Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.

ABSTRACT Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31°C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11,550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

Three interferon-inducible Mr genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mr in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mr mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mr cDNA gene probe was used to determine whether there was a correlation between Mr mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mr mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mr mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mr protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mr locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mr locus and greater susceptibility to IHN-caused mortality.

Expression of the Mr protein of rainbow trout was analyzed after induction by poly I:C dsRNA and infectious hematopoietic necrosis virus (IHNV). Poly I:C dsRNA-treated rainbow trout gonad (RTG-2) cells expressed Mx protein that was detectable by immunoblot analysis at 24 h post induction. Increased expression was observed at 48 h and then declined by 72 h post-induction. In contrast, IHNV was not an efficient inducer of Mr protein in cells in culture. An immunocytochemical assay was also established to detect Mr proteins after transient transfection of chinook salmon embryo (CHSE-214) cells with trout Mr cDNA expression clones. Using these same techniques, endogenous Mr protein was detected in RTG-2 cells induced with 5 and 50 mu g ml(-1) of poly I:C dsRNA at 48 h post induction. In vivo, the appearance of Mr protein in rainbow trout after infection with IHNV (isolated at Rangen Research, Hagerman, ID, USA) was demonstrated in the immunoblots of kidney extracts of 4 out of 4 fish at 2 d post infection. Immunohistochemical staining of the kidney tissue from 3 out of 3 rainbow trout fry infected with the RB-76 strain of IHNV (isolated at Rounded Butte Hatchery, OR, USA) confirmed the production of Mr protein in the kidney tubules at 2 d post infection. These results suggest that Mr is a useful marker for the induction of fish interferon.

Current efforts to develop vaccines, particularly for aquacultured species, have turned largely to biotechnology because it provides the means to inexpensively produce sufficient quantities of the immunoprotective antigen. These efforts have resulted in several prototype vaccines for fish and the publication of a large number of articles on the subject. However, there are only a few recombinant DNA-based vaccines for aquaculture in the licensing pipeline. Continued funding of research on recombinant DNA vaccines comes from the recognition by industry and government funding agencies that this research can lead to an increased understanding of the mechanisms in protective immunity. This is especially important for fish and shellfish species since our knowledge of the immune mechanisms in these animals is pitifully meagre. This presentation discusses the relative merits of the different recombinant DNA technologies that have been used to produce viral vaccines for fish and the promising approaches that are under consideration to increase the efficacy of these vaccines. There are many approaches to antigen production by recombinant DNA techniques including: (i) the preparation of purified antigenic proteins produced from the cloned viral genes in a variety of vector/host expression systems, (ii) chemical synthesis or the use of fusion vectors to produce peptides corresponding to known epitopes, (iii) defined attenuations, i.e. specific genetic alterations, of live virus vaccines, (iv) the use of live bacterial or viral vectors to deliver resistance genes or viral antigens, (v) anti-idiotype antibodies, and (vi) DNA vaccines where purified plasmid DNA expressing the pathogen gene under a eucaryotic promoter is injected. All of these technologies have been used more or less successfully in the development of vaccines for aquacultured species. However, the requirements for safety, effectiveness, ease of application and low cost/dose restrict their commercial development for aquaculture. The ideal viral vaccine for aquaculture must be effective in preventing death, be inexpensive to produce and license, provide immunity of long duration, and be easily administered. In addition, these vaccines must not only provide protection against the lethal effects of virus infection but prevent the formation of virus persistence. This is especially true for infectious haematopoietic necrosis virus (IHNV) which has been shown to persist in survivors in the presence of high antibody levels. Since resolution of virus persistence is thought to be correlate with cell-mediated immunity, vaccines designed to augment the cell-mediated immunity must be developed for fish. Approaches that are being considered include the use of cytokines in combination with subunit vaccines and the use of specific MHC-I inducer adjuvants with the vaccine. The "tailoring" of vaccine immunogenicity using different combinations of antigen and adjuvant will be presented.

