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The bluegill sunfish (Lepomis macrochirus) is a notorious exotic fish, which causes serious damages to freshwater ecosystems in the world. Therefore, it is a matter of urgency to control this species for the conservation of ecosystem. New microsatellite loci were developed for L. macrochirus using high throughput 454 GS-FLX+ pyrosequencing. We selected 24 primer pairs that were tested on 47 individuals from Lake Biwa, which encompasses the oldest and largest population of L. macrochirus in Japan. Allele per locus varied 2–17. Expected and observed heterozygosities ranged from 0.185 to 0.888 and 0.120 to 0.872, respectively. Applied to L. macrochirus in Lake Biwa, these markers could detect genetic differentiation among populations, which showed a pattern of isolation by distance. These markers are expected to be useful in the estimation of population structure and ecology of L. macrochirus.

Coho salmon (Oncorhynchus kisutch) is a Pacific salmon species that is commercially farmed in Japan, and is susceptible to erythrocyte inclusion body syndrome (EIBS), which is characterized by anemia, erythrocyte inclusion bodies, and cardiovascular muscle necrosis. At present, no vaccine for this disease is available. Piscine orthoreovirus-2 (PRV-2) is the causative agent of EIBS in Japan, and is one of three genotypes of PRV that induce the formation of erythrocyte inclusion bodies. However, due to a deficiency in cell lines able to culture PRV-2, the development of vaccines for diseases caused by PRV-2 has been difficult. A recent strategy involved the combination of a DNA vaccine, a σ1, and a non-structural protein gene for moderate PRV-1 protection. In the present study, analysis of antiserum obtained from PRV-2 infection survivors identified σ1 as a PRV-2 antigenic protein. Since a DNA vaccine incorporated with only σ1 did not prove efficacious against PRV-2 infection in a preliminary study, the present study tested the enhanced protective effect of the DNA vaccine with various sequences linked to the σ1 gene. Antigenic PRV-2 gene products were first screened using recombinant PRV-2 proteins and serum from coho salmon with a history of EIBS. Six vaccine groups of DNA vaccines were then created by the linkage of several sequences to the PRV-2 gene. Two trials were conducted to identify effective vaccines and a third vaccination trial confirmed the reproducibility of an effective vaccine. Trial I showed insignificant differences in the mean antibody level after 42 days. However, an outlier test indicated significantly higher antibody levels in coho salmon vaccinated in signal-linked σ1 gene DNA vaccine group and chaperone-linked σ1 gene DNA vaccine group. Trial II evaluated the effects of individual DNA vaccines and showed that individuals in the Sec-σ1, TF-σ1, and mixed vaccine group had significantly higher antibody levels and significantly lower virus loads. Trial III confirmed the suppression of viral replication and increase in antibody levels of the TF-σ1 DNA vaccine. However, this DNA vaccine was only effective for increasing antibody levels and inhibiting viral growth in some individuals, indicating the necessity for further experimentation involving analyses of the effect of individual differences on the efficacy of the vaccine.

Four major genotypes of viral haemorrhagic septicaemia virus (VHSV), which have been isolated from many marine and freshwater fish species, are known to differ in virulence. While fast and low-cost genotyping systems based on monoclonal antibodies (MAbs) have been developed for typing of VHSV virulence, there is a need for supplementing the knowledge. In particular, 2 field isolates from viral haemorrhagic septicaemia (VHS) outbreaks in sea-reared rainbow trout Oncorhynchus mykiss in Sweden, SE-SVA-14 and SE-SVA-1033 (both genotype Ib), have yielded contradictory reactions. In the present study, upon cloning by limited dilution, both isolates appeared to be heterogeneous in terms of reactivity with nucleo (N)-protein-specific MAbs as well their gene sequences. Infection trials in rainbow trout further revealed differences in the virulence of these virus clones derived from the same primary isolate. Based on a comparative analysis of the entire genome of the clones tested, we suggest that the differences in virulence are tentatively linked to substitutions of amino acids (aa) in the N-protein region covered by aa 43-46 and aa position 168, or a combination of the two. The fact that such minor naturally occurring genetic differences affect the virulence implies that even low-virulent VHSV isolates in the marine environment should be considered as a potential threat for the trout farming industry. The described MAbs can represent useful tools for initial risk assessment of disease outbreaks in farmed trout by marine VHSV isolates.

