Nitazoxanide protects cats from feline calicivirus infection and acts synergistically with mizoribine in vitro

Abstract

Feline calicivirus (FCV) is a highly contagious pathogen that causes acute upper respiratory infections and oral disease in cats, thus seriously endangering feline health. Recently, there have been outbreaks of particularly virulent variant strains of FCV, which can cause both acute symptoms and fatal systemic disease. The discovery of effective antiviral agents to treat FCV infection is, therefore, gradually assuming increased importance. In this study, we showed that both nitazoxanide and mizoribine had antiviral activity in F81 cells infected with different strains of FCV and also demonstrated a synergistic effect between the two drugs. Experiments in cats challenged with FCV showed that nitazoxanide significantly reduced the clinical symptoms of FCV infection, reduced viral load in the trachea and lungs, and reduced viral shedding. Our results showed that nitazoxanide and mizoribine could potentially be used as therapeutic agents to treat FCV infection.

Keywords: Antiviral; Feline calicivirus; In vivo; Mizoribine; Nitazoxanide.

Fig. 1

Evaluation of antiviral effects of nitazoxanide and mizoribine against FCV. (A and E) Solutions of MZR (100 mM) and NTZ (100 mM) in DMSO were diluted with MEM to provide test solutions of different concentration. The solutions were mixed with virus (100 × TCID₅₀); the fluorescence was measured and the optical density was determined statistically. The mock group contained 0.4% DMSO. (B and F) The TCID₅₀ of FCV was measured after incubation with different concentrations of MZR and NTZ for 28 h. (C and G) Relative RT-qPCR was used to measure the expression of FCV RNA. (D and H) EC₅₀ curves for MZR and NTZ. Unless otherwise stated, all experiments were simulated with 0.4% DMSO and 100 × TCID₅₀ FCV, and all were repeated three times. *p < 0.0332; **p < 0.0021; ***p < 0.0002; ****p < 0.0001.

Fig. 2

Evaluation of antiviral effects of nitazoxanide and mizoribine against other FCV strains and their effects in combination. (A) Once the antiviral activity of NTZ and MZR against the FCV CH-JL2 strain had been established, CH-JL1, CH-JL3, CH-JL4 and CH-SH strains (diluted to 100 × TCID₅₀) were treated with NTZ (20 μM) or MZR (40 μM). TCID₅₀ values were measured after 28 h. (B) Relative RT-qPCR was used to assess changes in gene levels of different FCV strains. (C and D) Virus TCID₅₀ values were calculated to determine the combined effect of different concentrations of NTZ (0–2.5 μM) and MZR (0–20 μM) on FCV. The ZIP model in SynergyFinder was used to analyze the combined effects of the drugs and plot the results. The synergy score for the ZIP model was expressed as the average of all δ scores in the dose-response landscape, and the red portion of the graph indicates synergy. All experiments were repeated three times. *p < 0.0332; **p < 0.0021.

Fig. 3

Nitazoxanide reduces clinical symptoms in cats infected with FCV. Data are shown for the three oral treatment groups (A, B and C) used to test the prophylactic effect of NTZ (dosing one day before infection, −1 dpi) and the three groups (D, E and F) used to test the therapeutic effect of NTZ (dosed three days post infection, 3 dpi), together with the respective control groups. (A and E) Rectal temperature measured every 2 days from 0 dpi. (B and F) Daily changes in body weight, converted to rate of weight change compared with 0 dpi. (C and G) Clinical scores at 0 dpi and 7 dpi, calculated using Clinical Scores Table. (D and H) Survival rates for different groups. n = 4 cats/group; NS p > 0.1234; ***p < 0.0002; ****p < 0.0001.

Fig. 4

Nitazoxanide reduces virus shedding and viral load. (A and B) FCV viral shedding in cat's mouth and nose test paper on 0 dpi, measured by RT-qPCR. (C and D) At 14 dpi, one cat was euthanized in each group, the trachea, lung, spleen, liver and kidney were collected, and shedding of FCV from the different tissues was measured. (E) Cats' tracheas and lungs were sectioned (400-fold) and the sections were treated with cat anti-FCV primary antibody and HRP-labeled rabbit anti-cat secondary antibody, followed by color development. Brown areas indicated by black arrows are FCV-positive. (F) Image-Pro Plus 6.0 was used to analysis optical density in two selected slices (400-fold); n = 4 cats/group; −1 dpi, 10 mg/kg NTZ; 3 dpi, 10 mg/kg NTZ; Control, 500 μL PBS (to simulate 10 mg/kg NTZ orally); NS p > 0.1234; *p < 0.1234; **p < 0.0021; ***p < 0.0002; ***p < 0.0001.

Fig. 5

Complete blood count and biochemical analysis. After oral NTZ treatment, two cats in groups B and E were selected randomly and venous blood samples were collected every two days for complete blood count and biochemical analysis. (A) White Blood Cell (WBC) counts. (B) Lymphocyte (LYM) percentages. (C) MID cells (MID) counts. (D) MID percentages. (E) Granulocytes (GRAN) percentages. (F) Platelets (PLT) counts. (G) Alkaline phosphatase (ALKP) levels. (H) Alanine transaminase (ALT) levels. (I) Amylase (AMYL) levels. (J) Total bilirubin (TBIL) levels. (K) Urea (UREA) levels. (L) Glucose (GLU) levels.

S1

Nitazoxanide and Mizoribine IFA results. A solution of NTZ in DMSO (100 mM) was diluted to different concentrations (200 μM, 100 μM, 80 μM, 60 μM, 40 μM, 20 μM, 0 μM) with MEM. Solutions with different concentrations were then added to cells, together with 100 × TCID FCV. The mock group was treated with the same volume of MEM containing 0.4% DMSO. (A) NTZ (B) MZR.

S2

Reduction of cat oral ulcers.A cat in group E that started oral NTZ at 3 dpi developed a large area of oral ulcers at 3 dpi but the ulcerated area gradually decreased with time. A cat in group B that started oral NTZ at −1 dpi had no visible oral ulcers at 7 dpi. A cat in the control group had a mouth ulcer at 3 dpi.

S3

Nitazoxanide and Mizoribine cytotoxicity results A solution of NTZ and MZR in DMSO (100 mM) was diluted to different concentrations (1000 μM, 500 μM, 200 μM, 100 μM, 80 μM, 60 μM, 40 μM, 20 μM, 0 μM) with MEM. Solutions with different concentrations were then added to cells. The cells were incubated for 28 or 72 h at 37°C under an atmosphere containing 5% CO₂. Cell viability was calculated using the following equation:Cell viability = [OD (Compound) - OD (blank)] / [OD (control) - OD (blank)] × 100%. (a) MZR (b) NTZ.