The First Isolation in North America of Infectious,Hematopoietic Necrosis Virus (IHNV) and Viral Hemorrhagic Septicemia Virus (VHSV) in Coho Salmon from the Same Watershed

Abstract

In November 1989, infectious hematopoietic necrosis virus (IHNV) was found for the first time in the Soleduck River at the Washington Department of Fisheries Soleduck Hatchery. The virus was isolated from ovarian fluid and kidney-spleen tissue pools from chinook salmon Oncorhynchus tshawytscha and ovarian fluid pools from coho salmon O. kisutch returning to the Soleduck Hatchery. The virus was identified as IHNV by neutralization assays. In December 1989, the virus causing viral hemorrhagic septicemia (VHSV) was found in ovarian fluid and milt pools from wild coho salmon obtained from the Soleduck and Bogachiel rivers and held at the Soleduck Hatchery. The virus was identified as VHSV by neutralization and immunoblot assays. These findings and their implications for routine broodstock sampling are discussed.     

Biochemical and Antigenic Properties of the First Isolates of Infectious Hematopoietic Necrosis Virus from Salmonid Fish in Europe

Abstract

The first isolates of infectious hematopoietic necrosis virus (IHNV) recovered from rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) in France and Italy were compared to six representative strains from North America by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of virion polypeptides and neutralization by monoclonal antibodies (MAbs). All three IHNV isolates from Europe had similar polypeptide profiles when compared by SDS-PAGE. An analysis of the antigenic relatedness of the European isolates to representative strains from North America showed that they were clearly different from viruses obtained from salmonids in California. The RB/B5 MAb, which was developed against virus isolated from adult steelhead (anadromous rainbow trout) reared in central Oregon, neutralized all isolates examined. The 193–110/B4 MAb, developed against IHNV isolated from infected yearling rainbow trout in southern Idaho, neutralized all isolates tested except those from California. The SRCV/A4 MAb, developed against Sacramento River chinook virus (SRCV) isolated from adult spring chinook salmon O. tshawytscha in central California, was the least reactive, and strong neutralization was observed only with the SRCV strain of IHNV from California. However, partial reactivity of the virus isolates from France with the SRCV/A4 MAb distinguished them from the virus recovered from salmonids in Italy.     

Prevalence of Infectious Pancreatic Necrosis Virus on Salmonid Fish Farms in Spain

Abstract

In Spain, salmonid fish farming was commercially developed in the 1960s, and now there are 140 private farms that depend heavily on imported embryonate eggs. Infectious pancreatic necrosis was first clinically diagnosed in Spain in 1970, but the virus (IPNV) was not isolated and identified until 1980. Since that time, researchers have isolated IPNV from other samples in Spain. A diagnostic survey was conducted to determine how prevalent IPNV is on fish farms in Spain and whether the virus has been responsible for some of the major financial losses occurring every year on these farms. In total, 236 samplings of rainbow trout Oncorhynchus mykiss from 31 farms in eight hydrographic areas were done over a 3-year period. Infectious pancreatic necrosis virus was isolated in 94 cases, and serotyping of the viral strains revealed that 81% of these isolates were strain Sp and 19% were strain Ab. Neither IPNV strain VR-299 nor rhabdovirus (as infectious hematopoietic necrosis virus or viral hemorrhagic septicemia virus) was detected in any of the samples.     

Swine Interferon I. Induction in Porcine Cell Cultures with Viral and Synthetic Inducers

The production of interferon by porcine kidney (PK₁₅) cell culture in response to viral and synthetic inducers was studied. The inducers used included a synthetic double-stranded polyribonucleotide, polyriboinosinic-polyribocytidylic acid (Poly I:C), swine influenza virus and three strains of pseudorabies virus. Following exposure to these inducers cell culture fluids were examined for interferon by the plaque-reduction method.

The Poly I:C and the swine influenza virus induced production of interferon by PK₁₅ cell cultures, whereas, all three strains of pseudorabies virus at the two concentrations tested failed to induce production of interferon in vitro.

The antiviral substance produced in PK₁₅ cells was identified as an interferon because it was pH stable, non-dialyzable, sensitive to trypsin, non-sedimentable, relatively heat stable, host-species specific and it possessed broad-spectrum antiviral activity. The latter was demonstrated by inhibition of vesicular stomatitis, vaccinia and pseudorabies viruses. Differences in interferon activity against the different viruses were observed.

