Experimental horizontal transmission of viral hemorrhagic septicemia virus (VHSV) in Japanese flounder Paralichthys olivaceus.

Infection by viral hemorrhagic septicemia virus (VHSV) has recently occurred among wild and farmed Japanese flounder Paralichthys olivaceus in Japan. In the present study, horizontal transmission of VHSV among Japanese flounder was experimentally demonstrated by immersion challenge. Exposure to a flounder isolate (Obama25) of VHSV revealed a dose-response, with higher mortality (81 and 70%) at the 2 higher exposure levels (6.0 and 4.0 log10 TCID50 ml(-1)). In a second experiment, high titers of VHSV were expressed from moribund and dead flounder based on virus detection in holding-tank waters 2 to 3 d prior to death of the fish and 1 d after death. The virus could not be detected in tank waters 2 d after death. Finally, a third cohabitation experiment in small tanks demonstrated horizontal transmission of VHSV from experimentally infected to uninfected fish.

Apoptosis does not play an important role in the resistance of 'immune' Penaeus japonicus against white spot syndrome virus.

We previously demonstrated that kuruma shrimp, Penaeus japonicus, exposed to white spot syndrome virus (WSSV) became resistant ('immune' shrimp) to subsequent challenge with the virus. The present study investigated the role of apoptosis in the 'immune' shrimp during a secondary challenge with WSSV. When naive kuruma shrimp were intramuscularly injected with WSSV at a high or low dose, apoptosis was often detected by TUNEL assay in the lymphoid organ (LO), mainly in the early stage of the infection. A significantly higher incidence of apoptosis was observed in the LO of the shrimp injected with the high dose of WSSV (cumulative mortality: 100%) than in the shrimp injected with the low dose (cumulative mortality: 0%). When 'immune' and naive shrimp were injected with an equal dose of WSSV, the incidence of apoptosis was significantly lower in the 'immune' shrimp than in the naive shrimp. This difference is assumed to result from a substantial reduction of the virus by humoral neutralizing factor in the 'immune' shrimp. These results suggest that apoptosis is not a principal protective factor in 'immune' shrimp.

A time-course study on the resistance of Penaeus japonicus induced by artificial infection with white spot syndrome virus.

The onset and duration of resistance in experimental survivors of Penaeus japonicus produced by an intramuscular injection with white spot syndrome virus (WSSV) were surveyed by re-challenge tests with the virus conducted at weeks 1-4 and months 1-3 post initial exposure (PIE) to the virus. Virus neutralising activity in the survivors' plasma was also examined. Plasma-treated WSSV was separated from the plasma by centrifugation and then injected into naïve shrimp, in parallel with each re-challenge test. Re-challenge tests of the survivors conducted at weeks 1-4 PIE revealed that the resistance commenced at week 3 (relative percent survival, RPS: 39%) and almost fully developed at week 4 (RPS: 58%), because statistically significant differences in survival rates were observed between the test (previously virus exposed) and control groups at weeks 3 and 4. Re-challenge at months 1-3 PIE resulted in RPS values of 67, 54 and 6%, respectively, indicating the resistance persisted until month 2. RPS values in neutralisation tests performed at weeks 1-4 and months 1-3 PIE were -5, 14, 36, 50, 100, 38 and 6%, respectively, which coincided with the RPS values in each re-challenge test conducted in parallel. The present results demonstrated that resistance of P. japonicus against the viral pathogen developed 3 or 4 weeks after an exposure to the virus, and it persisted for another month at 24 degrees C. The resistance was paralleled by a humoral neutralising factor(s) in the plasma of shrimp.

Genetic relatedness among Japanese, American and European isolates of viral hemorrhagic septicemia virus (VHSV) based on partial G and P genes.

Molecular virological analyses of 8 Japanese VHSV (viral hemorrhagic septicemia virus) isolates from wild and farmed Japanese flounder Paralichthys olivaceus were performed to investigate their genetic relatedness to American and European isolates of VHSV. Phylogenetic analyses based on the partial nucleotide sequences of G and P genes revealed that there are 2 genogroups of VHSV in Japan. The first one represented by the Obama25 isolate is closely related to the American isolates (Genogroup I) while the other, the KRRV9601 isolate, is closely related to the traditional European isolates (Genogroup III). The 2 types of Japanese VHSV showed differences in the relative mobility of the G protein and intensity of the antibody reaction on the P and M proteins. The Obama25 type of VHSV is widely distributed as a native virus in the coastal areas of western Japan and has been responsible for the occurrence of VHSV infection in farmed Japanese flounder while the KRRV9601 isolate is considered to have been introduced from a foreign country.

