Feline calicivirus strain 2280 p30 antagonizes type I interferon-mediated antiviral innate immunity through directly degrading IFNAR1 mRNA.

Feline calicivirus (FCV) belongs to the Caliciviridae, which comprises small RNA viruses of both medical and veterinary importance. Once infection has occurred, FCV can persist in the cat population, but the molecular mechanism of how it escapes the innate immune response is still unknown. In this study, we found FCV strain 2280 to be relatively resistant to treatment with IFN-ß. FCV 2280 infection inhibited IFN-induced activation of the ISRE (Interferon-stimulated response element) promoter and transcription of ISGs (Interferon-stimulated genes). The mechanistic analysis showed that the expression of IFNAR1, but not IFNAR2, was markedly reduced in FCV 2280-infected cells by inducing the degradation of IFNAR1 mRNA, which inhibited the phosphorylation of downstream adaptors. Further, overexpression of the FCV 2280 nonstructural protein p30, but not p30 of the attenuated strain F9, downregulated the expression of IFNAR1 mRNA. His-p30 fusion proteins were produced in Escherichia coli and purified, and an in vitro digestion assay was performed. The results showed that 2280 His-p30 could directly degrade IFNAR1 RNA but not IFNAR2 RNA. Moreover, the 5'UTR of IFNAR1 mRNA renders it directly susceptible to cleavage by 2280 p30. Next, we constructed two chimeric viruses: rFCV 2280-F9 p30 and rFCV F9-2280 p30. Compared to infection with the parental virus, rFCV 2280-F9 p30 infection displayed attenuated activities in reducing the level of IFNAR1 and inhibiting the phosphorylation of STAT1 and STAT2, whereas rFCV F9-2280 p30 displayed enhanced activities. Animal experiments showed that the virulence of rFCV 2280-F9 p30 infection was attenuated but that the virulence of rFCV F9-2280 p30 was increased compared to that of the parental viruses. Collectively, these data show that FCV 2280 p30 could directly and selectively degrade IFNAR1 mRNA, thus blocking the type I interferon-induced activation of the JAK-STAT signalling pathway, which may contribute to the pathogenesis of FCV infection.

Assay validation and determination of in vitro binding of mefloquine to plasma proteins from clinically normal and FIP-affected cats.

The antimalarial agent mefloquine is currently being investigated for its potential to inhibit feline coronavirus and feline calicivirus infections. A simple, high pressure liquid chromatography assay was developed to detect mefloquine plasma concentrations in feline plasma. The assay's lower limit of quantification was 250 ng/mL. The mean ± standard deviation intra- and inter-day precision expressed as coefficients of variation were 6.83 ± 1.75 and 5.33 ± 1.37%, respectively, whereas intra- and inter-day accuracy expressed as a percentage of the bias were 11.40 ± 3.73 and 10.59 ± 3.88%, respectively. Accordingly, this validated assay should prove valuable for future in vivo clinical trials of mefloquine as an antiviral agent against feline coronavirus and feline calicivirus. However, the proportion of mefloquine binding to feline plasma proteins has not been reported. The proportion of drug bound to plasma protein binding is an important concept when developing drug dosing regimens. As cats with feline infectious peritonitis (FIP) demonstrate altered concentrations of plasma proteins, the proportion of mefloquine binding to plasma proteins in both clinically normal cats and FIP-affected cats was also investigated. An in vitro method using rapid equilibrium dialysis demonstrated that mefloquine was highly plasma protein bound in both populations (on average > 99%).

Nitazoxanide protects cats from feline calicivirus infection and acts synergistically with mizoribine in vitro.

Feline calicivirus (FCV) is a highly contagious pathogen that causes acute upper respiratory infections and oral disease in cats, thus seriously endangering feline health. Recently, there have been outbreaks of particularly virulent variant strains of FCV, which can cause both acute symptoms and fatal systemic disease. The discovery of effective antiviral agents to treat FCV infection is, therefore, gradually assuming increased importance. In this study, we showed that both nitazoxanide and mizoribine had antiviral activity in F81 cells infected with different strains of FCV and also demonstrated a synergistic effect between the two drugs. Experiments in cats challenged with FCV showed that nitazoxanide significantly reduced the clinical symptoms of FCV infection, reduced viral load in the trachea and lungs, and reduced viral shedding. Our results showed that nitazoxanide and mizoribine could potentially be used as therapeutic agents to treat FCV infection.

