[en] Cyvirus cyprinidallo2 (CyHV-2) is the causative agent of herpesviral hematopoietic necrosis in several economically important farmed freshwater fish species of the genus Carassius. Despite several CyHV-2 strains being isolated and fully sequenced, there is a lack of detailed characterization and consistent information on strains that exhibit high virulence in adult goldfish through viral challenge by immersion, particularly in the context of European strains and host populations. Strains that can cause highly virulent disease via this inoculation route are much more compatible with experimental designs that are representative of natural infection; thus, their utilization provides greater biological relevance. Consequently, in this study, we isolated three novel strains of CyHV-2 (designated NL-1, NL-2, and NL-3), originating from outbreaks in The Netherlands. Full-length genome sequencing and phylogenetic analyses revealed that these newly isolated strains are distinct from known strains and from each other. Significant differences were observed between the strains, in terms of in vitro growth kinetics, with NL-2 exhibiting stable passaging and superior fitness in vitro. Importantly, the challenge of adult Shubunkin goldfish with the NL-2 strain via immersion (2000 PFU/mL) induced an average mortality of ~40%, while parallel experiments with the CyHV-2 reference strain ST-J1 resulted in no mortality. Taken together, this study represents the characterization of a new CyHV-2 in vivo infection model, much more compatible with experimental designs that are required to be representative of natural infection. This model will be extremely useful in many aspects of CyHV-2 research in the future. Importantly, the genetic and phenotypic characterization performed in this study generates hypotheses on the potential roles of CyHV-2 genes in adaptation of the virus in vitro or in vivo.
Disciplines :
Veterinary medicine & animal health
Author, co-author :
He, Bo ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH)
Sridhar, Arun ; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Santé publique vétérinaire
Thiry, Marc ; Université de Liège - ULiège > Département des sciences de la vie > Service collectif des enseignements de biologie en bachelier
Haenen, Olga; National Reference Laboratory for Fish Diseases, Wageningen Bioveterinary Research, Wageningen University Research, P.O. Box 65, 8200 AB Lelystad, The Netherlands
Vanderplasschen, Alain ✱; Université de Liège - ULiège > Département des maladies infectieuses et parasitaires (DMI) > Immunologie vétérinaire ; WEL Research Institute, Avenue Pasteur 6, B-1300 Wavre, Belgium
Donohoe, Owen ✱; Université de Liège - ULiège > Fundamental and Applied Research for Animals and Health (FARAH) > FARAH: Santé publique vétérinaire ; Bioscience Research Institute, Technological University of the Shannon, N37 HD68 Athlone, Co., Westmeath, Ireland
✱ These authors have contributed equally to this work.
Language :
English
Title :
Genomic and Phenotypic Characterization of a Novel Virulent Strain of Cyvirus cyprinidallo2 Originating from an Outbreak in The Netherlands
ULiège - University of Liège F.R.S.-FNRS - Fonds de la Recherche Scientifique WELBIO - Walloon Excellence in Life Sciences and Biotechnology Walloon region
Hanson L.A. Doszpoly A. van Beurden S. de Oliveira Viadanna P.H. Waltzek T. Chapter 8—Alloherpesviruses of Fish Aquaculture Virology 2nd ed. Kibenge F.S.B. Godoy M.G. Academic Press Cambridge, MA, USA 2024 165 189 978-0-323-91169-6
