key: cord-0283979-my4n0vye
authors: Valle-Casuso, José Carlos; Gaudaire, Delphine; Martin-Faivre, Lydie; Madeline, Anthony; Dallemagne, Patrick; Pronost, Stéphane; Munier-Lehmann, Hélène; Zientara, Stephan; Vidalain, Pierre-Olivier; Hans, Aymeric
title: Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors
date: 2020-04-10
journal: bioRxiv
DOI: 10.1101/2020.04.10.035402
sha: 417724ea7cd1473ca223a7b1581f0c1221b5151a
doc_id: 283979
cord_uid: my4n0vye

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.

Viruses with a RNA genome infect both animals and plants, and dominate the eukaryotic virome by 57 their diversity and deleterious effects 1 Previous works exploring EAV inhibitors have proposed to use morpholino or peptide-conjugated 80 morpholino oligomers specifically designed against the EAV genome as a therapy to impair viral 81 replication 8 . However, this promising therapy is not easily applicable for use in vivo in large animal 82 such as horses. Although, two effective EAV vaccines have been available since 1985, the proportion 83 of horses vaccinated against EAV in the equine population remains insufficient to control the disease 84 9 . We think that a complementary approach using antiviral molecules would be ideal to complement 85 vaccination, reduce viral spreading in equine structures and contain EAV outbreaks. This is supported 86 by a recent study showing that a global strategy based on vaccination combined with drug therapies 87 enhances protection against foot-and-mouth disease 10 . In our study, we have developed a high-88 throughput cell-viability assay based on infected equine dermal cells. Then, we took advantage of this 89 new assay to explore the potential of broad-spectrum antivirals ( replication through multiple direct and indirect mechanisms. In particular, ribavirin inhibits inosine 5'-103 monophosphate dehydrogenase (IMPDH), a key enzyme catalyzing the first committed and rate-104 limiting step in the de novo synthesis of guanine nucleotides. As such, IMPDH inhibition interferes with 105 the production of GTP that is necessary for the synthesis of viral RNA molecules 22 , but other 106 mechanisms have been reported such as the direct inhibition of viral polymerases or the lethal 107 mutagenesis of viral genomes 23,24 . Besides ribavirin, we also tested two newly designed BSA that 108 target the pyrimidine biosynthesis pathway, namely GAC50 and IPPA17-A04. These two molecules 109 are inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth and rate-limiting enzyme of the 110 de novo pyrimidine biosynthesis pathway, and were shown to impair the replication of many different 111 positive-strand and negative-strand RNA viruses [25] [26] [27] . In this study, we have shown, for the first time, 

EAV is a cytopathic virus. In this study, we took advantage of this specificity to develop a microplate 119 assay for the identification of EAV inhibitors. As a cellular model, we selected the ED cell line that 120 originates from equine dermis and thus matches the host specificity of EAV. To setup our in vitro 121 assay, we first analyzed the proliferation of non-infected ED cells in 96-well plates. To determine the 122 number of viable cells in each culture well, we quantified ATP which parallels the number of 123 metabolically active, living cells. Assays were performed using a commercial reagent based on the 124 principle that firefly luciferase luminescence is proportional and dependent on ATP concentrations 125 (CellTiter-Glo Luminescent Cell Viability Assay; Promega). In non-infected cells, ATP levels increased 126 by 41% in the first 24 h as a consequence of cellular proliferation, but then plateaued at 48 h as cell 127 cultures reached confluence ( Figure 1A ). Same experiment was performed using cell cultures infected 128 with the Bucyrus strain of EAV at a multiplicity of infection (MOI) of 0.5. As shown in Figure 1A 

Previous studies on broad-spectrum antiviral molecules showed that ribavirin inhibits the replication of 144 different nidoviruses, including Arteriviridae such as porcine reproductive and respiratory syndrome 145 virus (PRRSV) 28 . We thus tested the antiviral effects of ribarivin on EAV using the cell-viability assay 146 described above. As a prerequisite, we first evaluated the cytotoxicity of ribavirin on ED cells. Cells 147 were incubated with ribavirin at different concentrations (0.5, 1, 2, 5, 10, 15, 20, 50 and 100 µg/ml), 148 and the number of viable cells was determined after 1 h without treatment or after 48 h with or without 149 treatment. No significant cell death was reported at 10 µg/ml or lower concentrations of ribavirin when 150 compared to the initial number viable cells seeded in the culture wells ( Figure 2A ). However, ribavirin 151 was clearly cytostatic at these concentrations. Cell viability at 20 µg/ml was 72%, and decreased to 152 63% and 52% when treating cells with 50 µg/ml and 100 µg/ml of ribavirin, respectively ( Figure 2A) . 153

