Establishment and Characterization of Ornamental Fish Cell Lines and Development of Methods for Detection of Koi Herpesvirus Disease

Abstract

Establishing ornamental fish cell lines is essential for researching the viral diseases that affect these ornamental fish, particularly ornamental fish species that can be artificially bred. Typically, because of high-density breeding and the flow of international trade, these fishes cause rapid viral disease spread and transmission, resulting in severe economic losses. This study reports the first successful cultivation of cell lines from the fin tissue of the marine ornamental fish species Apolemichthys trimaculatus, that is, the three-spot angelfish (TSAF). We successively cultivated 7 Carassius auratus or red carp (RC) cell lines from the fin (F), gill (G), heart (H), head kidney (HK), spleen (S), and ovum (O) tissues, yielding RCF, RCG, RCH, RCK, RCHK, RCS, and RCO cell lines. An RCHK macrophage (RCHKM) cell line was also cultivated from RC head kidney tissues. We also established 5 Cyprinus carpio (Koi) cell lines (KF, KG, KH, KB, and KO) from the fin, gill, heart, air bladder, and ovum tissues of koi. These cell lines can be continually reproduced and grown in simple cultivation conditions of 20°C-30°C and 10% FBS-L15. TSAF cell lines were highly susceptible to the eel herpesvirus in Formosa (EHVF), which ideally grows in 30°C environments, and to the infectious pancreatic necrosis virus (IPNV), endogenous viral elements (EVEs), and hippeastrum chlorotic ringspot virus (HCRV), which ideally grow in 18°C environments. Therefore, TSAF cell lines are extremely appropriate as virus detectors for cold and warm water fishes. The RCO, RCHKM, KF, KG, and KB cell lines were all susceptible to koi herpesvirus (KHV), generating the cytopathic effect commonly presented in herpesvirus. Hence, these cell lines can be used to mass-proliferate KHV. Accordingly, cell lines are the optimal tools for researching fish virology, pathology, molecular biology, and conducting environmental testing. A mutated KHV cell line, named NKV2, was identified at a koi breeding ground in Southern Taiwan. NKV2 cannot be detected using various KHV primers, carp interstitial nephritis and gill necrosis virus (CNGV) primers, thymidine kinase (TK) primers, and Gray primers, but can be detected using the carp pox cyprinid herpesvirus 1 (CyHV-1) helicase primer and CyHV-1 triplex primers, which are used to detect KHV. We designed and developed two new primers (i.e., the NKV2-2K and NKV2-1.5K primers) based on KHV primer. The proposed primers can be employed to detect both KHV and NKV2. An additional primer (i.e., the NKV2-TK primer) was designed and developed based on TK primer. This primer can be used to detect both KHV and NKV2 according to the introns of NKV2 are larger compared with those of KHV. The developed primers can be adopted in polymerase chain reaction (PCR) to detect KHV and NKV2 in the same time.