Comparative Immunology, Microbiology and Infectious Diseases
Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus
Introduction
Koi herpesvirus [KHV or cyprinid herpesvirus 3 (CyHV-3)] infections in carp are a serious problem worldwide [1], [2]. KHV was identified in 1998 as the causative agent of mass mortality among juvenile and adult koi carp, and among common carp cultured in Israel, USA, and Germany [1], [3], [4]. The virus has now been spread to most continents, including countries throughout Europe, USA, Japan, Indonesia, South Africa, Thailand, Taiwan, Malaysia, China and Korea [5], [6], [7]. To date, three types of herpesviruses have been reported in cyprinid fish, including pox herpesvirus (CHV or CyHV-1) [8], haematopoietic necrosis herpesvirus of goldfish (GFHNV or CyHV-2) [9], and KHV [10]. These viruses have common antigens [11], and so identifying specific antigens from KHV may help to control the disease.
KHV has been diagnosed with a polymerase chain reaction (PCR) [12], [13], [14], TaqMan PCR [15], and loop-mediated isothermal amplification (LAMP) [16], [17], [18]. However, these methods do not work well in the case of persistent or latent infections. PCR methods to detect KHV were unable to detect the virus in the tissues of fish that survived experimental or natural KHV infections [12], [13]. Furthermore, these methods are not suitable for field works since they require laboratory equipment such as a thermal cycler and heat block. Thus, a method or tool, which are capable of definitively detecting the virus from the infected tissues, e.g. immuno-chromatography kit with a specific monoclonal antibody in field, need to be required. What is needed is a monoclonal antibody that is highly specific for KHV.
We previously sequenced the genomes of three cyprinid herpesvirus strains from Japan, USA and Israel [19]. The sizes of the genomes were 295 kbp and each contained a 22-kbp terminal direct repeat. The viruses appeared to have arisen from a wild-type parent encoding 156 unique protein-coding genes, 8 of which are duplicated in the terminal repeat. The KHV genes were closely related to CHV and CyHV-2, and these three cyprinid viruses were related, albeit more distantly, to channel catfish virus (Ictalurid herpesvirus 1, IcHV-1) [10].
Major specific antigens can be identified by immunoscreening a viral genomic library using antisera that do not cross react with allied viral species [20]. Here, we used immunoscreening to identify two KHV-specific antigens from a KHV genomic phage library using a KHV-specific rabbit polyclonal antibody. The antigenic regions corresponded to open reading frames (ORF) 62 and ORF68. We then generated monoclonal antibodies (mAbs) against the recombinant ORF62 and ORF68 proteins. The specificity of one of the mAbs for KHV was confirmed by indirect fluorescent antibody (IFA) test and immunohistochemical (IHC) examination.
Section snippets
Extraction of KHV genomic DNA
KHV strain TUMSAT 1 was inoculated to the koi fin (KF-1) cell line in a 75 cm2 flask. The cells had been incubated at 23 °C for 2 weeks until the cytopathic effect (CPE) was observed in 90% of the cells. The infected cells were stored at −80 °C for 24 h, thawed at room temperature, and then homogenized in 10 ml of minimum essential medium (MEM). Cellular debris in the homogenate was removed by 3500 × g centrifugation at 10 °C for 20 min. The supernatant was centrifuged at 95,300 × g for 90 min at 10 °C in a
Antigenic protein encoding region
A phage display library including approximately 7.0 × 108 phage clones (approximately 12,000 times coverage of the KHV genome) was constructed. Seventeen positive clones were obtained as a result of immunoscreening with anti-KHV rabbit serum (polyclonal antibody) from approximately 85,000 phage clones (approximately 70 times coverage of KHV genome size). The 17 clones encoded two partial regions of antigenic proteins in KHV genome. Fifteen of the 17 clones were located at 114,797–116,503 bp (1707
Acknowledgements
We thank Dr. Hideo Fukuda (Fish Pathology Laboratory, Tokyo University of Marine Science and Technology) for providing anti-KHV rabbit serum, Dr. Satoshi Miwa (National Research Institute of Aquaculture, Fisheries Research Agency) for CHV, Dr. Ronald P. Hedrick (School of Veterinary Medicine, University of California, Davis) for CyHV-2, and Dr. Olga L.M. Haenen (Institute for Animal Disease Control, Netherlands) for the CCB cell lines. This work was supported by a grant from the World Class
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2019, Microbial PathogenesisCitation Excerpt :In herpesviruses, protease precursors undergo auto-processing and cleavage in order to achieve cleavage of subsequent polyproteins during capsid maturation and shell assembly [22], which can also lead to multiple bands being recognized by individual MAbs [23]. Previous characterization of a MAb detecting the product of CyHV-3 ORF68 resulted in 3 bands, which were thought to be the result of ORF68 encoding a polyprotein and thus the three bands detected were either a result of cleavage by proteases, or alternative splicing or glycosylation of the protein after cleavage [24]. Both MAb IB7IB4 and 7C72F7 showed similar multiple banding by western blot.
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2018, Chemical Engineering JournalCitation Excerpt :Direct methods detect the actual virus, or “pieces” of the virus. By contrast, indirect methods determine whether a fish has mounted an immune response against KHV following exposure to the virus by measuring the KHV antibody levels in the blood [6–8]. Direct methods for KHV identification include: 1) virus isolation and identification (i.e., growing the virus) using a susceptible cell line such as the Koi Fin (KF-1) cell line, and 2) polymerase chain reaction (PCR) techniques (i.e., testing for the presence of KHV DNA).