further phosphorylation of CPP-III was conducted by dry heating with sodium pyrophosphate, whereas dephosphorylation was performed enzymatically using alkaline phosphatase and alkaline treatment. Feline calicivirus (FCV) strain F9, a typical norovirus surrogate, and Crandell Rees feline kidney cells were used as the target virus and host cells, respectively. Antiviral activity was determined based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and quantitative polymerase chain reaction quantification of antiviral cytokine mRNA expression. Higher cell viability was observed in the host cells treated with phosphorylated CPP-III, and a significant up-regulation of type 1 interferon expression was induced compared to that treated with native CPP-III. However, dephosphorylation of CPP-III resulted in a decrease in the anti-FCV effect. CONCLUSION: The CPP effect was enhanced by the introduction of additional phosphates and conversely weakened by their elimination. Therefore, CPP-III phosphorylation represents an emerging approach for the production of food-grade antiviral agents. © 2016 Society of Chemical Industry.

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.

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.

Virucidal effect of cold atmospheric gaseous plasma on feline calicivirus, a surrogate for human norovirus.

Minimal food-processing methods are not effective against foodborne viruses, such as human norovirus (NV). It is important, therefore, to explore novel nonthermal technologies for decontamination of foods eaten fresh, minimally processed and ready-to-eat foods, and food contact surfaces. We studied the in vitro virucidal activity of cold atmospheric gaseous plasma (CGP) against feline calicivirus (FCV), a surrogate of NV. Factors affecting the virucidal activity of CGP (a so-called radio frequency atmospheric pressure plasma jet) were the plasma generation power, the exposure time and distance, the plasma feed gas mixture, and the virus suspension medium. Exposure to 2.5-W argon (Ar) plasma caused a 5.55 log10 unit reduction in the FCV titer within 120 s. The reduction in the virus titer increased with increasing exposure time and decreasing exposure distance. Of the four plasma gas mixtures studied (Ar, Ar plus 1% O2, Ar plus 1% dry air, and Ar plus 0.27% water), Ar plus 1% O2 plasma treatment had the highest virucidal effect: more than 6.0 log10 units of the virus after 15 s of exposure. The lowest virus reduction was observed with Ar plus 0.27% water plasma treatment (5 log10 unit reduction after 120 s). The highest reduction in titer was observed when the virus was suspended in distilled water. Changes in temperature and pH and formation of H2O2 were not responsible for the virucidal effect of plasma. The oxidation of viral capsid proteins by plasma-produced reactive oxygen and nitrogen species in the solution was thought to be responsible for the virucidal effect. In conclusion, CGP exhibits virucidal activity in vitro and has the potential to combat viral contamination in foods and on food preparation surfaces.

Antiviral effect of lithium chloride on feline calicivirus in vitro.

Feline calicivirus (FCV) is a highly contagious pathogen that causes oral and upper respiratory tract disease in cats. Despite widespread vaccination, the prevalence of FCV remains high. Furthermore, a high gene mutation rate has led to the emergence of variants, and some infections are lethal. To date, there is no effective antiviral drug available for treating FCV infection. Here, we show that lithium chloride (LiCl) effectively suppresses the replication of FCV strain F9 in Crandell-Reese feline kidney (CRFK) cells. The antiviral activity of LiCl occurred primarily during the early stage of infection and in a dose-dependent manner. LiCl treatment also inhibited the cytopathic effect. LiCl treatment exhibited a strong inhibitory effect against a panel of other two reference strains and two recent FCV isolates from China. These results demonstrate that LiCl might be an effective anti-FCV drug for controlling FCV disease. Further studies are required to explore the antiviral activity of LiCl against FCV replication in vivo.

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.

Inactivation of murine norovirus and feline calicivirus during oyster fermentation.