Boutier M. Morvan L. Delrez N. Origgi F. Doszpoly A. Vanderplasschen A. Fish and Amphibian Alloherpesviruses (Herpesviridae) Encyclopedia of Virology 4th ed. Bamford D.H. Zuckerman M. Academic Press Oxford, UK 2021 306 315 978-0-12-814516-6
Pradeepkiran J.A. Aquaculture Role in Global Food Security with Nutritional Value: A Review Transl. Anim. Sci. 2019 3 903 910 10.1093/tas/txz012 32704855
ICTV Available online: https://ictv.global/taxonomy (accessed on 5 September 2024)
Donohoe O. Zhang H. Delrez N. Gao Y. Suárez N.M. Davison A.J. Vanderplasschen A. Genomes of Anguillid Herpesvirus 1 Strains Reveal Evolutionary Disparities and Low Genetic Diversity in the Genus Cyprinivirus Microorganisms 2021 9 998 10.3390/microorganisms9050998 34063135
Streiff C. He B. Morvan L. Zhang H. Delrez N. Fourrier M. Manfroid I. Suárez N.M. Betoulle S. Davison A.J. et al. Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses Viruses 2023 15 768 10.3390/v15030768 36992477
Davison A.J. Kurobe T. Gatherer D. Cunningham C. Korf I. Fukuda H. Hedrick R.P. Waltzek T.B. Comparative Genomics of Carp Herpesviruses J. Virol. 2013 87 2908 2922 10.1128/JVI.03206-12
Padhiary S. Paul A. Tripathy D.K. Nayak D. Mohanty J. Pillai D. Rejish Kumar V.J. Krishnan R. Haridas L. Abdul Majeed S. et al. Current understanding of Cyprinid herpesvirus 2, and prospects in its management Indian J. Anim. Health 2023 62 49 60 10.36062/ijah.2023.spl.02523
Jung S.J. Miyazaki T. Herpesviral Haematopoietic Necrosis of Goldfish, Carassius auratus (L.) J. Fish Dis. 1995 18 211 220 10.1111/j.1365-2761.1995.tb00296.x
Thangaraj R.S. Nithianantham S.R. Dharmaratnam A. Kumar R. Pradhan P.K. Thangalazhy Gopakumar S. Sood N. Cyprinid Herpesvirus-2 (CyHV-2): A Comprehensive Review Rev. Aquac. 2021 13 796 821 10.1111/raq.12499
Zhang H. Sridhar A. Delrez N. He B. Fourny S. Gao Y. Donohoe O. Vanderplasschen A.F.C. Development Using Bioluminescence Imaging of a Recombinant Anguillid Herpesvirus 1 Vaccine Candidate Associated with Normal Replication In Vitro but Abortive Infection In Vivo Vaccines 2024 12 1423 10.3390/vaccines12121423
Boutier M. Ronsmans M. Ouyang P. Fournier G. Reschner A. Rakus K. Wilkie G.S. Farnir F. Bayrou C. Lieffrig F. et al. Rational Development of an Attenuated Recombinant Cyprinid Herpesvirus 3 Vaccine Using Prokaryotic Mutagenesis and In Vivo Bioluminescent Imaging PLoS Pathog. 2015 11 e1004690 10.1371/journal.ppat.1004690
Saito H. Okamura T. Shibata T. Kato G. Sano M. Development of a Live Attenuated Vaccine Candidate against Herpesviral Hematopoietic Necrosis of Goldfish Aquaculture 2022 552 737974 10.1016/j.aquaculture.2022.737974
Sun Y. Xu C. Wang H. Qiao G. Wang Z. Li Z. Li Q. Wei C. An Attenuated Strain of Cyprinid Herpesvirus 2 as a Vaccine Candidate against Herpesviral Hematopoietic Necrosis Disease in Gibel Carp, Carassius auratus gibelio Fish Shellfish Immunol. 2023 138 108826 10.1016/j.fsi.2023.108826 37201732
He B. Sridhar A. Streiff C. Deketelaere C. Zhang H. Gao Y. Hu Y. Pirotte S. Delrez N. Davison A.J. et al. In Vivo Imaging Sheds Light on the Susceptibility and Permissivity of Carassius auratus to Cyprinid Herpesvirus 2 According to Developmental Stage Viruses 2023 15 1746 10.3390/v15081746 37632088
Ito T. Kurita J. Haenen O.L.M. Importation of CyHV-2-Infected Goldfish into the Netherlands Dis. Aquat. Org. 2017 126 51 62 10.3354/dao03157 28930085
Kurobe T. Kurita J. Haenen O. Voorbergen-Laarman M. Ito T. Mass Mortality Events Associated with Cyprinid Herpesvirus 2 (CyHV-2) Infection in Wild Prussian Carp Carassius gibelio in the Netherlands, and Molecular Biology of Virus Strains J. Fish Dis. 2024 47 e13868 10.1111/jfd.13868
