The sequences of all generated plasmid DNAs and the recombined regions within the recombinant vaccinia viruses were verified at the nucleotide level by standard sequencing analysis

The sequences of all generated plasmid DNAs and the recombined regions within the recombinant vaccinia viruses were verified at the nucleotide level by standard sequencing analysis. == Recovery of recombinant FCoV. host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein. Coronaviruses are enveloped positive-stranded RNA viruses that infect a wide range of vertebrate species including livestock, companion animals, and humans. Coronaviruses are associated mainly with respiratory and enteric infections that can cause, in conjunction with systemic and/or persistent infection, severe diseases in animals and humans (17,18,32). Many coronaviruses have adopted a pronounced tropism for monocytic cells such as conventional dendritic cells (cDCs) and macrophages that can be attributed to particular receptor specificities of coronavirus surface (S) glycoproteins (1,18,22,28,33). On the cellular level, infection of macrophages results in a host cell transcriptional response that virtually lacks type I interferon (IFN) expression but is characterized mainly by a markedly increased level of expression of inflammatory cytokines (6,18,20,25,28). These inflammatory stimuli initiate and contribute to the pathogenesis of coronavirus-induced severe diseases. Apioside For example, overshooting inflammatory cytokine responses are associated with (i) mouse hepatitis virus-induced acute viral hepatitis in mice (6), (ii) lethal feline coronavirus (FCoV)-induced infectious peritonitis that Apioside is characterized by widespread granulomatous inflammatory lesions in multiple organs (8,13), and (iii) severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV)-induced extensive tissue damage to the lung parenchyma of fatal SARS cases (16,23,28). Feline infectious peritonitis (FIP) is a highly lethal, immunopathological disease accompanied by the development of vasculitis, anemia, Apioside neutrophilia, lymphopenia, and multifocal granulomatous inflammatory lesions in various organs (8). It is now widely accepted that the causative agent, feline infectious peritonitis virus (FIPV), represents a highly pathogenic FCoV mutant that arises during persistent, mostly benign FCoV infection. Notably, there are two serotypes of FCoVs, namely, type I and type II FCoVs, and both can infect wild and domesticFelidae. Both FCoV serotypes are genetically and antigenetically closely related to the group 1 coronaviruses canine coronavirus (CCoV) and porcine transmissible gastroenteritis virus (TGEV) (2,8,15). Furthermore, both FCoV serotypes have been associated with the development of pathogenic FIPV, although type I FCoV prevails in the field, with a seropositivity of 20 to 60% for domestic cats and up to 90% for animal shelters or multicat households. Consequently, type I FCoV-derived FIPVs are responsible for about 70 to 85% of FIP cases for domestic cats (14). Accumulating genetic evidence supports the idea that type SHCB II FCoVs have arisen by homologous recombination between type I FCoV and CCoV, and as a result of this recombination, the spike (S) gene and adjacent regions of type I FCoV were replaced by the corresponding region of the CCoV genome (2,8,9,15). Genetic analyses of several type II FIPV isolates revealed that the 5 and 3 recombination sites for different type II isolates can vary slightly, strongly suggesting that these type II variants have originated from independent recombination events and that interspecies transmission of FCoV and/or CCoV may occur frequently (8,9). The coronavirus S protein is the main determinant.