Fermented seafood is popular in Asian countries. This study examined the survival of feline calicivirus (FCV) and murine norovirus (MNV) during oyster fermentation. Oysters spiked with FCV and MNV were fermented with 5% or 10% salt at 18 °C for 15 days, and MNV and FCV titers, lactic acid bacteria (LAB) populations, pH, and enzymatic activity were measured at 0, 1, 3, 5, 7, 10, and 15 days post-fermentation (DPF). Reductions in MNV and FCV were greater in 5% NaCl-supplemented oysters than in 10% NaCl-supplemented oysters. In 5% NaCl oysters, MNV and FCV titers significantly decreased by 1.60 log and 3.01 log, respectively, at 15 DPF. Populations of LAB increased from 3.62 log10 colony-forming units/g at 0 DPF to 8.77 log10 colony-forming units/g at 15 DPF during oyster fermentation supplemented with 5% NaCl supplementation, and the pH decreased gradually from 5.38 at 0 DPF to 4.17 at 15 DPF. During oyster fermentation, α-amylase, proteinase, and lipase were produced at higher levels in 5% salted oysters than in 10% salted oysters (P < 0.01). We concluded that many of the antimicrobial factors produced in fermented oysters could contribute to a reduction in foodborne viruses.

Differential association between circulating testosterone and infection risk by several viruses in natural cat populations: a behavioural-mediated effect?

Testosterone is involved in the development and expression of physiological, morphological and behavioural traits. High levels are often associated with high infection risk and/or intensity, suggesting a trade-off between sexual traits and immunity. Classically invoked mechanisms are immunological or behavioural, i.e., testosterone increases susceptibility or resistance to parasites via an impact on immunity or modulates behaviours involved in parasite transmission. However, studies report contrasted patterns. Given its modes of action and the diversity of host-parasite interactions, testosterone should not act similarly on all interactions. To reduce host and context diversity, we studied 3 viruses in the same cat population: the aggressively transmitted Feline Immunodeficiency virus (FIV), and the Feline Calicivirus (FCV) and Herpesvirus (FHV) both transmitted during friendly contacts. Testosterone had a strong effect on the probability of being positive to FIV whereas its effect was significantly weaker on FCV and FHV. These findings demonstrate that testosterone can be differentially associated with parasites of the same type (viruses). The difference we observed was consistent with a behavioural-mediated effect (increased aggressiveness), supporting the idea that the testosterone effect on infection risk is at least partially driven by behavioural mechanisms in our system. Further investigations (e.g., individual immunity measures) are required to confirm this hypothesis.

Nucleolin interacts with the feline calicivirus 3' untranslated region and the protease-polymerase NS6 and NS7 proteins, playing a role in virus replication.

Cellular proteins play many important roles during the life cycle of all viruses. Specifically, host cell nucleic acid-binding proteins interact with viral components of positive-stranded RNA viruses and regulate viral translation, as well as RNA replication. Here, we report that nucleolin, a ubiquitous multifunctional nucleolar shuttling phosphoprotein, interacts with the Norwalk virus and feline calicivirus (FCV) genomic 3' untranslated regions (UTRs). Nucleolin can also form a complex in vitro with recombinant Norwalk virus NS6 and -7 (NS6/7) and can be copurified with the analogous protein from feline calicivirus (p76 or NS6/7) from infected feline kidney cells. Nucleolin RNA levels or protein were not modified during FCV infection; however, as a consequence of the infection, nucleolin was seen to relocalize from the nucleoli to the nucleoplasm, as well as to the perinuclear area where it colocalizes with the feline calicivirus NS6/7 protein. In addition, antibodies to nucleolin were able to precipitate viral RNA from feline calicivirus-infected cells, indicating a direct or indirect association of nucleolin with the viral RNA during virus replication. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a reduction of the cytopathic effect and virus yield in CrFK cells. Taken together, these results demonstrate that nucleolin is a nucleolar component that interacts with viral RNA and NS6/7 and is required for feline calicivirus replication.

Inactivation of feline calicivirus and murine norovirus during Dongchimi fermentation.

Among the traditional fermented vegetables in Korea, Dongchimi is a type of kimchi with a large water base. We aimed to investigate the survival of norovirus surrogates during Dongchimi fermentation. Dongchimi spiked with feline calicivirus (FCV) or murine norovirus (MNV) was prepared following a traditional recipe. Dongchimi was initially fermented at room temperature overnight and then kept at 4 °C. The number of lactic acid bacteria, pH, acidity, and virus titer were measured 0, 1, 2, 3, 5, 7, 10, 15, 20 days after fermentation. During the fermentation process, lactic acid bacteria and acidity increased. At the end of the fermentation, population of FCV and MNV decreased about 4.12 and 1.47 log units, respectively. Based on the significant reduction of norovirus surrogate during Dongchimi fermentation, we conclude that the risk of norovirus in Dongchimi may be low.

