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.

Evaluation of Drug Uses for Calicivirus and Panleukopenia Treatment in Bogor Animal Clinic on 2017 and 2018

Drug therapy in cases of infections by viruses is only to prevent secondary infections. The use of drugs for each action must be evaluated through a drug use evaluation program (EPO) in order to guarantee a rational and effective drug. This study aims to examine the drugs in case for handling and the effectiveness of drug use in cases of infections caused by viruses, in this study the therapeutic use of drugs in cases of feline calicivirus and feline panleukopenia. This research is a descriptive study using data from 543 patients who came to get treatment of clinic in Bogor City during 2017 and 2018, 29 of which were infected with feline calicivirus and 32 infected with feline panleukopenia. Evaluation of drug use in these two viral diseases is done descriptively by comparing research data with literature. The results showed the use of drugs in the case of feline calicivirus there were 12 types of drug treatment, while in the case of feline panleukopenia there were 9 types of drug treatment. The use of drugs most often used is based on the records of veterinary clinical records, namely the preparation of metronidazole combined with cefadroxil to handle cases of feline calicivirus and metronidazole alone to handle cases of feline panleukopenia.

Molecular Characterization and Cross-Reactivity of Feline Calicivirus Circulating in Southwestern China

Feline calicivirus (FCV) is an important pathogen of cats that has two genogroups (GI and GII). To investigate the prevalence and molecular characteristics of FCVs in southwestern China, 162 nasal swab samples were collected from cats in animal shelters and pet hospitals. In total, 38 of the clinical samples (23.46%) were identified as FCV positive using nested RT-PCR. Phylogenetic analyses using 10 capsid protein VP1 sequences revealed that 8 GI and 2 GII strains formed two independent clusters. Additionally, three separated FCVs that were not clustered phylogenetically (two GI and one GII strains) were successfully isolated from clinical samples and their full-length genomes were obtained. Phylogenetic and recombinant analyses of a GI FCV revealed genomic breakpoints in ORF1 and ORF2 regions with evidence for recombinant events between GI sub-genogroups, which is reported in China for the first time. Furthermore, sera obtained from mice immunized independently with the three FCV isolates and a commercial vaccine were used to evaluate the cross-reactivity of neutralizing antibodies. The three separate FCVs were neutralized by each other at a 1:19 to 1:775 titer range, whereas the triple-inactivated vaccine was at a titer of 1:16, which suggested that different genogroup/sub-genogroup FCV strains exhibit significantly different titers of neutralizing antibodies, including the commercial FCV vaccine. Thus, our study revealed the genetic diversity and complex cross-reactivity levels of FCVs in southwestern China, which provides new insights for application in vaccination strategies.

Molecular Investigation of Feline Calicivirus Infection in China, 2019-2020

Feline calicivirus (FCV) is a highly contagious viral pathogen of upper respiratory infections and oral disease in cats. To investigate the prevalence and gene characteristic of FCV in China, a total of 1739 clinical swabs of cat eyes and nasal were collected from 19 cities in China from 2019 to 2020. The FCV from clinical samples were isolated in F81 cells, and the gene sequences of the isolated FCV’s capsid proteins were phylogenetically analyzed by constructing the phylogenetic tree with the FCV vaccine strain F9 and reference strains of other countries. Results revealed a prevalence of 13.0% (226/1739) for FCV in China in this study, and samples from Langfang showed the highest prevalence in the cities. The 74 FCV strains isolated from clinical samples shared the nucleotide identity of 73.4%-79.1% and the amino acid identity of 83%-90% comparing with the F9 strain. Phylogenetic analysis reveals two branches of these FCV strains from China, which distinct from the vaccine strains of F9 and 255, and other reference strains. Structurally, the highly variable sites of capsid protein were exposed on the protein surface between circulating strains in China and the vaccine strain F9. Overall, this study would promote the understanding of the FCV prevalence and gene characteristics in China.

