{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T14:55:45Z","timestamp":1788188145541,"version":"build-2803163510"},"reference-count":34,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2025,2,15]],"date-time":"2025-02-15T00:00:00Z","timestamp":1739577600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Research Council of Norway","award":["#280847\/E40"],"award-info":[{"award-number":["#280847\/E40"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Background: Pancreas disease (PD) is a serious disease in European salmonid aquaculture caused by salmonid alphavirus (SAV), of which six genotypes (SAV1\u20136) have been described. The use of inactivated virus and DNA PD vaccines is common in marine salmonid aquaculture and has contributed to a reduction of the occurrence of disease; however, outbreaks are still frequent. Methods: In this study, we compared the long-term protection after immunization of Atlantic salmon (Salmo salar) with three different clones of attenuated infectious SAV3. The clones were made by site-directed mutagenesis targeting the glycoprotein E2 to disrupt the viral attachment and\/or nuclear localization signal (NLS) of the capsid protein to disrupt the viral suppression of cellular nuclear-cytosol trafficking. The resulting clones (Clones 1\u20133) were evaluated after injection of Atlantic salmon for infection dynamics, genetic stability, transmission, and protection against a subsequent SAV3 challenge. Results: Attenuated clones demonstrated reduced virulence, as indicated by lower viral RNA loads, diminished transmission to cohabitant fish, and minimal clinical symptoms compared to the virulent wild-type virus. The clones mutated in both capsid and E2 exhibited the most attenuation, observed as rapid clearing of the infection and showing little transmission, while the clone with glycoprotein E2 mutations displayed greater residual virulence but provided stronger protection, seen as reduced viral loads upon subsequent challenge with SAV3. Despite their attenuation, all viral clones caused significant reductions in weight gain. Conclusions: Despite promising attenuation and protection, this study highlights the trade-offs between virulence and immunogenicity in live vaccine design. Concerns over environmental risks, such as the shedding of genetically modified virus, necessitate further evaluation. Future efforts should optimize vaccine candidates to balance attenuation, immunogenicity, and minimal side effects.<\/jats:p>","DOI":"10.3390\/vaccines13020190","type":"journal-article","created":{"date-parts":[[2025,2,17]],"date-time":"2025-02-17T04:40:25Z","timestamp":1739767225000},"page":"190","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Long-Term Protection in Atlantic Salmon (Salmo salar) to Pancreas Disease (PD) Can Be Achieved Through Immunization with Genetically Modified, Live Attenuated Salmonid Alphavirus 3"],"prefix":"10.3390","volume":"13","author":[{"given":"Stine","family":"Braaen","sequence":"first","affiliation":[{"name":"Unit of Virology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 1433 \u00c5s, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0811-7305","authenticated-orcid":false,"given":"\u00d8ystein","family":"Wessel","sequence":"additional","affiliation":[{"name":"Unit of Virology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 1433 \u00c5s, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4683-0088","authenticated-orcid":false,"given":"H\u00e5vard","family":"Bj\u00f8rgen","sequence":"additional","affiliation":[{"name":"Unit of Anatomy, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 1433 \u00c5s, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9911-5948","authenticated-orcid":false,"given":"Espen","family":"Rimstad","sequence":"additional","affiliation":[{"name":"Unit of Virology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, 1433 \u00c5s, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1128\/JVI.74.1.173-183.2000","article-title":"Rainbow trout sleeping disease virus is an atypical alphavirus","volume":"74","author":"Villoing","year":"2000","journal-title":"J. Virol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"811","DOI":"10.1111\/j.1365-2761.2008.00944.x","article-title":"Phylogenetic analyses and molecular epidemiology of european salmonid alphaviruses (sav) based on partial e2 and nsp3 gene nucleotide sequences","volume":"31","author":"Fringuelli","year":"2008","journal-title":"J. Fish Dis."