Predicting antigens that would be protective is crucial for the development of recombinant vaccine using genome based vaccine development, also known as reverse vaccinology. High-throughput antigen screening is effective for identifying vaccine target genes, particularly for pathogens for which minimal antigenicity data exist. Using red sea bream iridovirus (RSIV) as a research model, we developed enzyme-linked immune sorbent assay (ELISA) based RSIV-derived 72 recombinant antigen array to profile antiviral antibody responses in convalescent Japanese amberjack (Seriola quinqueradiata). Two and three genes for which the products were unrecognized and recognized, respectively, by antibodies in convalescent serum were selected for recombinant vaccine preparation, and the protective effect was examined in infection tests using Japanese amberjack and greater amberjack (S. dumerili). No protection was provided by vaccines prepared from gene products unrecognized by convalescent serum antibodies. By contrast, two vaccines prepared from gene products recognized by serum antibodies induced protective immunity in both fish species. These results indicate that ELISA array screening is effective for identifying antigens that induce protective immune responses. As this method does not require culturing of pathogens, it is also suitable for identifying protective antigens to un-culturable etiologic agents.

Cyprinid herpesvirus 2 (CyHV-2) is known as the causative agent of herpesviral haematopoietic necrosis in goldfish Carassius auratus auratus. However, the virus has also been detected in Prussian carp C. gibelio and crucian carp C. carassius from European and Asian countries. To prevent spread of the causative virus to other areas, investigation of the risk factors of spread of this virus is important. In this study, 8 batches of goldfish imported into the Netherlands by airfreight from Asia and the Middle East were investigated for the presence of the virus. CyHV- 2 DNA was detected by PCR in the pooled kidneys of 4 of the 8 imported goldfish batches, of which 1 was from a CyHV-2 disease case at a Dutch importer's quarantine facility. Sequence analysis of the CyHV-2 strains from this study and from previous reports showed that there were at least 6 different lengths in the mA region, resulting in tentatively at least 4 genotypes. Virus isolation was positive for only 1 (Amsterdam Schiphol-1 [AMS-1]) of the 8 samples. It was shown that the AMS-1 isolate was highly virulent to Ryukin goldfish after 100.3 TCID50 fish-1 intraperitoneal injection. The viral titre of the AMS-1 isolate for goldfish fin cells at several temperatures was similar to that of a Japanese CyHV-2 isolate. Our results prove that one of the routes of spread of various CyHV-2 strains is through the global trade of apparently healthy infected goldfish.

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20°C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25°C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.

Lactate dehydrogenase (LDH)-release assay was applied to estimate the toxic potential of harmful algal species at the cellular level. African green monkey kidney (Vero), yellowtail fin epithelia (MJF), and rainbow trout gill (RTgill-W1) cells were used as target cells. A live cell suspension of Karenia mikimotoi (SUO-1) induced the release of LDH from these cell lines, while the activity of another strain, FUK, was much lower. The cell-free culture supernatants and ruptured cell suspensions of both strains of K. mikimotoi were less effective on LDH-release assay. Exposure experiments against abalone and shrimp revealed that SUO-1 showed much stronger lethal effects on these organisms than FUK. Among six phytoplankton species, three species known to be harmful algal species induced the release of LDH to different extents depending on the cell line, whereas the other three species, known to be non-toxic, showed no effects on any cell lines. These results suggest that LDH-release assay is a useful micro-plate assay for estimation of the toxic potential of harmful phytoplankton.

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n = 56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.

