Concentration of Infectious Aquatic Rhabdoviruses from Freshwater and Seawater Using Ultrafiltration

Abstract

Infectious hematopoietic necrosis virus (IHNV), viral hemorrhagic septicemia virus, and spring viremia of carp virus were concentrated and detected from freshwater and seawater samples by using hollow-fiber ultrafiltration. Within 60 min, virus in a 50-L freshwater or saltwater sample was concentrated more than 70-fold, and virus retention efficiencies were consistently greater than 88%. Retention efficiency was highly dependent upon concentrations of column blocking and sample stabilization solutions. A large column with a surface area of 1.15 m² and a filtration capacity of 5–200 L exhibited optimal viral retention when blocked with 2% fetal bovine serum (FBS) and when the samples were supplemented with 0.1% FBS. Conversely, a small column with 100-fold less surface area and a filtering capacity of 0.5–2.0 L was optimized when blocked with 1% FBS and when the samples were supplemented with 0.1% FBS. The optimized ultrafiltration procedure was further validated with water from a tank that contained IHNV-exposed juvenile sockeye salmon Oncorhynchus nerka, resulting in an average virus retention efficiency of 91.6 ± 4.1% (mean ± SE). Virus quantification of concentrated samples demonstrated that IHNV shedding in sockeye salmon preceded mortality; shedding of the virus was observed to increase significantly as early as 7 d postchallenge and peaked at day 14, when virus levels reached 4.87 × 10³ plaque-forming units/mL. We conclude that ultrafiltration is a reliable and effective method for concentrating viable aquatic rhabdoviruses from large volumes of water and has application for the analysis of environmental water samples.

Received April 22, 2011; accepted August 4, 2011     

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.     

Infectious Hematopoietic Necrosis Virus (IHNV) in Coho Salmon

Abstract

Infectious hematopoietic necrosis virus (IHNV) causes important losses of chinook salmon Oncorhynchus tshawytscha, sockeye salmon Oncorhynchus nerka, and rainbow trout and steelhead Oncorhynchus mykiss on the west coast of North America. Although coho salmon Oncorhynchus kisutch are considered resistant to IHNV infection, the virus was detected in numerous adult coho salmon returning to Trinity River Hatchery, California, in 1985 and 1986. The virus was isolated from internal organs and ovarian fluids of these fish. Antigenic and structural polypeptides of the viruses were identical in adult coho and chinook salmon collected at the same location. Chinook salmon and rainbow trout alevins exhibited high degrees of susceptibility to IHNV obtained from adult coho and chinook salmon. Coho salmon alevins were resistant to both virus isolants.     

Survivability of Infectious Hematopoietic Necrosis Virus in Fertilized Eggs of Masu and Chum Salmon

Abstract

The possibility of vertical transmission of infectious hematopoietic necrosis virus (IHNV) was studied with the eggs of masu (cherry) salmon Oncorhynchus masou and chum salmon O. keta. The surfaces of eggs and sperm were contaminated with IHNV (10^3.8-10^4.8 50% tissue culture infective dose [TCID50]/egg) and then the eggs were fertilized. Eggs just after fertilization and embryonated eggs also were infected by injection with IHNV (10^3.8 TCID50/egg) directly into the yolk. During incubation, eggs were held in running water at 10°C. Mortality of the eggs or hatched progeny was determined and isolation of IHNV on the surface or inside of the eggs was determined during the incubation period. No mortality occurred and no virus was detected in fertile eggs from contaminated gametes. For injected eggs, IHNV was not detected on the surface of masu and chum salmon eggs after 1 d of incubation. Infectivity of IHNV inside the eggs decreased gradually and could not be detected after 1 month of incubation. This rate of IHNV reduction in the fertilized egg was similar to that found in a mixture of IHNV and homogenized yolk contents. Several individual yolk components also showed anti-IHNV activity. When eyed eggs were injected with IHNV, the embryos of both masu and chum salmon became infected, and the concentration of virus increased rapidly and reached more than 10^6.5 TCID50/fish. The cumulative mortality of eggs injected at the eyed stage for both masu and chum salmon was 90%. The susceptibilities of hatched-out larvae of masu and chum salmon to IHNV were different; cumulative mortality was more than 90% in masu salmon and 20–30% in chum salmon artificially infected with the virus. We concluded that vertical transmission of IHNV is doubtful because the virus is apparently unable to survive in eggs before the eyed stage.     

