{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,28]],"date-time":"2026-02-28T04:29:53Z","timestamp":1772252993173,"version":"3.50.1"},"reference-count":50,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2021,3,18]],"date-time":"2021-03-18T00:00:00Z","timestamp":1616025600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001711","name":"Swiss National Science Foundation","doi-asserted-by":"publisher","award":["179260"],"award-info":[{"award-number":["179260"]}],"id":[{"id":"10.13039\/501100001711","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>The ruminant-associated influenza D virus (IDV) has a broad host tropism and was shown to have zoonotic potential. To identify and characterize molecular viral determinants influencing the host spectrum of IDV, a reverse genetic system is required. For this, we first performed 5\u2032 and 3\u2032 rapid amplification of cDNA ends (RACE) of all seven genomic segments, followed by assessment of the 5\u2032 and 3\u2032 NCR activity prior to constructing the viral genomic segments of a contemporary Swiss bovine IDV isolate (D\/CN286) into the bidirectional pHW2000 vector. The bidirectional plasmids were transfected in HRT-18G cells followed by viral rescue on the same cell type. Analysis of the segment specific 5\u2032 and 3\u2032 non-coding regions (NCR) highlighted that the terminal 3\u2032 end of all segments harbours an uracil instead of a cytosine nucleotide, similar to other influenza viruses. Subsequent analysis on the functionality of the 5\u2032 and 3\u2032 NCR in a minireplicon assay revealed that these sequences were functional and that the variable sequence length of the 5\u2032 and 3\u2032 NCR influences reporter gene expression. Thereafter, we evaluated the replication efficiency of the reverse genetic clone on conventional cell lines of human, swine and bovine origin, as well as by using an in vitro model recapitulating the natural replication site of IDV in bovine and swine. This revealed that the reverse genetic clone D\/CN286 replicates efficiently in all cell culture models. Combined, these results demonstrate the successful establishment of a reverse genetic system from a contemporary bovine IDV isolate that can be used for future identification and characterization of viral determinants influencing the broad host tropism of IDV.<\/jats:p>","DOI":"10.3390\/v13030502","type":"journal-article","created":{"date-parts":[[2021,3,18]],"date-time":"2021-03-18T22:19:36Z","timestamp":1616105976000},"page":"502","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Establishment of a Reverse Genetic System from a Bovine Derived Influenza D Virus Isolate"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9814-7793","authenticated-orcid":false,"given":"Melle","family":"Holwerda","sequence":"first","affiliation":[{"name":"Institute of Virology and Immunology, 3012 Bern, Switzerland"},{"name":"Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"},{"name":"Institute for Infectious Diseases, University of Bern, 3001 Bern, Switzerland"},{"name":"Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laura","family":"Laloli","sequence":"additional","affiliation":[{"name":"Institute for Infectious Diseases, University of Bern, 3001 Bern, Switzerland"},{"name":"Graduate School for Cellular and Biomedical Sciences, University of Bern, 3012 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Manon","family":"Wider","sequence":"additional","affiliation":[{"name":"Institute for Infectious Diseases, University of Bern, 3001 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lutz","family":"Sch\u00f6necker","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Bacteriology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"},{"name":"Clinic for Ruminants, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"},{"name":"Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jens","family":"Becker","sequence":"additional","affiliation":[{"name":"Clinic for Ruminants, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"},{"name":"Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mireille","family":"Meylan","sequence":"additional","affiliation":[{"name":"Clinic for Ruminants, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"},{"name":"Department of Clinical Veterinary Science, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0320-2743","authenticated-orcid":false,"given":"Ronald","family":"Dijkman","sequence":"additional","affiliation":[{"name":"Institute of Virology and Immunology, 3012 Bern, Switzerland"},{"name":"Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland"},{"name":"Institute for Infectious Diseases, University of Bern, 3001 Bern, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/mBio.00031-14","article-title":"Characterization of a novel influenza virus strain in cattle and swine: Proposal for a new genus in the Orthomyxoviridae family","volume":"5","author":"Hause","year":"2014","journal-title":"MBio"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"King, A.M.Q., Lefkowitz, E.J., Mushegian, A.R., Adams, M.J., Dutilh, B.E., Gorbalenya, A.E., Harrach, B., Harrison, R.L., Junglen, S., and Knowles, N.J. (2018). Changes to Taxonomy and the International Code of Virus Classification and Nomenclature Ratified by the International Committee on Taxonomy of Viruses (2018), Springer.","DOI":"10.1007\/s00705-018-3847-1"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Hause, B.M., Ducatez, M., Collin, E.A., Ran, Z., Liu, R., Sheng, Z., Armien, A., Kaplan, B., Chakravarty, S., and Hoppe, A.D. (2013). Isolation of a Novel Swine Influenza Virus from Oklahoma in 2011 Which Is Distantly Related to Human Influenza C Viruses. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003176"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2012","DOI":"10.3201\/eid2407.171937","article-title":"Influenza D Virus Circulation in Cattle and Swine, Luxembourg, 2012\u20132016","volume":"24","author":"Snoeck","year":"2018","journal-title":"Emerg. Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1016\/j.virol.2015.08.030","article-title":"Influenza D virus infection in Mississippi beef cattle","volume":"486","author":"Ferguson","year":"2015","journal-title":"Virology"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Horimoto, T., Hiono, T., Mekata, H., Odagiri, T., Lei, Z., Kobayashi, T., Norimine, J., Inoshima, Y., Hikono, H., and Murakami, K. (2016). Nationwide distribution of bovine influenza D virus infection in Japan. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0163828"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1177\/1040638720934056","article-title":"Seroprevalence of influenza D virus in bulls in Argentina","volume":"32","author":"Alvarez","year":"2020","journal-title":"J. Vet. Diagn. Investig."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2015","DOI":"10.3201\/eid2309.170342","article-title":"Serologic Evidence for Influenza C and D Virus among Ruminants and Camelids, Africa, 1991\u20132015","volume":"23","author":"Salem","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"368","DOI":"10.3201\/eid2102.141449","article-title":"Influenza d virus in cattle, France, 2011\u20132014","volume":"21","author":"Ducatez","year":"2015","journal-title":"Emerg. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2184","DOI":"10.1111\/tbed.13273","article-title":"Detection of influenza D virus in bovine respiratory disease samples, UK","volume":"66","author":"Dane","year":"2019","journal-title":"Transbound. Emerg. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-017-12012-3","article-title":"Influenza D in Italy: Towards a better understanding of an emerging viral infection in swine","volume":"7","author":"Foni","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5636","DOI":"10.1128\/JVI.03122-15","article-title":"Pathogenesis of Influenza D virus in Cattle","volume":"90","author":"Ferguson","year":"2016","journal-title":"J. Virol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1020","DOI":"10.3201\/eid2406.172102","article-title":"Influenza D Virus Infection in Feral Swine Populations, United States","volume":"24","author":"Ferguson","year":"2018","journal-title":"Emerg. Infect. Dis."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1392","DOI":"10.3201\/eid2308.170059","article-title":"Influenza D Virus in Animal Species in Guangdong province, Southern China","volume":"23","author":"Zhai","year":"2017","journal-title":"Emerg. Infect. Dis."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Oliva, J., Eichenbaum, A., Belin, J., Gaudino, M., Guillotin, J., Alzieu, J.P., Nicollet, P., Brugidou, R., Gueneau, E., and Michel, E. (2019). Serological evidence of influenza D virus circulation among cattle and small ruminants in france. Viruses, 11.","DOI":"10.3390\/v11060516"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"281","DOI":"10.1016\/j.vetmic.2015.09.005","article-title":"Serological evidence for the presence of influenza D virus in small ruminants","volume":"180","author":"Quast","year":"2015","journal-title":"Vet. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1016\/j.jcv.2016.05.017","article-title":"Serologic evidence of exposure to influenza D virus among persons with occupational contact with cattle","volume":"81","author":"White","year":"2016","journal-title":"J. Clin. Virol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Holwerda, M., Kelly, J., Laloli, L., St\u00fcrmer, I., Portmann, J., Stalder, H., and Dijkman, R. (2019). Determining the Replication Kinetics and Cellular Tropism of Influenza D Virus on Primary Well-Differentiated Human Airway Epithelial Cells. Viruses, 11.","DOI":"10.3390\/v11040377"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1534","DOI":"10.1126\/science.1213362","article-title":"Airborne Transmission of Influenza A\/H5N1 Virus Between Ferrets","volume":"336","author":"Herfst","year":"2012","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1038\/nature16474","article-title":"Species difference in ANP32A underlies influenza A virus polymerase host restriction","volume":"529","author":"Long","year":"2016","journal-title":"Nature"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"12797","DOI":"10.1073\/pnas.1608821113","article-title":"Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism","volume":"113","author":"Moreira","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"420","DOI":"10.1038\/nature10831","article-title":"Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA\/H1N1 virus in ferrets","volume":"486","author":"Imai","year":"2012","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"a038547","DOI":"10.1101\/cshperspect.a038547","article-title":"Influenza Reverse Genetics\u2014Historical Perspective","volume":"11","author":"Neumann","year":"2020","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3389\/fimmu.2018.01581","article-title":"Influenza A virus cell entry, replication, virion assembly and movement","volume":"9","author":"Dou","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Payne, S. (2017). Family Orthomyxoviridae. Viruses: From Understanding to Investigation, Elsevier.","DOI":"10.1016\/B978-0-12-803109-4.00023-4"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"9345","DOI":"10.1073\/pnas.96.16.9345","article-title":"Generation of influenza A viruses entirely from cloned cDNAs","volume":"96","author":"Neumann","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"6108","DOI":"10.1073\/pnas.100133697","article-title":"A DNA transfection system for generation of influenza A virus from eight plasmids","volume":"97","author":"Hoffmann","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"9679","DOI":"10.1128\/JVI.73.11.9679-9682.1999","article-title":"Rescue of Influenza A Virus from Recombinant DNA","volume":"73","author":"Fodor","year":"1999","journal-title":"J. Virol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"8431","DOI":"10.1128\/JVI.79.13.8431-8439.2005","article-title":"Attenuation of Equine Influenza Viruses through Truncations of the NS1 Protein","volume":"79","author":"Quinlivan","year":"2005","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"11411","DOI":"10.1073\/pnas.172393399","article-title":"Rescue of influenza B virus from eight plasmids","volume":"99","author":"Hoffmann","year":"2002","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"239","DOI":"10.4172\/1747-0862.1000042","article-title":"A seven plasmid-based system for the rescue of influenza C virus","volume":"4","author":"Pachler","year":"2010","journal-title":"J. Mol. Genet. Med."