Multiple Correspondence Analysis on Amino Acid Properties within the Variable Region of the Capsid Protein Shows Differences between Classical and Virulent Systemic Feline Calicivirus Strains.

Feline calicivirus (FCV) is a widespread and highly prevalent pathogen of domestic cats, responsible for mild upper respiratory tract disease. Outbreaks of severe virulent systemic disease (VSD) associated with FCV infection have been reported worldwide. VSD FCV strains have a broader tropism and cause a systemic vascular compromise. Despite clear differences in the pathogenesis of VSD and oral respiratory infections, attempts to identify specific molecular markers of VSD strains on the major capsid protein VP1 have failed. Region E of VP1 is responsible for the interaction with the cell receptor Junctional Adhesion Molecule JAM-1 (FeJAM-1) and with VP2 minor capsid protein during the entry of the virus. We carried out an original analysis on the sequences from region E of VSD and classical strains. A Multiple Correspondence Analysis was performed on a Boolean matrix built by coding sequences on the basis of their amino acid properties. For the first time, this approach was able to differentiate VSD and classical FCV. Seven remarkable residue positions were shown to be statistically significant for pathotype differentiation, mainly located in the N-terminal hypervariable part of region E. As structural analysis suggested an interaction of these residues with FeJAM-1 or VP2, post-binding events, and specific conformational changes may explain the difference of pathogenesis between pathotypes.

Eligibility of Feline Calicivirus for a Surrogate of Human Norovirus in Comparison with Murine Norovirus, Poliovirus and Coxsackievirus.

Feline calicivirus (FCV) is frequently used as a surrogate of human norovirus. We investigated eligibility of FCV for anti-viral assay by investigating the stability of infectivity and pH sensitivity in comparison with other viruses. We found that infectivities of FCV and murine norovirus (MNV) are relatively unstable in infected cells compared with those of coxsackievirus (CoV) and poliovirus (PoV) , suggesting that FCV and MNV have vulnerability. Western blotting indicated that inactivation of FCV was not due to viral protein degradation. We also demonstrated sensitivity of FCV to low pH, the 50% inhibitory pH value being ca. 3.9. Since human norovirus is thought to persist longer, in infectivity and to be a resistant virus, CoV, which is robust and not restrained in use as PoV, may be more appropriate as a test virus for disinfectants, rather than FCV and MNV.

Isolation and molecular characterization of a virulent systemic feline calicivirus isolated in China.

In this study, a virulent systemic (VS) feline calicivirus (FCV) strain, SH, was isolated from a household cat with severe systemic clinical signs, and its full-length genome was determined and analyzed. Through immunofluorescence assays (IFA) and western blotting assays, we found that FCV SH strain, like other isolates, can stably proliferate in Crandell feline kidney (CRFK) cells. Moreover, the typical morphology of FCV particles, with a diameter of about 35 nm, was observed using electron microscopy. The full-length genome of FCV strain SH was sequenced and determined to be 7704 nucleotides (nt) in length with a 5'-terminal untranslated region (UTR) of 19 nt and a 3'-terminal UTR of 67 nt. Three open reading frames (ORF1, ORF2, and ORF 3) were found within the genome, coding for a polypeptide, a capsid precursor (VP1) and a minor structural protein (VP2), respectively. Amino acid sequence comparison revealed diversity (from 82.2% to 88.5% homology) between the VP1 protein sequences of the SH/14 isolate and those of 33 reference isolates from different regions. Phylogenetic analyses using alignments of VP1 protein sequences showed that the SH/14 isolate shares the highest sequence homology with the reported VS-FCV George strain (88.5%), and is located in the same clade as other reported VS-FCV isolates, indicating that the FCV SH/14 strain is a VS-FCV isolate. However, the SH/14 strain does not belong to the same lineage as most other Chinese FCV isolates, suggesting that, in China, a very large geographical entity, the virulent systemic FCV might has emerged.

