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      Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye


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          A sensitive reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid visual detection of turkey coronavirus (TCoV) infection. The reaction is performed in one step in a single tube at 65 °C for 45 min, with hydroxynaphthol blue (HNB) dye added prior to amplification. The detection limit of the RT-LAMP assay was approximately 10 2 EID 50/50 μl TCoV genome, and no cross-reaction with other avian viruses was observed. The assay was evaluated further in tissue suspensions prepared from the ileum and ileum–caecal junctions of infected turkey embryos; 100% of these samples were positive in the RT-LAMP assay. All individual feces samples collected in the field were considered positive by both conventional RT-PCR and RT-LAMP. In conclusion, RT-LAMP with HNB dye was shown to be a sensitive, simple assay for the rapid diagnosis of TCoV infection, either directly from feces or in association with virus isolation methods.

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          Most cited references12

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          Coronaviruses in poultry and other birds.

          The number of avian species in which coronaviruses have been detected has doubled in the past couple of years. While the coronaviruses in these species have all been in coronavirus Group 3, as for the better known coronaviruses of the domestic fowl (infectious bronchitis virus [IBV], in Gallus gallus), turkey (Meleagris gallopavo) and pheasant (Phasianus colchicus), there is experimental evidence to suggest that birds are not limited to infection with Group 3 coronaviruses. In China coronaviruses have been isolated from peafowl (Pavo), guinea fowl (Numida meleagris; also isolated in Brazil), partridge (Alectoris) and also from a non-gallinaceous bird, the teal (Anas), all of which were being reared in the vicinity of domestic fowl. These viruses were closely related in genome organization and in gene sequences to IBV. Indeed, gene sequencing and experimental infection of chickens indicated that the peafowl isolate was the H120 IB vaccine strain, while the teal isolate was possibly a field strain of a nephropathogenic IBV. Thus the host range of IBV does extend beyond the chicken. Most recently, Group 3 coronaviruses have been detected in greylag goose (Anser anser), mallard duck (Anas platyrhynchos) and pigeon (Columbia livia). It is clear from the partial genome sequencing of these viruses that they are not IBV, as they have two additional small genes near the 3' end of the genome. Twenty years ago a coronavirus was isolated after inoculation of mice with tissue from the coastal shearwater (Puffinus puffinus). While it is not certain whether the virus was actually from the shearwater or from the mice, recent experiments have shown that bovine coronavirus (a Group 2 coronavirus) can infect and also cause enteric disease in turkeys. Experiments with some Group 1 coronaviruses (all from mammals, to date) have shown that they are not limited to replicating or causing disease in a single host. SARS-coronavirus has a wide host range. Clearly there is the potential for the emergence of new coronavirus diseases in domestic birds, from both avian and mammalian sources. Modest sequence conservation within gene 1 has enabled the design of oligonucleotide primers for use in diagnostic reverse transcriptase-polymerase chain reactions, which will be useful for the detection of new coronaviruses.
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            Emergence of a group 3 coronavirus through recombination

            Analyses of turkey coronavirus (TCoV), an enteric disease virus that is highly similar to infectious bronchitis virus (IBV) an upper-respiratory tract disease virus in chickens, were conducted to determine the adaptive potential, and genetic changes associated with emergence of this group 3 coronavirus. Strains of TCoV that were pathogenic in poults and nonpathogenic in chickens did not adapt to cause disease in chickens. Comparative genomics revealed two recombination sites that replaced the spike gene in IBV with an unidentified sequence likely from another coronavirus, resulting in cross-species transmission and a pathogenicity shift. Following emergence in turkeys, TCoV diverged to different serotypes through the accumulation of mutations within spike. This is the first evidence that recombination can directly lead to the emergence of new coronaviruses and new coronaviral diseases, emphasizing the importance of limiting exposure to reservoirs of coronaviruses that can serve as a source of genetic material for emerging viruses.
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              Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006.

              Intestinal samples collected from 43 commercial broiler and 33 commercial turkey flocks from all regions of the United States during 2005 and 2006 were examined for the presence of astrovirus, rotavirus, reovirus, and coronavirus by reverse transcription-polymerase chain reaction (PCR), and for the presence of groups 1 and 2 adenovirus by PCR. Phylogenetic analysis was performed to further characterize the viruses and to evaluate species association and geographic patterns. Astroviruses were identified in samples from 86% of the chicken flocks and from 100% of the turkey flocks. Both chicken astrovirus and avian nephritis virus (ANV) were identified in chicken samples, and often both viruses were detected in the same flock. Turkey astrovirus type-2 and turkey astrovirus type-1 were found in 100% and 15.4% of the turkey flocks, respectively. In addition, 12.5% of turkey flocks were positive for ANV. Rotaviruses were present in 46.5% of the chicken flocks tested and in 69.7% of the turkey flocks tested. Based upon the rotavirus NSP4 gene sequence, the chicken and turkey origin rotaviruses assorted in a species-specific manner. The turkey origin rotaviruses also assorted based upon geographical location. Reoviruses were identified in 62.8% and 45.5% of chicken and turkey flocks, respectively. Based on the reovirus S4 gene segment, the chicken and turkey origin viruses assorted separately, and they were distinct from all previously reported avian reoviruses. Coronaviruses were detected in the intestinal contents of chickens, but not turkeys. Adenoviruses were not detected in any chicken or turkeys flocks. Of the 76 total chicken and turkey flocks tested, only three chicken flocks were negative for all viruses targeted by this study. Most flocks were positive for two or more of the viruses, and overall no clear pattern of virus geographic distribution was evident. This study provides updated enteric virus prevalence data for the United States using molecular methods, and it reinforces that enteric viruses are widespread in poultry throughout the United States, although the clinical importance of most of these viruses remains unclear.

                Author and article information

                Mol Cell Probes
                Mol. Cell. Probes
                Molecular and Cellular Probes
                Elsevier Ltd.
                21 August 2010
                December 2010
                21 August 2010
                : 24
                : 6
                : 415-417
                UNESP – São Paulo State University, Laboratory of Animal Virology, Campus de Araçatuba, São Paulo, Brazil
                Author notes
                []Corresponding author. Departamento de Apoio, Produção e Saúde Animal, Curso de Medicina Veterinária, Rua Clóvis Pestana, 793. 16.050-680 Araçatuba, SP, Brazil. Tel.: +55 18 3663200. tcardoso@ 123456fmva.unesp.br
                Copyright © 2010 Elsevier Ltd. 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.

                : 11 May 2010
                : 4 August 2010
                : 9 August 2010

                turkey coronavirus,hydroxynaphthol blue,lamp assay,spike gene


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