Functional and Stress Response Analysis of Heat Shock Proteins 40 and 90 of Giant River Prawn (Macrobrachium rosenbergii) under Temperature and Pathogenic Bacterial Exposure Stimuli.

The aims of this research were to perform molecular characterization and biofunctional analyses of giant river prawn Hsp40 and Hsp90 genes (Mr-hsp40 and Mr-hsp90) under various stress conditions. Comparisons of the nucleotide and amino acid sequences of Mr-hsp40 and Mr-hsp90 with those of other species showed the highest similarity scores with crustaceans. Under normal conditions, expression analysis using quantitative real-time RT-PCR (qRT-PCR) indicated that Mr-hsp40 was highly expressed in the gills and testis, and Mr-hsp90 expression was observed in all tissues, with the highest expression in the ovary. The expression patterns of Mr-hsp40 and Mr-hsp90 transcripts under Aeromonas hydrophila challenge and heat-cold shock conditions were examined in gills, the hepatopancreas and hemocytes, at 0, 3, 6, 12, 24, 48 and 96 h by qRT-PCR. Under bacterial challenge, Mr-hsp40 displayed variable expression patterns in all tissues examined during the tested periods. In contrast, upregulated expression of Mr-hsp90 was quickly observed from 3 to 12 h in the gills and hepatopancreas, whereas obviously significant upregulation of Mr-hsp90 was observed in hemocytes at 12-96 h. Under temperature shock conditions, upregulation of Mr-hsp40 expression was detected in all tested tissues, while Mr-hsp90 expression was quickly upregulated at 3-48 h in all tissues in response to 35 °C conditions, and conditions of 35 and 25 °C stimulated its expression in gills and the hepatopancreas at 12 and 48 h, respectively. Silencing analyses of these two genes were successfully conducted under normal, high-temperature (35 °C) and A. hydrophila infection conditions. Overall, knockdown of Mr-hsp40 and Mr-hsp90 effectively induced more rapid and higher mortality than in the PBS control and GFP induction groups in temperature and infectious treatments. Evidence from this study clearly demonstrated the significant functional roles of Mr-hsp40 and Mr-hsp90, which are crucially involved in cellular stress responses to both temperature and pathogenic bacterial stimuli.

Mos1 transposon-based transformation of fish cell lines using baculoviral vectors.

Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes located between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.

Protection of Japanese flounder Paralichthys olivaceus from viral hemorrhagic septicemia (VHS) by Poly(I:C) immunization.

In immunization of fish with polyinosinic-polycytidylic acid (poly[I:C], a synthetic double-stranded RNA, injection of Poly(I:C) followed by challenge with a live virus induces a transient, non-specific antiviral state by interferon activity. When exposed to a virus while in this antiviral state, the fish acquire a specific and protective immunity against the corresponding viral disease and survive. In the present study, the effiacy of Poly(I:C) immunization was investigated in japanese flounder Paralichthys olivaceus using viral hemorrhagic septicemia virus (VHSV) as a model; the minimum dose of Poly(I:C) required for inducing protection and the duration of the antiviral state were determined, and a potentially curative effect of Poly(I:C) administration was assessed. The antiviral state was induced by administration of Poly(I:C) doses ranging from 12.5 to 200 microg fish(-1). Minimum dose to induce the antiviral state (relative percentage survival, RPS: 90%) was 12.5 microg fish(-1). No curative effect of Poly(I:C) was observed in fish pre-infected with VHSV. Fish injected with 200 microg Poly(I:C) fish(-1) were highly protected (RPS: 100%) from an artificial challenge with VHSV, and specific antibodies against VHSV were detected. The corresponding high level of antiviral state against VHSV was attained 1 d post Poly(I:C) injection, lasted for 6 d and susequently decreased. Moreover, the surviving fish were highly protected from re-challenge with VHSV (RPS: 100%). Thus, it was considered that an immunity against viral hemorrhagic septicemia was induced in the Japanese flounder by injecting live VHSV following Poly(I:C) administration.

Protection of rainbow trout from infectious hematopoietic necrosis (IHN) by injection of infectious pancreatic necrosis virus (IPNV) or poly(I:C).