     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

Evaluation of cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, and vesicular stomatitis virus in vitro

OBJECTIVE: To evaluate cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against cytopathic and noncytopathic bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), and vesicular stomatitis virus (VSV) in vitro. SAMPLE POPULATION: Primary bovine testicular cells and Mardin Darby bovine kidney cells. PROCEDURES: To evaluate cytotoxicity, cells were added to serial dilutions of each interferon. To evaluate antiviral activity of each interferon, interferons were serially diluted 1:10, and tissue culture cells were added; virus was then added at 3 time points. Prevention of viral infection by interferon was defined as failure to induce cytopathologic effect for VSV, IBRV, and cytopathic BVDV and failure to detect virus immunohistochemically for cytopathic and noncytopathic BVDV. RESULTS: No evidence of cytotoxicity in either cell line was detected after incubation with interferon alfa-2a or interferon alfa-B/D. However, reduced growth rates of tissue culture cells were detected for each interferon when undiluted interferon was tested. Comparable and profound antiviral activities against cytopathic and noncytopathic BVDV were evident for each interferon. Interferon alfa-2a and interferon a-B/D had comparable antiviral activities against VSV. Neither interferon had antiviral activity against IBRV. CONCLUSIONS AND CLINICAL RELEVANCE: The safety and marked in vitro antiviral activity against noncytopathic BVDV, cytopathic BVDV, and VSV suggest that interferons alfa-2a and alfa-B/D may be useful for treatment of natural disease after infection with these viruses.     

Enhanced Detection of Infectious Hematopoietic Necrosis Virus and Other Fish Viruses by Pretreatment of Cell Monolayers with Polyethylene Glycol

Abstract

To improve quantification of very low levels of infectious hematopoietic necrosis virus (IHNV) in samples of tissue, ovarian fluid, or natural water supplies, we tested the ability of polyethylene glycol (PEG) to enhance the sensitivity and speed of the plaque assay system. We compared 4, 7, and 10% solutions of PEG of molecular weight 6,000, 8,000, or 20,000 applied at selected volumes and for various durations. When cell monolayers of epithelioma papulosum cyprini (EPC), fathead minnow (FHM), chinook salmon embryo (CHSE-214), and bluegill fry (BF2) were pretreated with 7% PEG-20,000, they produced 4-17-fold increases in plaque assay titers of IHNV. The plaque assay titers of viral hemorrhagic septicemia virus, chum salmon reovirus, and chinook salmon paramyxovirus were also enhanced by exposure of CHSE-214 cells to PEG, but the titers of infectious pancreatic necrosis virus and Oncorhynchus masou virus were not substantially changed. Plaques formed by IHNV on PEG-treated EPC cells incubated at 15°C had a larger mean diameter at 6 d than those on control cells at 8 d; this suggests the assay could be shortened by use of PEG. Pretreatment of EPC cell monolayers with PEG enabled detection of IHNV in some samples that appeared negative with untreated cells. For example, when ovarian fluid samples from chinook salmon Oncorhynchus tshawytscha were inoculated onto untreated monolayers of EPC cells, IHNV was detected in only 11 of 51 samples; 17 of the samples were positive when PEG-treated EPC cells were used.     

DNA Vaccination Partially Protects Muskellunge against Viral Hemorrhagic Septicemia Virus (VHSV-IVb)

