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.

Molecular epidemiology and phylogenetic analysis of feline calicivirus in Kunshan, China.

Feline calicivirus (FCV) is a highly contagious virus in cats, which typically causes respiratory tract and oral infections. Despite vaccination against FCV being a regular practice in China, new FCV cases still occur. Antigenic diversity of FCV hinders the effective control by vaccination. This is first report which aims to investigate the molecular epidemiology and molecular characteristics of FCV in Kunshan, China. The nasopharyngeal swabs were collected from cats showing variable clinical signs from different animal clinics in Kunshan from 2022 to 2023. Preliminary detection and sequencing of the FCV capsid gene were performed to study genetic diversity and evolutionary characteristics. FCV-RNA was identified in 52 (26%) of the samples using RT-PCR. A significant association was found between FCV-positive detection rate, age, gender, vaccination status and living environment, while a non-significant association was found with breed of cats. Nucleotide analysis revealed two genotypes, GI and GII. GII predominated in Kunshan, with diverse strains and amino acid variations potentially affecting vaccination efficacy and FCV detection. Notably, analysis pinpointed certain strains' association with FCV-virulent systemic disease pathotypes. This investigation sheds light on FCV dynamics, which may aid in developing better prevention strategies and future vaccine designs against circulating FCV genotypes.

High-resolution melting analysis for simultaneous detection and discrimination between wild-type and vaccine strains of feline calicivirus.

High-resolution melting (HRM) analysis, a post-polymerase chain reaction (PCR) application in a single closed tube, is the straightforward method for simultaneous detection, genotyping, and mutation scanning, enabling more significant dynamic detection and sequencing-free turnaround time. This study aimed to establish a combined reverse-transcription quantitative PCR and HRM (RT-qPCR-HRM) assay for diagnosing and genotyping feline calicivirus (FCV). This developed method was validated with constructed FCV plasmids, clinical swab samples from living cats, fresh-frozen lung tissues from necropsied cats, and four available FCV vaccines. We performed RT-qPCR to amplify a 99-base pair sequence, targeting a segment between open reading frame (ORF) 1 and ORF2. Subsequently, the HRM assay was promptly applied using Rotor-Gene Q® Software. The results significantly revealed simultaneous detection and genetic discrimination between commercially available FCV vaccine strains, wild-type Thai FCV strains, and VS-FCV strains within a single PCR reaction. There was no cross-reactivity with other feline common viruses, including feline herpesvirus-1, feline coronavirus, feline leukemia virus, feline immunodeficiency virus, and feline morbillivirus. The detection limit of the assay was 6.18 × 101 copies/µl. This study, therefore, is the first demonstration of the uses and benefits of the RT-qPCR-HRM assay for FCV detection and strain differentiation in naturally infected cats.

Genetic and pathogenicity analysis for the two FCV strains isolated from Eastern China.

In this study, the diversity and regularity of two new feline calicivirus (FCV) isolates, QD-7 and QD-164, were investigated. The genomes of these new strains were compared with 39 strains from the NCBI database including isolates from China, United States, Germany, South Korea, the United Kingdom and Japan. The nucleotide sequence identities ranged from 75-88%, indicating a high degree of variability. These variations were not related to distributions of the virus by time of isolation and geographical location. Cats that were experimentally infected with the new isolate QD-164 showed typical clinical symptoms of sneezing, fever and conjunctivitis and all recovered within 30 days. In contrast, QD-7 infections were asymptomatic and the virus was cleared within 16 days. These results indicate that QD-7 and QD-164 were naturally attenuated strains. NNS mutations characteristic of highly virulent strains at positions 441-443 were absent in QD-7 while QD-164 possessed an N at position 442. This indicated that mutations in regions 441-443 may be linked to disease severity.

Broad-Spectrum, Potent, and Durable Ceria Nanoparticles Inactivate RNA Virus Infectivity by Targeting Virion Surfaces and Disrupting Virus-Receptor Interactions.

