Classification of genotypes based on the VP1 gene of feline calicivirus and study of cross-protection between different genotypes

Abstract

Feline calicivirus (FCV) causes upper respiratory tract diseases and even death in cats, thereby acting as a great threat to feline animals. Currently, FCV prevention is mainly achieved through vaccination, but the effectiveness of vaccination is limited. In this study, 105 FCV strain VP1 sequences with clear backgrounds were downloaded from the NCBI and subjected to a maximum likelihood method for systematic evolutionary analysis. Based on the genetic analysis results, FCV-positive sera were prepared using SPF mice and Chinese field cats as target animals, followed by a cross-neutralization assay conducted on the different genotype strains and in vivo challenge tests were carried out to further verify with the strain with best cross-protection effect. The results revealed that FCV was mainly divided into two genotypes: GI and GII. The GI genotype strains are prevalent worldwide, but all GII genotype strains were isolated from Asia, indicating a clear geographical feature. This may form resistance to FCV prevention in Asia. The in vitro neutralization assay conducted using murine serum demonstrated that the cross-protection effect varied among strains. A strain with broad-spectrum neutralization properties, DL39, was screened. This strain could produce neutralizing titers (10 × 2^3.08-10 × 2^0.25) against all strains used in this study. The antibody titers against the GI strains were 10 × 2^3.08-10 × 2^0.5 and those against the GII strains were 10 × 2^0.75-10 × 2^0.25. Preliminary evidence suggested that the antibody titer of the DL39 strain against GI was higher than that against GII. Subsequent cross-neutralization assays with cat serum prepared with the DL39 strain and each strain simultaneously yielded results similar to those described above. In vivo challenge tests revealed that the DL39 strain-immunized cats outperformed the positive controls in all measures. The results of several trials demonstrated that strain DL39 can potentially be used as a vaccine strain. The study attempted to combine the genetic diversity and phylogenetic analysis of FCV with the discovery of potential vaccines, which is crucial for developing highly effective FCV vaccines.

Keywords: FCV; VP1; broad-spectrum neutralization; challenge tests; cross-neutralization assay; genotype.

Figure 1

ML phylogenetic tree estimated from 105 FCV capsid (VP1) gene nucleotide sequences (A) and amino acid sequences (B) including the sequences of strains isolated in our laboratory and those downloaded from the NCBI. Model: JTT + G + I; Bootstrap: 1000 replicates. “·” represents the sequence used in this study. Each genotype is shaded by a different color and clearly marked. Strains isolated from different continents are color-coded for easier study. The scale bar indicates the mean number of nucleotide or amino acid substitutions per site. Phylogenetic tree modification: https://www.chiplot.online/.

Figure 2

Preparation of immune antigens. (A) Determination of dynamic growth curves of the 13 FCV strains used in this study. (B) CRFK cells were infected with FCV strains. (C) FCV was purified, counterstained, and visualized through electron microscopy (left image is the untreated sample, right image is the purified sample). (D) Image of plaque test results of FCV strains (The results in above panel (B-D) are presented with the FCV-2280 strain as an example).

Figure 3

Monitoring and identification of mouse serum. (A) Dynamic monitoring of IgG antibody in immunized mice. (B) Neutralizing titers of pooled mouse serum against the parental virus. (C) Indirect immunofluorescent assay results based on each parent virus.

Figure 4

Cross neutralization assay of positive serum in mice. (A) Results of the cross-neutralization test between FCV strains. Each figure represents the result of a cross -neutralization assay for a serum (the name of the serum is mentioned above the legend, the ordinate is the titer of the virus, 10 (log2) is used to calculate the final value, and the horizontal coordinate is the name of the virus used in the assay). (B) Box and violin plots of cross-neutralization titers for each strain. (C) Box and violin plots of cross-neutralization titers for each strain of GI and GII genotypes. (D) Cross-neutralization test results of the DL39 strain in different cat cell lines.

Figure 5

Serological test results of cats immunized after inactivation of DL39 strain. (A) Determination of serum IgG at different time points. (B) The titer of the neutralization test was determined with the parental virus at different time points. (C) Neutralization test titers with 13 FCV strains after the completion of immunization in different cat cell lines.

Figure 6

FCV challenge assay after immunization with DL39 strain [(A) DL39 strain challenge test group. (B) The DL39 strain challenged the experimental group after immunization with the DL39 strain. (C) HRB48 strain challenge test group. (D) The HRB48 strain challenged the experimental group after immunization with the DL39 strain. (E) FB-NJ-13 strain challenge test group. (F) The FB-NJ-13 strain challenged the experimental group after immunization with the DL39 strain. (G) Control group; “ns” no significance, “*“p < 0.05, “**“p < 0.01, “***“p < 0.001. (A) Histogram of clinical symptom scores for different groups after the DL39 challenge, with higher scores representing more severe clinical symptoms. (B) Body temperature statistics of each group. (C) Body weight statistics of each group. (D) Detection of viral RNA in oral swabs through RT-qPCR after the DL39 challenge. (E) Detection of viral RNA in anal swabs through RT-qPCR after the DL39 challenge. (F) Detection of viral RNA in blood through RT-qPCR after the DL39 challenge. (G) Determination of IgM in serum. (H) Determination of IgG in serum.