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

ベータノダウイルスが増殖可能な上限温度

RGNNV はベータノダウイルスの中で最も高温に適応している。本研究では世界各地から収集した RGNNV 16株を30℃,32℃,35℃あるいは37℃で培養し,これらの株の増殖上限温度を調べた。その結果,増殖上限温度は株に依存して30℃未満から35℃と様々であったが,37℃で増殖する株は存在しなかった。また,ウイルス増殖とウイルス RNA 複製との間には正の相関が見られ,温度はウイルス RNA 複製かそれより早期の感染・増殖過程に影響すると考えられた。37℃での非増殖性はヒトへのベータノダウイルス感染が起こらないことを示唆する。Among the four types of betanodaviruses, redspotted grouper nervous necrosis virus (RGNNV) has the highest optimum temperature (25-30°C) for its multiplication. We tested 16 RGNNV isolates for their temperature sensitivity in cultured cells and demonstrated that their upper temperature limits ranged from less than 30°C to 35°C. At the temperatures over the upper limits, viral RNA replication was inhibited similarly. These results indicate that temperatures mainly affect RNA replication or earlier virus multiplication processes. The incompetence of betanodaviruses at 37°C suggests their avirulence in human

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

Serological relationships among genotypic variants of betanodavirus.

Betanodaviruses, the causative agents of viral nervous necrosis or viral encephalopathy and retinopathy, are divided into 4 genotypes based on the coat protein gene (RNA2). In the present study, serological relationships among betanodavirus genotypic variants were examined by virus neutralization tests using rabbit antisera raised against purified virions of strains representative of each genotype. All 20 isolates examined shared epitopes for neutralizing, but they fell into 3 major serotypes (A, B, C). This sero-grouping is in part consistent with their genotypes, i.e. Serotype A for striped jack nervous necrosis virus (SJNNV) genotype, Serotype B for tiger puffer nervous necrosis virus (TPNNV) genotype, and Serotype C for both redspotted grouper nervous necrosis virus (RGNNV) and barfin flounder nervous necrosis virus (BFNNV) genotypes. The serological relatedness between RGNNV and BFNNV genotypes may result from their relatively higher similarity in RNA2 sequences. In neutralization tests using antisera of kelp grouper Epinephelus moara, which were raised against recombinant coat proteins representing each genotype, anti-SJNNV and anti-TPNNV sera neutralized only the homologous strain, and anti-RGNNV and anti-BFNNV sera reacted with both RGNNV and BFNNV strains. The present serological findings will be important in investigating the infectivity and host-specificity of betanodaviruses and in developing vaccines for the disease

Identification of RNA regions that determine temperature sensitivities in betanodaviruses

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The larger genomic segment, RNA1 (similar to 3.1 kb), encodes an RNA-dependent RNA polymerase (protein A), and the smaller genomic segment RNA2 (similar to 1.4 kb) codes for the coat protein. These viruses can be classified into four genotypes, designated striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV), and barfin flounder nervous necrosis virus (BFNNV), based on similarities in their partial RNA2 sequences. The optimal temperatures for the growth of these viruses are 20-25A degrees C (SJNNV), 25-30A degrees C (RGNNV), 20A degrees C (TPNNV), and 15-20A degrees C (BFNNV). However, little is known about the mechanisms underlying the temperature sensitivity of these viruses. We first constructed two reassortants between SJNNV and RGNNV to test their temperature sensitivity. The levels of viral growth and RNA replication of these reassortants and parental viruses in cultured fish cells were similar at 25A degrees C. However, the levels of all of the viruses but RGNNV were markedly reduced at 30A degrees C. These results indicate that both RNA1 and RNA2 control the temperature sensitivity of betanodaviruses by modulating RNA replication or earlier viral growth processes. We then constructed ten mutated RGNNVs, the RNA1 segments of which were chimeric between SJNNV and RGNNV, and showed that only chimeric viruses bearing the RGNNV RNA1 region, encoding amino acid residues 1-445, grew similarly to the parental RGNNV at 30A degrees C. This portion of protein A is known to serve as a mitochondrial-targeting signal rather than functioning as an enzymatic domain