Misao Arimoto

Vaccination trials with Penaeus japonicus to induce resistance to white spot syndrome virus

Crustaceans do not possess an adaptive immune response with immunoglobulins; however, recently, “quasi-immune response” has been reported by which kuruma shrimp (Penaeus japonicus) surviving from natural or experimental white spot syndrome virus (WSSV) infections possess a resistance against challenge with WSSV. In this study, efficacy of vaccines made of inactivated WSSV with or without immunostimulants (β-1,3-glucan or killed Vibrio penaeicida) and of recombinant proteins of WSSV (rVP26, rVP28) were tested by intramuscular vaccination followed by intramuscular challenge of kuruma shrimp with WSSV. The shrimp vaccinated with formalin-inactivated WSSV showed a resistance to the virus on 10th day post-vaccination (dpv) but not on 30th dpv. Heat-inactivated WSSV did not induce a resistance in the shrimp even on 10th dpv. Additional injections with glucan or V. penaeicida enhanced the efficacy of formalin-inactivated WSSV vaccine; however, the relative percent survival (RPS) values did not exceed 60% even when shrimp were vaccinated three times. On the other hand, two injections with rVP26 or rVP28 induced a higher resistance, with RPS values 60% and 95%, respectively, in the shrimp on 30th dpv. These results indicate the possibility of vaccination of kuruma shrimp with recombinant proteins against WSSV.

Effect of chemical and physical treatments on the inactivation of striped jack nervous necrosis virus (SJNNV)

Abstract The effects of some chemical disinfectants, organic solvents, hydrogen ions, heat, ultraviolet (UV) irradiation and ozone on the inactivation of striped jack nervous necrosis virus (SJNNV) were investigated. SJNNV was inactivated by contact with a final concentration of 50 ppm of sodium hypochlorite, calcium hypochlorite, benzalkonium chloride and iodine for 10 min at 20 °C. Cresol concentrations of more than 10 000 ppm were required to inactivate SJNNV, and no inactivation of SJNNV by formalin was detected at any concentration tested. The effective concentrations of ethanol and methanol were 60% and 50%, respectively, but SJNNV was resistant to ether and chloroform. SJNNV was inactivated by high alkalinity, pH 12 for 10 min at 20 °C, and also inactivated by heat treatment at 60 °C for 30 min. Inactivation of SJNNV by UV irradiation was observed at an intensity of 410 μW cm −2 for 244 s. Ozone at 0.1 μg ml −1 as a total residual oxidant was required to inactivate SJNNV for 2.5 min. Washing fertilized eggs and the treatment of sea water with ozone decreased the rate of occurrence of VNN.

Identification of Host-Specificity Determinants in Betanodaviruses by Using Reassortants between Striped Jack Nervous Necrosis Virus and Sevenband Grouper Nervous Necrosis Virus

ABSTRACT Betanodaviruses, the causal agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNAs as genomes. The larger genomic segment, RNA1 (3.1 kb), encodes an RNA-dependent RNA polymerase, and the smaller genomic segment, RNA2 (1.4 kb), codes for the coat protein. Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among betanodaviruses. However, no mechanism underlying the host specificity has yet been reported. To evaluate viral factors that control host specificity, we first constructed a cDNA-mediated infectious RNA transcription system for sevenband grouper nervous necrosis virus (SGNNV) in addition to that for striped jack nervous necrosis virus (SJNNV), which was previously established by us. We then tested two reassortants between SJNNV and SGNNV for infectivity in the host fish from which they originated. When striped jack and sevenband grouper larvae were bath challenged with the reassortant virus comprising SJNNV RNA1 and SGNNV RNA2, sevenband groupers were killed exclusively, similar to inoculation with SGNNV. Conversely, inoculations with the reassortant virus comprising SGNNV RNA1 and SJNNV RNA2 killed striped jacks but did not affect sevenband groupers. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that both of the reassortants multiplied in the brains, spinal cords, and retinas of infected fish, similar to infections with parental virus inoculations. These results indicate that viral RNA2 and/or encoded coat protein controls host specificity in SJNNV and SGNNV.

Failure of the Polymerase Chain Reaction (PCR) Method to Detect Striped Jack Nervous Necrosis Virus (SJNNV) in Striped Jack Pseudocaranx dentex Selected as Spawners

Abstract The causative agent of viral nervous necrosis (VNN) in striped jack Pseudocaranx dentex is transmitted from female and male spawners to larvae, and elimination of carrier spawners, determined by the detection of striped jack nervous necrosis virus (SJNNV) via polymerase chain reaction (PCR), has been used to prevent transmission of the disease in larval production facilities. However, some outbreaks of VNN occurred in larvae obtained from SJNNV-negative spawners. We compared the occurrence of infection between groups of larvae obtained from SJNNV-negative spawners and those from SJNNV-positive spawners. Viral nervous necrosis occurred in all seven groups of larvae obtained from the virus-positive spawners between the 3rd and 7th day of rearing. The virus was detected only occasionally after 2 weeks in four of six groups obtained from the virus-negative spawners. These results confirmed vertical transmission of the virus and revealed that a very small amount of SJNNV in spawners escaped PCR detect...

The movement protein gene is involved in the virus-specific requirement of the coat protein in cell-to-cell movement of bromoviruses.

Summary. Brome mosaic virus (BMV) requires the coat protein (CP) for cell-to-cell movement whereas Cowpea chlorotic mottle virus (CCMV), from the same genus, does not. Chimeric viruses created by exchanging the movement protein (MP) gene between the viruses can move from cell to cell. We show that interference in CP expression impaired the movement of the chimeric CCMV with the BMV MP gene but not of the chimeric BMV with the CCMV MP gene. We thus conclude that the MP gene plays a crucial role in determination of the virus-specific CP requirement in bromovirus cell-to-cell movement.

Characterization and partial cloning of the genomic DNA of a baculovirus from Penaeus japonicus (PjNOB = BMNV)

Abstract Many particles of a non-occluded baculovirus with a membrane 70 × 330 nm in size were observed in the nuclei of the mid-gut glands of larval Kuruma shrimp ( Penaeus japonicus ) with mid-gut necrosis using an electron microscope. Nucleotides of the baculovirus were isolated from the diseased larvae in the presence of Triton X-100. The nucleocapsid was a bacilliform virion, approximately 50 nm in width and 260 nm in length. From the purified preparations of nucleocapsids of the virus, two major proteins with molecular weights of 35 and 14 kDa were identified by SDS-polyacrylamide gel electrophoretic analysis. The viral genomic DNA extracted from the nucleocapsids was digested with Bam HI and Sau 3AI, but not with Eco RI, Pst I, Xho I and Sal I. The molecular weight of the DNA was estimated to be approximately 85.1 × 10 6 from the agarose gel electrophoretic pattern of the Bam HI fragments. A part of the Bam HI fragments was cloned, labeled with 32 P-CTP and used for Southern blot analysis as a probe. The fragment hybridized to DNA isolated from the diseased larvae, but not to DNA isolated from healthy larvae.