Integrated molecular analysis of the inactivation of a non-enveloped virus, feline calicivirus, by UV-C radiation.

UV-C treatment has been shown to be a powerful way to inactivate non-enveloped viruses in water samples. However, little is known about how the viruses were inactivated by UV-C radiation. In this study, we investigated the inactivation mechanism of a single-stranded RNA (ssRNA) non-enveloped virus, feline calicivirus (FCV), as a surrogate for the human norovirus, using UV-C radiation with different wavelengths. Integrated molecular analyses using RT-qPCR, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and mass spectrometry were employed to evaluate the extent of ssRNA genome and protein degradation. UV-C radiation of FCV efficiently impaired the infectivity of FCV in mammalian cells. We also identified degradation of the RNA genome, whose copy numbers decreased from 48% to 56% following UV255 or UV281 radiation. Significant degradation of capsid protein was not observed, whereas oxidation of amino acid residues in the major capsid protein VP-1 was determined. Our results suggest that damage to the RNA genome is primarily responsible for the observed decrease in FCV infectivity of CRFK cells. This study provides not only relevant baseline data but also an overview and possible mechanism for the disinfection of non-enveloped ssRNA viruses using UV-C radiation.

[Isolation and phylogenetic analysis of feline calicivirus in Siberia.]

The results of the study of the distribution of calicivirus infection in a population of domestic cats of different breeds, contained individually or the group method, the virus isolation in the cell culture and a comparative phylogenetic analysis of their nucleotide sequences with published sequences of reference feld and vaccine strains of Feline calicivirus (FCV) from other countries: USA, Germany, Japan, China and Korea are presented. Clinical signs of infection were found in 14.3% of the animals examined. After several passages in the primary kidney cells of the kitten embryo, seven cytopathogenic isolates FCV were isolated: 1 - from a cat with an acute infection, 5 - subclinical infection, 1 - systemic infection. They were adapted to continuous FK-81 cells in which they reached a maximum infectious activity of 10.0 ± 1.15 lg TCD 50 / cm3. Based on the sequence analysis of the open reading frame 2 region of the viral genome Eshli strain showed a close relationship with strain KM016908 from China with the identity of the nucleotide sequences between them of 81.0%. The results of the investigations showed that FCV isolates obtained from animals on the territory of Siberia are genetically different from strains included to imported vaccines used to prevent disease in Russian Federation and also among themselves. This causes a decrease in the effectiveness of preventive measures. In nurseries that do not have contacts and connections between themselves but located in the same geographic region FCV populations may have some genetic differences. A close relationship of some feld isolates with strains from other countries geographically located so far from the Siberian region has been revealed. Studies on the molecular epizootology of caliciviruses are important in the development of test systems and the monitoring of the spread of strains in Russia.

Molecular characterization of a feline calicivirus isolated from tiger and its pathogenesis in cats.

Feline calicivirus (FCV) is a virus that causes respiratory disease in cats. In this study, the FCV TIG-1 was isolated from Siberian tiger feces collected in 2014 in Heilongjiang Province, China. Phylogenetic analysis among TIG-1 and other FCVs showed that TIG-1 does not share the same lineage with other FCV isolates from Heilongjiang or other regions in China but is located in the same cluster with the FCV strain Urbana, which was isolated from the United States. The growth kinetics in vitro and the pathogenicity in cats between TIG-1 and the domestic cat-origin FCV strain F9 (vaccine strain) and strain 2280 were compared. We found that the growth kinetics of strains TIG-1 and 2280 were faster than that of strain F9 from 12h to 36h post-infection, indicating that strains TIG-1 and 2280 produce infectious virions and reach peak yields earlier. Challenge experiments in cats showed that TIG-1 grew faster than the other two strains in the lungs of cats and that TIG-1 is a virulent FCV with 100% morbidity and lethality. In addition, the histopathological results showed that the virulent TIG-1 strain directly led to severe lung tissue damage and indirectly led to intestinal damage. The results presented here show that a tiger-origin FCV exhibits high virulence in cats.

N-Terminal Domain of Feline Calicivirus (FCV) Proteinase-Polymerase Contributes to the Inhibition of Host Cell Transcription.