The genus Megalocytivirus, represented by red sea bream iridovirus (RSIV), the first identified and one of the best characterized megalocytiviruses, Infectious spleen and kidney necrosis virus (ISKNV), the type species of the genus, and numerous other isolates, is the newest genus within the family Iridoviridae. Viruses within this genus are causative agents of severe disease accompanied by high mortality in multiple species of marine and freshwater fish. To date outbreaks of megalocytivirus-induced disease have occurred primarily in south-east Asia and Japan, but infections have been detected in Australia and North America following the importation of infected ornamental fish. The first outbreak of megalocytiviral disease was recorded in cultured red sea bream (Pagrus major) in Japan in 1990 and was designated red sea bream iridovirus disease (RSIVD). Following infection fish became lethargic and exhibited severe anemia, petechiae of the gills, and enlargement of the spleen. Although RSIV was identified as an iridovirus, sequence analyses of RSIV genes revealed that the virus did not belong to any of the four known genera within the family Iridoviridae. Thus a new, fifth genus was established and designated Megalocytivirus to reflect the characteristic presence of enlarged basophilic cells within infected organs. Indirect immunofluorescence tests employing recently generated monoclonal antibodies and PCR assays are currently used in the rapid diagnosis of RSIVD. For disease control, a formalin-killed vaccine was developed and is now commercially available in Japan for several fish species. Following the identification of RSIV, markedly similar viruses such as infectious spleen and kidney necrosis virus (ISKNV), dwarf gourami iridovirus (DGIV), turbot reddish body iridovirus (TRBIV), Taiwan grouper iridovirus (TGIV), and rock bream iridovirus (RBIV) were isolated in East and Southeast Asia. Phylogenetic analyses of the major capsid protein (MCP) and ATPase genes indicated that although these viruses shared considerable sequence identity, they could be divided into three tentative species, represented by RSIV, ISKNV and TRBIV, respectively. Whole genome analyses have been reported for several of these viruses. Sequence analysis detected a characteristic difference in the genetic composition of megalocytiviruses and other members of the family in reference to the large and small subunits of ribonucleotide reductase (RR-1, RR‑2). Megalocytiviruses contain only the RR-2 gene, which is of eukaryotic origin; whereas the other genera encode both the RR-1 and RR-2 genes which are thought to originate from Rickettsia-like α-proteobacteria.

We evaluated the prevalence of the akoya oyster disease (AOD) in Japan and the pathogenicity of the unidentified causative agent. We obtained naive Japanese pearl oysters Pinctada fucata martensii from a site with no history of AOD. In separate experiments, the oysters were either challenged by intramuscular injection with the hemolymph of infected oysters or reared in several major pearl oyster farms throughout Japan. Both groups of oysters experienced high mortality and exhibited discoloration of the adductor muscle and histopathological changes characterizing AOD. Our data suggest that AOD is prevalent in pearl oyster culture fields throughout Japan and that the causative pathogen remains highly pathogenic to P. fucata martensii. When groups of oysters produced by repeated selective breeding against AOD were reared in different pearl oyster fields alongside uninfected naive oysters, the selectively bred oysters had significantly lower mortality than naive oysters. The recent decline in outbreaks of AOD can be attributed, in part, to the introduction of selectively bred oysters that are resistant to this disease.

Viral hemorrhagic septicemia virus (VHSV) infection has been one of the major obstacles for Japanese flounder Paralichthys olivaceus culture both in inland tank systems and open sea net-pens in Japan. The mortality due to the disease in cultured Japanese flounder occurs in the cold-water season at temperatures mostly below 15 °C. We examined the effect on the disease by shifting the rearing-water temperature to a higher temperature (20 °C) as a potential control measure. Japanese flounder reared in aquariums were infected with a VHSV isolate JF00Ehi1, and the water temperature was shifted from 14 °C to 20 °C (Experiment I) and from 20 °C to 15 °C (Experiment II). In Experiment I, earlier shifting resulted in lower cumulative mortality rates. In Experiment II, the group of fish reared for a longer period at 20 °C showed lower cumulative mortalities. In a third set of experiments, fish inoculated with the virus were reared at 20 °C or at 25 °C (a viral non-permissive temperature), observing no mortality in either group. Viral multiplication was detected at a low level in the fish at 20 °C but not in those at 25 °C. At 21 dpi, these fish were challenged with the virus and reared at 14 °C, yielding no mortality in the 20 °C group, and 92% in the 25 °C group. These results indicate that rearing at 20 °C reduced mortality in the Japanese flounder experimentally infected with VHSV, probably inducing protective immune response against subsequent infection.

