High permissivity of the fish cell line SSN-1 for piscine nodaviruses.

Seventeen isolates of piscine nodavirus from larvae or juveniles of 13 marine fish species affected with viral nervous necrosis (VNN) were examined for their infectivity to a fish cell line SSN-1. Based on cytopathic effects (CPE) and virus antigen detection by fluorescent antibody technique (FAT) after incubation at 25°C, the infectivity of these virus isolates was divided into 4 groups. Group 1, including 9 virus isolates from 4 species of grouper, 2 species of sea bass, barramundi, rock porgy, and Japanese flounder showed CPE characterized by rounded, granular cells with heavy cytoplasmic vacuoles within 3 d post-incubation (p.i.), and the monolayer partially or completely disintegrated over 3 to 6 d p.i. Scattered FAT-positive cells appeared at 3 h p.i. and spread through the cell sheet with an increasing fluorescence signal over 24 h p.i. Group 2, consisting of 3 virus isolates from striped jack, induced CPE with thin or rounded, granular, refractile cells without conspicuous vacuole formation, and extensive FAT-positive reaction was observed in a time course similar to that of Group 1. Cells inoculated with Group 3 (1 isolate from tiger puffer) developed no distinct CPE but viral infection was evidenced by localized FAT-positive cells. There were no FAT-positive cells in Group 4, which included 4 isolates from Japanese flounder, Pacific cod and Atlantic halibut. However, when incubation was performed at 20°C, the SSN-1 cells inoculated with the Group 3 isolate showed CPE similar to that of Group 1 and extensive FAT-positive reaction. Evidence of virus proliferation at 20°C was also obtained in Group 4 isolates. The virus titers in the infected fish varied from 1011 to 1016 tissue culture infectious dose (TCID50) g-1 of fish. There is a good correlation between these infectivities to the SSN-1 cells and the coat protein gene genotypes of the isolates. The present results indicate that SSN-1 cells are useful for propagating and differentiating genotypic variants of piscine nodavirus

Cloning of the fish cell line SSN-1 for piscine nodaviruses.

Six cell clones were derived from the SSN-1 cell line, which is composed of a mixed cell population and persistently infected with a C-type retrovirus (SnRV). These clones were susceptible to 4 piscine nodavirus strains belonging to different genotypes (SJNNV, RGNNV, TPNNV and BFNNV [striped jack, redspotted grouper, tiger puffer and barfin flounder nervous necrosis viruses]). Three clones, designated A-6, E-9, and E-11, were highly permissive to nodavirus infection and production. The virus-induced cytopathic effects appeared as cytoplasmic vacuoles and intensive disintegration at 3 to 5 d post-incubation. These observations were highly reproducible and formed the basis for a successful virus titration system. Quantitative analysis using the cloned E-11 cell line clearly revealed differences in the optimal growth temperatures among the 4 genotypic variants: 25 to 30°C for strain SGWak97 (RGNNV), 20 to 25°C for strain SJNag93 (SJNNV), 20°C for strain TPKag93 (TPNNV), and 15 to 20°C for strain JFIwa98 (BFNNV). Electron microscopy demonstrated SnRV retrovirus particles only in A-6 and E-9 cells, but PCR amplification for the pol gene and LTR region of the proviral DNA indicated the presence of the retrovirus in the other clones, including E 11. The cell clones obtained in the present study will be more useful for qualitative and quantitative analyses of piscine nodaviruses than the SSN-1 cell line

Detection of antibodies against striped jack nervous necrosis virus (SJNNV) from brood stocks of striped jack.

It has been indicated that parental spawners were a source of infection of a viral disease, “viral nervous necrosis", in larval striped jack Pseudocaranx dentex. In this study, the prevalence of antibodies against the causative virus (SJNNV) was examined among brood stocks of striped jack by indirect ELISA. Indirect ELISA using purified SJNNV, rabbit anti-SJNNV serum, and enzyme-conjugated goat anti-rabbit IgG antibody was employed for antibody detection from the plasma. The plasma IgM partially purified by ion-exchange chromatography was used for ELISA detection. The antibody to SJNNV was detected at high frequency (65%) in plasma samples collected from brood stocks reared at various facilities regardless of their sex or origin (wild or domestic). This indicates that the virus is prevalent among cultured populations of this fish species

シマアジ稚魚のPasteurella piscicida感染症

1991年2月に長崎県下で中間育成されていたシマアジ稚魚が大量斃死した。斃死は約1か月続き, オキシテトラサイクリンおよびオキソリン酸の径口投与により終息した(死亡率34%)。病魚には顕著な外見的症状は認められなかったが, 臓器から一種類の細菌が分離され, 生物学的・血清学的性状からPasteurella piscicida に同定された。分離菌は健康なシマアジ稚魚に強い病原性を示し, またシマアジおよびマダイに致死性を有する菌体外毒素を産生した。A mass mortality due to an infectious disease occurred in juvenile striped jack (Pseudocaranx dentex) reared at a station of the Japan Sea-Farming Association in Nagasaki Prefecture in February, 1991 and a total of about 10,000 juveniles (34%) were lost for about one month. Administrations of oxytetracycline and oxolinic acid with food were effective to decrease the mortality. One species of bacteria was purely or dominantly isolated from diseased juveniles and identified as Pasteurella piscicida based on its morphological, biochemical, genetical (G+C contents: 40.7-41.0 mol %), and serological characteristics. An experimental infection revealed that a selected isolate was pathogenic to juvenile striped jack and yound red sea bream, the LD_50 by intraperitoneal injection for the two fish species being about 10^3 and 10^7 CFU/fish, respectively. It was also demonstrated that the extracellular products (ECP) of the isolate were lethal to both fishes when it was injected intraperitoneally

中華人民共和国におけるウイルス性神経壊死症の発生

中華人民共和国の種苗生産施設で生産された孵化7-45日齢のチャイロマルハタとキジハタに異常遊泳を特徴とする大量死が発生した。病魚の脳および網膜組織には空胞形成が認められ, 神経細胞などに直径25-28nmのエンベロープを持たない球形ウイルス粒子が観察された。また, 病魚から魚類ノダウイルスの外被タンパク質抗原および遺伝子が検出されたことから, 本病はウイルス性神経壊死症(VNN)と診断された。本報告は中華人民共和国における VNN の初記載である。The viral etiology of mass mortalities of groupers, Epinephelus coioides and E. akaara, cultured in the People's Republic of China was examined. Disease outbreaks occurred in 7 to 45 day-old fish with erratic swimming motion and marked vacuolation was observed in the brain and retina of the affected fish. The piscine nodavirus (the Betanodavirus), the causative agent of viral nervous necrosis (VNN), was detected in the affected tissues by electron microscopy, indirect fluorescent antibody test and reverse transcription-polymerase chain reaction. This paper is the first record of the agent in the People's Republic of China