Effects of shrimp density on transmission of penaeid acute viremia in Penaeus japonicus by cannibalism and the waterborne route.

To investigate the effects of shrimp density on mortalities of Penaeus japonicus in experimental penaeid acute viremia (= white spot syndrome), shrimp injected intramuscularly with penaeid rod-shaped DNA virus (PRDV) were reared at different densities. In Expt 1, challenged (10(-6) dilution of a PRDV preparation) shrimp were reared collectively in a tank or individually in separate chamber units. A significant difference in cumulative mortalities was found between collectively (75.6%) and individually (1.2%) reared groups after 30 d. In Expt 2, effects of density on mortality were clearly shown when challenged (10(-5) dilution) shrimp were reared collectively in tanks at high (260 shrimp m(-2)), middle (135 shrimp m(-2)) and low densities (73 shrimp m(-2)). The cumulative mortalities for 14 d in the high, middle and low density groups were 72, 46 and 18%, respectively. In Expt 3, challenged (10(-5) dilution) shrimp were reared collectively in 3 tanks (Groups A, B and C) at the same high density (260 shrimp m(-2)): Group A, dead shrimp were immediately removed to avoid transmission of the pathogen through cannibalism and the waterborne route; Group B, dead shrimp were removed at scheduled times but were separated from living shrimp by a net partition to avoid cannibalism; and Group C, dead shrimp were removed twice a day at scheduled times. Resulting cumulative mortalities for 20 d in Groups A, B and C were 4, 24 and 64 %, respectively. These results show that the higher mortalities occur in P. japonicus reared at the higher densities in experimental PRDV infection, and this phenomenon is caused mainly by a higher opportunity of horizontal transmission of the virus through cannibalism and the waterborne route.

Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae.

Recurrent outbreaks of a disease leading to mass mortalities in an oyster (Crassostrea gigas) hatchery located in western Japan were investigated. The disease occurred regularly in 2- to 8-d-old larvae and has been experimentally controlled in the hatchery by treating the larval rearing water with streptomycin, without ascertaining the etiological agent. The signs of the disease and the course of infection resembled bacillary necrosis reported in oysters and other bivalve molluscs in the USA and Europe. Quantitative and qualitative examinations of the bacterial flora of hatchery samples including source water, broodstock, larval feed and larvae revealed a very high total bacterial load and presumptive vibrios in diseased larvae. Further, the bacterial profile revealed that Vibrio spp. constituted approximately 60 to 95% of the bacteria isolated from infected larvae and most isolates were identified as V. splendidus biovar II and V. harveyi, suggesting their possible role in the disease. However, experimental challenges proved the pathogenicity of V. splendidus II. Several isolates of V. splendidus II from infected larvae were highly pathogenic, producing 100% mortality at levels of 10(5) cfu ml-1 in 24 h, while isolates from other sources demonstrated a low degree of virulence. Detection of V. splendidus II from broodstock, especially in the gonad of a few breeders, suggests the probability that broodstock could be the source and route of transmission of this pathogen.

Genomic classification of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene.

A molecular phylogenetic analysis of 25 isolates of fish nodaviruses, the causative agents of viral nervous necrosis of marine fish, was performed based on the nucleotide sequences (427 bases) of the coat protein gene. These fish nodaviruses were classified into four clusters: tiger puffer nervous necrosis virus, striped jack nervous necrosis virus, berfin flounder nervous necrosis virus, and red-spotted grouper nervous necrosis virus.

Comparison of the coat protein genes of five fish nodaviruses, the causative agents of viral nervous necrosis in marine fish.

