Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

José-Carlos Valle-Casuso,Stephan Zientara,Aymeric Hans,Hélène Munier-Lehmann,Patrick Dallemagne,Delphine Gaudaire,Lydie Martin-Faivre,Anthony Madeline,Pierre-Olivier Vidalain,Stéphane Pronost

doi:10.1038/s41598-020-66944-4

Abstract

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

Highlights

RNA viruses are responsible for a large variety of animal infections
When cells were analyzed by phase-contrast microscopy, infected and non-infected cultures looked similar at 24 h, but most cells in infected wells were dead at 48 h post-infection (h.p.i) as a result of Equine Arteritis Virus (EAV) cytopathic effects
Addition of uridine at 30 μg/ml in the culture medium of EAV-infected ED cells abolished the cytoprotective effect of IPPA17-A04 (Fig. 6A) and restored viral replication (Fig. 6B). These results indicate that the antiviral activity of IPPA17-A04 in ED cells relies on the inhibition of the de novo pyrimidine biosynthesis pathway and depletion of the pool of pyrimidines. It remains unknown when the EAV outbreak will occur, but since 1953, when the EAV was isolated for the first time, EAV has regularly reemerged around the world

Summary

Introduction

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. We tested two newly designed BSA that target the pyrimidine biosynthesis pathway, namely GAC50 and IPPA17-A04 These two molecules are inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth and rate-limiting enzyme of the de novo pyrimidine biosynthesis pathway, and were shown to impair the replication of many different positive-strand and negative-strand RNA viruses[25,26,27]. We have shown, for the first time, that both ribavirin and pyrimidine biosynthesis inhibitors inhibit EAV in equine dermal cell cultures

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