Development of a Diagnostic Method and Characterization for Koi Herpesvirus Isolated in Taiwan

鯉科第三型疱疹病毒（CyHV-3），又名錦鯉疱疹病毒，是一種造成亞洲、歐洲、北美、南非地區錦鯉及鯉魚（Cyprinus carpio）大量死亡的致命病原。本研究的目的為開發一株能供病毒分離及增殖的細胞株，及探討台灣鯉科第三型疱疹病毒的抗原及分子親緣特性。 由錦鯉的鰓開發出一株新細胞株，命名為KoG。本細胞由上皮樣及纖維母樣細胞組成；可培養在25oC，添加10%胎牛血清的L-15培養液中。染色體分析顯示，本細胞在第46代時具有43個二倍體染色體。以台灣分離的錦鯉疱疹病毒對本細胞進行病毒感受性試驗；透過間接免疫螢光染色及穿透式電子顯微鏡檢視，發現病毒感染的細胞在細胞質內出現空泡化，細胞核內出現染色質著邊現象。病毒多步驟生長曲線發現在接種後第五天可產生最高力價病毒（106.2 TCID50/mL）。在研究台灣與其他分離株親緣關係時，針對兩個分子標誌的對偶基因序列分析發現台灣株具有I++II+對偶基因（第二基因型），屬於亞洲族群；與日本分離株相同，均屬於亞洲基因第一亞型（Asian genotype variant 1）。綜合以上，結果顯示KoG細胞可以作為錦鯉疱疹病毒的增殖工具，而台灣株屬於典型的亞洲基因型。 針對台灣錦鯉疱疹病毒ORF72及ORF92基因，運用分子選殖、基因表現技術進行鯉科第三型疱疹病毒結構蛋白抗原性分析。進一步分析分子選殖的ORF72及ORF92核酸序列，結果顯示台灣株具有與日本株完全相同的序列，與美國及以色列分離株則具有99%的相同性。上述結果暗示台灣株與日本株（亞洲基因型）親緣關係非常相近。在抗原性分析方面，發現未純化的ORF72及ORF92重組核衣蛋白可以與感染錦鯉疱疹病毒發病後殘存魚隻體內血清的抗體結合，表示這些重組核衣蛋白可能足以模擬野生型錦鯉疱疹病毒的表面抗原，去誘發感染宿主體內的免疫反應。這些結果顯示ORF72及ORF92蛋白能夠在鯉魚感染錦鯉疱疹病毒後誘發體液免疫反應。 再以由病魚分離之台灣錦鯉疱疹病毒株製備單株抗體。以完整病毒顆粒免疫小鼠，再透過間接酵素連結法及西方轉漬法確認，篩出一株MAb-B2單株抗體。透過間接螢光抗體染色法，本抗體可以在感染錦鯉疱疹病毒的細胞內偵測到病毒，但是不會對未感染細胞反應。抗體中和試驗顯示MAb-B2單株抗體能夠中和病毒。此外，運用液相層析串聯質譜儀及西方轉製法確認，發現MAb-B2單株抗體辨識的病毒蛋白為錦鯉疱疹病毒ORF72蛋白。另外利用免疫組織化學方法，MAb-B2單株抗體可以在臨床樣品中檢測出錦鯉疱疹病毒。整體而言，上述結果表示MAb-B單株抗體可以用來開發診斷錦鯉疱疹病毒的套組；另外ORF72蛋白或許可成為未來疫苗開發的潛力目標。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.中文摘要---------------------------------------------------I ABSTRACT---------------------------------------------------III TABLE OF CONTENTS------------------------------------------VI LIST OF TABLES---------------------------------------------IX LIST OF FIGURES---------------------------------------------X CHAPTER I. INTRODUCTION------------------------------------1 CHAPTER II. LITERATURE REVIEW------------------------------5 2.1 History and Geographic Distribution--------------------5 2.2 Characteristics and Taxonomy---------------------------6 2.2.1 Genome-----------------------------------------------6 2.2.2 Proteome---------------------------------------------7 2.2.3 Taxonomy---------------------------------------------9 2.2.4 Genotypes-------------------------------------------10 2.3 Susceptible Hosts---------------------------------------------------------- 11 2.4 Carriers---------------------------------------------------------------------- 11 2.5 Temperature Restriction--------------------------------------------------- 12 2.6 Clinical Signs--------------------------------------------------------------- 13 2.7 Histopathology------------------------------------------------------------- 13 2.8 Transmission---------------------------------------------------------------- 13 2.9 Pathogenesis---------------------------------------------------------------- 14 2.9.1 Portal of entry----------------------------------------------- 14 2.9.2 Latency------------------------------------------------------- 15 2.10 Diagnosis------------------------------------------------------------------- 16 2.11 Vaccination---------------------------------------------------------------- 18 2.12 Immune responses after CyHV-3 infection---------------------------- 19 CHAPTER III. DEVELOPMENT OF A CELL LINE FROM THE GILL OF KOI AND MOLECULAR CHARACTERIZATION OF KHV ISOLATED IN TAIWAN----------------------------------------------------22 Introduction-------------------------------------------------------------------------- 22 Materials and Methods -------------------------------------------------------- 24 Primary culture and passages------------------------------------------------- 24 Cell growth characterization ------------------------------------------------- 25 Chromosome analysis-------------------------------------------------------- 26 Production