We tried to establish a subculture system for cells from the Oka organ (lymphoid tissue) of the grass prawn Penaeus monodon. The basic culture medium was tested for osmolality, serum concentration, serum sources and pH. It was found that Leibovitz's L-15 medium supplemented with 10% fetal bovine serum, 5 g l−1 NaCl, pH 7.63–8.1, with final osmolality at 470–500 mmol kg−1 allowed for enhanced cell attachment and growth; however, cells could not be maintained for more than 5 days. The supplementary nutrients were also tested for carbohydrates, amino acids, L-ascorbic acid, Buffalo rat liver(BRL) -condition medium and selenium. The basic culture medium + l g l−1 glucose were found to enhance cell attachment and growth. Our collected lymphoid cells required 3 days of incubation to obtain 80% confluency; however, cells did not grow in subcultures. Several growth factors were tested for developing a subculture system of shrimp cells. Epidermal growth factor (EGF) or transforming growth factor β (TGF β) did not foster cell growth. Cells treated with insulin or insulin-like growth factor I (IGF I) were capable of being subcultured, but resultant cells differed in terms of feeder layer, and were eventually discarded due to yeast contamination. Cells treated with basic fibroblast growth factor (bFGF) 20 ng ml−1 (F-20) could be subcultured for more than 90 passages without a feeder layer. Some F-20-treated cells were capable of extending their extracellular matrices for cell attachment and piled up; some of them became suspended and lost their anchorage-dependent and contact inhibition properties.

Multiple approaches to control viral infections in fish are being employed on an experimental basis in many fish disease laboratories. They include techniques to monitor fish populations for viruses by tissue culture infectivity, tagged antibody reagents to detect viral proteins, and nucleic acid probes for in situ hybridization or polymerase chain reaction amplification. The specificity and resolution of these detection methods are being constantly improved to increase their ease‐of‐use and sensitivity. In addition, scientists are developing prophylactic treatments in the form of traditional vaccines and subunit, peptide and genetic vaccines using molecular biological techniques. The success of all these approaches is obviously dependent on an understanding not only of the molecular structure of the virus and its genome but on the pathogenic mechanisms that lead to disease in the host animal as well. It is at this level, where there is so little known, that the formulation of appropriate control strategies has been difficult. For example, molecular techniques have provided evidence that the survivors of infection with infectious haematopoietic necrosis virus are long‐term carriers of the virus. This finding raises questions regarding the policy of releasing anadromous fish that have survived the disease. Viral vaccines have been shown to work in preventing virus‐induced mortalities in rainbow trout fry in laboratory trials, but no determination has been made on whether vaccination also prevents the formation of a virus‐carrier state in the survivors. More importantly, is there a vaccine formulation that will prevent carrier formation?

. Steelhead trout, Oncorhynchus mykiss (Walbaum), fry were experimentally infected with infectious haematopoietic necrosis virus (IHNV) Round Butte 1983 (Type 1). Fry were sampled daily, before and during the epizootic. Fish tissues were tested for infectious virus by tissue culture assay and for IHNV nucleocapsid protein by alkaline phosphatase immunohistochemistry (APIH). The progression of virus through the tissues was followed by APIH until the fourteenth day. Viral infection progressed from two major sites: from the gills into the circulatory system; and from the oral region into the gastrointestinal tract and then into the circulatory system. Once in the blood, virus was disseminated to virtually every organ. Progression of IHNV within and between organs is discussed.

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 102 plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 106 pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 104 pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.

A description of the types of viral vaccines that are being developed for fish is presented in this review. All three types of vaccines, i.e. killed, attenuated, and subunit vaccines, have worked to some extent in fish under controlled laboratory conditions. However, with the exception of a killed vaccine for spring viremia of carp, there are no commercially available viral vaccines for fish. The introduction of these vaccines to aquaculture will require an understanding of immune recognition in fish and the development of cost-effective ways of producing a safe, immunogenic vaccine. A review of available information on vaccines for infectious pancreatic necrosis virus, viral hemorrhagic septicemia virus, infectious hematopoietic necrosis virus, spring viremia of carp virus, and channel catfish virus is presented. These are the main viruses that have been investigated for vaccine development. Very little information is available on vaccines for other viral diseases of fish.

