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Real-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samples

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dc.contributor.authorButt, Salman L.en
dc.contributor.authorErwood, Eric C.en
dc.contributor.authorZhang, Jianen
dc.contributor.authorSellers, Holly S.en
dc.contributor.authorYoung, Kelsey T.en
dc.contributor.authorLahmers, Kevin K.en
dc.contributor.authorStanton, James B.en
dc.contributor.departmentBiomedical Sciences and Pathobiologyen
dc.date.accessioned2021-01-06T13:38:20Zen
dc.date.available2021-01-06T13:38:20Zen
dc.date.issued2020-03en
dc.description.abstractInfectious bronchitis (IB) causes significant economic losses in the global poultry industry. Control of IB is hindered by the genetic diversity of the causative agent, infectious bronchitis virus (IBV), which has led to the emergence of several serotypes that lack complete serologic cross-protection. Although serotyping requires immunologic characterization, genotyping is an efficient means to identify IBVs detected in samples. Sanger sequencing of the S1 subunit of the spike gene is currently used to genotype IBV; however, the universal S1 PCR was created to work from cultured IBV, and it is inefficient at detecting multiple viruses in a single sample. We describe herein a MinION-based, amplicon-based sequencing (AmpSeq) method that genetically categorized IBV from clinical samples, including samples with multiple IBVs. Total RNA was extracted from 15 tracheal scrapings and choanal cleft swab samples, randomly reverse transcribed, and PCR amplified using modified S1-targeted primers. Amplicons were barcoded to allow for pooling of samples, processed per manufacturer's instructions into a 1D MinION sequencing library, and then sequenced on the MinION. The AmpSeq method detected IBV in 13 of 14 IBV-positive samples. AmpSeq accurately detected and genotyped both IBV lineages in 3 of 5 samples containing 2 IBV lineages. Additionally, 1 sample contained 3 IBV lineages, and AmpSeq accurately detected 2 of the 3 lineages. Strain identification, including detection of different IBVs from the same lineage, was also possible with this AmpSeq method. Our results demonstrate the feasibility of using MinION-based AmpSeq for rapid and accurate identification and lineage typing of IBV from oral swab samples.en
dc.description.notesOur project was supported by grant 5T35OD010433-12 from the Office of Research Infrastructure Programs (ORIP), a component of the National Institutes of Health (NIH; the contents are solely the responsibility of the authors and do not necessarily represent the official view of ORIP or NIH); University of Georgia Research Foundation 10-21-RX064-681; and Agriculture and Food Research Initiative Competitive Grant 2018-67015-28306 from the USDA National Institute of Food and Agriculture. S. L. Butt was funded for his PhD studies by the Fulbright Foreign Student program by the U.S. State Department.en
dc.description.sponsorshipOffice of Research Infrastructure Programs (ORIP), a component of the National Institutes of Health (NIH)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [5T35OD010433-12]; University of Georgia Research Foundation [10-21-RX064-681]; Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture [2018-67015-28306]; Fulbright Foreign Student program by the U.S. State Departmenten
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1177/1040638720910107en
dc.identifier.eissn1943-4936en
dc.identifier.issn1040-6387en
dc.identifier.other1040638720910107en
dc.identifier.pmid32133932en
dc.identifier.urihttp://hdl.handle.net/10919/101757en
dc.language.isoenen
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectgenotypeen
dc.subjectinfectious bronchitis virusen
dc.subjectMinIONen
dc.subjectnanopore sequencingen
dc.subjectrapid sequencingen
dc.subjectRNAen
dc.titleReal-time, MinION-based, amplicon sequencing for lineage typing of infectious bronchitis virus from upper respiratory samplesen
dc.title.serialJournal of Veterinary Diagnostic Investigationen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.dcmitypeStillImageen

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