It was recently reported that prophylaxis against infectious hematopoietic necrosis virus (IHNV) in fish was induced by pre-exposure to the infectious pancreatic necrosis virus (IPNV). Here the establishment of IHNV immunity in rainbow trout Oncorhynchus mykiss was investigated by IHNV challenge following non-lethal pre-infection with IPNV. Also, synthetic double-stranded RNA polyinosinic polycytidylic acid, Poly(I:C), an inducer for interferon (IFN), was evaluated as a substitute for IPNV induction of the non-specific antiviral state and subsequent IHNV-specific immunity in fish. Rainbow trout pre-infected with IPNV were protected from IHNV challenge 7 d later (relative percentage survival, RPS: 68.8%), and IHNV-specific antibodies were detected in sera from the survivors. Moreover, these surviving fish showed 91.6% RPS when re-challenged with IHNV 28 d after the primary IHNV challenge. Thus, fish appear to acquire IHNV-specific immunity through the IHNV challenge following pre-injection with IPNV. Fish pre-injected with Poly(I:C) were also highly protected from IHNV challenge 2 d later (RPS: 95.2%), and IHNV-specific antibodies were also detected amongst survivors. The survivors showed a 100% survival rate following re-challenge with IHNV both 21 and 49 d after the primary IHNV challenge. Thus, IHNV immunity in rainbow trout is induced by challenge with live IHNV following pre-injection with either IPNV or Poly(I:C). The use of Poly(I:C) to induce an anti-viral state protecting rainbow trout from an otherwise lethal vaccination dose of IHNV may have application to a wider range of fish species and fish pathogenic viruses.

Fish immunization using a synthetic double-stranded RNA Poly(I:C), an interferon inducer, offers protection against RGNNV, a fish nodavirus.

Viral nervous necrosis (VNN), caused by a fish nodavirus, is one of the most serious fish diseases worldwide. Here we report a unique vaccination method in sevenband grouper Epinephelus septemfasciatus using a synthetic double-stranded RNA polyinosinic polycytidylic acid (Poly(I:C)), an interferon inducer, followed by challenge with a live fish nodavirus. Fish injected with Poly(I:C) at 200 microg fish(-1) were highly protected from artificial challenge with red-spotted grouper nervous necrosis virus (RGNNV) (relative percentage survival, RPS: 100%), and specific antibodies against RGNNV were detected in sera from survivors. Moreover, the surviving fish were protected from rechallenge with RGNNV (relative percent survival RPS: 100%). Thus, it was confirmed that specific immunity against RGNNV was established in sevenband grouper by injection with live RGNNV following Poly(I:C) administration. Antiviral state was induced in fish by injection with Poly(I:C) at > or = 50 microg fish(-1), but no toxic response was observed in the fish even if Poly(I:C) was injected at a dose of 200 microg fish . In fish injected with Poly(I:C) at 200 pg fish(-1), a high level of antiviral state of > 90% RPS against RGNNV challenge lasted for at least 4 d after Poly(I:C) injection. However, no curative effect by Poly(I:C) injection was observed in fish already infected with RGNNV. It is considered that the present immunization method using Poly(I:C) followed by a live virus injection could offer protection against various viral infections in a broader range of fish species.

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells.

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.

Teleost TLR22 recognizes RNA duplex to induce IFN and protect cells from birnaviruses.

TLR22 occurs exclusively in aquatic animals and its role is unknown. Herein we show that the fugu (Takifugu rubripes) (fg)TLR3 and fgTLR22 link the IFN-inducing pathway via the fg Toll-IL-1R homology domain-containing adaptor protein 1(fgTICAM-1, or TRIF) adaptor in fish cells. fgTLR3 resides in endoplasmic reticulum and recognizes relatively short-sized dsRNA, whereas fgTLR22 recognizes long-sized dsRNA on the cell surface. On poly(I:C)-stimulated fish cells, both recruit fgTICAM-1, which in turn moves from the TLR to a cytoplasmic signalosome region. Thus, fgTICAM-1 acts as a shuttling platform for IFN signaling. When fish cells expressing fgTLR22 are exposed to dsRNA or aquatic dsRNA viruses, cells induce IFN responses to acquire resistance to virus infection. Thus, fish have a novel TICAM-1-coupling TLR that is distinct from the mammalian TLR3 in cellular localization, ligand selection, and tissue distribution. TLR22 may be a functional substitute of human cell-surface TLR3 and serve as a surveillant for infection with dsRNA virus to alert the immune system for antiviral protection in fish.