Feline Calicivirus (FCV) infection results in the inhibition of host protein synthesis, known as "shut-off". However, the precise mechanism of shut-off remains unknown. Here, we found that the FCV strain 2280 proteinase-polymerase (PP) protein can suppress luciferase reporter gene expression driven by endogenous and exogenous promoters. Furthermore, we found that the N-terminal 263 aa of PP (PPN-263) determined its shut-off activity using the expression of truncated proteins. However, the same domain of the FCV strain F9 PP protein failed to inhibit gene expression. A comparison between strains 2280 and F9 indicated that Val27, Ala96 and Ala98 were key sites for the inhibition of host gene expression by strain 2280 PPN-263, and PPN-263 exhibited the ability to shut off host gene expression as long as it contained any two of the three amino acids. Because the N-terminus of the PP protein is required for its proteinase and shut-off activities, we investigated the ability of norovirus 3C-like proteins (3CLP) from the GII.4-1987 and -2012 isolates to interfere with host gene expression. The results showed that 3CLP from both isolates was able to shut off host gene expression, but 3CLP from GII.4-2012 had a stronger inhibitory activity than that from GII.4-1987. Finally, we found that 2280 PP and 3CLP significantly repressed reporter gene transcription but did not affect mRNA translation. Our results provide new insight into the mechanism of the FCV-mediated inhibition of host gene expression.

Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage.

UNLABELLED: In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. IMPORTANCE: Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to understand norovirus biology. Recent studies have suggested that the assembly of stress granules is central in orchestrating stress and antiviral responses to restrict viral replication. Overall, our study provides the first insight on how caliciviruses impair stress granule assembly by targeting the nucleating factor G3BP1 via the viral proteinase NS6(Pro) This work provides new insights into host-pathogen interactions that regulate stress pathways during FCV infection.

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.

Tenacity of human norovirus and the surrogates feline calicivirus and murine norovirus during long-term storage on common nonporous food contact surfaces.

The transfer of human norovirus (hNV) to food via contaminated surfaces is highly probable during food production, processing, and preparation. In this study, the tenacity of hNV and its cultivable surrogates feline calicivirus (FCV) and murine norovirus (MNV) on two common nonporous surface materials at two storage temperatures was directly compared. Virus titer reduction on artificially inoculated stainless steel and plastic carriers was monitored for 70 days at room temperature and at 7°C. Viruses were recovered at various time points by elution. Genomes from intact capsids (hNV, FCV, and MNV) were quantified with real-time reverse transcription (RT) PCR, and infectivity (FCV and MNV) was assessed with plaque assay. RNase treatment before RNA extraction was used to eliminate exposed RNA and to assess capsid integrity. No significant differences in titer reduction were found between materials (stainless steel or plastic) with the plaque assay or the real-time quantitative RT-PCR. At room temperature, infectious FCV and MNV were detected for 7 days. Titers of intact hNV, FCV, and MNV capsids dropped gradually and were still detectable after 70 days with a loss of 3 to 4 log units. At 7°C, the viruses were considerably more stable than they were at room temperature. Although only MNV infectivity was unchanged after 70 days, the numbers of intact capsids (hNV, FCV, and MNV) were stable with less than a 1-log reduction. The results indicate that hNV persists on food contact surfaces and seems to remain infective for weeks. MNV appears to be more stable than FCV at 7°C, and thus is the most suitable surrogate for hNV under dry conditions. Although a perfect quantitative correlation between intact capsids and infective particles was not obtained, real-time quantitative RT-PCR provided qualitative data about hNV inactivation characteristics. The results of this comparative study might support future efforts in assessment of foodborne virus risk and food safety.

Antiviral Effects of Lactococcus lactis on Feline Calicivirus, A Human Norovirus Surrogate.