The pars tuberalis has been generally understood as a simple forward elongation of the glandular pituitary that develops into an architecturally similar structure. In rats, it is also believed that the pars tuberalis completely encircles the infundibular stalk at the ventral portion of hypothalamus. Intact Wistar-Imamichi rats were used in the present study, and frontal and sagittal serial sections of the pituitary gland/brain complex were prepared. Serial sections were stained with hematoxylin-eosin and with antibodies to LH-RH, S-100 protein or LH-RH receptor. Electron microscopic analyses were also conducted. The anatomical end of the epithelial structure of the pars tuberalis was observed at approximately 1 mm behind the optic chiasm. At this point, both the capillaries and the pars tuberalis spread and interdigitated over the median eminence. The pars tuberalis did not, however, completely encircle the pituitary stalk. Electron microscopic observations revealed some profiles of neuroendocrine nerve elements that were also present between the agranular cells of the pars tuberalis. A few LH-RH-positive nerve fibers were initially found just behind the optic chiasm, while the number of LH-RH-positive reactions increased around the capillary vessels. These reactions then decreased significantly and were sparsely distributed both in the diencephalon and in the area near the pituitary stalk where the two separated. LH-RH receptors were observed on S-100 protein-positive cells in the pars tuberalis. The present observations show the significance of the pars tuberalis and its relationship between the anterior pituitary gland and the hypothalamus as organs that possibly form another collaborating mechanism for the regulation of hormone secretion of the anterior pituitary gland.

ヒラメから分離されたウイルス性出血性敗血症ウイルス (VHSV) に対する各種市販消毒剤の殺ウイルス効果を検討した。 角消毒剤の効果は処理ウイルス液を培養細胞に接種することにより判定した。 PBS (-)希釈した場合は, VHSV は1-プロパノール, クレゾール, 塩素剤, ヨード剤および逆性石鹸剤で安易に不活性化されたが, メタノール, エタノールおよびフェノールの効果は低かった。 一方, 人工海水で希釈した場合は, クレゾールと塩素剤の効力が明らかに低下した。

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

In 2004, a massive mortality of wild common carp ( Cyprinus carpio ) due to CyHV-3 infection occurred in Lake Biwa. Although common carp of two different mitochondrial types (Japanese indigenous and domesticated Eurasian) occur in the lake, the majority of the dead fish seemed to be the indigenous type. The apparent high mortality in the indigenous type implies a higher susceptibility of this type to CyHV-3. To test the hypothesis that the susceptibility of indigenous and Eurasian types differ, we performed experimental infections with CyHV-3 among 2 groups of the indigenous type, and for the Eurasian type 4 groups of domesticated common carp and 4 groups of koi carp. Fish were immersed in CyHV-3 isolate and kept at 24 °C. Both groups of the indigenous type died more rapidly compared with the 8 groups of the Eurasian type. Cumulative mortality in both indigenous groups reached 95–100%, whereas the cumulative mortalities of domesticated common carp (30–95%) and koi carp (35–100%) were more varied. CyHV-3 genome in the organs of the indigenous type increased more rapidly after the viral exposure and reached higher peak levels than those of the domesticated strain. These findings revealed that susceptibility of the indigenous type of carp to CyHV-3 can be considered especially high.

The susceptibility of carp Cyprinus carpio to koi herpesvirus (KHV) was examined by experimental infections. Larvae of two strains of common carp were exposed to KHV by immersing the fish in water containing the virus at a dose of 101.6TCID50/mL measured using KF-1 cells. No mortality by KHV disease was observed among any larvae (average TL: 7.5 and 8.7 mm). On the other hand, high mortalities (69% and 100%) were observed in common carp juveniles (TL: 13.8 and 29.2 mm). Koi carp larvae (TL: 6.9 mm) were challenged in the same experimental conditions as described above, in which no larva died. Two months later, the same fish (TL: 48.2 mm) were exposed to the virus again. This time, all fish died of KHV. These results suggest that carp larvae are not susceptible to KHV, and that they become susceptible as they grow up.