of antiserum against KHV envelope protein---------------- 27 Susceptibility of the KoG cell line to koi herpesvirus and indirect immunofluorescence assay-------------------------------------------------- 28 Electron microscopy---------------------------------------------------------- 29 KHV replication in KoG cells----------------------------------------------- 30 Phylogenetic analysis--------------------------------------------------------- 30 Results-------------------------------------------------------------------------------- 32 Establishment of KoG cell line---------------------------------------------- 32 Cell growth characterization------------------------------------------------- 33 Susceptibility of KoG cells to KHV---------------------------------------- 33 Replication of KHV in KoG cells------------------------------------------- 34 Phylogenetic analysis--------------------------------------------------------- 35 Discussion---------------------------------------------------------------------------- 36 CHAPTER IV. CLONING, EXPRESSION AND PRELIMINARY ANTIGENICITY ANALYSIS OF STRUCTURAL PROTEINS OF A KOI HERPESVIRUS ISOLATE FROM KOI, CPRINUS CARPIO IN TAIWAN---- 48 Introduction--------------------------------------------------------------------------- 48 Materials and Methods-------------------------------------------------------------- 49 Genomic DNA preparation-------------------------------------------------- 49 Cloning and Expression------------------------------------------------------ 50 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of crude recombinant proteins produced by E. Coli-------------------------- 52 Antigenicity analysis of recombinant proteins by Western blot--------- 53 Homology analysis of cloned ORF72 and ORF92 gene------------------ 54 Results--------------------------------------------------------------------------------- 54 Constructions and homology analysis of bacterial expression clones-- 54 Small scale expression------------------------------------------------------- 55 Antigenicity analysis of recombinant proteins by Western blot--------- 56 Discussion---------------------------------------------------------------------------- 56 CHAPTER V. PRODUCTION OF MONOCLONAL ANTIBODY AGAINST ORF72 OF KOI HERPESVIRUS ISOLATED IN TAIWAN----------------------- 66 Introduction--------------------------------------------------------------------------- 66 Materials and Methods-------------------------------------------------------------- 67 Virus preparation for immunization---------------------------------------- 67 Electron microscope examination------------------------------------------ 67 Immunization of mice and screening for MAb against KHV---------- 68 Western blot assay ----------------------------------------------------------- 69 Indirect immunofluorescence assay---------------------------------------- 70 Identification of MAb-reacted protein by 1D gel/nanoLC-MS/MS --- 71 Neutralizing test with MAb-B2--------------------------------------------- 73 Immunohistochemistry (IHC)----------------------------------------------- 73 Isotype determination-------------------------------------------------------- 74 Results--------------------------------------------------------------------------------- 75 Discussion---------------------------------------------------------------------------- 77 CHAPTER VI. GENERAL DISCUSSION------------------------------------------- 87 BIBLIOGRAPHY------------------------------------------------------------------------- 93 ORIGINAL PUBLICATIONS---------------------------------------------------------- 10