The half maximal toxic concentration (TC 50 ), which is the concentration that kills 50% of the cells in 154 culture, was estimated to be >100 µg/ml for ribavirin. Then, we tested the effect of ribavirin in our in 155 vitro EAV infection model at the non-cytotoxic concentration range of 0.5 to 20 µg/ml. As shown in 156 Figure 3A , ATP quantification in infected culture wells at 48 h showed that only 35% of cells were alive 157 in the absence of ribavirin as opposed to 67% in presence of the drug at 0.5 µg/ml. The viability of 158 infected cells was above 80% when treated with ribavirin at 2, 5 or 10 µg/ml ( Figure 3A ). These results 159 were used to calculate the half maximal inhibitory concentration (IC 50 ) of ribavirin that was estimated 160 to 0.90 µg/ml, i.e. 3.7 µM. This demonstrates that ribavirin is able to protect ED cells against EAV-161 associated cytopathic effects. 162

We were also interested in exploring the antiviral capacity of two new BSA, IPPA17-04 and GAC50 163 that impair viral replication through inhibition of de novo pyrimidine biosynthesis in host cells. Using 164 our in vitro EAV infection assay, we tested first the cytotoxicity of both compounds. IPPA17-A04 and 165 GAC-50 treatment did not show any sign of cytotoxicity on ED cells. At 100 µg/ml, cell viability was 166 above 80% after 48h of treatment, but cytostatic effects were observed as expected for this class of 167 drug ( Figure 2B and 2C) . These results indicate that TC 50 for IPPA17-A04 and GAC-50 is over 100 168 µg/ml in ED cells. We then explored the cytoprotective effect of these two compounds in EAV-infected 169 cultures at different concentrations. Our results showed that after 48 h of culture, the viability of 170 infected ED cells reached 79% when treated with 1 µg/ml of IPPA17-A04, and was above 80% when 171 treated with concentrations >5 µg/ml ( Figure 3B ). The IC 50 of IPPA17-A04 was estimated at 0.70 172 µg/ml, i.e. 1.74 µM. GAC50 is not as effective since viability of ED infected cells treated with GAC50 is 173 >80% only at the highest concentrations of 20 or 50 µg/ml ( Figure 3C ) with an IC 50 at 7.2 µg/ml, i.e. 174 30.2 µM. These results show that cell cultures treated with pyrimidine biosynthesis inhibitors did not 175 exhibit cytopathic effects associated to EAV infection, suggesting that EAV replication is also impaired. 176 177

To verify that ribavirin, IPPA17-A04 and GAC50 actually block EAV replication, viral genome copies 180 were quantified at 48 h.p.i. Culture supernatants of infected ED cells treated or not with the 181 compounds at different concentrations were analyzed by RT-qPCR. Results showed that ribavirin at 5 182 and 10 µg/ml significantly reduced the number of EAV genome copies in culture supernatants 183 compared to the untreated infected cells ( Figure 4A ). EAV replication was totally blocked at 20 µg/ml 184 as determined by comparison with the initial inoculum quantified at 1 h.p.i. These results confirmed 185 that ribavirin impairs EAV replication in ED cells, thus explaining the positive effect on cell survival. In 186 parallel, we also measured viral genome copies present in the supernatant of infected cells treated 187 with pyrimidine biosynthesis inhibitors. As expected, IPPA17-A04 and GAC50 both reduced the 188 number of viral genome copies in culture supernatants when treated with concentrations >20 µg/ml 189 and fully blocked EAV replication at 50 µg/ml ( Figure 4B and 4C) . 

To rank the three molecules that we characterized as EAV inhibitors in this study, we calculated for 203 each molecule the Selectivity Index (SI) that corresponds to the TC 50 /IC 50 ratio, and thus reflects the 204 activity of a drug while taking into account cytotoxic effects. TC 50 and IC 50 values were determined 205 from the dose response experiments presented in Figures 2 and 3 . SI values for ribavirin, IPPA17-A04 206 and GAC50 were >111, >143 and >14 respectively. In conclusion, this confirms that IPPA17-A04 is, 207 over ribavirin and GAC50, a lead molecule of interest for developing potential antiviral therapies 208 against EAV. 209 

Equine viral arteritis: A respiratory and 360 reproductive disease of significant economic importance to the equine industry. Equine 361 Veterinary Education

Curing of HeLa cells persistently 363 infected with equine arteritis virus by a peptide-conjugated morpholino oligomer

Serological evidence of equine arteritis virus infection and phylogenetic 366 analysis of viral isolates in semen of stallions from Serbia

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Dihydroorotate Dehydrogenase (DHODH)

Inhibition of pyrimidine biosynthesis pathway suppresses viral growth 405 through innate immunity

Respiratory syncytial virus infection in macaques is not suppressed by 407 intranasal sprays of pyrimidine biosynthesis inhibitors

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We would like to thank Yves Janin from the Institut Pasteur, Paris, France and Daniel Dauzonne from 436 CNRS, UMR3666, INSERM, U1143, Institut Curie, Centre de Recherche, Paris, France who kindly 437 provided IPPA-A14 and GAC-50, respectively. We are also very grateful to Fanny Lecouturier and 438Gabrielle Bouet for their technical assistance. 439