Characterization and immunogenic evaluation of feline calicivirus epidemic strains

Figures

• 

  Fig. 1. Isolation and identification of the FCV strain. (A) CRFK cells were infected with FCV023 and uninfected medium at 12 h post-infection. Scale bar = 100 μm. (B) The FCV023 isolate was verified through RT-PCR with a 450 bp target fragment. (C) The 62 kD target protein of FCV023 isolates was identified by western blot using an anti-Feline Calicivirus [1D7] antibody. (D) Specific indirect immunofluorescence analysis (IFA, green fluorescence) of CRFK cells isolated by FCV023 at 12 hpi using anti-feline calicivirus antibody. Mock indicates the negative control. Scale bar = 100 μm. (E) TEM identified FCV023 particles (black arrow) with a diameter of approximately 37 nm. Scale bar = 100 nm. NC, nagative control; FCV, feline calicivirus; DAPI, 4ʹ,6-diamidino-2-phenylindole; CRFK, Crandell-Reese feline kidney.

• 

  Fig. 2. Phylogenetic tree analysis of the VP1 nucleotide sequences of the FCV strains. A phylogenetic tree based on 30 VP1 gene sequences from isolated strains in the authors’ laboratory and 32 reference FCV strains (14 Chinese isolates and 18 foreign isolates) was constructed using the maximum likelihood method using the MEGA 7.0 software. The bootstrap values were calculated according to 1,000 replicates of the alignment (FastQC, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). VSD, virulent systemic disease; FCV, feline calicivirus.

• 

  Fig. 3. Comparison of the characteristic amino acid site analysis of the partial E region in VP1 protein. FCV, feline calicivirus; CHN, China; USA; United States of America; FR, France; UK, United Kingdom; GER, Germany; JPN, Japan.

• 

  Fig. 4. Clinical symptoms, histopathology, and immunohistochemistry of cats inoculated with FCV023. (A) Relatively body weight loss within 14 days post-infection. The weight on day 0 before the challenge was 0%. (B) The temperature response to viral infection. (C) Daily scores of the cats’ clinical signs based on the scoring sheet. Viral-specific qRT-PCR detection of the viral loads from oral swabs (D) and eye swabs (E) of cats in the experimental group during the infection period. (F) Gross anatomy of the experimental cats during an FCV023 infection. Gross anatomy indicated gross lesions consistent with FCV, such as mucosal ulceration and hemorrhagic spots in the trachea and lungs. (G) Histopathological examination staining of different tissue sections in the mock-infected and virus-infected cats. Lung consolidation with inflammatory cell infiltration, lingual epithelial mucosa degeneration and necrosis with abscess formation, laryngeal mucosa erosion, tracheal epithelial cell degeneration, and increased germinal centers in the splenic white pulp. The black arrows point to the inflammatory reaction. Scale bar = 200 μm. (H) Immunohistochemical analysis using an anti-feline calicivirus…

• 

  Fig. 5. Antibody level and neutralizing titer detection. (A) Dynamic monitoring of the FCV IgG antibody in immunized mice. (B) Determination of the titer of the neutralization test using the parental virus and positive serum samples collected 14 days after booster immunization from the indicated groups. (C) Results of the cross-neutralization test between the different positive serum and different strains (the ordinate is the titer of the serum; log2 is used to calculate the final value; the horizontal coordinate is the name of the virus used in the assay. (D) Dynamic monitoring of the IgG antibody in immunized cats. (E) Measurements of the titer of the neutralization test using the parental virus at different time points. (F) Results of the cross-neutralization test between the positive serum and seven FCV strains after immunization. Log2 was used to calculate the final value. The data were taken from the average of three individuals). FCV, feline calicivirus.