Journal of Aquatic Animal HealthVolume 5, Issue 4 p. 265-269 Article Serological Identification of Infectious Hematopoietic Necrosis Virus in Fixed Tissue Culture Cells by Alkaline Phosphatase Immunocytochemistry Barbara S. Drolet, Barbara S. Drolet Department of Microbiology and Laboratory for Fish Disease Research Oregon State University, Corvallis, Oregon, 97331-3804 USASearch for more papers by this authorJ. S. Rohovec, J. S. Rohovec Department of Microbiology and Laboratory for Fish Disease Research Oregon State University, Corvallis, Oregon, 97331-3804 USASearch for more papers by this authorJ. C. Leong, J. C. Leong Department of Microbiology and Laboratory for Fish Disease Research Oregon State University, Corvallis, Oregon, 97331-3804 USASearch for more papers by this author Barbara S. Drolet, Barbara S. Drolet Department of Microbiology and Laboratory for Fish Disease Research Oregon State University, Corvallis, Oregon, 97331-3804 USASearch for more papers by this authorJ. S. Rohovec, J. S. Rohovec Department of Microbiology and Laboratory for Fish Disease Research Oregon State University, Corvallis, Oregon, 97331-3804 USASearch for more papers by this authorJ. C. Leong, J. C. Leong Department of Microbiology and Laboratory for Fish Disease Research Oregon State University, Corvallis, Oregon, 97331-3804 USASearch for more papers by this author First published: 1 December 1993 https://doi.org/10.1577/1548-8667(1993)005<0265:SIOIHN>2.3.CO;2Citations: 9 To whom correspondence should be addressed. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Abstract An alkaline phosphatase immunocytochemical (APIC) assay was adapted for direct detection of infectious hematopoietic necrosis virus (IHNV) in infected tissue culture cells. The APIC assay provided a means of confirming the diagnosis of IHNV after the tissue culture plates had been fixed with formalin and stained with crystal violet. In cases where the original fish tissue samples had been discarded, the APIC assay was useful and was able to detect IHNV on plates that were more than 1 year old. The assay used a broadly reactive monoclonal antibody (lNDW14D) to the IHNV nucleoprotein to detect viral antigen in cells infected by virus isolates representing the five known IHNV types. Likewise, the type-2-specific monoclonal antibody (2NH105B) was able to distinguish type 2 IHNV in plaques that had been previously fixed. No cross-reactivity was seen with six other fish rhabdoviruses or with a fish birnavirus, infectious pancreatic necrosis virus (IPNV). Immunocytochernical staining was able to distinguish between the cytopathology produced by virus infection and that induced by the toxicity of tissue samples. In the latter case, no staining was observed. The staining was not affected by the age of the fixed and dried plates or by the polyethylene glycol pretreatment of cells. The ease, specificity, and sensitivity of the procedure made the APIC assay an attractive alternative to the standard serum neutralization or immunofluorescent identification of IHNV for diagnostic fish disease laboratories. Citing Literature Volume5, Issue41 December 1993Pages 265-269 RelatedInformation

The ribonucleoprotein gene of infectious hematopoietic necrosis virus (IHNV) has been expressed in Escherichia coli as a trpE fusion protein. This viral protein does not induce protective immunity to lethal IHNV infection in fish, and virus-neutralizing antibodies do not react with this viral protein. However, when it was administered with a bacterial lysate containing a region of the IHNV glycoprotein, there was enhanced resistance in immunized fish to lethal virus infection.

A characterization of the antigenic determinants (epitopes) of the glycoprotein (G) of infectious hematopoietic necrosis virus was made by expressing different regions of the G gene in Escherichia coli. A cDNA copy of the G gene was divided into four fragments by TaqI digestion, and the fragments were subcloned into pATH vectors, placing the expression of each G gene fragment under control of the trpE promoter. The resulting plasmids, pXL2, pXL3, and pXL7, encoded trpE-G fusion proteins subsequently detected with anti-infectious hematopoietic necrosis virus sera by Western immunoblots. A comparison of reactivities of the fusion proteins encoded by these plasmids was made by Western immunoblot and radioimmunoassay with a number of anti-G specific monoclonal antibodies (MAbs). The nonneutralizing MAb 136J reacted with the trpE-G fusion protein encoded by pXL3 and fusion proteins encoded by plasmids p52G and p618G, which were described in previous studies (R. D. Gilmore, Jr., H. M. Engelking, D. S. Manning, and J. C. Leong. Bio/Technology 6:295-300, 1988). Another nonneutralizing MAb, 2F, bound to the pXL3 fusion protein, and the neutralizing MAb RB/B5 recognized the pXL7 fusion protein. All fusion proteins were tested as vaccines in rainbow trout fry. Although significant protection was induced by all fusion proteins, the pXL3 fusion protein was most effective as a vaccine.

A cDNA clone of the large genomic segment of infectious pancreatic necrosis virus (IPNV) was inserted into transcription vectors and used for production of RNA transcripts of various lengths. These RNA transcripts were used to prime the synthesis of virus-specific polypeptides in a rabbit reticulocyte translation system. Full-length transcripts resulted in the synthesis of processed viral proteins pVP2, NS, and VP3. Transcripts which were deleted in the VP2 coding region did not affect the processing of NS or VP3 and similarly, deletions in VP3 did not affect the formation of NS or VP2. However, deletions which extended into the NS coding region resulted in a loss of protease activity and the production of truncated precursor polypeptides. The virus-specific proteolytic activity could not be inhibited by specific antisera and a trans activity for the viral proteases could not be demonstrated.

