Infectious Feline Calicivirus Research Articles

Comparative Recovery of Two Human Norovirus Surrogates, Feline Calicivirus and Murine Norovirus, with a Wet Vacuum System, Macrofoam-Tipped Swab, and Bottle Extraction Method from Carpets

Human noroviruses (HuNoV) are the leading cause of known foodborne illness in the United States, but direct detection during outbreak investigations is challenging. On the other hand, sampling both hard and soft environmental surfaces can be used to improve outbreak investigations. Currently, we lack virus recovery methods for soft surfaces, such as carpet, despite evidence suggesting that carpets contribute to HuNoV outbreaks. Our aim was to compare two recovery methods, wet vacuum and swabbing, for routine carpet sampling of HuNoV against a laboratory reference method known as bottle extraction (BE). Specifically, we compared the microbial vacuum (MVAC), macrofoam-tipped swab (MS), and BE by using HuNoV surrogates, feline calicivirus (FCV) and murine norovirus (MNV), inoculated on wool and nylon carpet carriers. The highest recovery of infectious FCV from wool was 5.51, 3.76, and 5.16 log PFU, whereas on nylon, recovery was 5.03, 3.62, and 4.75 log PFU by using BE, MS, and MVAC, respectively. On the other hand, the highest recovery of infectious MNV from wool was 6.10, 4.50, and 5.99 log PFU, while recovery on nylon was 6.07, 4.58, and 6.13 log PFU by using BE, MS, and MVAC, respectively. Significantly more PFU and genomic copies were recovered by using BE and MVAC compared with MS, while buffer type played a significant role in recovery of infectious FCV.

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.

Virucidal Activity of Fogged Chlorine Dioxide- and Hydrogen Peroxide-Based Disinfectants against Human Norovirus and Its Surrogate, Feline Calicivirus, on Hard-to-Reach Surfaces.

Human norovirus (NoV) is the leading cause of foodborne illnesses in the United States. Norovirus is shed in high numbers in the feces and vomitous of infected individuals. Contact surfaces contaminated with bodily fluids harboring infectious virus particles serve as vehicles for pathogen transmission. Environmental stability of NoV and its resistance to many conventional disinfectants necessitate effective inactivation strategies to control the spread of virus. We investigated the efficacy of two commercial disinfectants, hydrogen peroxide (7.5%) and a chlorine dioxide (0.2%)-surfactant-based product using a fogging delivery system against human NoV GI.6 and GII.4 Sydney strains as well as the cultivable surrogate, feline calicivirus (FCV) dried on stainless steel coupons. Log10 reductions in human NoV and FCV were calculated utilizing RNase RT-qPCR and infectivity (plaque) assay, respectively. An improved antiviral activity of hydrogen peroxide as a function of disinfectant formulation concentration in the atmosphere was observed against both GII.4 and FCV. At 12.4 ml/m3, hydrogen peroxide achieved a respective 2.5 ± 0.1 and 2.7 ± 0.3 log10 reduction in GI.6 and GII.4 NoV genome copies, and a 4.3 ± 0.1 log10 reduction in infectious FCV within 5 min. At the same disinfectant formulation concentration, chlorine dioxide-surfactant-based product resulted in a respective 1.7 ± 0.2, 0.6 ± 0.0, and 2.4 ± 0.2 log10 reduction in GI.6, GII.4, and FCV within 10 min; however, increasing the disinfectant formulation concentration to 15.9 ml/m3 negatively impacted its efficacy. Fogging uniformly delivered the disinfectants throughout the room, and effectively decontaminated viruses on hard-to-reach surfaces. Hydrogen peroxide delivered by fog showed promising virucidal activity against FCV by meeting the United States EPA 4-log10 reduction criteria for an anti-noroviral disinfectant; however, fogged chlorine dioxide-surfactant-based product did not achieve a 4-log10 inactivation. Future investigation aimed at optimizing decontamination practices is warranted.

