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J. R. Winton

Publications and source records attributed to J. R. Winton.

At least 73 records · Page 4Linked to original sources
USGS1992

In vitro infection of salmonid epidermal tissues by infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha ; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.

Journal of Aquatic Animal Health↗
USGS1992

Identification of viral hemorrhagic septicemia virus isolated from Pacific cod Gadus macrocephalus in Prince William Sound Alaska, USA

Ulcerative slun tissues from 2 Pacific cod Gadus rnacrocephalus caught in Prince William Sound, Alaska, USA, were examined for virus by Fish Pathology staff within the F.R.E.D. Division of the Alaska Department of Fish and Game. Six days after inoculation of Epitheliorna papulosum cyprini (EPC) cells at 14"C, diffuse rounding and lifting of cells from the monolayers suggestive of cytopathlc effect became visible in the lower sample dilutions. Ultrastructural examinations of affected EPC cells showed rhabdovirus particles within cytoplasmic vacuoles and on the cell surface membranes. Virus isolates from both cod were subsequently confirmed as viral hemorrhagic septicemia virus (VHSV) by serum neutralizabon and immunoblot assay. This is the first VHSV isolated from Pacific cod, which represents a new host species for the virus. Histologically, cod skin ulcers appeared to be caused by a foreign-body-type inflammatory response to foci of protozoa resembling X cells that also had plasmodial stages. Whether the rhabdovirus was incidental to the slun lesion or played a role in its etiology remains to be determined. The possible relationship between thls virus and the recent occurrences of VHSV in anadromous salmoruds from Washington State, USA, is discussed.

Diseases of Aquatic Organisms↗
USGS1991

Infectious hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS): Detection of the trout antibodies to the causative viruses by means of plaque neutralization, immunofluorescence, and enzyme-linked immunosorbent assay

Sera collected from cultured rainbow trout Oncorhynchus mykiss surviving outbreaks of infectious hematopoietic necrosis (IHN) or viral hemorrhagic septicemia (VHS) were examined for the presence of antibodies to both of the causative viruses, infectious hematopoietic necrosis virus (IHNV) and Egtved virus (viral hemorrhagic septicemia virus: VHSV). Sera were screened with three serological tests: 50% plaque neutralization test (PNT), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). In sera from 20 rainbow trout surviving IHN, antibodies to IHNV were detected in 9 fish by PNT, in 12 fish by IF, and in 9 fish by ELISA. In these sera, antibodies cross-reacting with VHSV were rare (detected in 0 fish by PNT, in 1 by IF, and in 1 by ELISA). In sera from 20 rainbow trout surviving VHS, antibodies to VHSV were detected in 9 fish by PNT, in 16 fish by IF, and in 18 fish by ELISA. A considerable percentage of the VHS-survivor sera contained antibodies that cross-reacted with IHNV, as detected by ELISA (16 fish) and 1F (7 fish) but not by PNT (0 fish). The three serological tests appear to be useful tools for IHNV and VHSV epidemiology; however, the presence of cross-reacting antibodies in some sera suggests caution when farms require specific pathogen-free certification for one of the viruses in the presence of the other.

Journal of Aquatic Animal Health↗
USGS1991

Development of a biotinylated DNA probe for detection of infectious hematopoietic necrosis virus

A nonrad~oact~ve DNA probe assay was developed to detect and ~dent~fy infect~ous hernatopoiet~c necrosls virus (IHNV) uslng a dot blot format The probe a synthet~c DNA oligonucleot~de labeled enzymatlcally w~th biotln hybnd~zed spec~f~cally w~th nucleocaps~d mRNA extracted from Infected cells early In the vlrus repl~cation cycle A rap~d guan~dln~um th~ocyanate based RNA extraction method uslng RNAzol B and rn~crocentrifuge tubes eff~c~ently pioduced h~gh qual~ty RNA from 3 commonly used f~sh cell llnes, CHSE-214, CHH-1, and EPC The probe reacted with 6 d~verse ~solates of IHNV, but d~d not react \nth 2 related rhabdovlruses of fish viral hemorrhagic septlcemla vlrus and H~rame rhabdovlrus The b~ot~nylated probe was sensltlve detect~ng plcogram levels of target mRNA Detect~on and ~dentif~cat~on of IHNV requ~red 2d when cells wele lnoculated at n~ultiplic~t~es of infect~on (MOI) greater than 2 Flve days were necessary to detect and identify IHNV In cells lnoculated at a MO1 of 0 0002

Diseases of Aquatic Organisms↗
USGS1990

Multiplication of infectious hematopoietic necrosis virus in rainbow trout following immersion infection: whole-body assay and immunohistochemistry

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.

