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.

The LC protein plays an important role in viral spread and can cause an activation of caspases and a cytopathic effect (CPE) in cell cultures (Abente et al. 2013). VP1 capsid protein has a functional role during the early replication of the calicivirus. The capsid precursor protein (LC-VP1) can further be divided into six regions, termed A to F. Regions B, D and F are more conserved, whereas regions A, C and E are variable. Region E contains two hypervariable regions that are separated by a conserved domain (Seal et al. 1993). The E region contains the major B cell epitopes and plays an important role in the formation of viral particle antigen structures. And conformational epitopes are probably more important than linear epitopes in viral neutralization (Radford et al. 2007;Sun et al. 2017). VP2 is essential for productive replication that results in the synthesis and maturation of infectious virions (Brunet et al. 2019;Sosnovtsev et al. 2005).
Since FCV was rstly isolated and identi ed in 1957, it has been distributed worldwide in all feline species, such as cats, tigers, lions, and cheetahs, with cats under one year old being the most susceptible (Kadoi et al. 1997;Radford et al. 1998).
FCV infection is closely related to upper respiratory tract disease, acute mouth ulceration, and chronic stomatitis. Moreover, most infected or clinically recovered cats can persistently excrete the virus. During the widespread use of commercial vaccines, the cross-protection between the FCV wild strains and the live vaccine strain F9 has been gradually weakened (Smith et al. 2020), and immunization failure occurs frequently. Although existing vaccines can reduce clinical symptoms and virus excretion, they cannot prevent infection from occurring (Berger et al. 2015). Therefore, the persistent surveillance should be performed for the prevalence of FCV isolates and can provide an important insight into the development of novel vaccines. In recent years, there have been some reports of Chinese FCV strains, but few reports of FCV strains from the Chinese stray cats were reported (Guo et al. 2018;Sun et al. 2017). In view of the larger sphere of activity for stray cats, it is easier to spread viruses. So, the primary aim of this study was to isolate FCV strains from some stray cats in Shanghai, China and analyze their genomic and evolutionary characterization.

Materials And Methods
Sample description and DNA extraction Page 3/9 27 fecal samples were collected from the stray cats of Shanghai in September 2019. All samples were respectively homogenized in 1 mL of phosphate-buffered saline and centrifuged at 12,000 × g for 10 min. The clari ed supernatant was ltered through 0.22 µm lters (Millipore). Viral RNA was then extracted from the clari ed suspension using a TIANamp Virus DNA/RNA Kit (Tiangen, Beijing, China ) and reverse transcribed into cDNA using the FastQuant RT Kit (with gDNase) (Tiangen) according to the manufacturer's instructions Primer design and Polymerase Chain Reaction(PCR) ampli cation of FCV Identi cation of viruses was performed by PCR using the highly speci c primers ( Table 1). The results showed three out of 27 samples were positive for FCV. And Crandell feline kidney (CRFK) cells were respectively inoculated with the positive supernatant at 37°C under 5% CO 2 in 25 cm 2 asks. When more than 80% of cells show obvious cytopathic effect, cell suspension was collected and stored at -80°C until use. To amplify the complete genome of FCV, 7 pairs of speci c primers (Table 1) were designed based on the conserved regions of the FCV genome sequences retrieved from NCBI. Reverse transcription PCR was performed in a 50 µL reaction mixture comprising 25 µL of Premix Taq (Takara Bio Inc., Shiga, Japan), 2 µL of each primer (10 µM), 2 µL of cDNA template, and 19 µL of ddH2O. The reaction conditions were as follows: initial denaturation at 95°C for 5 min;, 33 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min; and a nal elongation step of 72°C for 10min. In addition, the 3′ end of the genome was determined by using the method of rapid ampli cation of cDNA ends (RACE). The PCR products were visualized through electrophoresis on a 1% agarose gel and then puri ed using the FastPure Gel DNA Extraction Mini Kit (Vazyme) per the manufacturer's instructions.
The puri ed amplicons were cloned into the pMD19-T vector (Takara) for subsequent sequencing (TSINGKE). Table 1 Primers used for identi cation and PCR ampli cation of the complete genome of FCV.

Results And Discussion
After three serial passages, a mutated strain of FCV, referred to as SH1909, was isolated from the fecal specimen of a stray cat. has evolved signi cantly when compared with other FCV isolates and less similarity to the FCV vaccine strains than the eld isolates.
Phylogenetic trees based on the complete aa sequences of LC-VP1. As shown in Fig. 1, all Chinese FCV isolates can be divided into different evolutionary branches based on the phylogenetic analysis of LC-VP1. However, some FCV isolates including SH1909 were clustered in a monophyletic clade, which was distantly related to the FCV vaccine strains, indicating the genotype diversity of FCV isolates and the potential of some Chinese endemic isolates including SH1909 to escape from immune response induced by vaccine.  2). According to the alignment of amino acid sequences, it showed that peptides A and B were located in the conserved N-terminal of VP1, whereas peptides C and D were displayed in its hypervariable C-terminal (Fig. 3). Speci c amino acid mutation site 439 and the positive selection site 449 for SH1909 was included in the peptides D and C, respectively. These results indicated that amino acid mutations with high frequency in the predicted B cell epitopes (peptide C and D) may weaken the capacity of the cross-protection of vaccine. Moreover, the mutations at amino acid sites 439 and 449 may be closely related with SH1909 escaping from the immune response to FCV vaccine.

Conclusion
In conclusion, the complete genome sequence of a FCV strain SH1909 from a stray cat in China, was determined in this study. Phylogenetic analysis revealed that SH1909 was clustered into an independent evolutionary branch with some Chinese isolates and possessed the potential to escape from vaccine-elicited immunity. Sequence comparison and analysis showed that thirteen unique aa residues substitutions and single-aa insertion of N or Y were respectively observed in ORF1 and ORF2 of SH1909, which indicated that SH1909 was a novel and natural mutant of FCV. In addition, two (peptides C and D) out of four predicted B cell epitopes, located in the hypervariable regions of VP1, may result in the decreased immunological protection. Moreover, two speci c mutation sites 439 and 449 may be closely related with SH1909 escaping from the immune response to FCV vaccine. These results will provide an important insight into genetic variations involved in viral evolution and a reference for design of future FCV vaccines. Figure 1 Phylogenetic analysis of 60 FCVs based on LC-VP1 amino acid sequences using the neighbor-joining (NJ) method with the Jones-Taylor-Thornton (JTT) model with 1,000 bootstrap replicates. The reference sequences were retrieved from GenBank. SH1909 isolated in this study is labeled with a lled circle (•). The FCV eld isolates from China were indicated by a lled diamond (♦), the FCV vaccine strains are indicated by a lled triangle (▲). GenBank accession numbers, names of viruses and nations were indicated in the branches. Scale bars indicate amino acid substitutions per site. Clade 1: FCV eld strains and vaccine strains from different countries were assigned into a major branch of Clade 1.Clade 2: FCV eld isolates only from China were grouped into a minor branch of Clade 2.