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      Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs

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          Abstract

          Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10 1 to 10 8 copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.

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          Most cited references14

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          Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis

          Feline coronaviruses (FCoV) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (FIP). Independent of virulence variations they are separated into two different types, type I, the original FCoV, and type II, which is closely related to canine coronavirus (CCV). The prevalence of FCoV types in Austrian cat populations without FIP has been surveyed recently indicating that type I infections predominate. The distribution of FCoV types in cats, which had succumbed to FIP, however, was fairly unknown. PCR assays have been developed amplifying parts of the spike protein gene. Type-specific primer pairs were designed, generating PCR products of different sizes. A total of 94 organ pools of cats with histopathologically verified FIP was tested. A clear differentiation was achieved in 74 cats, 86% of them were type I positive, 7% type II positive, and 7% were positive for both types. These findings demonstrate that in FIP cases FCoV type I predominates, too, nonetheless, in 14% of the cases FCoV type II was detected, suggesting its causative involvement in cases of FIP.
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            Pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683.

            Two feline coronavirus isolates were characterized by their disease-causing potential in cats. The 79-1683 feline coronavirus isolate caused an inapparent-to-mild enteritis when given oronasally to specific-pathogen-free kittens and was not a cause of feline infectious peritonitis (FIP). Target tissues for the virus were the mature apical epithelium of the small intestine, mesenteric lymph nodes, tonsils, thymus, and (to a lesser extent) the lungs. Inoculated kittens shed high numbers of virus in their feces for 14 to 17 days, but remained infectious to susceptible kittens for longer periods of time, as evidenced by contact-exposure studies. Because the 79-1683 isolate induced only enteritis, it was designated feline enteric coronavirus (FECV) 79-1683. The 79-1146 feline coronavirus isolate induced effusive abdominal FIP in specific-pathogen-free kittens after oronasal and intraperitoneal inoculation. Clinical signs of disease appeared within 12 to 14 days in almost all inoculated kittens. Because this isolate caused FIP, it was designated FIP virus (FIPV) 79-1146. Cross-protective immunity was not induced by the various coronavirus infections. Kittens preimmunized with the UCD strain of FECV (FECV-UCD) or with FECV-79-1683 were not immune to infection with FIPV-79-1146. Likewise, kittens previously inoculated with FECV-79-1683 were not immune to infection with FIPV-UCD1. In fact, preexisting heterologous FECV-79-1683 immunity often accelerated and enhanced the severity of disease caused by inoculation with FIPV-UCD1.(ABSTRACT TRUNCATED AT 250 WORDS)
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              Genetic diversity of a canine coronavirus detected in pups with diarrhoea in Italy

              The sequence of the S gene of a field canine coronavirus (CCoV), strain Elmo/02, revealed low nucleotide (61%) and amino acid (54%) identity to reference CCoV strains. The highest correlation (77% nt and 81.7% aa) was found with feline coronavirus type I. A PCR assay for the S gene of strain Elmo/02 detected analogous CCoVs of different geographic origin, all which exhibited at least 92–96% nucleotide identity to each other and to strain Elmo/02. The evident genetic divergence between the reference CCoV strains and the newly identified Elmo/02-like CCoVs strongly suggests that a novel genotype of CCoV is widespread in the dog population.
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                Author and article information

                Contributors
                Journal
                J Virol Methods
                J. Virol. Methods
                Journal of Virological Methods
                Elsevier B.V.
                0166-0934
                1879-0984
                15 July 2005
                December 2005
                15 July 2005
                : 130
                : 1
                : 72-78
                Affiliations
                [a ]Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari, Strada prov per Casamassima Km 3, 70010 Valenzano, Bari, Italy
                [b ]Istituto Zooprofilattico Sperimentale di Puglia e Basilicata, via Manfredonia 20, 71100 Foggia, Italy
                [c ]Istituto Superiore di Sanità, viale Regina Elena 299, 00161 Rome, Italy
                Author notes
                [* ]Corresponding author. Tel.: +39 0804679833; fax: +39 0804679843. n.decaro@ 123456veterinaria.uniba.it
                Article
                S0166-0934(05)00198-9
                10.1016/j.jviromet.2005.06.005
                7112928
                16024100
                ab622594-77ea-45ca-b5cd-9d7361fddb57
                Copyright © 2005 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 14 April 2005
                : 31 May 2005
                : 9 June 2005
                Categories
                Article

                Microbiology & Virology
                Microbiology & Virology

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