Antiviral properties of silver nanoparticles against norovirus surrogates and their efficacy in coated polyhydroxyalkanoates systems

Silver nanoparticles (AgNP) have strong broad-spectrum antimicrobial activity and gained increased attention for the development of AgNP based products, including medical and food applications. Initially, the efficacy of AgNP and silver nitrate (AgNO3) was evaluated for inactivating norovirus surrogates, the feline calicivirus (FCV) and the murine norovirus (MNV). These norovirus surrogates were exposed to AgNO3 and AgNP solutions for 24 h at 25 °C and then analyzed by cell-culture assays. Both AgNP and silver ions significantly decreased FCV and MNV infectivity in a dose-dependent manner between concentrations of 2.1 and 21 mg/L. Furthermore, poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) films were prepared by depositing a coating of thermally post-processed electrospun PHBV18/AgNP fiber mats over compression moulded PHBV3 films. After 24 h exposure at 37 °C and 100% RH, no infectious FCV were recovered when in contact with the AgNP films while MNV titers decreased by 0.86 log. The morphology of the PHBV18 and PHBV18/AgNP fibers studied by SEM showed smooth and continuous fibers in both cases and the EDAX analysis confirmed the homogeneously distribution of AgNP into the coating and onto the PHBV3/PHBV18 layer. This study showed, for the first time, the suitability of the PHBV18/AgNP electrospun coating for antiviral surfaces.

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.

Feline calicivirus can tolerate gross changes of its minor capsid protein expression levels induced by changing translation reinitiation frequency or use of a separate VP2-coding mRNA.

Caliciviruses use reinitiation of translation governed by a ‘termination upstream ribosomal binding site’ (TURBS) for expression of their minor capsid protein VP2. Mutation analysis allowed to identify sequences surrounding the translational start/stop site of the feline calicivirus (FCV) that fine tune reinitiation frequency. A selection of these changes was introduced into the infectious FCV cDNA clone to check the influence of altered VP2 levels on virus replication. In addition, full length constructs were established that displayed a conformation, in which VP2 expression occurred under control of a duplicated subgenomic promoter. Viable viruses recovered from such constructs revealed a rather broad range of VP2 expression levels but comparable growth kinetics showing that caliciviruses can tolerate gross changes of the VP2 expression level.

Evaluation of silver-infused polylactide films for inactivation of Salmonella and feline calicivirus in vitro and on fresh-cut vegetables

There is a growing trend to develop packaging materials with an active role in guarantying that the quality and safety characteristics of packaged products will remain or improve from preparation throughout shelf-life.In the present study, 0.001–1.0wt.% silver ions were satisfactorily incorporated into polylactide (PLA) films by a solvent casting technique. Silver migration from the films was measured by voltamperometry and then correlated with its antimicrobial efficacy against Salmonella enterica and feline calicivirus (FCV), a human norovirus surrogate, by using the Japanese industrial standard (JIS Z 2801). The PLA-silver films showed strong antibacterial and antiviral activity in vitro, with increasing effects at higher silver concentrations. Moreover, results show that FCV was less susceptible to silver than Salmonella. When films were applied on food samples, antibacterial and antiviral activity was reduced as compared to in vitro. Antimicrobial activity was very much dependent on the food type and temperature. In lettuce samples incubated at 4°C during 6days, 4 log CFU of Salmonella was inactivated for films with 1.0wt.% and no infectious FCV was reported under the same conditions. On paprika samples, no antiviral effect was seen on FCV infectivity whereas films showed less antibacterial activity on Salmonella.

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.

In vitro assessment of the feline cell-mediated immune response against feline panleukopeniavirus, calicivirus and felid herpesvirus 1 using 5-bromo-2′-deoxyuridine labeling

In this study an in vitro assay was optimized to detect feline proliferating lymphocytes as an assessment for the cell-mediated immune response. For this purpose, 5-bromo-2′-deoxyuridine (BrdU) labeling was chosen because of its sensitivity and the possibility of further characterization of proliferating cells. The assay was optimized by selecting the best batch and concentration of fetal bovine serum, β-mercaptoethanol concentration, cell density, BrdU incubation time and antigen presenting cell type. Cats were vaccinated with the attenuated Nobivac vaccine Tricat and the peripheral blood lymphocyte proliferation responses were quantified upon in vitro restimulation with inactivated and infectious feline panleukopenia virus (FPV), feline calicivirus (FCV) and felid herpesvirus 1 (FeHV-1). Proliferation signals were detected with inactivated FeHV-1 in the CD8+ but not in the CD8− T lymphocyte population, with inactivated FCV and FPV in both CD8− and CD8+ T lymphocyte populations. Restimulation with infectious FCV caused significant proliferation in the CD8− T lymphocyte population only while infectious FPV and FeHV-1 seemed to suppress lymphocyte proliferation in both T cell populations. Additional IFN-γ quantification in the culture supernatant revealed a large correlation between the proliferation signals and IFN-γ production, indicating that BrdU labeling is a very reliable technique to assess and characterize feline lymphoproliferative responses to viral antigens in vitro.