The virucidal effect of disinfectants against spring viremia of carp virus was assessed using EPC cells. Five kinds of disinfectants, i. e. benzalkonium chloride (BZC), alkyltoluene (AKT), chlorhexidine gluconate (CHG), cresol and ethanol, were examined at graduated concentrations of the disinfectants for different reaction periods, 30 s and 20 min. The minimum concentration of each disinfectant demonstrating >99.99% inactivation of the virus was 100 ppm (20 min) for BZC, 500 ppm (30 s) and 350 ppm (20 min) for AKT, 175 ppm (30 s) and 100 ppm (20 min) for CHG, 0.25% (30 s) and 200 ppm (20 min) for cresol and 50% (30 s) and 40% (20 min) for ethanol.

The polymerase chain reaction (PCR) was used to amplify DNA of the red sea bream iridovirus (RSIV). Four oligonucleotide primer sets based on the ATPase gene, DNA polymerase gene, and a Pst I-restriction fragment of RSIV genomic DNA were synthesized. PCR products of the expected size were amplified with each primer set after 30 cycles using template DNA which was extracted from the supernatant of tissue-cultured GF cells infected with RSIV. These amplified products were shown to be specific for the genomic DNA of RSIV by Southern blot hybridization. In addition, PCR products were obtained from the DNAs of the spleen and blood of red sea bream, Pagrus major, artificially infected with RSIV. Furthermore, the PCR products were detected from the tissues of cultured marine fish naturally infected with RSIV and from the supernatant of tissue-cultured GF cells infected with RSIV isolated from cultured marine fish. However, PCR products were not obtained at 3 months post-challenge from the spleens of red sea bream infected by intraperitoneal inoculation of RSIV. None of the PCR products were obtained from the supernatant of tissue-cultured FHM cells infected with frog virus 3 or from the lymphocystis tissue of Japanese flounder, Paralichthys olivaceus, infected with fish lymphocystis disease virus.

The effectiveness of vaccination against red sea bream iridoviral disease (RSIVD) was evaluated using two kinds of vaccines in red sea bream (Pagrus major). For one vaccine, RSIV-infected GF cells were inactivated with formalin (1.0% v/v) for 10 days at 4°C and for the other the cell culture supernatant of RSIV-infected GF cells was inactivated with formalin (0.3% v/v) for 10 days at 4°C. Juvenile red sea bream were intraperitoneally injected with vaccines and after 10 days the fish were RSIV-challenged by intraperitoneal injection. Statistical analysis showed significantly high survival rates in the vaccinated groups than that of the respective control groups, indicating the protection is induced by the vaccination.In addition, the expression of the virus specific antigens in the spleen was examined for vaccinated and control fish after RSIV challenge by immunofluorescence test. The result that the expression of antigens was weaker in the vaccinated fish compared with the control fish supported the efficacy of the vaccination.

Improvement of a PCR method using the Sph I-5 primer set, which has been used for detection of KHV in Japan, was carried out to reduce negative factors, such as the appearance of non-specific reactions and the comparatively long reaction time. Moreover, there was a wrong sequence in the original reverse primer. The modified PCR protocol with the corrected primer set is as follows : initial denaturation at 94°C for 30s, 40 cycles of amplification (denaturation at 94°C for 30s, annealing at 63°C for 30s and elongation at 72°C for 30s) and final elongation at 72°C for 7min. The improved PCR reduced non-specific reactions and the total reaction time to almost half reguired by the original one.