There is intense interest in developing long-lasting, potent, and broad-spectrum antiviral disinfectants. Ceria nanoparticles (CNPs) can undergo surface redox reactions (Ce3+ ↔ Ce4+) to generate ROS without requiring an external driving force. Here, we tested the mechanism behind our prior finding of potent inactivation of enveloped and non-enveloped RNA viruses by silver-modified CNPs, AgCNP1 and AgCNP2. Treatment of human respiratory viruses, coronavirus OC43 and parainfluenza virus type 5 (PIV5) with AgCNP1 and 2, respectively, prevented virus interactions with host cell receptors and resulted in virion aggregation. Rhinovirus 14 (RV14) mutants were selected to be resistant to inactivation by AgCNP2. Sequence analysis of the resistant virus genomes predicted two amino acid changes in surface-located residues D91V and F177L within capsid protein VP1. Consistent with the regenerative properties of CNPs, surface-applied AgCNP1 and 2 inactivated a wide range of structurally diverse viruses, including enveloped (OC43, SARS-CoV-2, and PIV5) and non-enveloped RNA viruses (RV14 and feline calicivirus; FCV). Remarkably, a single application of AgCNP1 and 2 potently inactivated up to four sequential rounds of virus challenge. Our results show broad-spectrum and long-lasting anti-viral activity of AgCNP nanoparticles, due to targeting of viral surface proteins to disrupt interactions with cellular receptors.

Genetic Diversity and Evolution of Viruses Infecting Felis catus: A Global Perspective.

Cats harbor many important viral pathogens, and the knowledge of their diversity has been greatly expanded thanks to increasingly popular molecular sequencing techniques. While the diversity is mostly described in numerous regionally defined studies, there lacks a global overview of the diversity for the majority of cat viruses, and therefore our understanding of the evolution and epidemiology of these viruses was generally inadequate. In this study, we analyzed 12,377 genetic sequences from 25 cat virus species and conducted comprehensive phylodynamic analyses. It revealed, for the first time, the global diversity for all cat viruses known to date, taking into account highly virulent strains and vaccine strains. From there, we further characterized and compared the geographic expansion patterns, temporal dynamics and recombination frequencies of these viruses. While respiratory pathogens such as feline calicivirus showed some degree of geographical panmixes, the other viral species are more geographically defined. Furthermore, recombination rates were much higher in feline parvovirus, feline coronavirus, feline calicivirus and feline foamy virus than the other feline virus species. Collectively, our findings deepen the understanding of the evolutionary and epidemiological features of cat viruses, which in turn provide important insight into the prevention and control of cat pathogens.

A cDNA-based reverse genetics system for feline calicivirus identifies the 3' untranslated region as an essential element for viral replication.

Virulent systemic feline calicivirus (VS-FCV) is a newly emerging FCV variant that is associated with a severe acute multisystem disease in cats that is characterized by jaundice, oedema, and high mortality (approximately 70%). VS-FCV has spread throughout the world, but there are no effective vaccines or therapeutic options to combat infection. VS-FCV may therefore pose a serious threat to the health of felines. The genomic characteristics and functions of VS-FCV are still poorly understood, and the reason for its increased pathogenicity is unknown. Reverse genetics systems are powerful tools for studying the molecular biology of RNA viruses, but a reverse genetics system for VS-FCV has not yet been reported. In this study, we developed a plasmid-based reverse genetics system for VS-FCV in which infectious progeny virus is produced in plasmid-transfected CRFK cells. Using this system, we found that the 3' untranslated region (UTR) and poly(A) tail are important for maintaining the infection and replication capacity of VS-FCV and that shortening of the poly(A) tail to less than 28 bases eliminated the ability to rescue infectious progeny virus. Whether these observations are unique to VS-FCV or represent more-general features of FCV remains to be determined. In conclusion, we successfully established a rapid and efficient VS-FCV reverse genetics system, which provides a good platform for future research on the gene functions and pathogenesis of VS-FCV. The effects of the deletion of 3' UTR and poly(A) tail on VS-FCV infectivity and replication also provided new information about the pathogenesis of VS-FCV.

Distribution and genetic diversity of Feline calicivirus in Moscow metropolitan area.

