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      Diagnosis and characterization of canine distemper virus through sequencing by MinION nanopore technology

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          Abstract

          Prompt identification of the causative pathogen of an infectious disease is essential for the choice of treatment or preventive measures. In this perspective, nucleic acids purified from the brain tissue of a dog succumbed after severe neurological signs were processed with the MinION (Oxford Nanopore Technologies, Oxford UK) sequencing technology. Canine distemper virus (CDV) sequence reads were detected. Subsequently, a specific molecular test and immunohistochemistry were used to confirm the presence of CDV RNA and antigen, respectively, in tissues. This study supports the use of the NGS in veterinary clinical practice with potential advantages in terms of rapidity and broad-range of molecular diagnosis.

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          Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis

          Nádia Conceição-Neto, Mark Zeller, Hanne Lefrère … (2015)
          A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.
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            Metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis.

            Joseph Victoria, Amit Kapoor, Linlin Li … (2009)
            We analyzed viral nucleic acids in stool samples collected from 35 South Asian children with nonpolio acute flaccid paralysis (AFP). Sequence-independent reverse transcription and PCR amplification of capsid-protected, nuclease-resistant viral nucleic acids were followed by DNA sequencing and sequence similarity searches. Limited Sanger sequencing (35 to 240 subclones per sample) identified an average of 1.4 distinct eukaryotic viruses per sample, while pyrosequencing yielded 2.6 viruses per sample. In addition to bacteriophage and plant viruses, we detected known enteric viruses, including rotavirus, adenovirus, picobirnavirus, and human enterovirus species A (HEV-A) to HEV-C, as well as numerous other members of the Picornaviridae family, including parechovirus, Aichi virus, rhinovirus, and human cardiovirus. The viruses with the most divergent sequences relative to those of previously reported viruses included members of a novel Picornaviridae genus and four new viral species (members of the Dicistroviridae, Nodaviridae, and Circoviridae families and the Bocavirus genus). Samples from six healthy contacts of AFP patients were similarly analyzed and also contained numerous viruses, particularly HEV-C, including a potentially novel Enterovirus genotype. Determining the prevalences and pathogenicities of the novel genotypes, species, genera, and potential new viral families identified in this study in different demographic groups will require further studies with different demographic and patient groups, now facilitated by knowledge of these viral genomes.
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              Detection of canine distemper virus in dogs by real-time RT-PCR.

              Maria Lucente, Livia Di Renzo, Eleonora Lorusso … (2006)
              Canine distemper virus is the etiological agent of a severe disease in dogs and many other carnivores. Clinical diagnosis of canine distemper is difficult due to the broad spectrum of signs that may be confounded with other respiratory and enteric diseases of dogs. Accordingly, a laboratory confirmation is required for suspected cases. In this study a real-time RT-PCR assay was developed for detection and quantitation of canine distemper virus. The assay exhibited high specificity as all the negative controls (no-template-controls and samples from healthy sero-negative dogs) and other canine pathogens were not misdetected. Up to 1 x 10(2) copies of RNA were detected by the TaqMan assay, thus revealing a high sensitivity. Quantitative TaqMan was validated on clinical samples, including various tissues and organs collected from dogs naturally infected by canine distemper virus. Urines, tonsil, conjunctival swabs and whole blood were found to contain high virus loads and therefore proved to be suitable targets for detection of canine distemper virus RNA.
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                Author and article information

                Contributors
                Alessio Lorusso: a.lorusso@izs.it
                Journal
                Journal ID (nlm-ta): Sci Rep
                Journal ID (iso-abbrev): Sci Rep
                Title: Scientific Reports
                Publisher: Nature Publishing Group UK (London )
                ISSN (Electronic): 2045-2322
                Publication date (Electronic): 8 February 2019
                Publication date PMC-release: 8 February 2019
                Publication date Collection: 2019
                Volume: 9
                Electronic Location Identifier: 1714
                Affiliations
                [1 ]National Reference Center for Whole Genome Sequencing of microbial pathogens: database and bioinformatic analysis, Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise, Campo Boario, 64100 Teramo, Italy
                [2 ]Dipartimento di Medicina Veterinaria, strada provinciale per Casamassima Km 3, 70100 Valenzano, Bari Italy
                Author information
                http://orcid.org/0000-0003-4795-8323
                http://orcid.org/0000-0001-6862-1792
                Article
                Publisher ID: 37497
                DOI: 10.1038/s41598-018-37497-4
                PMC ID: 6368598
                PubMed ID: 30737428
                SO-VID: 72f093b8-35ea-4660-b788-027bc8033ae6
                Copyright © © The Author(s) 2019
                License:

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                Date received : 18 June 2018
                Date accepted : 6 December 2018
                Funding
                Funded by: Italian Ministry of Health, MSRCTE06/17
                Categories
                Subject: Article
                Custom metadata
                issue-copyright-statement © The Author(s) 2019

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