Mass mortality among common carp Cyprinus carpio, especially in large-sized fish cultured in Lake Kasumigaura, has occurred since early October 2003. The affected fish swam lethargically near the surface. A histopathological examination revealed hyperplasia and necrosis of the epithelial cells in the gills. DNA of the koi herpesvirus (KHV) was detected in the gills and kidneys of moribund fish by polymerase chain reaction (PCR) assay. The virus could not be isolated from the tissues, but the inoculation of gill homogenate from affected fish into healthy carp reproduced the disease conditions and killed the fish. KHV was detected from the gills of the experimentally infected fish by PCR, and the virus was isolated from those fish with KF-1 cells.

Interspecific hybrid females produced from reciprocal crosses between Oryzias latipes and O. curvinotus show reduced fertility, but they do lay eggs. When fertilized by sperm from one of the parental species, such eggs developed into triploid males and females. To confirm the ploidy of the eggs spawned by the hybrid females we examined the karyotypes of backcross and gynogenetic offspring. The karyotype of the China-West Korean medaka, O. latipes sinensis, was 2n, with 46 chromosomes, including a pair of “large” metacentrics, whereas O. curvinotus had 2n, 48, without such a chromosome pair. F1 hybrids had a karyotype 2n, 47, with one large chromosome. When the hybrid females were backcrossed to O.l. sinensis, the offspring had a karyotype of 3n, 70, with two large chromosomes. By contrast, when backcrossed to O. curvinotus, the progeny of the hybrid females had a karyotype of 3n, 71, with one large chromosome. After gynogenetic treatment with UV-irradiated sperm of rainbow trout, the eggs spawned by the hybrid females developed into diploid (2n, 47) fish with one large metacentric chromosome. These results indicate that the hybrid females spawn unreduced diploid eggs with one complete chromosome set for both parental species. A triploid female, in contrast to the diploid hybrid females, spawned eggs with reduced chromosome number, giving rise to hyperdiploid embryos when fertilized by sperm. Transplantation of scales and electrophoretic analysis of proteins indicated that a hybrid female and her gynogenetic offspring can be regarded as “clones” with identical sets of chromosomes. © 1995 Wiley-Liss, Inc.

Erythrocytic inclusion body syndrome (EIBS) causes mass mortality in farmed salmonid fish, including the coho salmon, Onchorhynchus kisutchi, and chinook salmon, O. tshawytscha. The causative agent of the disease is a virus with an icosahedral virion structure, but this virus has not been characterized at the molecular level. In this study, we sequenced the genome of a virus purified from EIBS-affected coho salmon. The virus has 10 dsRNA genomic segments (L1, L2, L3, M1, M2, M3, S1, S2, S3, and S4), which closely resembles the genomic organization of piscine orthoreovirus (PRV), the causative agent of heart and skeletal inflammation (HSMI) in Atlantic salmon and HSMI-like disease in coho salmon. The genomic segments of the novel virus contain at least 10 open reading frames (ORFs): lambda 1 (λ1), λ2, λ3, mu 1 (μ1), μ2, μNS, sigma 1 (σ1), σ2, σ3, and σNS. An additional ORF encoding a 12.6-kDa protein (homologue of PRV p13) occurs in the same genomic segment as σ3. Phylogenetic analyses based on S1 and λ3 suggest that this novel virus is closely related to PRV, but distinctly different. Therefore, we designated the new virus ‘piscine orthoreovirus 2’ (PRV-2). Reverse transcription–quantitative real-time PCR revealed a significant increase in PRV-2 RNA in fish blood after the artificial infection of EIBS-naïve fish but not in that of fish that had recovered from EIBS. The degree of anemia in each fish increased as the PRV-2 RNA increased during an epizootic season of EIBS on an inland coho salmon farm. These results indicate that PRV-2 is the probable causative agent of EIBS in coho salmon, and that the host acquires immunity to reinfection with this virus. Further research is required to determine the host range of PRV species and the relationship between EIBS and HSMI in salmonid fish.