BACKGROUND: Feline calicivirus (FCV) is widespread throughout the world. An FCV infection is associated with conjunctivitis, rhinitis, and mouth ulcers that can lead to the animal's death. Because vaccination is not always effective, it is necessary to monitor the infection regularly. OBJECTIVES: This study examined the FCV epizootic situation in the Moscow metropolitan area by conducting a molecular phylogenetic analysis of the virus isolates. METHODS: Samples from 6213 animals were examined by a reverse transcription polymerase chain reaction. For phylogenetic analysis, 12 nucleotide sequences obtained from animal samples were selected. Sequencing was performed using the Sanger method. Phylogenetic analysis was conducted using the Maximum Likelihood method. RESULTS: The FCV genome was detected in 1,596 (25.7%) samples out of 6,213. In 2018, calicivirus was detected in 18.9% of samples, 27.8% in 2019, 21.4% in 2020, and 32.6% in 2021. Phylogenetic analysis of the F ORF2 region and the ORF3 start region led to division into two FCV genogroups. Most of the isolates (8 out of 12) were close to the Chinese strains. On the other hand, there were isolates closely related to European and American strains. The isolates circulating in Moscow were not included in clusters with vaccine strains; their nucleotide similarity varied from 77% to 83%. CONCLUSIONS: This study revealed a high prevalence and genetic diversity of the FCV in Moscow. The epizootic situation remains stably tense because 24 viruses were detected in 25% of animals annually.

Development of a triple NanoPCR method for feline calicivirus, feline panleukopenia syndrome virus, and feline herpesvirus type I virus.

BACKGROUND: Feline calicivirus (FCV), Feline panleukopenia virus (FPV), and Feline herpesvirus type I (FHV-1) are the three most common pathogens in cats, and also are the main pathogens leading to the death of kittens. Here, by a combination of gold nanoparticles and conventional PCR, we established a novel triple NanoPCR molecular detection method for clinical detection. RESULTS: The triple NanoPCR molecular detection is able to detect 2.97 × 101copies/µL FCV recombinant copies plasmid per reaction, 2.64 × 104copies/µL FPV recombinant copies plasmid per reaction, and 2.85copies/µL FHV-1 recombinant copies plasmid per reaction at the same time. The sensitivity of each plasmid is 100 times, 10 times, and 100 times higher than conventional PCR, respectively. The clinical results showed that among the 38 samples, the positive rates of FCV, FPV, and FHV-1 in a NanoPCR test were 63.16, 31.58, and 60.53%, while in a conventional PCR were 39.47, 18.42, and 34.21%. CONCLUSIONS: In this report, it is the first time that NanoPCR assays are applied in the detection of FCV, FPV, and FHV-1 as well. This sensitive and specific NanoPCR assay can be widely used in clinical diagnosis and field monitoring of FCV, FPV, and FHV-1 infections.

Genetic and phylogenetic analysis of capsid gene of feline calicivirus in Nanjing, China.

Feline calicivirus (FCV) is a common and important pathogen in cats, typically resulting in upper respiratory tract disease or ulcerative oral lesions. Although there are large number of researches on FCV and vaccines against FCV have been widely used for years, the explanation for vaccination failure and further studies on the prevalence of FCV are still necessary in China. In this study, 86 nasopharyngeal swabs from pet cats with upper respiratory symptoms from several Nanjing animal hospitals were collected in 2020. Among them, 36 (41.86%) were positive for FCV. In addition, 13 FCV capsid genes were sequenced. The comparative analysis of linear B-cell epitopes of VP1 gene indicated that there were many amino acid variations existed among FCV vaccine strains and these strains currently circulating in Nanjing, which may relate to the failure of vaccination and maybe aid for future vaccine design. Besides, phylogenetic analysis of capsid gene revealed two genotypes. Except for the F86 strain, most of the strains were clustered with FCV I genotype, which indicated that FCV I genotype was the most prevalent genotype currently circulating in Nanjing. In conclusion, this study provided useful information as to the evolution and genetic variants of FCV in Nanjing, which is urgent for the future instructions of effective disease prevention and control strategies.

Identification of amino acid substitutions escaping from a broadly neutralizing monoclonal antibody of feline calicivirus.

Feline calicivirus (FCV) causes upper respiratory tract diseases in cats and has highly variable antigenicity for neutralization of each strain. Neutralizing epitopes of FCV are currently found in the hypervariable region (HVR) in the P2 domain of the major capsid protein VP1. Due to its unique ability to neutralize various FCV strains, 1D7 is a monoclonal antibody that may recognize a novel neutralizing epitope. While other neutralizing epitopes were characterized by producing neutralization-resistant variants, only 1D7-resistant variants could not be obtained, and its epitope has not been identified in the previous studies. In this study, we successfully generated these variants by multiple passaging of the FCV F4 strain in the presence of 1D7 and discovered that several amino acid substitutions (K638N, R662G, and T666I in the P1 domain of VP1) are involved in the decreased binding of 1D7. These substitution sites are also highly conserved among FCV strains compared with the substitution sites of other neutralization-resistant variants found in the HVR. Our results indicate that amino acid substitutions in the P1 domain, which are not responsible for direct interaction with the FCV receptor, are associated with neutralization escape. Since FCV can be conveniently cultured in vitro and the receptor required for infection is known, a detailed analysis of the 1D7 epitope could shed more light on the neutralization mechanism of the epitopes of viruses belonging to the Caliciviridae.

