{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T16:57:41Z","timestamp":1769187461513,"version":"3.49.0"},"reference-count":41,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T00:00:00Z","timestamp":1769126400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100018818","name":"National Research, Development, and Innovation Office","doi-asserted-by":"publisher","award":["FK154149"],"award-info":[{"award-number":["FK154149"]}],"id":[{"id":"10.13039\/501100018818","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100018818","name":"National Research, Development, and Innovation Office","doi-asserted-by":"publisher","award":["FK137778"],"award-info":[{"award-number":["FK137778"]}],"id":[{"id":"10.13039\/501100018818","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100018818","name":"National Research, Development, and Innovation Office","doi-asserted-by":"publisher","award":["RRF-2.3.1-21-2022-00001"],"award-info":[{"award-number":["RRF-2.3.1-21-2022-00001"]}],"id":[{"id":"10.13039\/501100018818","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Ministry of Culture and Innovation of Hungary from the National Research, Development, and Innovation Fund","award":["2024-2.1.1-EK\u00d6P"],"award-info":[{"award-number":["2024-2.1.1-EK\u00d6P"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The rapid evolution of coronaviruses (CoVs) requires researchers to develop specific yet broad-spectrum detection methods to monitor their constant genomic changes. The goal of the present study was to establish a current pan-coronavirus RT-PCR system capable of detecting a wide variety of CoVs and useful for the investigation of virus diversity and host spectrum. For optimization, one-step and two-step nested RT-PCRs with three RT enzymes were examined, amplifying a ~600 bp long product of the RNA-dependent RNA polymerase. As templates, the in vitro transcribed RNA of ten pathogenic CoVs (SARS-CoV, SARS-CoV-2, NL-63, OC43, feline CoV, porcine epidemic diarrhea virus or PEDV, transmissible gastroenteritis virus or TGEV, canine CoV, bat CoV, and infectious bronchitis virus) were applied instead of the often-used DNA standards. A limit of detection of 5\u201350 copies\/reaction was achieved with a random hexamer-primed two-step RT-PCR and a touchdown cycling profile, representing a lower detection limit and higher specificity compared to previously published primer sets. Swine origin pooled samples (n = 121), collected from apparently healthy herds in Hungary, were tested with the novel RT-PCR system. Sequences of porcine respiratory CoV\/TGEV and porcine hemagglutinating encephalomyelitis virus were identified in 24 oral fluid and nasal swab pools, demonstrating the circulation of these viruses in this country, as well as the suitability of the new PCR for their detection. The results highlighted the importance of adequate RT enzyme selection and the use of RNase inhibitors in sample preparation and conservation.<\/jats:p>","DOI":"10.3390\/ani16030358","type":"journal-article","created":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T14:58:40Z","timestamp":1769180320000},"page":"358","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Surveillance of Swine Coronaviruses in Hungarian Herds with a Newly Established Pan-Coronavirus RT-PCR System"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-7200-4823","authenticated-orcid":false,"given":"D\u00f3ra","family":"M\u00e1t\u00e9","sequence":"first","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest, Hung\u00e1ria krt. 23-25, H-1143 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0093-5418","authenticated-orcid":false,"given":"Ren\u00e1ta","family":"Varga-Kugler","sequence":"additional","affiliation":[{"name":"Ceva-Phylaxia Ltd., Sz\u00e1ll\u00e1s utca 5, H-1107 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5750-1872","authenticated-orcid":false,"given":"Eszter","family":"Kaszab","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest, Hung\u00e1ria krt. 23-25, H-1143 Budapest, Hungary"},{"name":"National Laboratory for Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, Istv\u00e1n utca 2, H-1078 Budapest, Hungary"},{"name":"Department of Bioinformatics, One Health Institute, Faculty of Health Sciences, University of Debrecen, Nagyerdei krt. 98, H-4032 Debrecen, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-5035-7701","authenticated-orcid":false,"given":"Henrik F\u00fcl\u00f6p","family":"K\u00e1rolyi","sequence":"additional","affiliation":[{"name":"National Laboratory