Seven mAbs with specific reaction patterns against each of the four genotypes and eight subtypes of viral hemorrhagic septicemia virus (VHSV) were produced, aiming to establish an immunoassay for typing VHSV isolates according to their genotype. Among the mAbs, VHS-1.24 reacted with all genotypes except genotype Ie, whilst mAb VHS-9.23 reacted with all genotypes except genotype III. mAb VHS-3.80 reacted with genotypes Ib, Ic, Id and II. mAb VHS-7.57 reacted with genotypes II and IVa, and mAb VHS-5.18 with genotype Ib only. Interestingly, mAb VHS-3.75 reacted with all of the genotype III isolates except a rainbow trout-pathogenic isolate from the west coast of Norway, and reacted in addition with the IVb isolate, CA-NB00-01, from the east coast of the USA. Finally, mAb VHS-1.88 reacted with all genotype IVb isolates from the Great Lakes, but not with CA-NB00-01. In conclusion, we can distinguish between all four genotypes and between five of eight subtypes of VHSV by testing isolates in immunoassay using a panel of nine mAbs. By Western blotting and transfection of cell cultures, it was shown that mAb VHS-1.24 recognized an epitope on the viral phosphoprotein (P), whilst all others recognized antigenic determinants on the nucleoprotein (N). From amino acid alignments of the various genotypes and subtypes of VHSV isolates, it was possible to determine the epitope specificity of mAb VHS-1.24 to be aa 32–34 in the P-protein; the specificities of mAbs VHS-3.80, VHS-7.57 and VHS-3.75 were found to be aa 43 and 45–48, aa 117 and 121, and aa 103, 118 and 121 of the N-protein, respectively.

An mRNA-specific reverse transcription (RT)-PCR primer set spanning the exon junction of a spliced putative terminase gene in the koi herpesvirus (KHV) was developed to detect the replicating stage of the virus. The proposed RT-PCR amplified a target gene from the RNA template, but not from a DNA template extracted from common carp brain (CCB) cells infected with KHV. In addition, the RT-PCR did not amplify the target gene of templates extracted from specific cell lines infected with either CyHV-1 or CyHV-2. RT-PCR detected mRNA from the scales of koi experimentally infected with KHV at 24 h post exposure (hpe). However, unlike conventional PCR, RT-PCR could not detect KHV DNA in fish at 0 hpe. The results indicate that the RT-PCR developed in this study is mRNA-specific and that the assay can detect the replicating stage of KHV from both fish and cultured cells infected with the virus.

アコヤガイ軟体部の赤変化を伴う大量死の原因を究明するため, 病貝の血リンパを健常貝に接種し疾病が再現されるかを給餌, 無給餌の条件下で検討した。 その結果, 接種2～3ヶ月後に病気が再現され, 感染実験系が確立された。 さらに, その原因体は病貝血リンパの血清, 血球の両方に存在し, 0.45μmのメンブレンフィルターを通過する濾過性病原体であると考えられた。

We examined the ability of developing Japanese flounder (Paralichthys olivaceus) to acquire protective immunity after exposure to viral hemorrhagic septicemia virus (VHSV). Juveniles measuring 9.8 cm average body length were not susceptible to infection with VHSV at 20 °C, while the smaller fish were susceptible. Mortality was not observed after secondary infection at 15 °C in the 9.8 cm cohort that had previously been exposed to the virus at 20 °C, while the smaller fish were susceptible to secondary infection. The expression of interferon (IFN)-related genes was shown to be better developed in larger fish upon virus infection and basal expression levels of the virus recognition proteins were higher in larger fish. Virus-specific antibody was detected in the larger fish, but not in smaller fish. These data indicate that the largest juvenile (9.8 cm) acquired immunity against VHSV infection at the first virus challenge, but smaller fish did not. The anti-viral immune system in the Japanese flounder matures when juveniles reach approximately 10 cm.