Journal of Aquatic Animal Health↗
USGS1990

Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus

The polymerase chain reaction [PCR) was used to amplify a portion of the nucleoprotein [NI gene of infectious hematopoietic necrosis virus (IHNV). Using a published sequence for the Round Butte isolate of IHNV, a pair of PCR pnmers was synthesized that spanned a 252 nucleotide region of the N gene from residue 319 to residue 570 of the open reading frame. This region included a 30 nucleotide target sequence for a synthetic oligonucleotide probe developed for detection of IHNV N gene messenger RNA. After 25 cycles of amplification of either messenger or genomic RNA, the PCR product (DNA) of the expected size was easily visible on agarose gels stained with ethidium bromide. The specificity of the amplified DNA was confirmed by Southern and dot-blot analysis using the biotinylated oligonucleotide probe. The PCR was able to amplify the N gene sequence of purified genomic RNA from isolates of IHNV representing 5 different electropherotypes. Using the IHNV primer set, no PCR product was obtained from viral hemorrhagic septicemia virus RNA, but 2 higher molecular weight products were synthesized from hirame rhabdovirus RNA that did not hybridize with the biotinylated probe. The PCR could be efficiently performed with all IHNV genomic RNA template concentrations tested (1 ng to 1 pg). The lowest level of sensitivity was not determined. The PCR was used to amplify RNA extracted from infected cell cultures and selected tissues of Infected rainbow trout. The combination of PCR and nucleic acid probe promises to provide a detection method for IHNV that is rapid, h~ghly specific, and sensitive.

Diseases of Aquatic Organisms↗
USGS1989

Preliminary characterization of a virus causing infectious anemia among stocks of salmonid fish in the western United States

Since 1982, anemias occurring in stocks of yearling coho ( Oncorhynchus kisutch ) and chinook salmon (Oncorhynchus tshawytscha ) have been associated with serious losses at hatcheries in the Pacific Northwest, USA. The anemia is often accompanied by infections with external fungus (e.g. Saprolegnia) or the bacterial pathogens Cytophaga psychrophila or Renibacterium salmoninarum (Holt and Rohovec 1984, Leek 1987). The losses associated with the anemia are thought to be caused by these secondary infections. Blood smears that were made from anemic fish and stained with Giemsa or pinacyanol chloride showed erythrocytic inclusions ranging in size from 1 to 8 um and varying in number per cell. Thin sections of infected red blood cells (RBC) examined by electron microscopy revealed virus particles approximately 70 nm in diameter. The virions were scattered in the cytoplasm of the RBC or contained within membrane bound organelles. These virus particles were morphologically distinct from the iridovirus, erythrocytic necrosis virus (ENV), which is also associated with anemia (Holt and Rohovec 1984, Leek 1987). Evidence suggests that the etiological agent of this new anemic disease, termed erythrocytic inclusion body syndrome (EIBS) by Leek (1987), is a previously undescribed virus infecting salmon. The purpose of this study was to experimentally transmit the disease to healthy fish, to determine the blood parameters associated with infection, and to investigate the nature of the virus associated with EIBS.

Book chapter↗
USGS1989

Concentration of infectious hematopoietic necrosis virus from water samples by tangential flow filtration and polyethylene glycol precipitation

Infectious hematopoietic necrosis virus (IHNV) was concentrated from water samples by polyethylene glycol (PEG) precipitation, tangential flow filtration (TFF), and by a combination of TFF followed by PEG precipitation of the retentate. Used alone, PEG increased virus titers more than 200-fold, and the efficiency of recovery was as great as 100%. Used alone, TFF concentrated IHNV more than 20-fold, and average recovery was 70%. When the two techniques were combined, 10-L water samples were reduced to about 300 mL by TFF and the virus was precipitated with PEG into a 1 to 2 g pellet; total recovery was as great as 100%. The combined techniques were used to isolate IHNV from water samples taken from a river containing adult sockeye salmon ( Oncorhynchus nerka ) and from a hatchery pond containing adult spring chinook salmon ( O . tshawytscha ). The combination of these methods was effective in concentrating and detecting IHNV from water containing only three infectious particles per 10-L sample.

Canadian Journal of Fisheries and Aquatic Sciences↗
USGS1989

Biochemical and antigenic properties of the first isolates of infectious hematopoietic necrosis virus from salmonid fish in Europe

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.

Journal of Aquatic Animal Health↗
USGS1989

Enhanced detection of infectious hematopoietic necrosis virus by pretreatment of cell monolayers with polyethylene glycol

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. PDF

Journal of Aquatic Animal Health↗
USGS1989

Picornaviruses and reoviruses of fishes

The number of fish viruses isolated in cell culture or observed by electron microscopy continues to increase rapidly. Until recently, most viruses that were isolated from finfish and characterized were found to be members of the Rhabdoviridae, Iridoviridae, or Herpesviridae (Wolf and Mann 1980). In a comprehensive review of fish viruses published in 1984, there were no picornaviruses and only two reoviruses listed (Wolf 1984). The expansion of aquaculture into the rearing of new species at high density in different geographic areas, and the use of improved methods of detection that include newly developed cell lines and increased sampling effort, have led to the discovery of fish viruses representing nearly all families of animal viruses. Among the newest additions, are a member of the family Picornaviridae and several new viruses that belong within the Reoviridae.

Book chapter↗