Abstract

A DNA vaccine containing the glycoprotein (G) gene of the North American viral hemorrhagic septicemia virus (VHSV) genotype IVb was developed to evaluate the immune response of fish following vaccination and evaluate its efficacy in protecting a susceptible species, the Muskellunge Esox masquinongy, against VHSV-IVb challenge. Seven weeks (539 degree-days) following vaccination with 10 μg of either pVHSivb-G or a control plasmid, Muskellunge were challenged by immersion with 10⁵ plaque-forming units (pfu)/mL of VHSV-IVb. Fish vaccinated with pVHSivb-G had a relative percent survival (RPS) of 45%. Vaccinated fish also had significantly lower mean viral titers in tissues (4.2 × 10² pfu/g) and viral prevalence (4%) than fish receiving the plasmid control vaccine (3.3 × 10⁵ pfu/g; 82%). Neutralizing antibodies were detected 28 d (308 degree-days) postchallenge (11 weeks postvaccination) in 100% of Muskellunge vaccinated with pVHSivb-G compared with only 12% of plasmid-control-vaccinated Muskellunge, suggesting robust induction of a secondary, adaptive immune response. In addition, pVHSivb-G–vaccinated Rainbow Trout Oncorhynchus mykiss challenged 7 d (100 degree-days) postvaccination with the heterologous novirhabdovirus, infectious hematopoietic necrosis virus (IHNV), experienced an RPS of 61%, compared to control fish, suggesting induction of an early and transient nonspecific antiviral immune response. This study provides an important starting point for VHSV-IVb vaccine development and useful information about the antiviral immune response elicited by DNA vaccination in a nondomesticated fish species.

Received May 1, 2016; accepted September 1, 2016     

Characterization of a Cell Line Derived from Inconnu

Abstract

Little is known about the diseases of the northern piscivorous salmonid inconnu Stenodus leucichthys (also known as sheefish). Fish health concerns surrounding transport and culture led to initiation of a cell line, now designated INEM-1, from inconnu embryonic tissue, to be used primarily for viral testing of inconnu. Cells were cultured at 16°C in Eagle's modified minimum essential medium with 10% fetal bovine serum. The fibroblastlike cells have now been passaged 57 times. Optimum growth occurs at 20°C, and doubling time is 1.5 d. Good growth also occurred at 16 and 24°C, but 30°C was rapidly lethal. Optimum density was 100,000 cells/ 35-mm-diameter plate, or 11,000 cells/cm². Chromosome analysis revealed a modal number of 76 chromosomes, two more than has been reported for blastulas of inconnu. The karyotype consists of 16 metacentric, 8 submetacentric, and 52 acrocentric chromosomes. Nucleolar organizing regions were identified on one chromosome pair. The INEM-1 cell line is susceptible to infectious pancreatic necrosis virus and infectious hematopoietic necrosis virus. No mycoplasmal contamination or indigenous viruses were detected.     

Unique Physicochemical Properties of the Occluded Penaeid Shrimp Baculoviruses and Their Use in Diagnosis of Infections

Abstract

Occlusion bodies in wet mounts of phloxine-stained tissue squashes and paraffin sections of penaeid shrimp infected with Baculovirus penaei and Penaeus monodon-type baculovirus fluoresced when viewed with an epifluorescent microscope. Inclusions of hepatopancreatic parvo-like virus, infectious hypodermal and hematopoietic necrosis virus, and Autographa californica nuclear polyhedrosis virus did not fluoresce. The use of phloxine stain (alone or as a component of an eosin preparation) and fluorescent microscopy may enhance detection of lowgrade infections by these disease-causing agents.     

Characterization of Three Continuous Cell Lines from Marine Fish

Abstract

Three continuous cell lines were established: JSKG from gonads of Japanese striped knife jaw Oplegnathus fasciatus, KRE from embryos of a hybrid of kelp Epinephelus moara and red spotted grouper E. akaara, and PAS from the skin of greater amberjack (also called purplish amberjack) Seriola dumerili; these cell lines were passed 60, 89, 120 times, respectively. Although initially cultured in Leibovitz's L-15 medium, two of the cell lines, JSKG and PAS, exhibited optimal growth response in Eagle's minimum essential medium buffered with a combination of tris and sodium bicarbonate. These cell lines were initiated at a higher NaCl concentration of 0.206 M but gradually adapted to the low NaCl concentration of 0.116 M after several subcultures. Optimum growth temperature was 25°C for JSKG and PAS cells, and 30°C for KRE cells. The modal chromosome number is 83 for the JSKG cell line, 92 for the KRE cell line, and 96 for the PAS cell line. Results for efficiency of plating indicate that all three cell lines are composed of transformed cells. Cell lines JSKG and PAS are susceptible to nine fish viruses, including channel catfish virus (CCV) and chum salmon virus (CSV). The KRE cell line is susceptible to CCV and fish rhabdoviruses of the vesiculovirus group. None of the cells showed cytopathic effect for Oncorhynchus masou virus (OMV) or Herpesvirus salmonis. Yields of infectious pancreatic necrosis virus (IPNV), infectious hematopoietic necrosis virus (IHNV), hirame rhabdovirus (HRV), and CSV were relatively low in these cell lines.     