of Virology, Szent\u00e1gothai Research Centre, University of P\u00e9cs, Ifj\u00fas\u00e1g \u00fatja 20, H-7624 P\u00e9cs, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1910-4024","authenticated-orcid":false,"given":"Tam\u00e1s","family":"G\u00f6rf\u00f6l","sequence":"additional","affiliation":[{"name":"National Laboratory of Virology, Szent\u00e1gothai Research Centre, University of P\u00e9cs, Ifj\u00fas\u00e1g \u00fatja 20, H-7624 P\u00e9cs, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9775-3065","authenticated-orcid":false,"given":"G\u00e1bor","family":"Kemenesi","sequence":"additional","affiliation":[{"name":"National Laboratory of Virology, Szent\u00e1gothai Research Centre, University of P\u00e9cs, Ifj\u00fas\u00e1g \u00fatja 20, H-7624 P\u00e9cs, Hungary"},{"name":"Institute of Biology, Faculty of Sciences, University of P\u00e9cs, Ifj\u00fas\u00e1g \u00fatja 6, H-7624 P\u00e9cs, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7372-3144","authenticated-orcid":false,"given":"Barbara","family":"Igriczi","sequence":"additional","affiliation":[{"name":"National Laboratory for Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, Istv\u00e1n utca 2, H-1078 Budapest, Hungary"},{"name":"Department of Pathology, University of Veterinary Medicine Budapest, Istv\u00e1n utca 2, H-1078 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2742-173X","authenticated-orcid":false,"given":"Gyula","family":"Balka","sequence":"additional","affiliation":[{"name":"National Laboratory for Infectious Animal Diseases, Antimicrobial Resistance, Veterinary Public Health and Food Chain Safety, Istv\u00e1n utca 2, H-1078 Budapest, Hungary"},{"name":"Department of Pathology, University of Veterinary Medicine Budapest, Istv\u00e1n utca 2, H-1078 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1823-9971","authenticated-orcid":false,"given":"Marianna","family":"Dom\u00e1n","sequence":"additional","affiliation":[{"name":"HUN-REN Veterinary Medical Research Institute, Hung\u00e1ria krt. 21, H-1143 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1d\u00e1m","family":"B\u00e1lint","sequence":"additional","affiliation":[{"name":"Vetcontrol Ltd., D\u00e9li-Bek\u00f6t\u0151 \u00fat 8, H-1211 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7312-618X","authenticated-orcid":false,"given":"Zolt\u00e1n","family":"Z\u00e1dori","sequence":"additional","affiliation":[{"name":"HUN-REN Veterinary Medical Research Institute, Hung\u00e1ria krt. 21, H-1143 Budapest, Hungary"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7778-9116","authenticated-orcid":false,"given":"Enik\u0151","family":"Feh\u00e9r","sequence":"additional","affiliation":[{"name":"Department of Microbiology and Infectious Diseases, University of Veterinary Medicine Budapest, Hung\u00e1ria krt. 23-25, H-1143 Budapest, Hungary"},{"name":"National Laboratory for Infectious Animal Diseases, 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Emerg. Dis."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Islam, A., Ferdous, J., Islam, S., Sayeed, M.A., Dutta Choudhury, S., Saha, O., Hassan, M.M., and Shirin, T. (2021). Evolutionary Dynamics and Epidemiology of Endemic and Emerging Coronaviruses in Humans, Domestic Animals, and Wildlife. Viruses, 13.","DOI":"10.3390\/v13101908"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e33040","DOI":"10.1016\/j.heliyon.2024.e33040","article-title":"The reverse zoonotic potential of SARS-CoV-2","volume":"10","author":"Milich","year":"2024","journal-title":"Heliyon"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1038\/s41579-021-00652-2","article-title":"Ecology, evolution and spillover of coronaviruses from bats","volume":"20","author":"McKee","year":"2022","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1080\/01652176.2022.2079756","article-title":"Swine coronaviruses (SCoVs) and their emerging threats to swine population, inter-species transmission, exploring the susceptibility of pigs for SARS-CoV-2 and zoonotic concerns","volume":"42","author":"Thakor","year":"2022","journal-title":"Vet. Q."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1016\/j.chom.2023.05.003","article-title":"The coronavirus recombination pathway","volume":"31","author":"Wells","year":"2023","journal-title":"Cell Host Microb."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1686","DOI":"10.7150\/ijbs.45472","article-title":"Zoonotic origins of human coronaviruses","volume":"16","author":"Ye","year":"2020","journal-title":"Int. J. Biol. Sci."