Inhibition of rubella virus replication by the broad-spectrum drug nitazoxanide in cell culture and in a patient with a primary immune deficiency

Abstract

Persistent rubella virus (RV) infection has been associated with various pathologies such as congenital rubella syndrome, Fuchs's uveitis, and cutaneous granulomas in patients with primary immune deficiencies (PID). Currently there are no drugs to treat RV infections. Nitazoxanide (NTZ) is an FDA-approved drug for parasitic infections, and has been recently shown to have broad-spectrum antiviral activities. Here we found that empiric 2-month therapy with oral NTZ was associated in the decline/elimination of RV antigen from lesions in a PID patient with RV positive granulomas, while peginterferon treatment had no effect. In addition, we characterized the effects of NTZ on cell culture models of persistent RV infection. NTZ significantly inhibited RV replication in a primary culture of human umbilical vein endothelial cells (HUVEC) and Vero and A549 epithelial cell lines in a dose dependent manner with an average 50% inhibitory concentration of 0.35 μg/ml (1.1 μM). RV strains representing currently circulating genotypes were inhibited to a similar extent. NTZ affected early and late stages of infection by inhibiting synthesis of cellular and RV RNA and interfering with intracellular trafficking of the RV surface glycoproteins, E1 and E2. These results suggest a potential application of NTZ for the treatment of persistent rubella infections, but more studies are required.

Keywords: Antivirals; Nitazoxanide; Patient with primary immune deficiency; Rubella virus; Rubella-positive granuloma.

Fig. 1

RV antigen in lesions of a PID patient. (A) Cutaneous skin lesions. (B) Hematoxylin and eosin staining of a cutaneous granuloma. (C) Histological immunofluorescent staining showing distribution of RV capsid protein (red) in granulomas and epidermis of the same skin lesion before and after 2-month treatments with interferon and oral NTZ spaced one month apart. Nuclei were stained with DAPI. Multiple clusters of RV-positive cells were easily detected prior to the NTZ treatment. Note the lack of RV staining in the epidermis and a single weakly positive cell (the white arrow) in the granuloma after the NTZ treatment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Antiviral effects of NTZ against RV in HUVECs. Cell monolayers were infected with RV-Dz at an MOI of 5 and then treated with NTZ at the indicated concentrations or vehicle control immediately after adsorption (Panels A and B). (A) Virus titer in the medium was determined at 48 hpi by a plaque assay. The data represent the means $\pm$ SD of two independent experiments each done in duplicate. The dashed line shows the lower detection limit of the assay. (B) The monolayers (10⁵ cells/well) were fixed at 48 hpi with methanol and immunostained for E1. Nuclei were counterstained with DAPI. HUVECs were treated with different concentrations of NTZ for 48 h (Panels C and D). (C) Cytotoxicity and growth inhibition were analyzed by a modified LDH assay. The data represent the means $\pm$ SD of two independent experiments each done in quadruplicate. (D) Phase contrast images of cells showing the effect of different NTZ concentrations on cell viability.

Fig. 3

Dose-dependent effects of NTZ in different cells cultures. A549, Vero or HUVEC monolayers were infected with RV-Dz at an MOI of 5 and then treated with different concentrations of NTZ immediately after virus adsorption. Virus titer in the medium was determined at 48 hpi by a plaque assay. RV-Dz titers in the DMSO-treated cultures (100%) were 7 × 10⁵ (A549), 3 × 10⁵ (Vero), and 6 × 10⁴ (HUVEC). The data, which are expressed as a percentage of untreated control, represent the mean $\pm$ SD of two (A549 and Vero) or three (HUVEC) independent experiments each done in duplicate. The dotted lines indicate the lC₅₀ values.

Fig. 4

Effect of NTZ treatment on different stages of RV infection. HUVECs were treated with 2.5 μg/ml NTZ at the indicated times prior (pre, black bars), post infection (post, white bars), or during adsorption only (Ad, the striped bar). The grey bar represents a DMSO control. Virus titers in the medium were determined at 48 hpi (pre, Ad) or 48 h post-treatment. The data represent the means $\pm$ SD of at least three independent experiments each done in duplicate. *, p < 0.05; **, p < 0.01 compared with the DMSO-treated cultures.

Fig. 5

Effects of NTZ on RV proteins in HUVECs. (A) WB analysis of rubella structural proteins. HUVECs were infected with RV-Dz at an MOI of 5 or mock-infected and then exposed to 0.5 or 1 μg/ml NTZ or vehicle for 48 h. Proteins were extracted with RIPA buffer, separated by a 4–12% non-reducing NuPage gel, and then the blots were probed with rubella specific antibodies to identify RV structural proteins E1, E2 and C (two C bands indicated by the arrows). The blots were also probed with β-actin MAb to demonstrate equal protein loading. (B) Immunofluorescence analysis (at 2 dpi) of rubella structural proteins in the HUVEC monolayers that were treated with 1 μg/ml NTZ following infection with RA27/3 (MOI = 5 pfu/well). Insert represents enlarged image of the E2 globular structures. Representative results of two independent experiments are shown in both A and B.

Fig. 6

Inhibition of genomic RV RNA synthesis by NTZ. HUVEC monolayers were infected with RV-Dz (A) or RA27/3 (B) at an MOI of 5 and then exposed to 2.5 μg/ml NTZ or vehicle for 48 h (A and B) Positive or negative strand RV RNA (red) and mRNA for GAPDH and β-actin (green) were detected by RNA-FISH. Nuclei were stained with DAPI. (C) Total fluorescence of each of six randomly picked macroscopic fields per each condition was determined by using ImageJ. Data are presented as a mean ± SD of fluorescence intensity in arbitrary units (AU) per a field. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Inhibition of nascent cellular RNA synthesis by NTZ. (A) HUVEC monolayers were treated with 5 μg/ml of NTZ or DMSO for 24 h. For a positive control, the cells were treated with an inhibitor of cellular RNA transcription, actinomycin D (ActD, 10 μg/ml) for 4 h. Following the treatments, the cells were metabolically labeled with 2 mM 5-ethynyl uridine. Newly synthesized RNA was detected by using click chemistry with azide-derivatized Alexa Fluor 488. Images were acquired using a Zeiss fluorescent microscope. Inserts represent enlarged images of the nuclei. Note the reduced total fluorescence intensity of the nuclei following the NTZ treatment in comparison to a vehicle control and the size reduction and dispersion of nucleoli (ribosomal RNA) in the NTZ treated cells. (B) Total fluorescence of each nucleus was determined using ImageJ. The averages (n = 50 nuclei for each condition) and standard deviation of the mean are shown. ****, p < 0.0001.