Detection of Norovirus and Feline Calicivirus in spiked molluscs subjected to heat treatments

Aim of the study was the evaluation of the effect of heat treatment in shellfish experimentally contaminated with human Norovirus (NoV). Feline Calicivirus (FCV), often used as surrogate for human NoV, was examined in parallel to test for virus infectivity after treatment. The experiments were performed subjecting suspensions and spiked mussels to heat treatment (60 °C and 80 °C) for various times. Analysis by rRT-PCR showed a limited reduction of NoV (less than 1 log RT-PCR units ml−1) and FCV (0.3–1.2 log TCID50 ml−1) both in virus suspensions and in spiked mussels, with the exception of NoV suspensions treated at 80 °C (reduction of 3 log RT-PCR units ml−1). Cell culture assay showed that the infectious FCV in suspensions decreased of 4 log after 3 min of treatment while a lower reduction (2 log) was obtained in spiked samples treated for 15 min. Data showed that mussel matrix plays a protective role against heat inactivation of viruses. Despite the efficacy of rRT-PCR for viral nucleic acid detection, its inability to provide information on virus infectivity, represents a limit to the evaluation of product safety. Caution should therefore be used in the evaluation of efficacy of heat treatments on virus-contaminated foods and when the judgment of food product safety is based exclusively on rRT-PCR results.

Inactivation of Norovirus by ozone gas in conditions relevant to healthcare

We evaluated the ability of ozone gas to inactivate Norovirus and its animal surrogate feline calicivirus (FCV) in dried samples placed at various locations within a hotel room, a cruise liner cabin and an office. Norovirus was measured by quantitative reverse transcriptase real-time polymerase chain reaction (QRT-PCR) assay, and FCV by a combination of QRT-PCR and virus infectivity assays. We were able to reduce the concentration of infectious FCV by a factor of more than 10(3), and in some cases beyond detection, under optimal conditions of ozone exposure with less than an hour of total operation. QRT-PCR assays indicated similar decreases in both viral RNAs. Virus-containing samples dried onto hard surfaces (plastic, steel and glass), and soft surfaces such as fabric, cotton and carpet, were equally vulnerable to the treatment. Our results show that Norovirus can be inactivated by exposure to ozone gas from a portable commercial generator in settings such as hotel rooms, cruise ship cabins and healthcare facilities.

Virucidal activity of a quaternary ammonium compound disinfectant against feline calicivirus: A surrogate for norovirus

Norovirus, formerly known as Norwalk virus, is an important cause of gastroenteritis outbreaks in hospitals, food services, schools, and cruise ships. Infection control practices by using disinfectants to eliminate noroviruses from surfaces and environmental samples reduce the morbidity and spread of virus outbreaks. There are not many commercial disinfectants effective against norovirus. Noroviruses cannot be cultivated in vitro. However, feline calicivirus can be used as a surrogate to determine disinfectant efficacy against noroviruses. Feline calicivirus was used in a virucidal effectiveness test protocol as a surrogate for norovirus to determine the virucidal efficacy of R-82, a quaternary ammonium compound disinfectant cleaner. Feline calicivirus suspensions containing at least 5% fetal bovine serum were dried on carriers and treated with 1:256 dilutions of R-82 disinfectant in water, with a hardness of 400 ppm as calcium carbonate, for 10 minutes. Hypochlorite concentrations of 100 +/- 10 and 1,000 +/- 10 ppm, respectively, were also analyzed as internal control standards. After contact period, the test agents were neutralized with 2 mL of appropriate neutralizer, and mixtures were scraped from carrier surfaces with a cell scraper. Selected dilutions of the neutralized inoculum/test agent mixtures were added to cultured cell monolayers of appropriate host cells. Postincubation, the infectious feline calicivirus was scored microscopically by observing virus-specific cytopathic effects produced by replicating infectious virus. The performance criterion was a minimum of 4-log(10) reduction in cytopathic effects of feline calicivirus. After a 10-minute contact time, formulation R-82 diluted 1:256 showed a 6.6- and 6.4-log(10) reductions in cytopathic effects of feline calicivirus during initial and confirmatory testing, respectively, demonstrating complete inactivation of the virus. A hypochlorite solution of 1,000 ppm exhibited similar log(10) reductions to the quaternary ammonium disinfectant, demonstrating the reproducibility of the protocol. Formulation R-82, a quaternary ammonium compound, is a 1-step disinfectant cleaner, which exhibited virucidal activity against feline calicivirus suspensions dried on hard surface carriers. Surfaces are vectors for virus transmission during outbreaks by transferring the virus to people or other environmental surfaces. Therefore, treatment of contaminated surfaces with formulation R-82 will optimize disinfection of noncritical and critical surfaces in health care institutions, reducing the possibility of virus transmission during outbreaks.

