key: cord-0334129-a4wnrhxa
authors: Cook, S.E.; Vogel, H.; Castillo, D.; Olsen, M.; Pedersen, N.; Murphy, B. G.
title: A rational approach to identifying effective combined anticoronaviral therapies against feline coronavirus
date: 2020-07-09
journal: bioRxiv
DOI: 10.1101/2020.07.09.195016
sha: d3ac1bbb603ff240da948ee8b3858d73aa83b414
doc_id: 334129
cord_uid: a4wnrhxa

Feline infectious peritonitis (FIP), caused by a genetic mutant of feline enteric coronavirus known as FIPV, is a highly fatal disease of cats with no currently available vaccine or FDA-approved cure. Dissemination of FIPV in affected cats results in a range of clinical signs including cavitary effusions, anorexia, fever and lesions of pyogranulomatous vasculitis and peri-vasculitis with or without central nervous system and/or ocular involvement. There is a critical need for effective and approved antiviral therapies against coronaviruses including FIPV and zoonotic coronaviruses such as SARS-CoV-2, the cause of COVID-19. With regards to SARS-CoV-2, preliminary evidence suggests that there may be potential clinical and pathological overlap with feline coronaviral disease including enteric and neurological involvement in some cases. We have screened 89 putative antiviral compounds and have identified 25 compounds with antiviral activity against FIPV, representing a variety of drug classes and mechanisms of antiviral action. Based upon successful combination treatment strategies for human patients with HIV or hepatitis C virus infections, we have identified combinations of drugs targeting different steps of the FIPV life cycle resulting in synergistic antiviral effect. Translationally, we suggest that a combined anticoronaviral therapy (cACT) with multiple mechanisms of action and penetration of all potential anatomic sites of viral infection should be applied towards other challenging to treat coronaviruses, like SARS-CoV-2. Author summary We have screened 89 compounds in vitro for antiviral activity against FIPV. The putative antiviral activity of these compounds was either purported to be a direct effect on viral proteins involved in viral replication or an indirect inhibitory effect on normal cellular pathways usurped by FIPV to aid viral replication. Twenty-five of these compounds were found to have significant antiviral activity. Certain combinations of these compounds were determined to be superior to monotherapy alone.

). We tested several 148 nucleoside analog compounds provided by Gilead Sciences structurally related to the 149 nucleoside analogs GS-441524 and Remdesivir for their antiviral properties and found several 150 with potential (included in the above reported 25 identified compounds) but did not pursue 151 these agents further. As a result, the total number of antiviral agents carried forward for 152 further analyses was 13. This total includes the previously identified 3-C protease inhibitor, GC-153 376 (Anavive). Table 3 ). Those with the least inhibitory effect on viral RNA 219 production include elbasvir, nelfinavir, and ritonavir. Ritonavir, a protease inhibitor, is used in 220 combination with lopinavir to treat HIV-1 infection (Kaletra, AbbVie). Lopinavir monotherapy 221 has poor oral bioavailability in people, however, when used in combination, Ritonavir has been 222 demonstrated to markedly improve lopinavir's plasma concentration [33] . Therefore, despite 223 the relatively minimal FIPV inhibition identified with ritonavir as monotherapy, this compound 224 was carried forward for additional testing, including combined anticoronaviral assessment. 252 chemical class-based ability to penetrate the blood-brain or blood-ocular barriers), and (iv) 253 minimal cytotoxicity (based on the CSP). For each cACT, any resulting decrease in FIPV copy 254 number over the calculated additive effect for each drug used as monotherapy was considered 255 to be synergistic (Table 4 ). The combination of antiviral agents with the greatest total fold 256 reduction in viral RNA as well as greatest synergistic effect was determined to be GC376 and 257 amodiaquine with a 76-fold decrease in viral RNA over the additive effect (Fig 6) . This particular 258 synergistic combination was one of the more interesting results given that amodiaquine alone 259 demonstrated limited inhibition of FIPV viral RNA copies determined by qRT-PCR. 519 520 Viral RNA knock-down assay 521 Real time RT-PCR assays were used to quantify compound inhibition of viral RNA production. 522 CRFK cells were cultured in a 6-well tissue culture plate (Genesee Biotek). At approximately 75-523 85% cellular confluency, the culture media was replaced with fresh media and the cells were 524 infected with FIPV serotype II at a MOI of 0.2 (MOI based upon the TCID50 bioassay/pfu). Plates 525 were incubated for one hour, with periodic gentle agitation every 15 minutes. FIPV-infected

The inconsistency between subjective visual 201 assessment of EIDD-treated wells and the fluorescence assay is enigmatic. It is possible that the 202 overall decreased cell number in EIDD-treated wells resulted in loss and degradation of nucleic 203 acid necessary for fluorescence binding and detection in the CellTox assay

Percent cytotoxicity bar graphs +/-standard deviation 206 (SD) for four compounds with anti-FIPV activity. Percent cytotoxicity values were determined by 207 normalizing cytotoxicity to the positive toxicity control wells (set to 100% cytotoxicity) and untreated 208 CRFK cells

Table 2. Percent cytotoxicity by compound and concentration

Past clinical successes using GS-441524 or GC-376 in cats with experimental and naturally 396 occurring FIP demonstrate that an effective cure for FIP is possible, however, challenges in 397 treating non-effusive (granulomatous), neurological and ocular FIP remain. The 3C-like protease 398 inhibitor, GC-376, appears to be relatively effective in the treatment of effusive FIPV infection 399 confined to body cavities but may be less effective in treating the neurological or ocular forms 400 of the disease