PseudoRHDV constructed with feline calicivirus genome as vector has the characteristics of well proliferation in vitro.

Rabbit hemorrhagic disease virus (RHDV) is a major member of the Caliciviridae. which is fatal to wild and domestic European rabbit. Because RHDV does not reproduce stably in vitro, molecular studies on this pathogen have been limited. Feline calicivirus (FCV), also a member of the Caliciviridae, reproduces well in vitro and is a good viral vector. As these viruses share similar genomic structures, we hypothesized that a chimeric infectious clone could be constructed by replacing the corresponding regions of the FCV genome with the structural proteins VP60 and VP10 and the 3' non-translated region of the RHDV genome. Transfection of the infectious clone into RK13 cells made it possible to rescue the chimeric virus, named pseudoRHDV, which reproduced in an RK13 cell line with high titer. An infectious pseudoRHDV was produced, which proliferated in RK13 cells to at least 15 generations. PseudoRHDV caused significant cytopathic changes in the RK13 cells, with a viral titer was 9.74 log10 TCID50 / mL. The pseudoRHDV constructed in this study will be helpful for investigating the molecular biology of RHDV, especially its interaction with the host. The model can also be used to explore some common laws between FCV and RHDV.

Natural cases of polyarthritis associated with feline calicivirus infection in cats.

The limping syndrome is occasionally reported during acute feline calicivirus (FCV) infections or as consequence of vaccination. In this retrospective study, three clinical cases of lameness in household cats naturally infected by FCV were described and phylogeny of the virus were investigated by analysing the hypervariable E region of the ORF2 viral gene. Cats were diagnosed with polyarthritis and FCV RNA or antigens were detected in symptomatic joints. One cat, euthanized for ethical reasons, underwent a complete post-mortem examination and was subjected to histopathological and immunohistochemical investigations. No phylogenetic subgrouping were evident for the sequenced FCV. Histopathology of the euthanized cat revealed diffuse fibrinous synovitis and osteoarthritis eight months after the onset of lameness and the first detection of FCV RNA, supporting the hypothesis of a persistent infection. FCV was demonstrated by immunohistochemistry in synoviocytes and fibroblasts of the synovial membranes. This study provides new data on the occurrence of polyarthritis in FCV-infected cats, demonstrates by immunohistochemistry the presence of FCV in the synovial membranes of a cat with persistent polyarthritis and supports the absence of correlation between limping syndrome and phylogenetic subgrouping of viruses.

The Feline Calicivirus Leader of the Capsid Protein Has the Functional Characteristics of a Viroporin.

The leader of the capsid (LC) protein is exclusive to the Vesivirus genus, and it is needed for successful feline calicivirus (FCV) replication, as well as an efficient apoptosis induction through the mitochondrial pathway. In this work, we aimed to determine if the LC protein from the FCV is a viroporin. Although lacking in a transmembrane domain or an amphipathic helix, the LC protein from the FCV is toxic when expressed in bacteria and it oligomerizes through disulfide bonds, which are both key characteristics of viroporins. An electron microscopy analysis of LC-expressing E. coli cells suggest that the protein induces osmotic stress. Moreover, we found that the previously studied C40A LC mutant, that fails to induce apoptosis and that hinders the replication cycle, also oligomerizes but it has a reduced toxicity and fails to induce osmotic stress in bacteria. We propose that the LC protein is a viroporin that acts as a disulfide bond-dependent antimicrobial peptide, similar to the Ebola virus delta peptide.

An Update on Feline Calicivirus.