Suppression of feline calicivirus replication using small interfering RNA targeted to its polymerase gene.

Feline calicivirus (FCV) is a pathogenic microorganism that causes upper respiratory diseases in cats. Recently, an FCV infection with a high mortality rate has been confirmed, and there is need to develop a treatment for cases of acute infection. We evaluated whether the replication of FCV could be prevented by RNA interference. For this study, we designed an siRNA targeted to the polymerase region of the strain FCV-B isolated from a cat that died after exhibiting neurological symptoms. Cells transfected with siR-pol dose-dependently suppressed the replication of FCV-B. siR-pol suppressed its replication by suppressing the target viral RNA.

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.

Feline calicivirus p32, p39 and p30 proteins localize to the endoplasmic reticulum to initiate replication complex formation.

In common with other positive-strand RNA viruses, replication of feline calicivirus (FCV) results in rearrangement of intracellular membranes and production of numerous membrane-bound vesicular structures on which viral genome replication is thought to occur. In this study, bioinformatics approaches have identified three of the FCV non-structural proteins, namely p32, p39 and p30, as potential transmembrane proteins. These proteins were able to target enhanced cyan fluorescent protein to membrane fractions where they behaved as integral membrane proteins. Immunofluorescence microscopy of these proteins expressed in cells showed co-localization with endoplasmic reticulum (ER) markers. Further electron microscopy analysis of cells co-expressing FCV p39 or p30 with a horseradish peroxidase protein containing the KDEL ER retention motif demonstrated gross morphological changes to the ER. Similar reorganization patterns, especially for those produced by p30, were observed in naturally infected Crandel-Rees feline kidney cells. Together, the data demonstrate that the p32, p39 and p30 proteins of FCV locate to the ER and lead to reorganization of ER membranes. This suggests that they may play a role in the generation of FCV replication complexes and that the endoplasmic reticulum may represent the potential source of the membrane vesicles induced during FCV infection.

In vitro inactivation of feline calicivirus (FCV) by chemical disinfectants: resistance variation among field strains.

Several commercial products commonly used for disinfection against feline calicivirus (FCV) have been validated using the vaccine strain F9. However, little information is available regarding the resistance of field strains of FCV to chemical inactivation. In this study, disinfection experiments were performed either on two laboratory-adapted strains or on seven field isolates using three alcohol mixtures and one containing a chlorine compound. For each biocide tested, considerable variability was found among the strains in their susceptibility to inactivation, demonstrating that the effectiveness of disinfection depends not only on the type of chemical compound and exposure time, but also on the FCV strain.

Inactivation of human enteric virus surrogates by high-intensity ultrasound.

Foodborne viruses, especially human noroviruses, are recognized as leading causes of nonbacterial gastroenteritis worldwide. Development of effective inactivation methods is of great importance to control their spread. In this study, the effect of high-intensity ultrasound (HIUS) on the infectivity of three foodborne virus surrogates was investigated. The three surrogates, murine norovirus (MNV-1), feline calicivirus (FCV-F9), and MS2 bacteriophage, were diluted in phosphate-buffered saline (PBS) or orange juice to a titer of approximately 6 log(10) PFU/mL or approximately 4 log(10) PFU/mL. The ultrasound treatment was performed in duplicate by immersing the HIUS probe in virus-containing solution that was cooled in ice-water and sonicated at 20 kHz for 2, 5, 10, 15, 20, and 30 min with 30 sec on and 30 sec off. The infectivity of the recovered viruses after each ultrasound treatment was evaluated in duplicate using standardized plaque assays and compared to untreated controls. The results show that HIUS effectiveness depended on the virus type, the initial titer of the viruses, and the virus suspension solution. At titers of approximately 4 log(10) PFU/mL in PBS, feline calicivirus (FCV)-F9, MS2, and murine norovirus (MNV)-1 required 5-, 10-, and 30-min treatment, respectively, for complete inactivation. At initial titers of approximately 4 log(10) PFU/mL in orange juice, FCV-F9 required a 15-min treatment for complete inactivation and only a 1.55 log(10) PFU/mL reduction was achieved for MNV-1 in orange juice after 30-min treatment. Thus, inactivation by HIUS in orange juice was much lower than in PBS. Experiments using titers of approximately 6 log(10) PFU/mL showed decreased effects compared to those using titers of approximately 4 log(10) PFU/mL. These results indicate that HIUS alone is not sufficient to inactivate virus in food. Hurdle technologies that combine HIUS with antimicrobials, heat, or pressure should be explored for viral inactivation.