After 72 hours of incubation at 37˚C, extensive cytopathic effect 411 (CPE) and large areas of cell clearing/detachment were noted. Flasks were then flash frozen at -412 70˚C for 8 minutes, thawed briefly at room temperature and the cells and supernatant were 413 then centrifuged at 1500g for 5 minutes followed by a second centrifugation step at 4000g for 5 414 minutes in order to isolate cell-free viral stocks. Supernatant containing the viral stock was 415 divided into 0.5-and 1.0-ml aliquots in 1.5 ml cryotubes (Nalgene) and archived at -70˚C

The tissue culture infectious dose-50 (TCID50) was determined using a viral plaquing assay

Genesee Scientific) until the CRFK cells 421 achieved approximately 75-85% confluency. Serial 10-fold dilutions were made of FIPV stock 422 and 200 L samples of each dilution were added to 10-well replicates. At 72 hours post-423 infection, the cells were fixed with methanol and stained with crystal violet

Individual wells were evaluated visually for virus-induced CPE, scored as CPE positive or 425 negative, and the TCID50 was determined based upon the equation log 10 TCID50 = [total # wells 426 CPE positive/# replicates] + 0.5 to reflect infectious virions per milliliter of supernatant

Quantification of FIPV by qRT-PCR

Cell-free viral RNA was isolated from the viral stock using the QIAamp Viral RNA Mini Kit 430 (Qiagen) following the manufacturer's instructions. The isolated RNA was DNase treated 431 (Turbo DNase, Invitrogen) and subsequently reverse transcribed using the High-Capacity RNA-432 to-cDNA Kit (Applied Biosystems) following the manufacturers' protocols. The copy number of 433 FIPV and feline GAPDH cDNA were determined using Applied Biosystems

Master Mix, following the manufacturer's protocol the FIPV forward primer, 5'-GGAAGTTTAGATTTGATTTGGCAATGCTAG, and the FIP reverse 439 primer, 5'-AACAATCACTAGATCCAGACGTTAGCT (terminal portion of the FIPV 7b gene

Real-time PCR for the feline GAPDH housekeeping gene was performed concurrently using the 441 primers, 5 GAPDH, 5'-AAATTCCACGGCACAGTCAAG, and 3 GAPDH

Cycling conditions for both FIPV and GAPDH amplicons were as 443 follows: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s, 58°C for 30 s, 444 72°C for 1 min. The final step included a dissociation curve to evaluate specificity of primer

In general, the drugs examined and described in this study were preexisting antiviral agents. In 451 contrast, the helicase enzyme of FIPV was cloned, expressed and utilized as a target for 452 coronavirus and enzyme specific viral discovery

HisTag was optimized and synthesized. The synthesized sequence was cloned

Adedeji) into vector pET30a with Avi-His tag for protein

The concentration was determined by Bradford protein 463 assay with BSA as standard. The protein purity and molecular weight were determined by SDSimmobilization was 10mM HEPES

Hits were selected based upon RU and kinetics and utilized for cellcontrols (uninfected cells) and treatment controls (infected cells treated with a known 25 in 96-well tissue culture plates (Genesee Scientific) containing 200 L culture media. At 484~75-85% cell confluency, the media in the uninfected control wells was aspirated and replaced 485 with 200 L of fresh media. The media in the infected wells was aspirated and replaced with 486 media inoculated with FIPV at a multiplicity of infection (MOI) of 0.004 infectious virions per 487 cell. The tissue culture plate was incubated for 1 hour with periodic gentle agitation ("figure 488 eight" manipulations) performed every 15 minutes to facilitate virus-cell interaction. At 1-hour 489 post-infection, each putative antiviral compound was added to six FIPV-infected wells (to assess 490 compound antiviral efficacy) and six uninfected control wells (to screen for compound 491 cytotoxicity in CRFK cells)

the EC50 was determined by performing a progressive 2-502 fold compound dilution series in the viral plaquing assay. For EC50 determination, CRFK cells was based upon the compounds' EC50 and ranged from 0.001-20 M. For each 529 experimental set, three culture wells with FIPV-infected and untreated CRFK cells acted as 530 virus-infected controls. The infected cell cultures were subsequently incubated for 24 hours and 531 cell-associated total RNA was isolated using the PureLink™ RNA mini kit (Invitrogen). The RNA 532 was treated with DNAse

Applied Biosystems) and FIPV cDNA and feline GAPDH cDNA were 534 measured using real time qRT-PCR, as described above. Fold reduction in viral titer was 535 determined by dividing the normalized average FIPV RNA copy number for untreated, FIPV-536 infected CRFK cells, into the normalized average FIPV RNA copy number for treated CRFK cells

Determination of Cytotoxicity Safety Profiles (CSP)

Compound cytotoxicity in feline cells was assessed using the commercially available kit

Untreated 545 CRFK cells were used as negative controls and cells treated with a cytotoxic solution provided 546 by the manufacturer was used as the positive toxicity control. Briefly, in addition to the control 547 wells, CRFK cells were plated in 96-well tissue culture plates (Genesee Scientific) in four well 548 replicates with 5, 10, 25, 50, or 100 M concentrations of the compound of interest and were 549 incubated for 72 hours. After 72 hours, the kit DNA binding dye was applied to all wells

552 Molecular Devices; Softmax Pro, Molecular Devices). binding of the dye to the DNA of degenerate, apoptotic or necrotic 555 cells. The cytotoxicity range was determined by setting the fluorescence value for cells treated 556 with the positive control reagent as 100% and the untreated feline cells as 0% cytotoxicity

We appreciate the funding provided by the Winn Feline Foundation (MTW 17-020; MTW 19-564 026) and the University of California, Davis Center for Companion Animal Health (CCAH; 2018-565 92-F; 2018-94-FE) through gifts specified for FIP research by multiple individual donors and 566 organizations (SOCK FIP

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