Serological Evidence of Infectious Hematopoietic Necrosis in Rainbow Trout from a French Outbreak of Disease

Abstract

Following the detection of infectious hematopoietic necrosis virus (IHNV) in France in April 1987, a serological survey was conducted of the rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) from an infected cultured stock previously known to be contaminated with viral hemorrhagic septicemia virus (VHSV) for 3 years. The work lasted from April to December 1987, at which time all the remaining fish were slaughtered. Serum samples were assayed by a plaque-reduction test and a simplified neutralization test that is more suitable for processing large numbers of serum samples. Such investigations revealed that IHNV neutralization by trout antibodies depended on trout complement, as did neutralization of VHSV. Incubation for 16 h at 4°C increased the sensitivity of the test compared to incubation for 1 h at 20°C. During the course of clinical IHN from April to June, young fish did not display any neutralizing activity, but in September, 29 of 50 of them exhibited significant anti-IHN neutralizing antibody titers ranging from 21 to over 160, and 18 of 46 of these same fingerlings did so in December. Similarly, fish that had undergone VHS infection in August began to develop anti-VHSV antibodies in December (5 of 50), demonstrating that one fish can harbor neutralizing antibodies to both IHNV and VHSV, and that these antibodies had required 14 weeks to appear under fish culture conditions at 10°C. As could be expected from seroneutralization tests, neutralizing antibodies to IHNV did not result in protection against VHS. Sera from 13 of 20 adult fish sampled in mid-June revealed neutralizing antibodies to IHNV, suggesting that they harbored the virus prior to the clinical infection that affected their progeny. Only two of the fish showed low anti-VHSV antibody titers. Similarly, neutralizing antibodies to IHNV were detected in 53 of 73 other adult fish sampled in late October, 10 months after they had spawned and 7 months after mortality had occurred among their progeny. Given the prevalence, level, and persistence of neutralizing antibody titers, the seroneutralization test would be worth investigating more thoroughly to define the conditions that could make it a reliable tool for checking the virus status of trout carriers.     

Communications: Bacteria in the Hemolymph of the Freshwater Prawn Macrobrachium rosenbergii

Abstract

Bacteria from the hemolymph of the freshwater prawn Macrobrachium rosenbergii were identified and quantified. Total bacterial counts ranged from 0.0 to 4.6 × 10⁵ cells/1.0 mL of hemolymph. Predominant bacteria isolated included Aeromonas spp., Bacillus sp., and Pseudomonas spp. No bacteria were found in the hemolymph of prawns without lesions. The predominant species of bacteria isolated from water samples of prawn culture ponds was a chitinoclastic Bacillus sp.     

Fluorescent Antibody Test for the Rapid Diagnosis of Infectious Hematopoietic Necrosis

Abstract

A fluorescent antibody test (FAT) was developed for the rapid detection of infectious hematopoietic necrosis virus (IHNV). Both polyclonal and monoclonal antisera prepared against IHNV were evaluated. Test variables investigated included type of fixative, dilution rate of antibody reagents, staining time, and type of fluorescent conjugate that would be optimal for detection of IHNV. Specificity tests of the FAT indicated no cross-reactivity of the two antisera with other viruses or with cell lines of salmonid and nonsalmonid origin. All strains of IHNV tested, which included different electropherotypes, those isolated from selected salmonids at different life stages, and those from different geographic regions, reacted with both antisera. The FAT has been used for the detection of IHNV in blood smears and organ imprints from clinically infected juveniles, and in IHNV-infected cells in ovarian fluid from adult carriers. With this FAT, IHNV was detected after 48 h in cell lines inoculated with infected fish tissue. The test was equal in sensitivity to the plaque assay method and required less time to obtain a definitive diagnosis.     