Feline Calicivirus VP2 Is Essential for the Production of Infectious Virions

The third open reading frame (ORF3) located at the 3' end of the genomic RNA of feline calicivirus (FCV) encodes a small (12.2-kDa) minor structural protein of 106 amino acids designated VP2. Point mutations and deletions were introduced into an infectious FCV cDNA clone in order to evaluate the functional importance of ORF3 and its encoded protein, VP2. Deletion of the entire ORF3 sequence was lethal for the virus, and evidence was found for strong selective pressure to produce the VP2 protein. Extended deletions in the 5' end and small deletions in the 3' end of ORF3, as well as the introduction of stop codons into the ORF3 sequence, were tolerated by the viral replication machinery, but infectious virus could not be recovered. Infectious virus particles could be rescued from a full-length FCV cDNA clone encoding a nonfunctional VP2 when VP2 was provided in trans from a eukaryotic expression plasmid. Our data indicate that VP2, a protein apparently unique to the caliciviruses, is essential for productive replication that results in the synthesis and maturation of infectious virions and that the ORF3 nucleotide sequence itself overlaps a cis-acting RNA signal at the genomic 3' end.

Mutagenesis of tyrosine 24 in the VPg protein is lethal for feline calicivirus.

The genome of feline calicivirus (FCV) is an approximately 7.7-kb single-stranded positive-sense RNA molecule that is polyadenylated at its 3' end and covalently linked to a VPg protein (calculated mass, 12.6 kDa) at its 5' end. We performed a mutational analysis of the VPg protein in order to identify amino acids potentially involved in linkage to the genome and replication. The tyrosine residues at positions 12, 24, 76, and 104 were changed to alanines by mutagenesis of an infectious FCV cDNA clone. Viruses were recovered when Tyr-12, Tyr-76, or Tyr-104 of the VPg protein was changed to alanine, but virus was not recovered when Tyr-24 was changed to alanine. Growth properties of the recovered viruses were similar to those of the parental virus. We examined whether the amino acids serine, threonine, and phenylalanine could substitute for the tyrosine at position 24, but these mutations were lethal as well. A tyrosine at this relative position is conserved among all calicivirus VPg proteins examined thus far, suggesting that the VPg protein of caliciviruses, like those of picornaviruses and potyviruses, utilizes tyrosine in the formation of a covalent bond with RNA.

Carrier-state infection of feline T-lymphoblastoid cells with feline calicivirus

The susceptibility of feline T lymphocytes to feline calicivirus (FCV) in vitro was investigated using feline T-lymphoblastoid cell lines, namely MYA-1 and FL74 cells. The virus titers of supernatants in FCV-infected MYA-1 and FL74 cell cultures increased rapidly, and FCV antigens were also detected in the FCV-infected cells. There were slight differences in the molecular weights of capsid proteins expressed in FCV-infected MYA-1, FL74 and Crandell feline kidney cells. MYA-1 and FL74 cells were productively and persistently infected with FCV, and FCV antigens were observed in the FCV-infected cells for more than one month. At 3 months post infection, FCV-infected FL74 cells that stopped producing infectious FCV could be reinfected with FCV. However, no cytopathic effects were observed.