Waltzek T.B. Kurobe T. Goodwin A.E. Hedrick R.P. Development of a Polymerase Chain Reaction Assay to Detect Cyprinid Herpesvirus 2 in Goldfish J. Aquat. Anim. Health 2009 21 60 67 10.1577/H08-045.1
Shibata T. Nanjo A. Saito M. Yoshii K. Ito T. Nakanishi T. Sakamoto T. Sano M. In Vitro Characteristics of Cyprinid Herpesvirus 2: Effect of Kidney Extract Supplementation on Growth Dis. Aquat. Org. 2015 115 223 232 10.3354/dao02885
BBTools Available online: https://jgi.doe.gov/data-and-tools/software-tools/bbtools/ (accessed on 5 October 2022)
Antipov D. Korobeynikov A. McLean J.S. Pevzner P.A. hybridSPAdes: An Algorithm for Hybrid Assembly of Short and Long Reads Bioinformatics 2016 32 1009 1015 10.1093/bioinformatics/btv688
Mikheenko A. Prjibelski A. Saveliev V. Antipov D. Gurevich A. Versatile Genome Assembly Evaluation with QUAST-LG Bioinformatics 2018 34 i142 i150 10.1093/bioinformatics/bty266
Wick R.R. Schultz M.B. Zobel J. Holt K.E. Bandage: Interactive Visualization of de Novo Genome Assemblies Bioinformatics 2015 31 3350 3352 10.1093/bioinformatics/btv383
SnapGene LLC SnapGene | Software for Everyday Molecular Biology Available online: https://www.snapgene.com/ (accessed on 7 May 2024)
Katoh K. Rozewicki J. Yamada K.D. MAFFT Online Service: Multiple Sequence Alignment, Interactive Sequence Choice and Visualization Brief. Bioinform. 2019 20 1160 1166 10.1093/bib/bbx108
Complete Genomes: Alloherpesviridae Available online: https://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=548682 (accessed on 7 August 2024)
Viral Genomes Available online: https://www.ncbi.nlm.nih.gov/genome/viruses/ (accessed on 7 August 2024)
Kumar S. Stecher G. Li M. Knyaz C. Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms Mol. Biol. Evol. 2018 35 1547 1549 10.1093/molbev/msy096 29722887
Lindenbaum P. JVarkit: Java-Based Utilities for Bioinformatics 2015 Available online: https://github.com/lindenb/jvarkit (accessed on 1 April 2025)
MsaToVcf Available online: http://lindenb.github.io/jvarkit/MsaToVcf.html (accessed on 27 June 2024)
Kim D. Paggi J.M. Park C. Bennett C. Salzberg S.L. Graph-Based Genome Alignment and Genotyping with HISAT2 and HISAT-Genotype Nat. Biotechnol. 2019 37 907 915 10.1038/s41587-019-0201-4 31375807
Pertea M. Kim D. Pertea G.M. Leek J.T. Salzberg S.L. Transcript-Level Expression Analysis of RNA-Seq Experiments with HISAT, StringTie and Ballgown Nat. Protoc. 2016 11 1650 1667 10.1038/nprot.2016.095 27560171
Danecek P. Bonfield J.K. Liddle J. Marshall J. Ohan V. Pollard M.O. Whitwham A. Keane T. McCarthy S.A. Davies R.M. et al. Twelve Years of SAMtools and BCFtools Gigascience 2021 10 giab008 10.1093/gigascience/giab008
Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. Mesirov J.P. Integrative Genomics Viewer Nat. Biotechnol. 2011 29 24 26 10.1038/nbt.1754
McLaren W. Gil L. Hunt S.E. Riat H.S. Ritchie G.R.S. Thormann A. Flicek P. Cunningham F. The Ensembl Variant Effect Predictor Genome Biol. 2016 17 122 10.1186/s13059-016-0974-4
Geneious | Bioinformatics Software Available online: https://www.geneious.com/ (accessed on 4 July 2024)
Baldwin S. Revanna R. Thomson S. Pither-Joyce M. Wright K. Crowhurst R. Fiers M. Chen L. Macknight R. McCallum J.A. A Toolkit for Bulk PCR-Based Marker Design from next-Generation Sequence Data: Application for Development of a Framework Linkage Map in Bulb Onion (Allium cepa L.) BMC Genom. 2012 13 637 10.1186/1471-2164-13-637
McCallum J. Cfljam/Galaxy-Pcr-Markers 2022 Available online: https://github.com/cfljam/galaxy-pcr-markers (accessed on 1 April 2025)
R Core Team R: A Language and Environment for Statistical Computing R Foundation for Statistical Computing Vienna, Austria 2022