Molecular Characterization of a Natural Mutant of Feline Calicivirus in China

During epidemiological surveillance of Feline calicivirus (FCV) isolates in Shanghai, China, a natural mutant of FCV, designated SH1909, was successfully isolated from a stray cat. The complete genome sequence of SH1909 was determined in this study. Sequence comparison and analysis showed that thirteen unique aa residues substitutions and single-aa insertion of N or Y were observed in SH1909, which indicated that SH1909 was a novel and natural mutant of FCV. Interestingly, phylogenetic analysis based on LC-VP1 showed SH1909 could be clustered into an independent evolutionary branch with some Chinese isolates and was more distantly related to vaccine strains, indicating its potential to escape from vaccine-elicited immunity. According to the predicted B-cell epitopes of LC-VP1, amino acid mutation sites and positive selective sites, peptide C (aa sites: 445–460) and, peptide D (aa sites: 425–440) located in the hypervariable regions of LC-VP1, may result in the decreased immunological protection. Moreover, amino acid sites 439 and 449 may be responsible for the potential immune escape of SH1909. This study provides an important insight into genetic variations of FCV and vaccine development.

Modified-Live Feline Calicivirus Vaccination Elicits Cellular Immunity against a Current Feline Calicivirus Field Strain in an Experimental Feline Challenge Study

Feline calicivirus (FCV) is a common cat virus associated with oral ulcerations and virulent-systemic disease. Efficacious FCV vaccines protect against severe disease but not against infection. The high genetic diversity of FCV poses a challenge in vaccine design. Protection against FCV has been related to humoral and cellular immunity; the latter has not been studied in detail. This study investigates the cellular and humoral immune response of specified pathogen-free (SPF) cats after modified-live FCV F9 vaccinations and two heterologous FCV challenges by the analysis of lymphocyte subsets, cytokine mRNA transcription levels, interferon (IFN)-γ release assays in peripheral blood mononuclear cells (PBMCs), anti-FCV antibodies, and neutralisation activity. Vaccinated cats developed a Th1 cytokine response after vaccination. Vaccination resulted in antibodies with neutralising activity against the vaccine but not the challenge viruses. Remarkably, IFN-γ-releasing PBMCs were detected in vaccinated cats upon stimulation with the vaccine strain and the first heterologous FCV challenge strain. After the first experimental infection, the mRNA transcription levels of perforin, granzyme B, INF-γ, and antiviral factor MX1 and the number of IFN-γ-releasing PBMCs when stimulated with the first challenge virus were higher in vaccinated cats compared to control cats. The first FCV challenge induced crossneutralising antibodies in all cats against the second challenge virus. Before the second challenge, vaccinated cats had a higher number of IFN-γ-releasing PBMCs when stimulated with the second challenge virus than control cats. After the second FCV challenge, there were less significant differences detected between the groups regarding lymphocyte subsets and cytokine mRNA transcription levels. In conclusion, modified-live FCV vaccination induced cellular but not humoral crossimmunity in SPF cats; innate immune mechanisms, secretory and membranolytic pathways, and IFN-γ-releasing PBMCs seem to be important in the host immune defence against FCV.

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 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 provided no support for 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 treatment of FCV-VSD is warranted.

Erratum: Investigation of Feline calicivirus infection in cats with upper respiratory tract disease in Diyarbakir, Turkey

Feline calicivirus is among the most common pathogenic microorganisms in upper respiratory tract disease (URTD) and oral lesions of cats. It leads to stomatitis, oral ulceration, ocular and nasal discharge, conjunctivitis, fever, lameness, anorexia, hypersalivation, pneumonia, respiratory distress, coughing, and depression in infected cats. This study aimed to determine the role of Feline calicivirus (FCV) in cats with the upper respiratory tract disease in the Diyarbakir region, Turkey, to provide treatment for infected cats and contribute to the disease prophylaxis. The study material consisted of 10 cats (control group) considered to be healthy according to the clinical examination and 20 cats with URTD that were not vaccinated against Feline calicivirus infection of different breeds, ages, and genders brought to Dicle University Veterinary Faculty Prof. Dr. Servet SEKIN Polyclinic with URTD. After routine clinical examinations of the animals, oral and conjunctival swabs and blood samples were taken. Hematological and biochemical analyzes of blood samples were performed. Swab samples were analyzed by the polymerase chain reaction (PCR) method for the diagnosis of the agent. Oral lesions, hypersalivation, ocular and nasal discharge, coughing, and breathing difficulties were seen in clinical examinations of cats with URTD. Feline calicivirus was detected in only one cat's conjunctival swab sample in PCR analyses. As a result, we found that Feline calicivirus infection was present in cats with URTD in the Diyarbakir region, and 5% positivity was found in cats with clinical symptoms according to PCR analysis.