The Effect Of Nitazoxanide Against Feline Calicivirus And Its Effect On Apoptosis Of Virus-infected F81 Cells

Feline calicivirus(FCV)belongs to Caliciviridae,the Vesicular virus genus.It is a respiratory pathogen that is widely prevalent among felines and is more harmful to kittens.It mainly proliferates in the oral cavity and upper respiratory tract,causing feline oral ulcers and upper respiratory tract diseases.At present,there is no specific treatment for this disease,and vaccination is still the primary method to prevent this disease.However,many reports indicate that vaccination cannot effectively control mutant strains'infection,so researchers are gradually valuing anti-FCV drugs'research.In this study,we first verified that nitazoxanide(NTZ)and mizoribine(MZR)have low cytotoxicity in vitro and later found that they affect FCV CH-JL2,CH-JL1,CH-JL3,CH-JL4 and CH-SH strains have antiviral effects.Among them,the FCV CH-JL2 strain has been proved to have good pathogenicity and strong immunogenicity in previous studies,so the FCV CH-JL2strain was selected as the follow-up experimental study.We found that when NTZ or MZR was added to the virus-infected cells at different time points,NTZ and MZR may play a role in the early stage of FCV replication.We subsequently found that the EC₅₀ of NTZ and MZR on FCV was 1.53?M and 12.68?M,indicating that NTZ has better antiviral effects on FCV than MZR.Through the Zero Interaction Efficacy(ZIP)model,we found that NTZ(0-2.5?M)and MZR(0-20?M)have a synergistic anti-FCV effect,and the good synergistic effect score is 45.5.Therefore,both NTZ and MZR have antiviral effects on FCV in vitro.First,NTZ was dissolved in PBS to make a suspension,and NTZ of different doses was orally administered to experimental cats 1 day before the challenge and 3 days after the challenge to evaluate their antiviral effects.We found that significantly improved the clinical symptoms of cats taking NTZ,and the mortality rate was lower than that of the simulated treatment group without oral drugs.At the same time,oral administration of NTZ can also reduce the cat's daily virus shedding and the viral load of the trachea and lungs,and it still has a therapeutic effect on cats infected with FCV after 3 days of the challenge.We further found that NTZ did not negatively affect the cat's body through complete blood count and blood biochemical analysis.Therefore,NTZ may be used as one of the sources of clinical anti-FCV drugs.Past studies have found that FCV can induce apoptosis after infecting cells,and some proteins activated during apoptosis play a crucial role in their proliferation.Therefore,subsequent research starts from apoptosis and further explores the possible anti-virus mechanism of NTZ.We found that after virus infection,NTZ treatment increased the expression levels of caspase-2,9,and BCL-2 genes and down-regulated the expression levels of caspase-3genes but had no effect on the expression levels caspase-7,8,10 genes.We further found that the treatment of NTZ did not affect the activity of caspase-2 and 9 proteins,but Western blot experiments found that the treatment of NTZ reduced the protein expression level of caspase-2and 9 and further showed that NTZ could inhibit FCV-induced F81 cells apoptosis.