John F. Sanford W. An R Companion to Applied Regression SAGE Publications, Inc. Los Angeles, CA, USA 2018 978-1-5443-3647-3
Lenth R.V. Least-Squares Means: The R Package Lsmeans J. Stat. Softw. 2016 69 1 33 10.18637/jss.v069.i01
Li L. Luo Y. Gao Z. Huang J. Zheng X. Nie H. Zhang J. Lin L. Yuan J. Molecular Characterisation and Prevalence of a New Genotype of Cyprinid Herpesvirus 2 in Mainland China Can. J. Microbiol. 2015 61 381 387 10.1139/cjm-2014-0796
Wang F. Xu Y. Zhou Y. Ding C. Duan H. Isolation and Characterization of a Cyprinid Herpesvirus Strain YZ01 from Apparently Healthy Goldfish after Rising Water Temperature bioRxiv 2021 10.1101/2021.07.05.451087
Yang J. Xiao S. Lu L. Wang H. Jiang Y. Genomic and Molecular Characterization of a Cyprinid Herpesvirus 2 YC-01 Strain Isolated from Gibel Carp Heliyon 2024 10 e32811 10.1016/j.heliyon.2024.e32811 39035518
Jeffery K.R. Bateman K. Bayley A. Feist S.W. Hulland J. Longshaw C. Stone D. Woolford G. Way K. Isolation of a Cyprinid Herpesvirus 2 from Goldfish, Carassius auratus (L.), in the UK J. Fish Dis. 2007 30 649 656 10.1111/j.1365-2761.2007.00847.x 17958608
Luo Y.Z. Lin L. Liu Y. Wu Z.X. Gu Z.M. Li L.J. Yuan J.F. Haematopoietic Necrosis of Cultured Prussian Carp, Carassius Gibelio (Bloch), Associated with Cyprinid Herpesvirus 2 J. Fish Dis. 2013 36 1035 1039 10.1111/jfd.12110
Stephens F.J. Raidal S.R. Jones B. Haematopoietic Necrosis in a Goldfish (Carassius auratus) Associated with an Agent Morphologically Similar to Herpesvirus Aust. Vet. J. 2004 82 167 169 10.1111/j.1751-0813.2004.tb12650.x
Adamek M. Hellmann J. Jung-Schroers V. Teitge F. Steinhagen D. CyHV-2 Transmission in Traded Goldfish Stocks in Germany—A Case Study J. Fish Dis. 2018 41 401 404 10.1111/jfd.12734
Boitard P.-M. Baud M. Labrut S. de Boisséson C. Jamin M. Bigarré L. First Detection of Cyprinid Herpesvirus 2 (CyHV-2) in Goldfish (Carassius auratus) in France J. Fish Dis. 2016 39 673 680 10.1111/jfd.12400
Daněk T. Kalous L. Vesel T. Krásová E. Reschová S. Rylková K. Kulich P. L M.P. Pokorová D. Knytl M. Massive Mortality of Prussian Carp Carassius gibelio in the Upper Elbe Basin Associated with Herpesviral Hematopoietic Necrosis (CyHV-2) Dis. Aquat. Organ. 2012 102 87 95 10.3354/dao02535
Doszpoly A. Benkő M. Csaba G. Dán Á. Lang M. Harrach B. Introduction of the Family Alloherpesviridae: The First Molecular Detection of Herpesviruses of Cyprinid Fish in Hungary Magy. Allatorvosok Lapja 2011 133 174 181
Giovannini S. Bergmann S.M. Keeling C. Lany C. Schütze H. Schmidt-Posthaus H. Herpesviral Hematopoietic Necrosis in Goldfish in Switzerland: Early Lesions in Clinically Normal Goldfish (Carassius auratus) Vet. Pathol. 2016 53 847 852 10.1177/0300985815614974
Haenen O. Way K. Gorgoglione B. Ito T. Paley R. Bigarré L. Waltzek T. Novel Viral Infections Threatening Cyprinid Fish Bull.-Eur. Assoc. Fish Pathol. 2016 36 11 23
Schiphouwer M. Kessel N. Matthews J. Leuven R.S.E.W. Koppel S. Kranenbarg J. Haenen O. Lenders H. Nagelkerke L. Van der Velde G. et al. Risk Analysis of Exotic Fish Species Included in the Dutch Fisheries Act and Their Hybrids Nederlands Expertise Centrum Exoten (NEC-E) Nijmegen, The Netherlands 2014 Available online: https://edepot.wur.nl/295342 (accessed on 1 April 2025)
Ito T. Kurita J. Ozaki A. Sano M. Fukuda H. Ototake M. Growth of Cyprinid Herpesvirus 2 (CyHV-2) in Cell Culture and Experimental Infection of Goldfish Carassius auratus Dis. Aquat. Org. 2013 105 193 202 10.3354/dao02627 23999703