Enhanced propagation of fish nodaviruses in BF-2 cells persistently infected with snakehead retrovirus (SnRV).

Fish nodaviruses are causative agents of viral nervous necrosis causing high mortality in cultured marine fishes around the world. The first successful isolation of fish nodavirus was made with SSN-1 cells, which are persistently infected with snakehead retrovirus (SnRV). In the present study, a BF-2 cell line persistently infected with SnRV (PI-BF-2) was established to evaluate the influence of SnRV on the production of fish nodavirus. The PI-BF-2 cells were slightly more slender than BF-2 cells, but no difference was observed in propagation rate between both cell lines. No difference was observed in production of SnRV between PI-BF-2 and SSN-1 cell lines. Although both PI-BF-2 and BF-2 cell lines showed no cytopathic effect (CPE) after inoculation of striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), these fish nodaviruses could be amplified in BF-2 cells, and moreover, production of fish nodaviruses in the PI-BF-2 cell line was more than 40 times higher than in BF-2 cells. Thus, it was concluded that BF-2 cell permissiveness to fish nodaviruses was enhanced by persistent infection with SnRV. Furthermore, homologous cDNA to genomic RNA of SJNNV was detected from both PI-BF-2 and SSN-1 cell lines persistently infected with SnRV. The amount of nodavirus cDNA in SJNNV-inoculated PI-BF-2 cells was clearly lower than that in SJNNV-inoculated SSN-1 cells.

Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28.

White spot syndrome virus (WSSV) is the causative agent of white spot disease (WSD), one of the most serious diseases affecting global shrimp farming. We compared WSSV infection induction in kuruma shrimp Marsupenaeus japonicus by oral, immersion, and intramuscular injection (IM) exposure methods and evaluated the oral vaccine prepared from the recombinant WSSV proteins rVP26 and rVP28. The 50% lethal doses (LD50) of WSSV by oral, immersion, and IM challenges were 10(-0.4), 109-4.4), and 10(-7.7) g shrimp(-1), respectively, indicating that WSSV infection efficiency by oral challenge was significantly less than the other 2 challenge routes. However, in shrimp farms it is believed that WSSV infection is easily and commonly established by the oral route as a result of cannibalization of WSSV-infected shrimp. Kuruma shrimp vaccinated orally with WSSV rVP26 or rVP28 were challenged with WSSV by oral, immersion, and IM routes to compare protection efficacy. The relative percent survival values were 100% for oral challenge, 70 to 71% for immersion, and 34 to 61% for IM. Thus, the protection against WSSV-infection that was induced in kuruma shrimp by oral vaccination with rVP26 or rVP28 seemed equivalent to that obtained through IM vaccination.

Genotyping and pathogenicity of viral hemorrhagic septicemia virus from free-living turbot (Psetta maxima) in a Turkish coastal area of the Black Sea.

Viral hemorrhagic septicemia (VHS) is one of the most serious fish viral diseases for cultured rainbow trout (Oncorhynchus mykiss), although VHS virus (VHSV) seems to be ubiquitous among marine fishes. In the present study, VHSV isolation was performed with free-living and cultured turbot (Psetta maxima) in the Trabzon coastal area of the Black Sea to evaluate participation of VHSV in mass mortalities of seed-produced turbot larvae. VHSV was detected in 14 of 66 free-living spawners (positive ratio, 21.2%), 1 of 65 free-living immature fish (1.5%) and 7 of 40 cultured brood stock (17.5%), respectively. Based on a partial glycoprotein gene nucleotide sequence, Turkish VHSV isolates were classified into the class I-e of genotype I and were the most closely related to the GE-1.2 isolate (>98% identity), which was found >20 years ago in Georgia. Thus, it was revealed that Turkish VHSV isolates were not introduced from European countries, it could be an indigenous type of VHSV distributing in the Black Sea environment. In pathogenicity tests, the Turkish isolates did not induce mortality in turbot larvae and rainbow trout fingerlings. Mass mortalities at a rate of approximately 90% occurred in turbot larvae produced by experimental seeding, although VHSV was not detected in any dead fish. Thus, it was concluded that mass mortality in the seed-produced turbot larvae was not caused by VHSV infection.