Purification of an acid-stable bovine leukocyte interferon

Bovine leukocyte interferon (BoL-IFN), produced in bovine peripheral blood leukocytes after priming and induction with Sendai virus, was concentrated by precipitation with KSCN (pH 3.5) and purified by gel column chromatography. Recovery of BoL-IFN from precipitation was higher when crude BoL-IFN containing more fetal bovine serum (FBS) was used. However, purity of BoL-IFN recovered from the gel filtration column was highest when crude BoL-IFN with no FBS was used. The use of 25% ethylene glycol in the column elution buffer resulted in over 93% recovery of the applied IFN activity, versus only 25% when buffer contained no ethylene glycol. Column-purified BoL-IFN was further concentrated by ultrafiltration and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in denaturing buffer. When crude BoL-IFN containing no FBS was used for purification, BoL-IFN from a selected column fraction applied to SDS-PAGE resulted in a single narrow band with an apparent molecular weight (MW) of 19,000 Da. Extraction of the SDS-PAGE gel resulted in a single peak of IFN activity indicating identity of the activity and the polypeptide. This proved to be a practical method for obtaining sufficient quantities of purified natural BoL-IFN for use in the production of monoclonal antibodies to BoL-IFN and other biological experiments.     

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.     

Genetic diversity of infectious hematopoietic necrosis virus from Feather River and Lake Oroville, California, and virulence of selected isolates for Chinook salmon and rainbow trout

Infectious hematopoietic necrosis virus (IHNV) is a significant pathogen of young salmonid fishes worldwide but particularly within the historical range of the Pacific Northwest and California. In the Sacramento and San Joaquin River drainages of California, IHNV outbreaks in juvenile Chinook salmon Oncorhynchus tshawytscha have been observed regularly at large production hatcheries, including Coleman National Fish Hatchery (established in 1941) and Feather River State Fish Hatchery (FRH; established in 1967), since facility operations began. Recent severe epidemics at the FRH in 1998 and 2000-2002 prompted investigations into the characteristics and potential sources of virus at this facility. Both phylogenetic analyses of a variable portion of the glycoprotein gene and serologic comparisons based on neutralization with three polyclonal rabbit sera were used to characterize 82 IHNV isolates from the Feather River watershed between 1969 and 2004. All isolates examined were in the L genogroup and belonged to one of three serologic groups typical of IHNV from California. The IHNV isolates from the Feather River area demonstrated a maximum nucleotide sequence divergence of 4.0%, and new isolates appeared to emerge from previous isolates rather than by the introduction of more diverse subgroups from exogenous sources. The earliest isolates examined from the watershed formed the subgroup LI, which disappeared coincidently with a temporal shift to new genetic and serologic types of the larger subgroup LII. Experimental challenges demonstrated no significant differences in the virulence for juvenile Chinook salmon and rainbow trout O. mykiss from selected isolates representing the principal types of IHNV found historically and from recent epidemics at FRH. While most isolates were equally virulent for both host species, one isolate was found to be more virulent for Chinook salmon than for rainbow trout.     

In Vitro Infection of Salmonid Epidermal Tissues by Infectious Hematopoietic Necrosis Virus and Viral Hemorrhagic Septicemia Virus

Abstract

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.     

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.     

