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

Abstract

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.

Keywords: 2′-C-methylcytidine; Caliciviridae; NITD-008; Vesivirus; nitazoxanide.

Figure 1

Evolutionary history of viruses discovered in this study. Maximum-likelihood trees were reconstructed based on (A) Whole-genome and (B) partial VP1 gene alignments, which included virus sequences obtained in this study (marked with solid red circles) as well as those obtained from the GenBank. The trees were mid-point rooted and bootstrap values of ≥70% are marked on the tree. Each sequence name contains the accession number, strain name, geographic location, host and isolation year, if available. Disease phenotype is colour coded.

Figure 2

Multiple correspondence analysis (MCA) graph depicting the results of MCA of 13 physical and chemical categorical variables of 7 amino-acid residues of the hypervariable E region of VSD and non-VSD FCV strains from Table 4. * Most common amino-acid configuration of FCV_VSD and FCV_URTD reference strains (see Table 4); E-ITA, enteric FCV strains (see Table 4).

Figure 3

Recombination analyses of Australian FCV strains identified in this study. The top panel shows similarity comparisons of ACT2 against representative FCV strains across the entire genome using a sliding window (window size: 500 bp, step size: 10 bp). The potential recombination breakpoints are shown as red vertical lines. The bottom panel shows phylogenetic trees based on each non-recombinant region separated by recombination breakpoints. The strain names are labelled with different colours to mark the representative strains used in SimPlot analyses, including the parental groups for the recombinants.

Figure 4

The antiviral activity of nitazoxanide (NTZ), NITD-008 and 2′-C-methylcytidine (2CMC) against four FCV strains in cell culture. The EC₅₀ values of the three compounds against each of FCV strain were calculated by fitting the dose–response curves from a plaque reduction assay. Triplicate values from at least two independent experiments are presented, and the mean ± SEM are shown.