An RNA replicon system to investigate promising inhibitors of feline coronavirus.

Feline infectious peritonitis (FIP) is a fatal feline disease, caused by a feline coronavirus (FCoV), namely feline infectious peritonitis virus (FIPV). We produced a baby hamster kidney 21 (BHK) cell line expressing a serotype I FCoV replicon RNA with a green fluorescent protein (GFP) reporter gene (BHK-F-Rep) and used it as an in vitro screening system to test different antiviral compounds. Two inhibitors of the FCoV main protease (Mpro), namely GC376 and Nirmatrelvir, as well as the nucleoside analog Remdesivir proved to be effective in inhibiting the replicon system. Different combinations of these compounds also proved to be potent inhibitors, having an additive effect when combined. Remdesivir, GC376, and Nirmatrelvir all have a 50% cytotoxic concentration (CC50) more than 200 times higher than their half-maximal inhibitory concentrations (IC50), making them important candidates for future in vivo studies as well as clinically implemented drug candidates. In addition, results were acquired with a virus infection system, where Felis catus whole fetus 4 (Fcwf-4) cells were infected with a previously described recombinant GFP-expressing FIPV (based on the laboratory-adapted serotype I FIPV strain Black) and treated with the most promising compounds. Results acquired with the replicon system were comparable to the results acquired with the virus infection system, demonstrating that we successfully implemented the FCoV replicon system for antiviral screening. We expect that this system will greatly facilitate future screens for anti-FIPV compounds and provide a non-infectious system to study and evaluate drug-resistant mutations that may emerge in the FIPV genome.IMPORTANCEFIPV is of great significance in the cat population around the world, causing 0.3%-1.4% of feline deaths in veterinary practices (2). As there are neither effective preventive measures nor approved treatment options available, there is an urgent need to identify antiviral drugs against FIPV. Our FCoV replicon system provides a valuable tool for drug discovery in vitro. Due to the lack of cell culture systems for serotype I FCoVs (the serotype most prevalent in the feline population) (2), a different system is needed to study these viruses. A viral replicon system is a valuable tool for studying FCoVs. Overall, our results demonstrate the utility of the serotype I feline coronavirus replicon system for antiviral screening as well as to study this virus in general. We propose several compounds representing promising candidates for future clinical trials and ultimately with the potential to save cats suffering from FIP.

Reverse Genetic Assessment of the Roles Played by the Spike Protein and ORF3 in Porcine Epidemic Diarrhea Virus Pathogenicity.

Porcine epidemic diarrhea virus is a swine pathogen that has been responsible for significant animal and economic losses worldwide in recent years. In this manuscript, we report the generation of a reverse genetics system C(RGS) for the highly virulent US PEDV strain Minnesota (PEDV-MN; GenBank accession number KF468752), which was based on the assembly and cloning of synthetic DNA, using vaccinia virus as a cloning vector. Viral rescue was only possible following the substitution of 2 nucleotides within the 5'UTR and 2 additional nucleotides within the spike gene, based on the sequence of the cell culture-adapted strains. Besides displaying a highly pathogenic phenotype in newborn piglets, in comparison with the parental virus, the rescued recombinant PEDV-MN was used to confirm that the PEDV spike gene has an important role in PEDV virulence and that the impact of an intact PEDV ORF3 on viral pathogenicity is modest. Moreover, a chimeric virus with a TGEV spike gene in the PEDV backbone generated with RGS was able to replicate efficiently in vivo and could be readily transmitted between piglets. Although this chimeric virus did not cause severe disease upon the initial infection of piglets, there was evidence of increasing pathogenicity upon transmission to contact piglets. The RGS described in this study constitutes a powerful tool with which to study PEDV pathogenesis and can be used to generate vaccines against porcine enteric coronaviruses. IMPORTANCE PEDV is a swine pathogen that is responsible for significant animal and economic losses worldwide. Highly pathogenic variants can lead to a mortality rate of up to 100% in newborn piglets. The generation of a reverse genetics system for a highly virulent PEDV strain originating from the United States is an important step in phenotypically characterizing PEDV. The synthetic PEDV mirrored the authentic isolate and displayed a highly pathogenic phenotype in newborn piglets. With this system, it was possible to characterize potential viral virulence factors. Our data revealed that an accessory gene (ORF3) has a limited impact on pathogenicity. However, as it is also now known for many coronaviruses, the PEDV spike gene is one of the main determinants of pathogenicity. Finally, we show that the spike gene of another porcine coronavirus, namely, TGEV, can be accommodated in the PEDV genome background, suggesting that similar viruses can emerge in the field via recombination.

Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP), one of the most important lethal infections of cats, is caused by feline infectious peritonitis virus (FIPV), the high-virulence biotype of feline coronaviruses (FCoVs). FIPVs are suggested to emerge from feline enteric coronaviruses (FECVs) by acquiring mutations in specific genes in the course of persistent infections. Although numerous studies identified mutations predicted to be responsible for the FECV-FIPV biotype switch, the presumed roles of specific genetic changes in FIP pathogenesis have not been confirmed experimentally. Reverse genetics systems established previously for serotype I and the less common serotype II FCoVs were based on cell culture-adapted FIPV strains which, however, were shown to be unsuitable for FIP pathogenesis studies in vivo To date, systems to produce and manipulate recombinant serotype I field viruses have not been developed, mainly because these viruses cannot be grown in vitro Here, we report the first reverse genetics system based on a serotype I FECV field isolate that is suitable to produce high-titer stocks of recombinant FECVs. We demonstrate that these recombinant viruses cause productive persistent infections in cats that are similar to what is observed in natural infections. The system provides an excellent tool for studying FCoVs that do not grow in standard cell culture systems and will greatly facilitate studies into the molecular pathogenesis of FIP. Importantly, the system could also be adapted for studies of other RNA viruses with large genomes whose production and characterization in vivo are currently hampered by the lack of in vitro propagation systems.IMPORTANCE The availability of recombinant serotype I FCoV field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of FIP, especially since previous studies using cell culture-adapted FIPVs had proven unsuccessful. To our knowledge, we report the first serotype I FECV field isolate-based reverse genetics system that allows the production of high-titer recombinant virus stocks that can be used for subsequent in vivo studies in cats. The system represents a milestone in FCoV research. It provides an essential tool for studying the molecular pathogenesis of FIP and, more specifically, the functions of specific gene products in causing a fundamentally different progression of disease following acquisition of specific mutations. The system developed in this study will also be useful for studying other coronaviruses or more distantly related RNA viruses with large genomes for which suitable in vitro culture systems are not available.

Identification and characterization of a Golgi retention signal in feline coronavirus accessory protein 7b.

Feline coronaviruses encode five accessory proteins with largely elusive functions. Here, one of these proteins, called 7b (206 residues), was investigated using a reverse genetic approach established for feline infectious peritonitis virus (FIPV) strain 79-1146. Recombinant FIPVs (rFPIVs) expressing mutant and/or FLAG-tagged forms of 7b were generated and used to investigate the expression, processing, glycosylation, localization and trafficking of the 7b protein in rFIPV-infected cells, focusing on a previously predicted ER retention signal, KTEL, at the C-terminus of 7b. The study revealed that 7b is N-terminally processed by a cellular signalase. The cleavage site, 17-Ala|Thr-18, was unambiguously identified by N-terminal sequence analysis of a 7b processing product purified from rFIPV-infected cells. Based on this information, rFIPVs expressing FLAG-tagged 7b proteins were generated and the effects of substitutions in the C-terminal 202KTEL206 sequence were investigated. The data show that (i) 7b localizes to and is retained in the medial- and/or trans-Golgi compartment, (ii) the C-terminal KTEL sequence acts as a Golgi [rather than an endoplasmic reticulum (ER)] retention signal, (iii) minor changes in the KTEL motif (such as KTE, KTEV, or the addition of a C-terminal tag) abolish Golgi retention, resulting in relocalization and secretion of 7b, and (iv) a KTEL-to-KDEL replacement causes retention of 7b in the ER of rFIPV-infected feline cells. Taken together, this study provides interesting new insights into an efficient Golgi retention signal that controls the cellular localization and trafficking of the FIPV 7b protein in virus-infected feline cells.