Jo-Ann C. Leong

DNA Vaccines Encoding Viral Glycoproteins Induce Nonspecific Immunity and Mx Protein Synthesis in Fish

ABSTRACT Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.

Infectious Hematopoietic Necrosis Virus Matrix Protein Inhibits Host-Directed Gene Expression and Induces Morphological Changes of Apoptosis in Cell Cultures

ABSTRACT Infectious hematopoietic necrosis virus (IHNV) infection in tissue culture cells has previously been shown to result in the shutdown of host protein synthesis, cell rounding, and cell death. We report here an investigation of the cytopathogenicity of the viral phosphoprotein (P or M1), matrix (M or M2), and nonvirion (NV) proteins in cultured fish cells. The expression of M alone potently inhibited reporter gene expression from a viral and an interferon (IFN)-inducible promoter, whereas P and NV did not produce a similar effect. Northern blot analysis further revealed a reduction in the steady-state level of reporter mRNA when the M gene was cotransfected into cells; conversely, M mRNA was not drastically reduced in the same cells. By immunofluorescence confocal microscopy, fragmented nuclei were found in some cells expressing M protein but not in cells expressing P, NV, or β-galactosidase protein. Electron microscopy revealed the morphological changes associated with apoptosis in the M-transfected cells. Furthermore, IHNV infection was shown to produce DNA “laddering” in cultured cells. Taken together, these data suggested at least two functions for M protein in an IHNV infection: down regulation of host transcription and the induction of programmed cell death. In the course of these experiments, we also discovered that NV expression was associated with cell rounding, the first biological effect on cells to be attributed to the NV gene.

Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

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.

Analysis of Salmonid Leukocytes Purified by Hypotonic Lysis of Erythrocytes

Abstract A technique that uses hypotonic lysis of erythrocytes was optimized for the purification of leukocytes from the peripheral blood and anterior kidney (pronephros) of rainbow trout Oncorhynchus mykiss. Comparisons of initial blood dilution (1:2, 1:4, and 1:6) and the time of exposure to hypotonic conditions (10, 20, and 40 s) revealed that a dilution of 1:2 provided the most complete hemolysis after 20 or 40 s in a hypotonic solution. For pronephros, a 1:5 (w:v) dilution and lysis in hypotonic solution for 10–40 s was effective in eliminating erythrocytes. Total leukocyte yield from the blood and pronephros by use of the hypotonic lysis method was comparable with that obtained by use of typical density gradient centrifugation, and cell viability was 97% or greater. Differential cell counts showed that hypotonic lysis resulted in a distribution of leukocyte cell types similar to that of density gradient separation. Hypotonic lysis of erythrocytes is a simple, rapid, and inexpensive method of purifyi...

Glycoprotein from Infectious Hematopoietic Necrosis Virus (IHNV) Induces Protective Immunity against Five IHNV Types

Abstract 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.

Serological Identification of Infectious Hematopoietic Necrosis Virus in Fixed Tissue Culture Cells by Alkaline Phosphatase Immunocytochemistry

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 disting...

Polymerase Chain Amplification of Infectious Hematopoietic Necrosis Virus RNA Extracted from Fixed and Embedded Fish Tissue

Abstract Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that infects salmonid fish and, as a result, causes great economic losses to fish hatcheries. The direct detection of IHNV RNA in infected tissue would be useful in diagnosing the disease and preventing its spread. In this report, we describe a sensitive method for detecting IHNV RNA in formalin-fixed, paraffinembedded tissues of rainbow trout and steelhead Oncorhynchus mykiss. The technique is capable of detecting viral RNA in samples that have remained at room temperature in 10% buffered formalin for over 2 years. The RNA extracted from the deparaffinized tissue sections by phenol and guanidinium thiocyanate was amplified with primers to the viral nucleocapsid (N) gene with a heatstable DNA polymerase capable of reading both DNA and RNA. Prolonged incubation of the firststrand complementary DNA synthesis reaction for 20 min at 70°C was optimal for the synthesis of an N-specific, 252-base-pair product.

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract 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% mortalit...