},{"key":"ref_3","unstructured":"Sommerset, I., Wiik-Nielsen, J., Moldal, T., Oliveira, V.H.S., Svendsen, J.C., Haukaas, A., and Brun, E. (2024). Fish Health Report, 2023, Norwegian Veterinary Institute."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1047","DOI":"10.1111\/jfd.12312","article-title":"Mortality and weight loss of atlantic salmon, salmon salar l., experimentally infected with salmonid alphavirus subtype 2 and subtype 3 isolates from norway","volume":"38","author":"Taksdal","year":"2015","journal-title":"J. Fish Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"511","DOI":"10.1111\/j.1365-2761.2007.00848.x","article-title":"Alphavirus infections in salmonids\u2014A review","volume":"30","author":"McLoughlin","year":"2007","journal-title":"J. Fish Dis."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.fsi.2020.12.002","article-title":"Effect of a novel DNA vaccine against pancreas disease caused by salmonid alphavirus subtype 3 in atlantic salmon (salmo salar)","volume":"108","author":"Thorarinsson","year":"2020","journal-title":"Fish Shellfish Immunol."},{"key":"ref_7","unstructured":"Sommerset, I., Wiik-Nielsen, J., De Oliveira, V.H.S., Moldal, T., Born\u00f8, B., Haukaas, A., and Brun, E.R. (2023). Fish Health Report 2022, Norwegian Veterinary Institute."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"e0214921","DOI":"10.1128\/jvi.02149-21","article-title":"Mutations at the alphavirus e1\u2019-e2 interdimer interface have host-specific phenotypes","volume":"96","author":"Ren","year":"2022","journal-title":"J. Virol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6206","DOI":"10.1016\/j.vaccine.2014.09.026","article-title":"Salmonid alphavirus glycoprotein e2 requires low temperature and e1 for virion formation and induction of protective immunity","volume":"32","author":"Hikke","year":"2014","journal-title":"Vaccine"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1094","DOI":"10.1099\/jgv.0.000412","article-title":"Role of envelope n-linked glycosylation in ross river virus virulence and transmission","volume":"97","author":"Nelson","year":"2016","journal-title":"J. Gen. Virol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Aksnes, I., Markussen, T., Braaen, S., and Rimstad, E. (2020). Mutation of n-glycosylation sites in salmonid alphavirus (sav) envelope proteins attenuate the virus in cell culture. Viruses, 12.","DOI":"10.3390\/v12101071"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"421","DOI":"10.1016\/j.fsi.2019.01.043","article-title":"Antiviral defense in salmonids\u2014Mission made possible?","volume":"87","author":"Dahle","year":"2019","journal-title":"Fish Shellfish Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Elmasri, Z., Nasal, B.L., and Jose, J. (2021). Alphavirus-induced membrane rearrangements during replication, assembly, and budding. Pathogens, 10.","DOI":"10.3390\/pathogens10080984"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"13552","DOI":"10.1128\/JVI.01576-07","article-title":"Analysis of venezuelan equine encephalitis virus capsid protein function in the inhibition of cellular transcription","volume":"81","author":"Garmashova","year":"2007","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Lundberg, L., Carey, B., and Kehn-Hall, K. (2017). Venezuelan equine encephalitis virus capsid-the clever caper. Viruses, 9.","DOI":"10.3389\/fmicb.2018.03126"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1128\/JVI.02252-14","article-title":"Venezuelan equine encephalitis virus variants lacking transcription inhibitory functions demonstrate highly attenuated phenotype","volume":"89","author":"Atasheva","year":"2015","journal-title":"J. Virol."},{"key":"ref_17","first-page":"1","article-title":"Successful vaccines","volume":"428","author":"Amanna","year":"2020","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Abeyratne, E., Freitas, J.R., Zaid, A., Mahalingam, S., and Taylor, A. (2018). Attenuation and stability of chikv-nols, a live-attenuated chikungunya virus vaccine candidate. Vaccines, 7.","DOI":"10.3390\/vaccines7010002"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"109","DOI":"10.3354\/dao018109","article-title":"Acquired protection in atlantic salmon salmo salar parr and post-smolts against pancreas disease","volume":"18","author":"Houghton","year":"1994","journal-title":"Dis. Aquat. Org."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1111\/jfd.13352","article-title":"Genetically modified attenuated salmonid alphavirus: A potential strategy for immunization of atlantic salmon","volume":"44","author":"Aksnes","year":"2021","journal-title":"J. Fish Dis."