},{"key":"ref_8","unstructured":"(2025, August 30). International Committee on Taxonomy of Viruses: ICTV. Available online: https:\/\/ictv.global\/taxonomy."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1002\/jmv.25681","article-title":"Emerging coronaviruses: Genome structure, replication, and pathogenesis","volume":"92","author":"Chen","year":"2020","journal-title":"J. Med. Virol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"105246","DOI":"10.1016\/j.jcv.2022.105246","article-title":"Human coronaviruses: Origin, host and receptor","volume":"155","author":"Tang","year":"2022","journal-title":"J. Clin. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.3201\/eid2006.140299","article-title":"MERS coronaviruses in dromedary camels","volume":"20","author":"Chu","year":"2014","journal-title":"Egypt. Emerg. Infect. Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1319","DOI":"10.1016\/S0140-6736(03)13077-2","article-title":"Coronavirus as a possible cause of severe acute respiratory syndrome","volume":"361","author":"Peiris","year":"2003","journal-title":"Lancet"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1814","DOI":"10.1056\/NEJMoa1211721","article-title":"Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia","volume":"367","author":"Zaki","year":"2012","journal-title":"N. Engl. J. Med."},{"key":"ref_14","first-page":"259","article-title":"The biology of coronaviruses, with special regards to SARS-CoV-2-and COVID-19","volume":"142","author":"Balka","year":"2020","journal-title":"Magy. Allatorvosok Lapja"},{"key":"ref_15","first-page":"323","article-title":"Vaccine developments and pharmacological treatment options against SARS-CoV-2 infection and the control of coronaviruses in domestic animals Literature review","volume":"142","author":"Balka","year":"2020","journal-title":"Magy. Allatorvosok Lapja"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Buonavoglia, A., Pellegrini, F., Decaro, N., Galgano, M., and Pratelli, A. (2023). A One Health Perspective on Canine Coronavirus: A Wolf in Sheep\u2019s Clothing?. Microorganisms, 11.","DOI":"10.3390\/microorganisms11040921"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s11259-021-09808-0","article-title":"Porcine enteric coronaviruses: An updated overview of the pathogenesis, prevalence, and diagnosis","volume":"45","author":"Liu","year":"2021","journal-title":"Vet. Res. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"106371","DOI":"10.1016\/j.tvjl.2025.106371","article-title":"Occurrence and epidemiology of bovine coronavirus in cattle in Kazakhstan","volume":"24","author":"Perfilyeva","year":"2025","journal-title":"Vet. J."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1016\/j.tvjl.2017.11.004","article-title":"Enteric coronavirus infection in adult horses","volume":"231","author":"Pusterla","year":"2018","journal-title":"Vet. J."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1016\/j.coviro.2018.12.001","article-title":"Emerging and re-emerging coronaviruses in pigs","volume":"34","author":"Wang","year":"2019","journal-title":"Curr. Opin. Virol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2876278","DOI":"10.1155\/2024\/2876278","article-title":"The Emergence of Novel Variants of the Porcine Epidemic Diarrhea Virus Spike Gene from 2011 to 2023","volume":"2024","author":"Zhang","year":"2024","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1089\/vbz.2014.1637","article-title":"Molecular survey of RNA viruses in Hungarian bats: Discovering novel astroviruses, coronaviruses, and caliciviruses","volume":"14","author":"Kemenesi","year":"2014","journal-title":"Vector Borne Zoonotic Dis."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5534854","DOI":"10.1155\/2024\/5534854","article-title":"Prevalence Estimation and Genetic Characterization of Porcine Parainfluenza Virus 1 (PPIV-1) in Hungary and the First Report of the Virus in Slovakia","volume":"2024","author":"Igriczi","year":"2024","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5117884","DOI":"10.1155\/2024\/5117884","article-title":"Comparative Prevalence Estimation and Phylogenetic Analysis of Novel Porcine Parvoviruses (PPV2-7) in Hungarian Pig Herds","volume":"2024","author":"Igriczi","year":"2024","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_25","unstructured":"(2025, December 12). Basic Local Alignment Search Tool: BLAST, Available online: https:\/\/blast.ncbi.nlm.nih.gov\/Blast.cgi."