Antiviral activity of nitazoxanide against Morbillivirus infections

Abstract

The measles virus (MeV) and canine distemper virus (CDV) belong to the genus Morbillivirus of the Paramyxoviridae family. They are enveloped viruses harboring a non-segmented negative-sense RNA. Morbilliviruses are extremely contagious and transmitted through infectious aerosol droplets. Both MeV and CDV may cause respiratory infections and fatal encephalitis, although a high incidence of brain infections is unique to CDV. Despite the availability of a safe and effective vaccine against these viruses, in recent years we are witnessing a strong resurgence of Morbillivirus infection. Measles still kills more than 100,000 people each year, and CDV causes widespread outbreaks, especially among wild animals, including non-human primates. No drugs are currently approved for MeV and CDV. Therefore, the identification of effective antiviral agents represents an unmet medical need. Here, we have investigated the potential antiviral properties of nitazoxanide (NTZ) against MeV and CDV. Antiviral activity was explored with live virus and cell-based assays. NTZ is a thiazolide that is approved by the FDA as an antiprotozoal agent for the treatment of Giardia intestinalis and Cryptosporidium parvum. Further, nitazoxanide and its metabolite tizoxanide have recently emerged as broad-spectrum antiviral agents. We found that NTZ blocks the MeV and CDV replication, acting at the post-entry level. Moreover, we showed that NTZ affects the function of the viral fusion protein (F), impairing viral spread. Our results indicate that NTZ should be further explored as a therapeutic option in measles and canine distemper virus treatment.

Keywords: Antiviral; Canine distemper virus; Measles; Morbillivirus; Nitazoxanide; Thiazolide.

Fig. 1

Antiviral activity of nitazoxanide (NTZ) against measles virus (MeV) and canine distemper virus (CDV). (A–B). MeV (A) and CDV (B) were incubated with the indicated concentrations of NTZ and added to Vero-hSLAM cells for 2 h to allow viral entry. After this time, infected cells were washed with PBS three times, and the medium containing carboxymethylcellulose was added. After 72 h, cells were fixed with 4% paraformaldehyde, and plaques were stained with crystal violet. Data represents the results from three independent experiments. (C–D) Vero-hSLAM cells were infected with 100 PFU/well of MeV (C) and CDV (D) viruses (96-well plate) and 2 h post-infection (p.i.), the medium was replaced with a complete medium containing different concentrations of NTZ. After 72 h, the supernatants were collected, and virus yield was determined by plaque assay. Data from three different experiments are shown. The error bars show the mean ± SEM.* p-value<0.05, **<0.01, ***<0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Effect of nitazoxanide (NTZ) on viral fusion and spread. (A) The fusion assay was performed in the presence or in the absence of NTZ (10 or 20 μg/ml) and in HEK-293T cells co-transfected with the measle virus (MeV) IC323–F-wt and MeV IC323–H-wt. Target cells were transfected with nectin-4 as a receptor. Effector cells were overlaid on target cells expressing the nectin-4 receptor and incubated overnight. NTZ was added to target cells 30 min before adding the effector cells. (B)PVero-hSLAM cells were infected with MeV IC323-EGFP in the presence of the indicated concentrations of NTZ. 72 h p.i the cells were fixed with 4 % paraformaldehyde, images were collected with a Nikon Ti2–U fluorescent microscope and the area of infection was measured in pixels using ImageJ software. (C) Graph showing the infection area in pixels measured with ImageJ software. * p-value<0.05.

Suppl. Fig. 1

Antiviral activity of TIZ on MeV. Vero-hSLAM cells were infected with MeV 100 plaque forming unit (PFU)/well and 2 h p.i. the medium was replaced with a complete medium containing the different concentrations of TIZ. At 72 h p.i., the supernatants were collected and virus yield was determined by plaque assay. Data from three different experiments are shown. Error bars show the mean ± SEM. * p-value<0.05, ***<0.001.

Suppl. Fig.2

Pretreatment of cells with NTZ does not affect MeV infection. Cells were treated with the indicated concentrations of NTZ for 3 or 6 h before infection with measles virus. After drug removal cells were washed three times with PBS, and infected with MeV for 2 h, after which time carboxymethylcellulose was added. At 72 h p.i., cells were fixed with 4% paraformaldehyde and the plaques were stained using crystal violet. Results of a representative experiment are shown.

Suppl. Fig.3

Decrease of CDV spread in the presence of NTZ. (A) Vero-hSLAM cells infected with CDV in the presence of different concentrations of NTZ. Images were randomly acquired with a Nikon Ti2-U inverted microscope and the area of infection in pixels was measured using ImageJ software. (B) Graph showing the infection area in pixels measured with ImageJ software.