INTRODUCTION: Feline Calicivirus (FCV) is one of the most common viral pathogens in domestic cats worldwide. The first report of FCV dates back to 1957, when FCV was isolated from the gastrointestinal tract of cats in New Zealand. Subsequent reports recognised FCV as a cause of respiratory disease in cats, and at present, feline practitioners worldwide are daily confronted with cats suffering from suspected FCV. The highly mutagenic nature of FCV and its high genetic plasticity enable the virus to successfully survive in the feline population, and pose a special challenge as regards the diagnosis, treatment, and prevention of FCV-induced disease. Upper respiratory tract disease has been considered a common clinical sign of FCV infection. A study from Switzerland demonstrated that oral ulcerations, salivation and gingivitis-stomatitis were more commonly associated with FCV infection than upper respiratory tract disease, and less than half of the cats suspected to have FCV infection were found to be FCV-positive. Furthermore, a study investigating FCV isolates from Switzerland found some evidence that the genetic background of cats might influence their susceptibility to FCV infection. This review article provides a comprehensive summary of the FCV literature, and integrates the results of recent research on FCV's genetic characteristics, the cellular and humoral immunity evoked by FCV vaccination and infection, the diagnosis of FCV, FCV prevention/vaccination, the risk factors associated with FCV, and the hygienic measures necessary in FCV-contaminated areas. After each section, the key points are summarised, and relevant information is outlined to help feline practitioners in FCV diagnosis, treatment and prevention.

INTRODUCTION: Le calicivirus félin (FCV) est l'un des agents pathogènes viraux les plus courants chez les chats domestiques dans le monde. Le premier signalement de FCV remonte à 1957, lorsque le FCV a été isolé du tractus gastro-intestinal de chats en Nouvelle-Zélande. Des rapports ultérieurs ont reconnu le FCV comme une cause de maladie respiratoire chez les chats et, à l'heure actuelle, les praticiens félins du monde entier sont quotidiennement confrontés à des chats suspectés de FCV. La nature hautement mutagène du FCV et sa haute plasticité génétique permettent au virus de survivre avec succès dans la population féline et posent un défi particulier en ce qui concerne le diagnostic, le traitement et la prévention de la maladie induite par le FCV. La maladie des voies respiratoires supérieures a été considérée comme un signe clinique courant d'infection par le FCV. Une étude réalisée en Suisse a démontré que les ulcérations buccales, la salivation et la gingivite-stomatite étaient plus fréquemment associées à une infection à FCV qu'à une autre maladie des voies respiratoires supérieures et moins de la moitié des chats suspectés d'avoir une infection à FCV se sont avérés positifs pour le FCV. De plus, une étude portant sur des isolats de FCV en Suisse a trouvé des preuves que le profil génétique des chats pourrait influencer leur sensibilité à l'infection par le FCV. Cet article de synthèse fournit un résumé complet de la littérature sur le FCV et intègre les résultats de recherches récentes sur les caractéristiques génétiques du FCV, l'immunité cellulaire et humorale évoquée par la vaccination et l'infection au FCV, le diagnostic du FCV, la prévention/vaccination contre le FCV, les facteurs de risque associés avec le FCV et les mesures d'hygiène nécessaires dans les zones contaminées par le FCV. Après chaque section, les points clés sont résumés et des informations pertinentes sont décrites pour aider les praticiens félins dans le diagnostic, le traitement et la prévention du FCV.

Establishment and application of ERA-LFD method for rapid detection of feline calicivirus.

Feline calicivirus (FCV) has a single-stranded, positive-sense RNA genome, and it is responsible for many infectious respiratory diseases in cats. In addition, more worryingly, highly virulent strains of FCV can cause high mortality in felines. Therefore, a rapid and reliable diagnosis tool plays an important role in controlling the outbreak of FCV. In this study, enzymatic recombinase amplification (ERA) assay combined with lateral flow dipstick (LFD) was developed for the detection of FCV, targeting a relatively conversed position of FCV-ORF1. The results showed that the optimal reaction condition was at 40 °C for 30 min. ERA-LFD method was highly sensitive with the detection limit as low as 3.2 TCID50 of FCV RNA per reaction. The specificity analysis demonstrated no cross-reactivity with feline parvovirus (FPV), feline herpesvirus (FHV) and feline infectious peritonitis virus (FIPV). ERA-LFD was highly repeatable and reproducible, with the intra-assay and inter-assay coefficients of variation for this method both less than 7%. The general test showed that all the recombinant plasmids with known mutant sites and FCV strains with different mutant sites stored in our laboratory were all detected by this method. Of the 23 samples, 14 samples were tested positive for FCV by ERA-LFD and RT-qPCR, respectively. In summary, ERA-LFD assay was a fast, accurate and convenient diagnosis tool for the detection of FCV. KEY POINTS: • The detection principle of ERA-LFD was introduced. • Almost all the currently known FCV strains can be detected. • ERA-LFD is easy to operate and can be used for field detection.