Foodborne viruses, particularly human norovirus (NV) and hepatitis virus type A, are a cause of concern for public health making it necessary to explore novel and effective techniques for prevention of foodborne viral contamination, especially in minimally processed and ready-to-eat foods. This study aimed to determine the antiviral activity of a probiotic lactic acid bacterium (LAB) against feline calicivirus (FCV), a surrogate of human NV. Bacterial growth medium filtrate (BGMF) of Lactococcus lactis subsp. lactis LM0230 and its bacterial cell suspension (BCS) were evaluated separately for their antiviral activity against FCV grown in Crandell-Reese feline kidney (CRFK) cells. No significant antiviral effect was seen when CRFK cells were pre-treated with either BGMF (raw or pH 7-adjusted BGMF) or BCS. However, pre-treatment of FCV with BGMF and BCS resulted in a reduction in virus titers of 1.3 log10 tissue culture infectious dose (TCID)50 and 1.8 log10 TCID50, respectively. The highest reductions in FCV infectivity were obtained when CRFK cells were co-treated with FCV and pH 7-adjusted BGMF or with FCV and BCS (7.5 log10 TCID50 and 6.0 log10 TCID50, respectively). These preliminary results are encouraging and indicate the need for continued studies on the role of probiotics and LAB on inactivation of viruses in various types of foods.

Characterization of an avirulent FCV strain with a broad serum cross-neutralization profile and protection against challenge of a highly virulent vs feline calicivirus.

Highly virulent, systemic strains of Feline calicivirus (vs FCV) have been described in recent years. These vs FCV isolates cause severe edema, cutaneous ulcers, lameness and other upper respiratory and oral clinical signs typically associated with FCV infection in cats. Vs FCV isolates can cause high mortality even in cats vaccinated with currently available commercial vaccines. This study reports identification and characterization of an avirulent FCV strain (FCV 21). This strain offers a broader serum cross-neutralization profile in comparison with the commonly used vaccine strain (FCV F9), as tested with two separate viral panels of FCV isolates. The first viral panel consists of 45 FCV strains isolated around 1993. The second viral panel consists of 26 FCV strains with most isolated around 2003. The potential of using this strain as a vaccine, in a 3-way (FCV+FHV+FPV) or 4-way (FCV+FHV+FPV+FCp) format, was tested by using a highly virulent vs FCV strain (FCV-33585) as a challenge virus. The mortality induced by this vs FCV in unvaccinated control cats was 78% (7 out of 9 cats). The mortality decreased to 44% (4 out of 9 cats) in cats vaccinated with a 4-way vaccine containing FCV F9. However, when this novel FCV vaccine strain (FCV 21) was used, either in combination with FCV F9 or by itself, the mortality decreased to 0% (0 out of 10 cats). The 3-way vaccine (FCV+FHV+FPV) that contained both FCV 21 and FCV F9 also had mortality of 0% (0 out of 10 cats). The clinical scores, as calculated taking into consideration the frequency and severity of various clinical signs, correlated with mortality data. The results suggested this FCV vaccine has the potential to be broadly protective against newly emergent FCV isolates, including complete protection against challenge with a highly virulent vs FCV 33585.

Virulent feline calicivirus disease in a shelter in Italy: a case description.

Feline calicivirus (FCV) is a common pathogen of cats that is particularly widespread in multi-cat environments such as shelters and catteries. FCV infections are usually associated with acute, mild and self-limiting upper respiratory tract disease characterized by oral vesicles/ulcers. Recently, virulent systemic disease (VSD) associated with FCV infection has been reported in the USA and Europe. This paper describes a case of VSD affecting one adult, FIV infected cat ("Oscar") living in a shelter located in Northern Italy; the clinical, post-mortem and laboratory findings indicate that this is the first case of suspected FCV-VSD in this country. Similar to a previous report (Meyer et al., 2011), the disease affected only one cat, while others remained asymptomatic, despite their direct contact with "Oscar". Phylogenetic analysis identified unique features in the "Oscar" FCV isolate. The FIV infection of the patient might have favoured the generation of the virulent FCV strains in this cat.

The influence of oral bacteria on tissue levels of Toll-like receptor and cytokine mRNAs in feline chronic gingivostomatitis and oral health.

