{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T21:42:27Z","timestamp":1777930947543,"version":"3.51.4"},"reference-count":32,"publisher":"SAGE Publications","issue":"1","license":[{"start":{"date-parts":[[2025,10,7]],"date-time":"2025-10-07T00:00:00Z","timestamp":1759795200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.sagepub.com\/page\/policies\/text-and-data-mining-license"}],"funder":[{"name":"The Thailand Science Research and Innovation Fund Chulalongkorn University","award":["HEA_FF_68_051_3100_009"],"award-info":[{"award-number":["HEA_FF_68_051_3100_009"]}]},{"name":"the Second Century Fund (C2F), Chulalongkorn University"}],"content-domain":{"domain":["journals.sagepub.com"],"crossmark-restriction":true},"short-container-title":["J VET Diagn Invest"],"published-print":{"date-parts":[[2026,1]]},"abstract":"<jats:p>\n                    Canine astrovirus (mamastrovirus 5 [MAstV5], formerly CaAstV; family\n                    <jats:italic toggle=\"yes\">Astroviridae<\/jats:italic>\n                    , taxon species\n                    <jats:italic toggle=\"yes\">Mamastrovirus canis<\/jats:italic>\n                    ) and canine kobuvirus (aichivirus A2 [AiV-A2], formerly CaKoV;\n                    <jats:italic toggle=\"yes\">Picornaviridae<\/jats:italic>\n                    ,\n                    <jats:italic toggle=\"yes\">Kobuvirus aichi<\/jats:italic>\n                    ) are emerging enteric viruses increasingly detected in both diarrheic and subclinical dogs. Although primarily associated with gastrointestinal illness, recent evidence suggests a potential for systemic dissemination, which remains insufficiently explored. To improve detection, we developed and validated a duplex quantitative real-time PCR (dqPCR) assay for simultaneous identification of MAstV5 and AiV-A2. Primers and TaqMan probes were designed based on conserved regions of the MAstV5\n                    <jats:italic toggle=\"yes\">ORF1b<\/jats:italic>\n                    and AiV-A2\n                    <jats:italic toggle=\"yes\">3D<\/jats:italic>\n                    polymerase genes. Assay optimization included primer-probe concentration titration and thermal gradient analysis. Analytical performance was assessed using synthetic plasmid standards and RNA from non-target viruses, including canine parvovirus (CPV), canine morbillivirus (CDV), and canine enteric coronavirus (CCoV). Our dqPCR assay had high linearity (\n                    <jats:italic toggle=\"yes\">R<\/jats:italic>\n                    <jats:sup>2<\/jats:sup>\n                    \u2009&gt;\u20090.99) and sensitivity, with limits of detection of 10 copies\/\u03bcL for MAstV5 and 100 copies\/\u03bcL for AiV-A2. No cross-reactivity or interference was observed in coinfection simulations across various target ratios. Intra- and inter-assay CV values were &lt;2.2%, indicating excellent reproducibility. Validation using 50 clinical rectal swabs and tissue samples from 25 autopsied dogs revealed 1 additional AiV-A2\u2013positive case undetected by RT-PCR but confirmed by sequencing. Importantly, viral RNA was also found in extraintestinal tissues\u2014spleen, liver, trachea, and mesenteric lymph nodes\u2014suggesting systemic distribution in naturally infected dogs. Our dqPCR assay provides a sensitive, specific, and efficient tool for the detection of MAstV5 and AiV-A2, supporting both clinical testing and epidemiologic studies.\n                  <\/jats:p>","DOI":"10.1177\/10406387251382915","type":"journal-article","created":{"date-parts":[[2025,10,8]],"date-time":"2025-10-08T06:09:47Z","timestamp":1759903787000},"page":"84-92","update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":0,"title":["Simultaneous detection of canine astrovirus and canine kobuvirus by duplex qPCR: validation and evidence of extraintestinal viral RNA in naturally infected dogs"],"prefix":"10.1177","volume":"38","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-4313-198X","authenticated-orcid":false,"given":"Tin","family":"Van Nguyen","sequence":"first","affiliation":[{"name":"The International Graduate Program of Veterinary Science and Technology (VST), Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand"},{"name":"Faculty of Animal Science and Veterinary Medicine, Nong Lam University, Ho Chi Minh City, Vietnam"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1895-8992","authenticated-orcid":false,"given":"Tanit","family":"Kasantikul","sequence":"additional","affiliation":[{"name":"Veterinary Diagnostic Laboratory, Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5148-4696","authenticated-orcid":false,"given":"Chutchai","family":"Piewbang","sequence":"additional","affiliation":[{"name":"Center of Excellence in Animal Virome and Diagnostic Development, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand"},{"name":"Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4888-2677","authenticated-orcid":false,"given":"Somporn","family":"Techangamsuwan","sequence":"additional","affiliation":[{"name":"Center of Excellence in Animal Virome and Diagnostic Development, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand"},{"name":"Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"179","published-online":{"date-parts":[[2025,10,7]]},"reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0022-2836(05)80360-2"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1373\/clinchem.2008.112797"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.tvjl.2017.12.009"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2013.02.014"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1186\/s12917-019-1994-6"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.3201\/eid2205.151807"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.yasa.2020.07.012"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijfoodmicro.2010.09.022"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.05317-11"},{"key":"e_1_3_2_11_2","first-page":"497","volume-title":"Viral Gastroenteritis","author":"Khamrin P","unstructured":"Khamrin P, et al. Molecular epidemiology of astroviruses. In: Svensson L, et al., eds. Viral Gastroenteritis. Academic Press, 2016:497\u2013522."},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1186\/s40064-016-3738-4"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1093\/molbev\/msy096"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2005.07.019"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.034611-0"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-017-3648-y"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1099\/vir.0.029025-0"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.virusres.2016.12.005"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1007\/s42770-019-00095-1"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2023.1278417"},{"key":"e_1_3_2_21_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-014-2136-x"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.3390\/v12040361"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.02268-23"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-016-3164-5"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-015-2405-3"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.vetmic.2009.04.031"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gks596"},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-024-62833-2"},{"key":"e_1_3_2_29_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.bios.2011.02.027"},{"key":"e_1_3_2_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2021.114066"},{"key":"e_1_3_2_31_2","doi-asserted-by":"publisher","DOI":"10.3389\/fvets.2021.710701"},{"key":"e_1_3_2_32_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-7-85"},{"key":"e_1_3_2_33_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00705-011-1022-z"}],"container-title":["Journal of Veterinary Diagnostic Investigation"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.sagepub.com\/doi\/pdf\/10.1177\/10406387251382915","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.sagepub.com\/doi\/full-xml\/10.1177\/10406387251382915","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.sagepub.com\/doi\/pdf\/10.1177\/10406387251382915","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T00:29:19Z","timestamp":1777681759000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.sagepub.com\/doi\/10.1177\/10406387251382915"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,10,7]]},"references-count":32,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2026,1]]}},"alternative-id":["10.1177\/10406387251382915"],"URL":"https:\/\/doi.org\/10.1177\/10406387251382915","relation":{},"ISSN":["1040-6387","1943-4936"],"issn-type":[{"value":"1040-6387","type":"print"},{"value":"1943-4936","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,10,7]]}}}