Co-circulation and evolution of genogroups I and II of respiratory and enteric feline calicivirus isolates in cats.

Feline calicivirus (FCV) is a highly infectious pathogen that causes upper respiratory tract disease (URTD), but the enteric FCVs raise concerns regarding their role of an enteric pathogen. In this study, between 2019 and 2020, 101 clinical samples from domestic cats with symptoms of URTD, with or without enteritis, were collected for FCV-specific detection. The FCV-positive rate reached to 42.4% (28/66) in cats with respiratory symptoms. The rates were 11.1% (3/27) and 12.5% (1/8) when faeces and serum samples were measured using reverse transcription polymerase chain reaction (RT-PCR), respectively. Ten FCV strains were successfully isolated from respiratory and enteric sources in domestic cats from Guangxi. Phylogenetic analysis based on the genome sequences of 11 isolates (including GX01-13 isolated in 2013) indicated that the newly characterized FCV strains had two recombinant events in comparison with other FCVs and were of respiratory and enteric origins. These strains displayed high genetic diversity, and they were divided into two genogroups (I and II). Of these, the GXNN02-19 isolate was grouped with previously published Chinese isolates that were identified as genogroup II, which contained three specific amino acid residues (377K, 539V and 557S) in the VP1 protein. In addition, the three enteric viruses appeared genetically heterogeneous to each other. All isolates were found to be more sensitive when exposed to low pH conditions, but they were resistant to treatment with trypsin and bile salts. Furthermore, there were no significant differences between the respiratory and enteric FCVs. Our results showed that the genetically distinct FCV strains with genogroups I and II from respiratory and enteric origins were co-circulating in this geographical area. Also, it was revealed that the potential recombinant events between the enteric and respiratory FCVs suggested an important role of enteric FCV during the evolution.

Caliciviruses induce mRNA of tumor necrosis factor α via their protease activity.

Calicivirus infection in patients and animals is associated with the production of multiple inflammatory cytokines, including tumor necrosis factor α (TNF-α). Here we studied the feline calicivirus (FCV) non-structural proteins and found that the FCV protease was a key factor for TNF-α gene expression in cultured cells. The expression of the TNF-α gene in cells expressing FCV, human norovirus, and rabbit hemorrhagic disease virus protease was compared, revealing that the induction of TNF-α could be a common phenomenon during the infection by the viruses in the Caliciviridae. The level of TNF-α mRNA in the cells expressing mutant proteases that lacked the active site was measured. These data indicate that the protease activity is crucial for TNF-α expression. These findings provide new insight into the induction of inflammation during calicivirus infection.

Feline Calicivirus Virulent Systemic Disease: Clinical Epidemiology, Analysis of Viral Isolates and In Vitro Efficacy of Novel Antivirals in Australian Outbreaks.

Feline calicivirus (FCV) causes upper respiratory tract disease (URTD) and sporadic outbreaks of virulent systemic disease (FCV-VSD). The basis for the increased pathogenicity of FCV-VSD viruses is incompletely understood, and antivirals for FCV-VSD have yet to be developed. We investigated the clinicoepidemiology and viral features of three FCV-VSD outbreaks in Australia and evaluated the in vitro efficacy of nitazoxanide (NTZ), 2'-C-methylcytidine (2CMC) and NITD-008 against FCV-VSD viruses. Overall mortality among 23 cases of FCV-VSD was 39%. Metagenomic sequencing identified five genetically distinct FCV lineages within the three outbreaks, all seemingly evolving in situ in Australia. Notably, no mutations that clearly distinguished FCV-URTD from FCV-VSD phenotypes were identified. One FCV-URTD strain likely originated from a recombination event. Analysis of seven amino-acid residues from the hypervariable E region of the capsid in the cultured viruses did not support the contention that properties of these residues can reliably differentiate between the two pathotypes. On plaque reduction assays, dose-response inhibition of FCV-VSD was obtained with all antivirals at low micromolar concentrations; NTZ EC50, 0.4-0.6 µM, TI = 21; 2CMC EC50, 2.7-5.3 µM, TI > 18; NITD-008, 0.5 to 0.9 µM, TI > 111. Investigation of these antivirals for the treatment of FCV-VSD is warranted.