Abstract
ABSTRACTFeline 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.
Highlights
IMPORTANCE The availability of recombinant serotype I Feline coronaviruses (FCoVs) field isolates that are amenable to genetic manipulation is key to studying the molecular pathogenesis of feline infectious peritonitis (FIP), especially since previous studies using cell culture-adapted feline infectious peritonitis virus (FIPV) had proven unsuccessful
The full-length genomic sequence was determined by sequence analyses of overlapping reverse transcription-PCR (RT-PCR) fragments amplified from viral RNA and was deposited in GenBank
Significant efforts by several laboratories have been made to identify genetic changes that are involved in FECVto-FIPV biotype switches
Summary
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. On the basis of extensive comparative sequence analyses of FECV and FIPV isolates, it was concluded that mutations in the S and accessory genes are involved in the development of FIP [25,26,27,28,29,30,31,32]. In reports of recent studies based on comparative sequence analyses of complete FECV and FIPV genomes, three substitutions in the S gene were suggested to discriminate FIPVs from FECV [26,27,28] Two of these changes were located in the fusion peptide (FP) and one in the heptad repeat 1 (HR1) region of the S protein. The idea that particular mutations in the FCoV S gene and/or accessory genes lead to a biotype switch from FECV to FIPV remains to be experimentally
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