The viral haemorrhagic septicaemia virus (VHSV) comprises 4 major genotypes and a number of subtypes with, in most cases, distinct geographical distribution. A quick and simple detection method that can discriminate the different genotypes is desirable for a quick and more efficient prevention of the spread of genotypes to new geographical areas. A monoclonal antibody (MAb) against VHSV genotype IVa was produced, with the aim of providing a simple method of discriminating this genotype from the other VHSV genotypes (I, II, III and IVb). Balb/c mice were injected with purified VHSV-JF00Ehil (genotype IVa) from diseased farmed Japanese flounder. Ten hybridoma clones secreting monoclonal antibodies (MAbs) against VHSV were established. One of these, MAb VHS-10, reacted only with genotype IVa in indirect fluorescent antibody technique (IFAT) and ELISA. Using cell cultures that were transfected with each of the viral protein genes, it was shown that the MAb VHS-10 recognizes a nonlinear genotype IVa-specific epitope on the VHSV N-protein.

Hybrid females between Oryzias latipes sinensis and O. curvinotus lay non-reductional diploid eggs. We tried to produce trigenomic allotriploid medaka by fertilizing the diploid eggs with the sperm of three different species of Oryzias, O. celebensis, O. melastigma, and O. javanicus. Viable offspring were obtained when the eggs were inseminated by O. celebensis. Karyotype analysis confirmed that these fish were trigenomic allotriploids with three different genomes from O. latipes sinensis, O. curvinotus, and O. celebensis. Electrophoretic patterns of proteins (soluble muscle protein and eight isozymes) of these trigenomic allotriploids showed a normal expression of genes from these three genomes.

New methods were developed for the detection of koi herpesvirus (KHV, CyHV-3) by LAMP, which were compared with the PCR for specificity and sensitivity. We designed two primer sets targeting a specific sequence within the 9/5 PCR amplicon (9/5 LAMP) and the upper region of the SphI-5 PCR amplicon (SphI-5 LAMP), including a sequence highly conserved among the strains. The amplification was monitored in real-time based on the increase in turbidity, with magnesium pyrophosphate as the by-product. The reactions were carried out under isothermal conditions at 65 degrees C for 60 min. The detection limit of both LAMP was six copies, equal to the modified SphI-5 PCR. No cross-reactivity with other fish pathogenic viruses and bacteria was observed. SphI-5 LAMP was found to have a quicker response in terms of the reaction velocity than 9/5 LAMP. Therefore, we consider SphI-5 LAMP to be superior for routine use. Additionally, LAMP was found applicable to crude extract from gills and other organs. LAMP methods are superior in terms of sensitivity, specificity, rapidity and simplicity, and are potentially a valuable diagnostic tool for KHV infections.

Three regions of koi herpesvirus (KHV) genomic DNA were compared for 34 samples from Japan, six from Indonesia, two from Taiwan, one from the Philippines, 13 from the Netherlands, one from the UK, one from the USA and one from Israel. The analyzed genomic regions included known PCR-detection targets (Sphl-5, 9/5 and the thymidine kinase gene). The KHVs from Asian countries were very homogeneous, although two variants were noted based on a single nucleotide polymorphism. In contrast, seven variants were found in KHVs from outside of Asia, and although closely related to one another, they were clearly distinct from those from Asian. The results suggest that a clear genetic distinction exists between Asian and European (including each single isolate from the USA and Israel) types of KHV, and that unique types of KHV were independently introduced or emerged in the respective geographic locations.

The authors present a tester capable of generating and measuring real-time-managed mixed signals by means of a dual master clock and master/slave multi-sequencing. This tester can test devices under conditions very close to their real working environment. The tester thus readily enhances testing reliability and improves testing throughput promptly. A powerful software package, combined with a library-oriented application environment, eases the job of developing tests for mixed-signal devices. >

The effectiveness of a vaccine made of the cell culture supernatant of RSIV (red sea bream iridovirus) infected cells inactivated with formalin (0.1% v/v) in yellowtail Seriola quinqueradiata, amberjack S. dumerili, kelp grouper Epinephlelus moara, striped jack Pseudocaranx dentex and spotted parrot fish Oplegnathus punctatus was evaluated under experimental conditions. Juvenile or young fish were intraperitoneally injected with the vaccine, and challenged with RSIV by intraperitoneal injection at 10th day post-vaccination. Statistical analysis showed significantly higher survival rates in the vaccinated groups of all tested fish species.