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      FCoV Viral Sequences of Systemically Infected Healthy Cats Lack Gene Mutations Previously Linked to the Development of FIP

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          Abstract

          Feline Infectious Peritonitis (FIP)—the deadliest infectious disease of young cats in shelters or catteries—is induced by highly virulent feline coronaviruses (FCoVs) emerging in infected hosts after mutations of less virulent FCoVs. Previous studies have shown that some mutations in the open reading frames (ORF) 3c and 7b and the spike (S) gene have implications for the development of FIP, but mainly indirectly, likely also due to their association with systemic spread. The aim of the present study was to determine whether FCoV detected in organs of experimentally FCoV infected healthy cats carry some of these mutations. Viral RNA isolated from different tissues of seven asymptomatic cats infected with the field strains FCoV Zu1 or FCoV Zu3 was sequenced. Deletions in the 3c gene and mutations in the 7b and S genes that have been shown to have implications for the development of FIP were not detected, suggesting that these are not essential for systemic viral dissemination. However, deletions and single nucleotide polymorphisms leading to truncations were detected in all nonstructural proteins. These were found across all analyzed ORFs, but with significantly higher frequency in ORF 7b than ORF 3a. Additionally, a previously unknown homologous recombination site was detected in FCoV Zu1.

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          Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

          Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.
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            Persistence and transmission of natural type I feline coronavirus infection.

            To examine the mode of natural transmission and persistence of feline coronavirus (FCoV), FCoV strains shed by domestic cats were investigated over periods of up to 7 years. An RT-PCR that amplified part of the 3' end of the viral spike (S) gene was devised to distinguish FCoV types I and II. All but 1 of 28 strains of FCoV from 43 cats were type I. Nucleotide identities of the amplified 320 bp product from 49 type I FCoVs ranged from 79 to 100 %. The consensus partial S sequence of isolates recovered from persistently infected cats at time intervals spanning years was generally conserved. While most cats were infected with a single strain, a few may have been infected by more than one strain. Cats that were transiently infected and ceased shedding could be re-infected with either the same, or a different, strain. In most cases, whether a cat became persistently or transiently infected was independent of the virus strain. However, one strain was unusual in that it infected the majority of cats in the household simultaneously and was still being shed 18 months later. Factors that influence whether FCoV establishes lifelong infection in some cats and not others are determined mainly by the host response to infection.
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              Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis.

              Feline infectious peritonitis (FIP) is a fatal immunity-mediated disease caused by mutants of a ubiquitous coronavirus. Since previous attempts to protect cats under laboratory and field conditions have been largely unsuccessful, we used our recently developed system of reverse genetics (B. J. Haijema, H. Volders, and P. J. M. Rottier, J. Virol. 77:4528-4538, 2003) for the development of a modified live FIP vaccine. With this objective, we deleted the group-specific gene cluster open reading frame 3abc or 7ab and obtained deletion mutant viruses that not only multiplied well in cell culture but also showed an attenuated phenotype in the cat. At doses at which the wild-type virus would be fatal, the mutants with gene deletions did not cause any clinical symptoms. They still induced an immune response, however, as judged from the high levels of virus-neutralizing antibodies. The FIP virus (FIPV) mutant lacking the 3abc cluster and, to a lesser extent, the mutant missing the 7ab cluster, protected cats against a lethal homologous challenge; no protection was obtained with the mutant devoid of both gene clusters. Our studies show that the deletion of group-specific genes from the coronavirus genome results in live attenuated candidate vaccines against FIPV. More generally, our approach may allow the development of vaccines against infections with other pathogenic coronaviruses, including that causing severe acute respiratory syndrome in humans.

                Author and article information

                Journal
                Pathogens
                Pathogens
                pathogens
                Pathogens
                MDPI
                2076-0817
                24 July 2020
                August 2020
                : 9
                : 8
                : 603
                Affiliations
                [1 ]Clinical Laboratory, Department of Clinical Diagnostics and Services and Center for Clinical Studies, Vetsuisse Faculty, University of Zurich, CH 8057 Zurich, Switzerland; lutz@ 123456immunology.uzh.ch (M.L.); aline_steiner@ 123456gmx.ch (A.R.S.); valentino.cattori@ 123456gmail.com (V.C.); rhofmann@ 123456vetclinics.uzh.ch (R.H.-L.); hanslutz@ 123456me.com (H.L.)
                [2 ]Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, CH 8057 Zurich, Switzerland; anja.kipar@ 123456uzh.ch
                Author notes
                [* ]Correspondence: mmeli@ 123456vetclinics.uzh.ch ; Tel.: +41-44-635-88-56
                Author information
                https://orcid.org/0000-0002-9949-2230
                https://orcid.org/0000-0001-6856-1347
                https://orcid.org/0000-0001-9750-4296
                https://orcid.org/0000-0001-7289-3459
                https://orcid.org/0000-0002-3609-2416
                Article
                pathogens-09-00603
                10.3390/pathogens9080603
                7459962
                32722056
                ada8999f-a90d-49b7-94f7-a580ae4d2525
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 31 May 2020
                : 20 July 2020
                Categories
                Article

                healthy fcov carriers,fip,mutations,s gene,orf3,orf7,experimental infection

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