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1093\/molbev\/msy096","article-title":"MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms","volume":"35","author":"Kumar","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Holbrook, M.G., Anthony, S.J., Navarrete-Macias, I., Bestebroer, T., Munster, V.J., and van Doremalen, N. (2021). Updated and Validated Pan-Coronavirus PCR Assay to Detect All Coronavirus Genera. Viruses, 13.","DOI":"10.3390\/v13040599"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1128\/JCM.02426-06","article-title":"Generic Detection of Coronaviruses and Differentiation at the Prototype Strain Level by Reverse Transcription-PCR and Nonfluorescent Low-Density Microarray","volume":"45","author":"Heiser","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Drzewniokov\u00e1, P., Festa, F., Panzarin, V., Lelli, D., Moreno, A., Zecchin, B., De Benedictis, P., and Leopardi, S. (2021). Best Molecular Tools to Investigate Coronavirus Diversity in Mammals: A Comparison. Viruses, 13.","DOI":"10.3390\/v13101975"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.jviromet.2018.02.021","article-title":"Development of a one-step RT-PCR assay for detection of pancoronaviruses (\u03b1-, \u03b2-, \u03b3-, and \u03b4-coronaviruses) using newly designed degenerate primers for porcine and avian fecal samples","volume":"256","author":"Hu","year":"2018","journal-title":"J. Virol. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"12815","DOI":"10.1128\/JVI.05838-11","article-title":"Avian coronavirus in wild aquatic birds","volume":"85","author":"Chu","year":"2011","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"104391","DOI":"10.1016\/j.jcv.2020.104391","article-title":"A RT-PCR assay for the detection of coronaviruses from four genera","volume":"128","author":"Xiu","year":"2020","journal-title":"J. Clin. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Olech, M., and Antas, M. (2025). Transmissible Gastroenteritis Virus (TGEV) and Porcine Respiratory Coronavirus (PRCV): Epidemiology and Molecular Characteristics-An Updated Overview. Viruses, 17.","DOI":"10.3390\/v17040493"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1556\/avet.2013.043","article-title":"Sporadic re-emergence of enzootic porcine transmissible gastroenteritis in Hungary","volume":"62","author":"Biksi","year":"2014","journal-title":"Acta Vet. Hung."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1556\/004.2017.025","article-title":"Porcine epidemic diarrhoea virus with a recombinant S gene detected in Hungary, 2016","volume":"65","author":"Biksi","year":"2017","journal-title":"Acta Vet. Hung."},{"key":"ref_36","first-page":"207","article-title":"Enteral coronaviruses of swine","volume":"140","author":"Tuboly","year":"2018","journal-title":"Magy. Allatorvosok Lapja"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1556\/004.2019.031","article-title":"Isolation and characterisation of porcine epidemic diarrhoea virus in Hungary","volume":"67","author":"Albert","year":"2019","journal-title":"Acta Vet. Hung."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"100042","DOI":"10.1016\/j.vas.2018.11.003","article-title":"Prevalence of antibodies against transmissible gastroenteritis virus (TGEV) in Hungary","volume":"7","author":"Bozsa","year":"2019","journal-title":"Vet. Anim. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2443","DOI":"10.7717\/peerj.2443","article-title":"The evidence of porcine hemagglutinating encephalomyelitis virus induced nonsuppurative encephalitis as the cause of death in piglets","volume":"15","author":"Li","year":"2016","journal-title":"PeerJ"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.3201\/eid2307.170019","article-title":"Porcine Hemagglutinating Encephalomyelitis Virus and Respiratory Disease in Exhibition Swine, Michigan, USA, 2015","volume":"23","author":"Lorbach","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref_41","first-page":"305","article-title":"A Disease of nursing pigs previously unreported in Ontario","volume":"22","author":"Roe","year":"1958","journal-title":"Can. J. Comp. Med. Vet. Sci."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/3\/358\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,23]],"date-time":"2026-01-23T15:12:54Z","timestamp":1769181174000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/3\/358"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,1,23]]},"references-count":41,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,2]]}},"alternative-id":["ani16030358"],"URL":"https:\/\/doi.org\/10.3390\/ani16030358","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,1,23]]}}}