An approach for genogrouping of Japanese isolates of aquabirnaviruses in a new genogroup, VII, based on the VP2/NS junction region.

Aquabirnaviruses, represented by Infectious pancreatic necrosis virus (IPNV), have been isolated from epizootics in salmonids and a variety of aquatic animals in the world; six genogroups of aquabirnaviruses have been identified. In comparisons of nucleotide sequences of the VP2/NS junction region, maximum nucleotide diversities of 30.8 % were observed among 93 worldwide aquabirnavirus isolates. A phylogenetic tree revealed the existence of a new genogroup, VII, for Japanese aquabirnavirus isolates from marine fish and molluscan shellfish. Nucleotide diversities between genogroups VII and I-VI were 18.7 % or greater. At the nucleotide level, Japanese IPNV isolates from epizootics in salmonids were nearly identical to a genogroup I strain from the USA or Canada. It is suggested that Japanese IPNV isolates belonging to genogroup I were originally introduced from North American sources, whereas Japanese aquabirnavirus isolates of genogroup VII were from marine aquatic animals indigenous to Japan.

Fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting reveal host-specific genetic diversity of Vibrio halioticoli-like strains isolated from the gut of Japanese abalone.

When analyzed by fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting, a total of 47 Vibrio halioticoli strains isolated from four Japanese abalone species and one turban shell species formed three clusters that roughly reflect the different species of host abalone from which they were isolated. The V. halioticoli isolates from turban shells were distributed evenly among the clusters. Representative isolates from two clusters were deemed separate species or subspecies by DNA-DNA hybridization.

Adsorption and Infectivity of Infectious Hematopoietic Necrosis Virus (IHNV) with Various Solids.

Adsorption of infectious hematopoietic necrosis virus (IHNV) to sea sand, Japanese acid clay, diatomaceous earth, kaolin, bentonite, quartz sand, chitin, cellulose powder, ion exchange hydrophobic Toyopeal and Cellulofine, alundum, active carbon, silica gel, glass, plastic, and bacterial cells was studied. The IHNV adsorbed to several clays (kaolin, bentonite, Japanese acid clay) and diatomaceous earth in sterilized water with a wide range of pH (5-11) at concentrations of 1, 10, and 100 mg/mL. Except for bentonite, infectivity of clay-adsorbed IHNV persisted for as long as 9 weeks. The clay-adsorbed IHNV also persisted in infectivity to rainbow trout Oncorhynchus mykiss, causing cumulative mortality rates of more than 73%. The results suggest that IHNV adsorbed to naturally occurring substances in various aquatic environments may provide a source of infection for susceptible fish inhabiting these environments.

Viral diseases of fish in Japan.

Viruses causing infectious pancreatic necrosis (IPN) and infectious hematopoietic necrosis (IHN) were first isolated in Japan during the 1970s and these two diseases remain among the most serious problems affecting cultured salmonids in Japan. In addition to IHN and IPN, four other viral diseases cause major economic losses among cultured fishes in Japan. These include viral pancreatic hepatic necrosis of yellowtail, rhabdovirus infection of Japanese flounder and black rock fish, Kuchishiro-sho of tiger puffer fish, and epidermal hyperplasia or necrosis of Japanese flounder. Over the years, a number of other viruses have been isolated from, or have been observed by electron microscopy, in moribund fish. Members of 10 of the existing families of animal viruses are represented in this group. Their importance to the aquaculture industry in Japan is reviewed here.