Susceptibility of Four New Salmonid Cell Lines to Infectious Hematopoietic Necrosis Virus

Abstract

Four salmonid cell lines, CoE 45, CoE 115, CoE 345, and RBTE 45, were established from embryonic tissues of coho salmon Oncorhynchus kisutch and rainbow trout O. mykiss. In vitro challenges of the new lines were conducted with four isolates of infectious hematopoietic necrosis virus (IHNV). Two of the IHNV isolates used for the challenges were derived from infected tissues of rainbow trout, one was derived from chinook salmon O. tshawytscha, and the other isolate was derived from coho salmon. To standardize the virus challenges of the new cell lines, several established piscine cell lines (EPC, CHSE 214, CSE-119, RTH-149, RTG, and RTS) were challenged in the same way as the new lines. Each of the lines was challenged with virus at a single low multiplicity of infection (0.01 plaque-forming unit per cell). Virus yields were quantitated by plaque assay on epithelioma papulosum cyprini (EPC) cells on day 3. Results of the challenge experiments revealed different levels of production of virus for each isolate on the various cell lines. Overall, the new cell line derived from rainbow trout, RBTE 45, was quite susceptible to all viruses tested. The three cell lines newly derived from coho salmon embryo were not as resistant to the replication of IHNV as was the established coho salmon cell line, CSE-119. An established cell line, EPC, derived from an epithelial tumor of common carp Cyprinus carpio, remained the most susceptible to all four IHNV isolates tested.     

Comparison of porcine reproductive and respiratory syndrome virus growth in media supplemented with fetal bovine serum or a serum replacement.

Commercially available serum replacements are often used in cell culture as a cheaper and less variable substitute for fetal bovine serum (FBS). The growth of porcine reproductive and respiratory syndrome virus (PRRSV) isolates in CRL11171 cells maintained in a medium supplemented with FBS was compared with virus propagation in the same cell line maintained in the same medium with a serum replacement. The PRRSV replicated significantly better when the cell culture medium was supplemented with FBS. The results of this study have implications for the use of serum replacement-supplemented medium for PRRSV diagnosis by virus isolation.     

Viremia, virus shedding, and antibody response during natural avian polyomavirus infection in parrots

OBJECTIVE: To determine rapidity of spread and onset and duration of viremia, virus shedding, and antibody production in parrots naturally infected with avian polyomavirus (APV). DESIGN: Case series. ANIMALS: 92 parrots in 2 aviaries. PROCEDURE: Blood samples were obtained from parrots naturally exposed to APV during a 3- to 4-month period for determination of serum virus neutralizing antibody and detection of viral DNA. Nestlings from the next year's hatch were monitored for APV infection. RESULTS: The first indication of inapparent infection was viremia, which developed simultaneously with or was followed within 1 week by cloacal virus shedding and antibody production. Cloacal virus shedding continued after viremia ceased. During viremia, viral DNA was detected continuously in blood samples. Viral DNA was detected in serial cloacal swab specimens in most birds, but it was detected inconsistently in 6 birds and not detected in 3 birds, even though these birds were viremic. Duration of cloacal virus shedding was < or = 4.5 months. In 1 aviary, prevalence of infection was 88% and dissemination of virus through the 3-room building required 4.5 months. In the second aviary, a single-room nursery, prevalence of infection was > or = 90%. For all affected birds, infection could be detected 18 days after the first death. CONCLUSIONS AND CLINICAL RELEVANCE: If a single sampling is used for polymerase chain reaction detection of viral DNA, blood and cloacal swab specimens are required. In nestling nonbudgerigar parrots, cloacal virus shedding may persist for 4.5 months. Management protocols alone are sufficient to prevent introduction of APV into a nursery.     

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.     

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.     

Early Life Stage Survival and Susceptibility of Brook Trout,Coho Salmon,Rainbow Trout,and Their Reciprocal Hybrids to Infectious Hematopoietic Necrosis Virus

Abstract

Triploid (heat-shocked) and diploid groups of rainbow trout Oncorhynchus mykiss, brook trout Salvelinus fontinalis, coho salmon Oncorhynchus kisutch, and reciprocal hybrids were produced, monitored for early life stage survival, and evaluated for susceptibility to infectious hematopoietic necrosis virus (IHNV). The female rainbow trout × male brook trout triploid hybrids had significantly greater (P < 0.01) survival than the diploid hybrids of this cross. The heat-shocked hybrid group of the female rainbow trout × male coho salmon also exhibited significantly greater survival to the eyed egg stage of development than the untreated group of this hybrid. Studies of the susceptibility of treatment groups to a 1990 IHNV isolate from the Hagerman Valley were conducted by using a standardized immersion exposure procedure at one or two different mean body weights. The diploid brook trout and coho salmon and two triploid hybrids (female rainbow trout × male brook trout or male coho salmon) were significantly less (P < 0.05) susceptible to IHNV than the pure-species diploid and triploid rainbow trout groups.     