A complete cDNA clone of the larger A segment of the genome of infectious pancreatic necrosis virus (IPNV) was expressed in Escherichia coli in an effort to develop a vaccine for IPNV in fish. When the cDNA insert was positioned in the correct orientation to the pUC19 lacZ promoter, the viral proteins VP2, NS, and VP3 were synthesized and processed as observed in infected cells. When the insert was placed in the opposite orientation, VP3 and a 38-kDa virus-specific polypeptide were also synthesized. In addition, specific deletions made from the 3' end into the NS gene of the cloned A segment led to inactivation of the NS proteolytic activity and subsequently, the synthesis of an unprocessed VP2-NS polyprotein precursor. Antiserum to this polyprotein distinguished NS (28.5 kDa) from VP3 (31 kDa) and led to the identification of a previously undescribed 38-kDa virus-specific polypeptide in infected cells. Thus, both internal translational initiation and proteolytic cleavage could lead to the synthesis of VP2, NS, and VP3 from a single mRNA with a single open reading frame. A trpE expression vector, pATH2, was used to synthesize large quantities of the A-segment-encoded proteins in bacteria. The resulting bacterial lysate was very effective in inducing protective immunity in rainbow trout fry.

Infectious hematopoietic necrosis virus (IHNV) is a fish pathogen that kills young salmon and trout. Outbreaks of the disease among hatchery-reared fish are a problem in the northwestern USA from northern California to Alaska. At least five biochemical types and several strains of differing host specificity of IHNV exist. Any vaccine developed to immunize fish must be able to elicit a response that will neutralize all strains of IHNV. This report shows that a single type of IHNV can induce a protective immune response in vivo to the five biochemical types of IHNV and indicates that, of the IHNV isolates examined, there is at least one common major neutralization epitope. Therefore, a vaccine developed against this common neutralization epitope will provide cross-protective immunity against these IHNV variants.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.

Abstract. A comparative study of immunological methods for detecting infectious haematopoietic necrosis virus (IHNV) was made. The anti–IHNV antibody titre was measured by solid phase direct binding assays with‘125iodinated Protein A from Staphylococcus aureus and with immunoperoxidase staining. The binding antibody titre was much higher than that obtained in the virus neutralization assay. The high binding antibody titre of rabbit anti–IHNV sera made the development of two immunological tests for IHNV possible. Virus–specific proteins were detected on nitrocellulose membranes after transfer from denaturing polyacrylamide gels. The immunological methods were highly specific, sensitive lo less than 10 ng of virus protein, and were useful in characterizing the different strains of IHNV.

The structural polypeptides of the Round Butte strain of infectious hematopoietic necrosis virus (IHNV) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and stained with silver nitrate. Five virion proteins were identified. The relative contribution of each protein species to the total protein content of the virus was determined. An approximation of the number of molecules of each protein per virion was calculated.The time of the intracellular appearance for each virion protein of IHNV was investigated during the infection cycle. The first protein for IHNV to appear in the course of infection was the N protein, at 2-3 h after infection. At 6-7 h after infection, the membrane proteins, M1 and M2, could be identified in the autoradiograms. The two forms of the glycoprotein, G1 and G2, were found at 9-10 h after infection. The results from both pulse and pulse-chase labeling experiments suggest that G2 is synthesized immediately and then is further glycosylated to form G1. Since cellular protein synthesis was not inhibited during early infection, it was difficult to distinguish the viral L protein from host protein in the early samples. The temporal synthesis of the viral polypeptides suggests that those polypeptides are the translation products of independently transcribed monocistronic mRNA.

The genome RNA and six mRNA species of infectious hematopoietic necrosis virus were analyzed by denaturing gel electrophoresis. The following molecular weights were determined: genome RNA, 3.7 X 10(6); mRNA 1, 2.26 X 10(6); mRNA 2, 5.63 X 10(5); mRNA 3, 4.84 X 10(5); mRNA 4 (containing two different mRNA species), 3.00 X 10(5); and mRNA 5, 1.95 X 10(5). Densitometer analyses of gels were used to calculate the molar ratios of the intracellular mRNA species: mRNA 1, 0.02; mRNA 2, 0.49; mRNA 3, 1.0; mRNA 4, 2.52; and mRNA 5, 0.41. Hybrid selection studies determined the mRNA coding assignments as follows: mRNA 1 encodes the viral polymerase, L; mRNA 2 encodes the glycoprotein, G; mRNA 3 encodes the nucleocapsid protein, N; mRNA 4 is composed of two comigrating mRNA species which encode the matrix proteins, M1 and M2; and mRNA 5 encodes a previously unrecognized viral protein which is induced in infected cells but is not present in mature virions. This nonvirion protein has been designated the NV protein.