The Lethal Dose of Largemouth Bass Virus in Juvenile Largemouth Bass and the Comparative Susceptibility of Striped Bass

Abstract

Largemouth bass virus (LMBV) is an iridovirus that was isolated from wild adult largemouth bass Micropterus salmoides in the southeastern United States in 1994. Although originally isolated from moribund wild fish, its virulence to juvenile largemouth bass is uncertain. To help clarify this point, two LMBV titrations were made in juvenile largemouth bass. Titers of LMBV in fathead minnow cells were 10^4.8 and 10^5.8 tissue culture infectious doses—50% cytopathic endpoint (TCID50) per milliliter, respectively. Tenfold serial dilutions of LMBV employed in each cell culture titration, injected intraperitoneally (0.1 mL/fish) into largemouth bass produced calculated lethal dose—50% mortality endpoints (LD50s) of 282 (10^2.45) and 288 (10^2.46) infectious doses in two consecutive infectivity trials. Virus yield of assayed infected fish averaged 10^8.5 TCID50/g and 10^7.7 TCID50/g in viscera of moribund and dead fish in the two trials and 10^6.5 TCID50/g in surviving exposed fish 14 d after infection. In a second experiment, largemouth bass had 100% mortality 5 d after injection while virus immersed fish had a significantly (P ≤ 0.005) lower mortality of 17% at 14 d. Similarly treated juvenile striped bass Morone saxatilis suffered 63% mortality after injection and significantly (P ≤ 0.005) lower mortality of 10% after immersion. In a third study of 25 d, 100% of injected largemouth bass died by 5 d after injection, and all of them were virus-positive. Injected striped bass had a significantly (P ≤ 0.005) lower mortality of 24%; all three fish were virus-positive initially, two fish were virus-positive at 18 d, and none were positive at 25 d. Juvenile largemouth bass were highly susceptible to LMBV injection and striped bass were moderately susceptible, but both species were only mildly susceptible when exposed by immersion.     

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC₅₀) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC₅₀ (concentration required to inhibit 50% of viral cytopathic effect). CC₅₀s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC₅₀ values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC₅₀ 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.     

Use of the Delphi Panel Method to Assess Expert Perception of the Accuracy of Screening Test Systems for Infectious Pancreatic Necrosis Virus and Infectious Hematopoietic Necrosis Virus

Abstract

Fifteen people, considered to be experts on fish virology, participated in a Delphi panel exercise to solicit opinion concerning the importance of factors that influence the ability of cell culture to detect infectious pancreatic necrosis virus (IPNV) or infectious hematopoietic necrosis virus (IHNV) in asymptomatic infected salmonids. Panelists rated many factors as having a strong impact on the sensitivity of cell culture and particularly emphasized the importance of technical and laboratory-related factors. Participants also provided their perceived estimates of the sensitivity and specificity of test systems—consisting of cell culture followed by serum neutralization, specific gene probes, enzyme-linked immunosorbent assay (ELISA), or fluorescent-antibody microscopy—for IPNV and IHNV in asymptomatic salmonids. The sensitivities estimated by panelists for optimal conditions were less than 70% for both IPNV and IHNV. There was substantial panelist uncertainty about the estimates, as indicated by large variances among individual responses. The system using serum neutralization for virus identification was perceived to have the highest sensitivity. All panelists estimated specificity to be very high. The importance of these findings with respect to the design of surveillance, quality assurance and control programs, and the interpretation of screening data are discussed.     

Improving siRNA design targeting nucleoprotein gene as antiviral against the Indonesian H5N1 virus

BackgroundSmall interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia.ObjectivesThe main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus.MethodsThe effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells.ResultsThe siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene.ConclusionsThese findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.     

Isolation and identification of trout viruses in South Africa

A total of 3,257 samples of diseased rainbow trout were examined for the presence of viruses from January 1983 to December 1987. A virus closely related to the VR 299 serotype of infectious pancreatic necrosis virus was isolated from 13 cases. An additional 7,228 viscera samples from asymptomatic fish were collected during the same period and a similar virus was isolated from 2 sites. During the same period 2,892 ovarian fluid samples were collected and a similar virus was isolated from 1 site. A similar virus was also isolated from one consignment of imported trout ova. A total of 5,550 ova was examined during this period. The viruses were identified by various tests as being closely related to the VR299 serotype of infectious pancreatic necrosis virus. All these samples tested negative for infectious haematopoietic necrosis virus, viral haemorrhagic septicaemia virus and herpesvirus salmonis.