Evaluation of a new sandwich enzyme-linked immunosorbent assay for detection of bovine viral diarrhea virus in unprocessed fetal bovine serum.

A new sandwich enzyme-linked immunosorbent assay (S-ELISA) kit that uses raw (unprocessed) fetal bovine serum (FBS) as the testing sample was evaluated for upstream bovine viral diarrhea virus (BVDV) testing. Pooled FBS samples (n = 84) were tested using the S-ELISA. Thirty serum samples originating from persistently infected (PI) calves that had been confirmed by virus isolation (VI) as BVDV positive and another 30 samples previously confirmed by VI as BVDV negative were also evaluated. Of the 84 field samples, the S-ELISA detected 13 (15.5%) BVDV-positive specimens. When these 13 positive samples were tested by VI and immunofluorescent assay, 11 (84.6%) were positive and 2 (15.4%) were negative. The S-ELISA was positive for all 30 PI samples (100%) and negative for all 30 negative samples (100%). These data indicate that the new kit is a relatively reliable diagnostic tool and can be considered for upstream detection of BVDV-contaminated raw FBS pools.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry

Abstract

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.     

Single-dilution enzyme-linked immunosorbent assay for quantification of antigen-specific salmonid antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed on the basis of testing a single dilution of serum to quantify the level of antibody to the p57 protein of Renibacterium salmoninarum in sockeye salmon (Oncorhynchus nerka). The levels of antibody were interpolated from a standard curve constructed by relating the optical densities (OD) produced by several dilutions of a high-titer rainbow trout (O. mykiss) antiserum to the p57 protein. The ELISA OD values produced by as many as 36 test sera on each microplate were compared with the standard curve to calculate the antigen-specific antibody activity. Repeated measurements of 36 samples on 3 microplates on each of 6 assay dates indicated that the mean intraassay coefficient of variation (CV) was 6.68% (range, 0-23%) and the mean interassay CV was 8.29% (range, 4-16%). The antibody levels determined for the serum sample from 24 sockeye salmon vaccinated with a recombinant p57 protein generally were correlated with the levels determined by endpoint titration (r2 = 0.936) and with results from another ELISA that was based on extrapolation of antibody levels from a standard curve (r2 = 0.956). The single-dilution antibody ELISA described here increases the number of samples that can be tested on each microplate compared with immunoassays based on analysis of several dilutions of each test serum. It includes controls for interassay standardization and can be used to test fish weighing <3 g.     

Long-lasting stability of vaccinia virus (orthopoxvirus) in food and environmental samples

Poxviruses are known to remain infectious in the scabs of patients for months to years. The aim of this study was to investigate viral stability in storm water, food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm water, storm water supplemented with either fetal calf serum (FCS) or potting soil was stored at two different temperatures (refrigerator, room temperature; 4 degrees C/25 degrees C). In addition, we analysed the viability of VACV M1 on the surface of bread, salad, sausages and gauze bandages stored at 4 degrees C. Samples were titrated in MA 104 cells and the presence of viral DNA was demonstrated by orthopoxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds of food, bandage and water samples except for storm water supplemented with potting soil was possible. Viral DNA was detected in almost all samples by PCR. Prolonged experiments with VACV M1-spiked storm water and storm water supplemented with FCS revealed that samples kept at 4.5 degrees C are infectious for up to 166 days. Our data demonstrate that VACV M1 has a longlasting stability in water and food. The results obtained during this study should be taken into account for risk assessment calculations for poxvirus transmission. Implying that variola virus and vaccinia virus behave in a similar way, our data call for sophisticated countermeasures in cases of a variola release in biological warfare.