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"6027","DOI":"10.1128\/JVI.03501-12","article-title":"A fully attenuated recombinant salmonid alphavirus becomes pathogenic through a single amino acid change in the e2 glycoprotein","volume":"87","author":"Merour","year":"2013","journal-title":"J. Virol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1016\/j.jviromet.2005.08.012","article-title":"Sensitive and specific detection of salmonid alphavirus using real-time pcr (taqman)","volume":"131","author":"Hodneland","year":"2006","journal-title":"J. Virol. Methods"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"125","DOI":"10.3354\/dao072125","article-title":"Virological, serological and histopathological evaluation of fish strain susceptibility to experimental infection with salmonid alphavirus","volume":"72","author":"McLoughlin","year":"2006","journal-title":"Dis. Aquat. Organ."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"599","DOI":"10.1139\/facets-2018-0008","article-title":"The same strain of piscine orthoreovirus (prv-1) is involved in the development of different, but related, diseases in atlantic and pacific salmon in british columbia","volume":"3","author":"Ferguson","year":"2018","journal-title":"Facets"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1038\/nbt.1635","article-title":"Rationalizing the development of live attenuated virus vaccines","volume":"28","author":"Lauring","year":"2010","journal-title":"Nature biotechnology"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Sharma, A., and Knollmann-Ritschel, B. (2019). Current understanding of the molecular basis of venezuelan equine encephalitis virus pathogenesis and vaccine development. Viruses, 11.","DOI":"10.3390\/v11020164"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"100063","DOI":"10.1016\/j.fsirep.2022.100063","article-title":"Effects of a DNA and multivalent oil-adjuvanted vaccines against pancreas disease in atlantic salmon (salmo salar) challenged with salmonid alphavirus subtype 3","volume":"3","author":"Thorarinsson","year":"2022","journal-title":"Fish Shellfish Immunol. Rep."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1016\/j.chom.2020.04.003","article-title":"Engineering the live-attenuated polio vaccine to prevent reversion to virulence","volume":"27","author":"Yeh","year":"2020","journal-title":"Cell Host Microbe"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1","DOI":"10.3354\/dao02719","article-title":"Detection of salmonid alphavirus rna in celtic and irish sea flatfish","volume":"109","author":"McCleary","year":"2014","journal-title":"Dis. Aquat. Organ."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"S6","DOI":"10.1038\/nmeth.1557","article-title":"Development and applications of single-cell transcriptome analysis","volume":"8","author":"Tang","year":"2011","journal-title":"Nat. Methods"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Powers, A., and Roehrig, J.T. (2011). Alphaviruses. Diagnostic Virology Protocols, Humana Press.","DOI":"10.1007\/978-1-60761-817-1_2"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1111\/j.1365-2761.2009.01038.x","article-title":"Salmonid alphavirus associated with lepeophtheirus salmonis (copepoda: Caligidae) from atlantic salmon, Salmo salar L.","volume":"32","author":"Petterson","year":"2009","journal-title":"J. Fish Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1111\/j.1365-2761.2009.01096.x","article-title":"Prospective longitudinal studies of salmonid alphavirus infections on two atlantic salmon farms in ireland; evidence for viral persistence","volume":"33","author":"Graham","year":"2010","journal-title":"J. Fish Dis."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"6429","DOI":"10.1128\/jvi.66.11.6429-6435.1992","article-title":"Persistence of viral rna in mouse brains after recovery from acute alphavirus encephalitis","volume":"66","author":"Levine","year":"1992","journal-title":"J. Virol."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/2\/190\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:34:58Z","timestamp":1760027698000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/13\/2\/190"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,15]]},"references-count":34,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2025,2]]}},"alternative-id":["vaccines13020190"],"URL":"https:\/\/doi.org\/10.3390\/vaccines13020190","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,15]]}}}