Feline chronic gingivostomatitis (FCGS) is an inflammatory disease of the oral cavity that causes severe pain and distress in affected cats. Treatment methods are currently very limited. The aims of this study were to assess the feline innate immune response by investigating the levels of cytokine and Toll-like receptor (TLR) mRNAs in tissue biopsies of cats with and without FCGS, and to relate this to the presence or absence of putative oral pathogens identified previously within these cats. Mucosal biopsies were collected from 28 cats with FCGS and eight healthy cats. The levels of TLR (TLR2, TLR3, TLR4, TLR7, TLR9) and cytokine (IL-1ß, IL-4, IL-6, IL-10, IL-12, TNF-α, IFN-γ) mRNA was determined using quantitative PCR. In the FCGS group a statistically significant increase was seen in TLR2, TLR7, TNF-α, IFN-γ, IL-1ß and IL-6 mRNA levels compared to the healthy group. In cats where Tannerella forsythia was present, statistically significant increases were seen in TLR2, TLR4, TLR7, TLR9, TNF-α and IL-1ß mRNA levels compared to cats where this putative pathogen was absent. Statistically significant increases in mRNA expression were also seen in cats harbouring feline calicivirus (FCV) (TLR2, IL-1ß, IL-6, IFN-γ) and Porphyromonas circumdentaria (TLR2, TLR3) compared to cats where these putative pathogens were absent. Pasteurella multocida subsp. multocida and Pseudomonas sp. did not significantly alter the expression of any TLR or cytokine mRNAs when compared to animals who tested negative for these species, while cats colonised with P. multocida subsp. septica demonstrated a statistically significant reduction in the expression of TLR7, TNF-α and IFN-γ mRNAs compared to cats free of this species. The expression of mRNA for several TLRs and cytokines is elevated in FCGS. A positive correlation was observed between clinical disease severity and the presence of FCV (p=0.001; Rho=0.58). Although the number of cats harbouring T. forsythia was low by comparison, 80% of samples in which it was present were from cases with the highest clinical disease severity. Positive correlations with clinical disease severity were seen for TLR2 (p=0.00086), TLR7 (p=0.049), TNF-α (p=0.027), IFN-γ (p=0.0015), IL-1ß (p=0.004) and IL-6 (p=0.00001) mRNAs. The putative pathogens FCV and T. forsythia may be important in stimulating a host immune response to FCGS and may play a role in the pathogenesis of this disease.

The feline calicivirus leader of the capsid protein is associated with cytopathic effect.

Open reading frame 2 (ORF2) of the feline calicivirus (FCV) genome encodes a capsid precursor that is posttranslationally processed to release the mature capsid protein (VP1) and a small protein of 124 amino acids, designated the leader of the capsid (LC). To investigate the role of the LC protein in the virus life cycle, mutations and deletions were introduced into the LC coding region of an infectious FCV cDNA clone. Three cysteine residues that are conserved among all vesivirus LC sequences were found to be critical for the recovery of FCV with a characteristic cytopathic effect in feline kidney cells. A cell-rounding phenotype associated with the transient expression of wild-type and mutagenized forms of the LC correlated with the cytopathic and growth properties of the corresponding engineered viruses. The host cellular protein annexin A2 was identified as a binding partner of the LC protein, consistent with a role for the LC in mediating host cell interactions that alter the integrity of the cell and enable virus spread.

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.

Investigation of the antiviral properties of copper iodide nanoparticles against feline calicivirus.

This study demonstrated the antiviral properties of copper iodide (CuI) nanoparticles against the non-enveloped virus feline calicivirus (FCV) as a surrogate for human norovirus. The effect of CuI nanoparticles on FCV infectivity to Crandell-Rees feline kidney (CRFK) cells was elucidated. The infectivity of FCV to CRFK cells was greatly reduced by 7 orders of magnitude at 1000µgml(-1) CuI nanoparticles. At the conditions, electron spin resonance (ESR) analysis proved hydroxyl radical production in CuI nanoparticle suspension. Furthermore, amino acid oxidation in the viral capsid protein of FCV was determined by nanoflow liquid chromatography-mass spectrometric (nano LC-MS) analysis. The use of CuI nanoparticles showed extremely high antiviral activity against FCV. The high antiviral property of CuI nanoparticles was attributed to Cu(+), followed by ROS generation and subsequent capsid protein oxidation. CuI nanoparticles could be proposed as useful sources of a continuous supply of Cu(+) ions for efficient virus inactivation. Furthermore, this study brings new insights into toxic actions of copper iodide nanoparticles against viruses.