Xiao Z. Xue M. Xu C. Jiang N. Luo X. Li Y. Fan Y. Meng Y. Liu W. Zeng L. et al. First Report of Cyprinid Herpesvirus 2 Isolated from the Golden Crucian Carp in China Aquaculture 2022 558 738361 10.1016/j.aquaculture.2022.738361
Xu J. Zeng L. Zhang H. Zhou Y. Ma J. Fan Y. Cyprinid Herpesvirus 2 Infection Emerged in Cultured Gibel Carp, Carassius auratus Gibelio in China Vet. Microbiol. 2013 166 138 144 10.1016/j.vetmic.2013.05.025
Jing H. Gao L. Zhang M. Wang N. Lin X. Zhang L. Wu S. Establishment from the Snout and Kidney of Goldfish, Carassius auratus, of Two New Cell Lines and Their Susceptibility to Infectious Pancreatic Necrosis Virus Fish Physiol. Biochem. 2016 42 303 311 10.1007/s10695-015-0138-6
Gao Y. Sridhar A. Bernard N. He B. Zhang H. Pirotte S. Desmecht S. Vancsok C. Boutier M. Suárez N.M. et al. Virus-Induced Interference as a Means for Accelerating Fitness-Based Selection of Cyprinid Herpesvirus 3 Single Nucleotide Variants in Vitro and in Vivo Virus Evol. 2023 9 vead003 10.1093/ve/vead003
Gao Y. Suárez N.M. Wilkie G.S. Dong C. Bergmann S. Lee P.-Y.A. Davison A.J. Vanderplasschen A.F.C. Boutier M. Genomic and Biologic Comparisons of Cyprinid Herpesvirus 3 Strains Vet. Res. 2018 49 40 10.1186/s13567-018-0532-z
Ahmad I. Wilson D.W. HSV-1 Cytoplasmic Envelopment and Egress Int. J. Mol. Sci. 2020 21 5969 10.3390/ijms21175969
Jambunathan N. Clark C.M. Musarrat F. Chouljenko V.N. Rudd J. Kousoulas K.G. Two Sides to Every Story: Herpes Simplex Type-1 Viral Glycoproteins gB, gD, gH/gL, gK, and Cellular Receptors Function as Key Players in Membrane Fusion Viruses 2021 13 1849 10.3390/v13091849
Al Rumaih Z. Tuazon Kels M.J. Ng E. Pandey P. Pontejo S.M. Alejo A. Alcamí A. Chaudhri G. Karupiah G. Poxvirus-Encoded TNF Receptor Homolog Dampens Inflammation and Protects from Uncontrolled Lung Pathology during Respiratory Infection Proc. Natl. Acad. Sci. USA 2020 117 26885 26894 10.1073/pnas.2004688117
Janeway C.A. Jr. Travers P. Walport M. Shlomchik M.J. Induced Innate Responses to Infection Immunobiology: The Immune System in Health and Disease 5th ed. Garland Science New York, NY, USA 2001
Netea M.G. Kullberg B.J. Van der Meer J.W.M. Circulating Cytokines as Mediators of Fever Clin. Infect. Dis. 2000 31 S178 S184 10.1086/317513
Rahman M.M. Lucas A.R. McFadden G. Viral TNF Inhibitors as Potential Therapeutics Madame Curie Bioscience Database [Internet] Landes Bioscience Austin, TX, USA 2013
Rakus K. Ronsmans M. Forlenza M. Boutier M. Piazzon M.C. Jazowiecka-Rakus J. Gatherer D. Athanasiadis A. Farnir F. Davison A.J. et al. Conserved Fever Pathways across Vertebrates: A Herpesvirus Expressed Decoy TNF-α Receptor Delays Behavioral Fever in Fish Cell Host Microbe 2017 21 244 253 10.1016/j.chom.2017.01.010 28182952
Chai W. Qi L. Zhang Y. Hong M. Jin L. Li L. Yuan J. Evaluation of Cyprinid Herpesvirus 2 Latency and Reactivation in Carassius gibel Microorganisms 2020 8 445 10.3390/microorganisms8030445 32245260
Liang L.-G. Xie J. Chen K. Bing X. Pathogenicity and Biological Characteristics of CyHV-2 Fish Pathol. 2015 35 85 93
Ouyang P. Zhou Y. Wang K. Geng Y. Lai W. Huang X. Chen D. Guo H. Fang J. Chen Z. et al. First Report of Cyprinid Herpesvirus 2 Outbreak in Cultured Gibel Carp, Carassius auratus Gibelio at Low Temperature J. World Aquac. Soc. 2020 51 1208 1220 10.1111/jwas.12681
Wen J. Xu Y. Su M. Lu L. Wang H. Susceptibility of Goldfish to Cyprinid Herpesvirus 2 (CyHV-2) SH01 Isolated from Cultured Crucian Carp Viruses 2021 13 1761 10.3390/v13091761
Løkka G. Gamil A.A.A. Evensen Ø. Kortner T.M. Establishment of an In Vitro Model to Study Viral Infections of the Fish Intestinal Epithelium Cells 2023 12 1531 10.3390/cells12111531
