Isolation, Molecular Characterization, and Pathogenicity Evaluation of a Genotype GI Feline Calicivirus Isolate

Figures

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  Figure 1. Sample Collection and Identification. (A) The sample collection site was labeled with a yellow pattern. The base layer of this modified map originated from National Earth System Science data center, National Science & Technology infrastructure of China (http://www.Geodata.cn) (B) PCR identification of FCV and other viruses (FPV, FHV, FIPV, FBoV, and FeAstV) in oral and nasal swabs. (C) NGS statistical results revealed FCV as the only identified virus in samples.

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  Figure 2. Isolation and characterization of FCV. (A) Virus-induced CPE in FCV-infected CRFK, F81 and Fc3Tg cells that are visible on light microscopy. (B,C) PCR detection of FCV in plaque-purified samples following serial passages. (D) Viral titers of FCV Strain HN/ZZ/2025 in CRFK, F81, and Fc3Tg cells determined by TCID50. Data are presented from three independent experiments (n = 3), with individual data points shown. (E,F) Growth curves of FCV in CRFK, F81, and Fc3Tg cells. Viral RNA copies at 0, 6, 12, 18, 24, 30, 36, 48 and 60 hpi determined by RT-qPCR and TCID50 assay. Data are presented from three independent experiments (n = 3), with individual data points shown. (G) Immunoblotting was used to detect the expression of viral protein VP1 in CRFK, F81, and Fc3Tg cells infected with FCV HN/ZZ/2025. (H) FCV-infected CRFK, F81, and Fc3Tg cells detected by IFA, the infected cells were stained with a rabbit serum raised against the FCV VP1 protein. Scale bar, 100 μm. ** p < 0.01.

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  Figure 3. Virus particle morphology and Physicochemical properties of the FCV strain HN/ZZ/2025. (A) Purification of virus by density gradient centrifugation with iodixanol. (B,C) Determination of viral nucleic acid and protein band positions by PCR and SDS-PAGE. 1: Less than the 20% iodixanol, 2: 20% iodixanol, 3: 30% iodixanol, 4: 40% iodixanol, 5: 50% iodixanol, and 6: 60% iodixanol; 7: purified FCV preparation collected after density-gradient purification; “+” and “−” indicate the positive and negative controls, respectively. (D) Electron microscopic examination of morphology of FCV particles. Negatively stained virions purified from FCV-infected CRFK cells. Scale bar, 200 nm and 500 nm. (E,F) Identification of physicochemical properties of FCV strain HN/ZZ/2025. The CPE in CRFK cells infected with FCV treated at 50 °C or exposed to acidic, alkaline, chloroform, or ether conditions were observed under a microscope (E), and viral titers were determined by TCID50 (F). Scale bar, 100 μm. ** p < 0.01, *** p < 0.001.

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  Figure 4. Phylogenetic analysis and amino acid sequence alignment of the isolates in this study. Phylogenetic analysis of isolates based on complete genome (A) and VP1 (B). The phylogenetic tree was generated using the maximum likelihood (ML) method implemented in the program MEGA 7.0. Bootstrap values are represented as a percentage based on 1000 replicates. (C) The genome organization of FCV HN/ZZ/2025 with open reading frames (ORF) 1–3, antigenic regions (A–F) of the VP1 capsid precursor protein, hypervariable and conserved regions of antigenic region E. VPg, virus protein, genome linked; LC, leader of capsid protein; HVR, hypervariable region. (D) Amino acid sequence alignment based on VP1. The abbreviation nt denotes nucleotides. (E–G) The conservation of amino acids in the VP1 E region, including the 5′ hypervariable region (E), the conserved region (F), and the 3′ hypervariable region (G), was analyzed using WebLogo.

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  Figure 5. Clinical sign monitoring in cats infected with FCV HN/ZZ/2025. (A) Schematic diagram of the experimental infection and monitoring schedule following intranasal inoculation with FCV HN/ZZ/2025. (B) Changes in rectal temperature of cats following intranasal inoculation with FCV HN/ZZ/2025 or MEM (control). (C) Body weight changes in infected and control cats during the observation period. (D) Clinical score dynamics based on body temperature, body weight, mental status, appetite, ocular discharge, nasal discharge, oral ulcers, and survival status. (E) White blood cell (WBC) counts in infected and control cats at 14 dpi. * p < 0.05.

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  Figure 6. Pathological lesions and tissue tropism of FCV strain HN/ZZ/2025 in experimentally infected cats. (A) Clinical signs in cats, and macroscopic examination of lung lesions. (B–D) Necropsy examinations, histopathology, IHC and IFA of the lungs, trachea, kidneys, and spleen of the FCV challenge cats at 21 dpi. (B) Histopathological changes in tissues of FCV-infected cats revealed by hematoxylin and eosin (H&E) staining. (C) IHC and IFA detection FCV VP1 antigen-positive signals in the corresponding tissues of infected cats. Scale bar, 50 μm and 100 μm.

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  Figure 7. Viral shedding and tissue distribution of FCV HN/ZZ/2025 in infected cats. (A,B) Dynamic viral shedding in oral and nasal swabs collected from infected cats following intranasal inoculation, as determined by RT-qPCR and TCID50 assay. (C,D) Cats were euthanized at 21 dpi, and different organs were collected for determination of viral RNA loads by RT-qPCR (C) and infectious viral titers by TCID50 assay (D). (ND, not Detected). (E) Immunoblotting analysis showing the expression of FCV VP1 protein in tissues of infected cats. (F) Densitometric analysis of VP1 protein expression in tissues based on Image J, normalized to GAPDH. arb. units, arbitrary units. *** p < 0.001.