Striped jack nervous necrosis virus (SJNNV), a nodavirus, is the causative agent of viral nervous necrosis (VNN) in larval striped jack fish. In the present study, the SJNNV coat protein gene was sequenced and compared with that of four known insect nodaviruses and with four other fish nodaviruses causing VNN. The SJNNV coat protein gene was 1410 bases in length and contained a single ORF of 1023 bases encoding a protein of 340 amino acids. The sequence similarities between the coat protein gene of SJNNV and four insect nodaviruses were 28.6% or less at the nucleotide level and 10.6% or less at the amino acid level. A portion of the coat protein gene from four additional fish VNN viruses was amplified by PCR using primers designed for SJNNV and the amplified fragments (870-876 bases) were sequenced. The sequence similarities among SJNNV and the four VNN viruses were 75.8% or greater at the nucleotide level and 80.9% or greater at the amino acid level. In the fish nodaviruses a highly conserved region of 134 amino acids with sequence similarity of 92.5% or greater was detected. This conserved sequence was not found in the coat protein of insect nodaviruses. These results indicate that the fish nodaviruses that cause VNN are closely related to each other but are quite different from insect nodaviruses.

Philadelphia-chromosome-positive, monosomy 7 biphenotypic acute mixed lineage leukemia in adults: a pluripotent stem cell disorder.

Two adult patients with acute mixed lineage leukemia (AMLL) having combined Philadelphia chromosome (Ph1) positivity and monosomy 7 are presented. The phenotypes of leukemic blasts from both cases were almost same (early B-lymphoid lineage and myeloid lineage); CD10+, CD13+, CD19+. HLA-DR+, and dual-color analysis showed simultaneous expression of CD10 (CD19) and CD13 antigens in individual blasts (biphenotypic) in both cases. On molecular analysis, the leukemic blasts showed rearrangement in the first intron of the BCR gene with breakpoint just outside of 3' end of m-BCR-2 (bcr 3) in case 1, and in the M-BCR in case 2. Immunoglobulin heavy chain gene (IgH) rearrangement was noted in both cases, but rearrangement of the T-cell receptor beta-chain gene (TCR beta) was detected only in case 1. Clinically, both cases achieved complete remission by the combination chemotherapy consisting of L-asparaginase, doxorubicin, vincristine, and prednisolone (L-AdVP). In remission, all these molecular abnormalities disappeared in both patients. These results suggest that the Ph1-positive and monosomy 7 AMLL in adults is de novo acute leukemia with both early B-lymphoid and myeloid phenotypes and may arise from malignant transformation of pluripotent stem cell, and expresses a heterogenous rearrangement pattern of the BCR gene.

Vibrio cholerae non-O1 isolated from ayu fish (Plecoglossus altivelis) in Japan.

A fish pathogen, Vibrio cholerae non-O1, was isolated from diseased ayu fish (Plecoglossus altivelis) collected from rivers in eight prefectural districts of Japan. This organism was found to have biochemical characteristics similar to those of V. cholerae non-O1, except that our isolates were negative for ornithine decarboxylase. Antiserum against an ayu isolate did not agglutinate with the majority of environmental V. cholerae non-O1 isolates, but a major O antigen was common among the ayu isolates. All strains were hemolytic to sheep erythrocytes, and oral administration of culture supernatants induced fluid accumulation in suckling mice. However, the crude toxin was not lethal to adult mice, and no cholera toxin-like enterotoxins were detected.

Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis.

Spherical virus particles were purified from larval striped jack (Pseudocaranx dentex) with nervous necrosis. The virus consists of nonenveloped particles, about 25 nm in diameter, and contains two single-stranded, positive-sense RNA molecules with molecular weights of 1.01 x 10(6)Da (RNA 1) and 0.49 x 10(6)Da (RNA 2), respectively. The RNAs do not have poly(A) sequences at the 3' terminus. Virus structural proteins consist of two proteins with molecular weights of 42 and 40 kDa. When translated into cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of the 1a protein (100 kDa), whereas RNA 2 synthesized the 2a protein (42 kDa), which is probably the coat protein of the virus, and a polypeptide of 40 kDa which appears to be the processed form of the 42-kDa protein. Under electron microscopic observation, the virus particles were found in the tissues of the central nervous system of the affected larval striped jack. From morphological and biochemical properties of the virus, we identified this virus as a new member of the family of Nodaviridae and designated it striped jack nervous necrosis virus.