Development of a Diagnostic Method and Characterization for Koi Herpesvirus Isolated in Taiwan
Date Issued
2014
Date
2014
Author(s)
Tu, Chien
Abstract
Cyprinid herpesvirus-3 (CyHV-3), also known as koi herpesvirus (KHV), is a lethal pathogen causing mass mortality of koi and common carp, Cyprinus carpio, in Asia, Europe, North America and South Africa. The purpose of this study was to develop a cell line for the isolation and propagation of CyHV-3, and to demonstrate the characteristics of antigenicity and the phylogenetic lineage of a Taiwanese CyHV-3 isolate. A new cell line (KoG) was carried out from the gill of koi, Cyprinus carpio. KoG cells were composed of epithelial-like cells and fibroblast-like cells. The cells were maintained well at 25oC in Leibovitz-15 medium supplemented with 10% fetal calf serum. Chromosome analysis showed that the KoG cells had 43 diploid chromosomes at passage 46. The KoG cells were used to test the susceptibility of a koi herpesvirus strain isolated in Taiwan. The KHV-infected KoG cells revealed vacuolation in the cytoplasm and marginal hyperchromation in the nucleus, which was confirmed by an indirect immunofluorescence assay and transmission electron microscopy. A multistep growth curve showed the virus reaching the highest titer of 106.2 TCID50/mL at five days post inoculation. In the study of the phylogenetic relationship between the Taiwanese isolate and others, sequence analysis targeting the alleles of the two molecular markers indicated that the Taiwanese isolate displayed the I++II+ allele (the second genotype) of the Asian lineage, and was identical to a Japanese strain (Asian genotype variant 1). Collectively, our results demonstrate that KoG cells may provide a tool for the propagation of KHV and that the Taiwanese isolate belongs to a typical Asian genotype. The antigenicity of structure proteins of CyHV-3 was evaluated by the cloning, expression and characterization of the ORF72 and ORF92 proteins of a Taiwanese CyHV-3 isolate. Sequence analysis revealed that both clones of ORF72 and ORF 92 from the Taiwanese KHV strain had 100% homology with those of the Japanese KHV strain, and 99% homology with those of the United States KHV strain and the Israeli KHV strain in terms of nucleotides. Results suggest that the Taiwanese KHV isolate in is more closely related to the Japanese strain (Asian genotype). As part of an antigenicity analysis, the crude recombinant ORF 72 and ORF 92 capsid proteins reacted with the anti-CyHV-3 antibody in sera of the survival fish after a KHV outbreak, indicating that these recombinant capsid proteins might mimic surface antigens of the wild type KHV to induce an immune response in the infected host. It is shown that ORF72 and ORF92 proteins can induce the humeral immune response in carp after CyHV-3 infection. The monoclonal antibody against CyHV-3 was generated using purified whole CyHV-3 virions isolated from diseased koi in Taiwan. A clone of MAb-B2 was obtained by immunizing mice with whole virus particles and further identified using an indirect enzyme-linked immunosorbent assay (ELISA) and a Western blot assay. In addition, as demonstrated by indirect immunofluorescence assay, KHV was detected in KHV-infected cells but not in those mock-infected. In the neutralization test, results showed that MAb-B2 neutralized KHV. Interestingly, MAb-B2 recognized the ORF72 protein of KHV, as revealed by LC-MS/MS and Western blot assays. Additionally, MAb-B2 was used as a diagnostic tool for the detection of KHV in clinical samples by immunohistochemistry. Collectively, our results indicate that MAb-B2 could be used in the development of a detection kit for diagnosis of KHV infections and that the ORF72 protein of KHV may be a potential candidate for future development of associated vaccine.
Subjects
錦鯉疱疹病毒
鯉科第三型疱疹病毒
單株抗體
鯉魚
抗原性
基因型
細胞株
SDGs
Type
thesis
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