Effects of famciclovir in cats with spontaneous acute upper respiratory tract disease.

OBJECTIVES: The aim of this study was to assess the effects of famciclovir administration in cats with spontaneously acquired acute upper respiratory tract disease. METHODS: Twenty-four kittens with clinical signs of acute upper respiratory tract disease were randomly allocated to receive doxycycline (5 mg/kg PO q12h) alone (group D; n = 12) or with famciclovir (90 mg/kg PO q12h; group DF; n = 12) for up to 3 weeks. Clinical disease severity was scored at study entry and daily thereafter. Oculo-oropharyngeal swabs collected at study entry and exit were assessed using quantitative PCR for nucleic acids of feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica and Mycoplasma felis. RESULTS: The median (range) age of cats was 1.5 (1-6) months in group D vs 1.6 (1-5) months in group DF (P = 0.54). Pathogens detected in oculo-oropharyngeal swabs at study entry included FCV (n = 13/24; 54%), M felis (n = 8/24; 33%), FHV-1 (n = 7/24; 29%), C felis (n = 7/24; 29%) and B bronchiseptica (n = 3/24; 12%). Median (range) duration of clinical signs was 11.5 (3-21) days in group DF and 11 (3-21) days in group D (P = 0.75). Median (range) total disease score at the end of the study did not differ between groups (group D 1 [1-1] vs group DF 1 [1-3]; P = 0.08). CONCLUSIONS AND RELEVANCE: This study revealed no significant difference in response to therapy between cats treated with doxycycline alone or with famciclovir; cats improved rapidly in both groups. However, identification of FHV-1 DNA was relatively uncommon in this study and clinical trials focused on FHV-1-infected cats are warranted to better evaluate famciclovir efficacy.

Effect of chitosan on the infectivity of murine norovirus, feline calicivirus, and bacteriophage MS2.

Chitosan is known to inhibit microorganisms of concern to plants, animals, and humans. However, the effect of chitosan on human enteric viruses of public health concern has not been extensively investigated. The purpose of this study was to determine the effect of chitosan on three human enteric viral surrogates: murine norovirus 1 (MNV-1), feline calicivirus F-9 (FCV-F9), and (ssRNA) bacteriophage MS2 (MS2). Chitosan oligosaccharide lactate (molecular weight of 5,000) and water-soluble chitosan (molecular weight of 53,000) at concentrations of 1.4, 0.7, and 0.35% were incubated at 37 degrees C for 3 h with equal volumes of each virus at high (approximately 7 log PFU/ml) and low (approximately 5 log PFU/ml) titers. Chitosan effects on each treated virus were evaluated with standardized plaque assays in comparison to untreated virus controls. The water-soluble chitosan at 0.7% decreased the FCV-F9 titer by approximately 2.83 log PFU/ml, with decreasing effects at lower concentrations, and also decreased MS2 at high titers by approximately 1.18 to 1.41 log PFU/ml, regardless of the concentration used. Chitosan treatments at the concentrations studied had no effect on MNV-1 at high titers. Chitosan oligosaccharide showed similar trends against the viruses, but to a lesser extent compared with that of water-soluble chitosan. When lower virus titers (approximately 5 log PFU/ml) were used, plaque reduction was observed for FCV-F9 and MS2, but not MNV-1. The use of higher-molecular-weight chitosan and at higher concentrations with longer incubation may be necessary to inactivate MNV-1. These results in the plaque reduction of human enteric virus surrogates by chitosan treatment show promise for its potential application in the food environment.