title: Expression of immunogenic structural proteins of cyprinid herpesvirus 3 in vitro assessed using immunofluorescence.
abstract: Cyprinid herpesvirus 3 (CyHV-3), also called koi herpesvirus (KHV), is the aetiological agent of a fatal disease in carp and koi (Cyprinus carpio L.), referred to as koi herpesvirus disease. The virus contains at least 40 structural proteins, of which few have been characterised with respect to their immunogenicity. Indirect immunofluorescence assays (IFAs) using two epitope-specific monoclonal antibodies (MAbs) were used to examine the expression kinetics of two potentially immunogenic and diagnostically relevant viral antigens, an envelope glycoprotein and a capsid-associated protein. The rate of expression of these antigens was determined following a time-course of infection in two CyHV-3 susceptible cell lines. The results were quantified using an IFA, performed in microtitre plates, and image analysis was used to analyse confocal micrographs, enabling measurement of differential virus-associated fluorescence and nucleus-associated fluorescence from stacks of captured scans. An 8-tenfold increase in capsid-associated protein expression was observed during the first 5 days post-infection compared to a ≤ 2-fold increase in glycoprotein expression. A dominant protein of ~100 kDa reacted with the capsid-associated MAb (20F10) in western blot analysis. This band was also recognised by sera obtained from carp infected with CyHV-3, indicating that this capsid-associated protein is produced in abundance during infection in vitro and is immunogenic to carp. Mass spectrometry carried out on this protein identified it as a previously uncharacterised product of open reading frame 84. This abundantly expressed and immunogenic capsid-associated antigen may be a useful candidate for KHV serological diagnostics.
authors.affiliation: Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. s.j.monaghan@stir.ac.uk.
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Monaghan
authors.affiliation: Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. Kim.Thompson@moredun.ac.uk.|Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ, UK. Kim.Thompson@moredun.ac.uk.
authors.forename: Kim D
authors.initials: KD
authors.lastname: Thompson
authors.affiliation: Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. j.e.bron@stir.ac.uk.
authors.forename: James E
authors.initials: JE
authors.lastname: Bron
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Infectology, Greifswald, Insel-Riems, Germany. Sven.Bergmann@fli.bund.de.
authors.forename: Sven M
authors.initials: SM
authors.lastname: Bergmann
authors.affiliation: Laboratory of Aquatic Animal Diseases, Institute of Animal Science, College of Veterinary Medicine, Gyeongsang National University, Jinju, Gyeongnam, South Korea. jungts@gnu.ac.kr.
authors.forename: Tae S
authors.initials: TS
authors.lastname: Jung
authors.affiliation: Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 513, Wasedatsurumaki-cho, Shinjuku-ku, Tokyo, 162-0041, Japan. aokitaka@aoni.waseda.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Aoki
authors.affiliation: Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. k.f.muir@stir.ac.uk.
authors.forename: K Fiona
authors.initials: KF
authors.lastname: Muir
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Infectology, Greifswald, Insel-Riems, Germany. malte.dauber@bfav.rie.de.
authors.forename: Malte
authors.initials: M
authors.lastname: Dauber
authors.affiliation: Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Infectology, Greifswald, Insel-Riems, Germany. sven.reiche@fli.bund.de.
authors.forename: Sven
authors.initials: S
authors.lastname: Reiche
authors.affiliation: Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. diana_chee@ava.gov.sg.|Aquatic Animal Health Section, Animal Health Laboratory Department, Laboratories Group, Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore. diana_chee@ava.gov.sg.
authors.forename: Diana
authors.initials: D
authors.lastname: Chee
authors.affiliation: Aquatic Animal Health Section, Animal Health Laboratory Department, Laboratories Group, Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore. chong_shin_min@ava.gov.sg.
authors.forename: Shin M
authors.initials: SM
authors.lastname: Chong
authors.affiliation: Virology Section, Animal Health Laboratory Department, Laboratories Group, Agri-Food and Veterinary Authority of Singapore, Singapore, Singapore. chen_jing@ava.gov.sg.
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.affiliation: Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling, FK9 4LA, UK. alexandra.adams@stir.ac.uk.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Adams
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D002347:Carps; D002460:Cell Line; D019084:Fluorescent Antibody Technique, Indirect / Q000662:veterinary*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006564:Herpesviridae / Q000145:classification*; D018613:Microscopy, Confocal; D013997:Time Factors; D015678:Viral Structural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000956:Antigens, Viral; D006023:Glycoproteins; D015678:Viral Structural Proteins