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3284","DOI":"10.1016\/j.celrep.2019.05.048","article-title":"N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses","volume":"27","author":"Broszeit","year":"2019","journal-title":"Cell Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.virol.2016.11.004","article-title":"Serological evidence for high prevalence of Influenza D Viruses in Cattle, Nebraska, United States, 2003\u20132004","volume":"501","author":"Luo","year":"2017","journal-title":"Virology"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1036","DOI":"10.1128\/JVI.02718-14","article-title":"Cocirculation of Two Distinct Genetic and Antigenic Lineages of Proposed Influenza D Virus in Cattle","volume":"89","author":"Collin","year":"2015","journal-title":"J. Virol."},{"key":"ref_35","unstructured":"Maier, H.J., and Bickerton, E. (2020). Well-Differentiated Primary Mammalian Airway Epithelial Cell Cultures. Coronaviruses: Methods and Protocols, Springer."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Barger, C.J., Branick, C., Chee, L., and Karpf, A.R. (2019). Pan-cancer analyses reveal genomic features of FOXM1 overexpression in cancer. Cancers, 11.","DOI":"10.3390\/cancers11020251"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"676","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: An open-source platform for biological-image analysis","volume":"9","author":"Schindelin","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/JVI.01767-19","article-title":"Establishment of a Reverse Genetics System for Influenza D Virus","volume":"94","author":"Ishida","year":"2020","journal-title":"J. Virol."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Benkaroun, J., Robertson, G.J., Whitney, H., and Lang, A.S. (2018). Analysis of the variability in the non-coding regions of influenza a viruses. Vet. Sci., 5.","DOI":"10.3390\/vetsci5030076"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"11282","DOI":"10.1128\/JVI.00910-07","article-title":"Rescue of influenza C virus from recombinant DNA","volume":"81","year":"2007","journal-title":"J. Virol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1330","DOI":"10.1128\/JCM.03265-13","article-title":"Universal influenza B virus genomic amplification facilitates sequencing, diagnostics, and reverse genetics","volume":"52","author":"Zhou","year":"2014","journal-title":"J. Clin. Microbiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"10309","DOI":"10.1128\/JVI.01109-09","article-title":"Single-Reaction Genomic Amplification Accelerates Sequencing and Vaccine Production for Classical and Swine Origin Human Influenza A Viruses","volume":"83","author":"Zhou","year":"2009","journal-title":"J. Virol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"M\u00e4nz, B., Dornfeld, D., G\u00f6tz, V., Zell, R., Zimmermann, P., Haller, O., Kochs, G., and Schwemmle, M. (2013). Pandemic Influenza A Viruses Escape from Restriction by Human MxA through Adaptive Mutations in the Nucleoprotein. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003279"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1080\/22221751.2019.1575700","article-title":"Genetic diversity and host adaptation of avian H5N1 influenza viruses during human infection","volume":"8","author":"Welkers","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"17863","DOI":"10.1073\/pnas.1201797109","article-title":"Temperature dependence of protein folding kinetics in living cells","volume":"109","author":"Guo","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1128\/JVI.01186-19","article-title":"Development and Characterization of a Reverse-Genetics System for Influenza D Virus","volume":"93","author":"Yu","year":"2019","journal-title":"J. Virol."},{"key":"ref_47","first-page":"1","article-title":"Non coding extremities of the seven influenza virus type C vRNA segments: Effect on transcription and replication by the type C and type a polymerase complexes","volume":"5","author":"Barbezange","year":"2008","journal-title":"Virol. J."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"153","DOI":"10.1016\/j.virusres.2003.11.013","article-title":"Biological significance of the U residue at the -3 position of the mRNA sequences of influenza A viral segments PB1 and NA","volume":"100","author":"Maeda","year":"2004","journal-title":"Virus Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"5142","DOI":"10.1128\/jvi.63.12.5142-5152.1989","article-title":"Promoter analysis of influenza virus RNA polymerase","volume":"63","author":"Parvin","year":"1989","journal-title":"J. Virol."},{"key":"ref_50","first-page":"162","article-title":"Genome Plasticity of Influenza Viruses","volume":"2","author":"Stertz","year":"2014","journal-title":"Genome Plast. Infect. Dis."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/3\/502\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:37:45Z","timestamp":1760161065000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/3\/502"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,18]]},"references-count":50,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2021,3]]}},"alternative-id":["v13030502"],"URL":"https:\/\/doi.org\/10.3390\/v13030502","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/2021.02.10.430577","asserted-by":"object"}]},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,18]]}}}