Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus,a rhabdovirus from salmonid fish

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon‐farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk‐derived proteins as bovine caseins or casein‐derived peptides at different stages during the course of IHNV infection. The results indicate that the 3‐h fraction of casein and α_S2‐casein hydrolysates reduced the yield of infectious IHNV in a dose‐dependent manner and impaired the production of IHNV‐specific antigens. Hydrolysates of total casein and α_S2‐casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein‐treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV‐inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.     

Comparative sensitivity of five fish cell lines to wild type infectious haematopoietic necrosis virus from two Oregon sources

Abstract. Five fish cell lines (CHSE-214, STE-137, RTG-2, EPC and FHM) were compared for sensitivity to infectious haematopoietic necrosis virus (IHNV) from samples obtained from naturally-infected fish. Infectious ovarian fluids were obtained from steelhead trout, Salmo gairdneri Richardson, at the Round Butte Hatchery in central Oregon and tissue homogenates were prepared from chinook salmon, Oncorhynchus tshawytscha (Walbaum), alevins during an IHN virus epizootic at the Elk River Hatchery in coastal Oregon. The only lines to show characteristic viral cytopathology by plaque or end-point dilution assay for the steelhead trout virus isolate were the EPC and FHM cell lines. The chinook salmon isolates produced CPE in CHSE-214, STE-137, FHM and EPC cells. The titre of the salmon virus isolate was 10-50-fold higher on FHM and EPC cells by both assay methods. Neither by end-point nor plaque assay did the Round Butte or Elk River isolates produce CPE on RTG-2 cells. With both virus isolants both cell lines showed that greater sensitivity was obtained with plaque assay than with end-point titration. Pre-treatment of the cells with the polycation, polybrene, did not increase the virus titre in either assay. However, a transient enhancement in virus titre was observed in polybrene-treated STE-137 and CHSE-214 cells.     

Cross‐neutralization studies with salmonid alphavirus subtype 1–6 strains: results with sera from experimental studies and natural infections

The serological reactivity between strains of each of the six currently genetically defined subtypes of salmonid alphavirus (SAV) was examined by comparison of homologous and heterologous virus neutralization titres on sera from experimentally infected fish. With the exception of the level of SAV subtype 6 neutralization by heterologous sera, good cross‐neutralization was detected between all subtypes, albeit with variation in geometric mean titres when each subtype‐specific serum set was tested against the panel of virus subtypes. A similar pattern was evident with field sera, except that heterologous neutralization of the SAV6 strain was more evident. In only 23% of available pairwise comparisons was the homologous titre recorded with an experimentally derived serum fourfold or greater than the heterologous titre, and in only two instances was this difference demonstrated in both directions. No virus strains consistently met the old serology‐based criteria (Sub‐committee on Inter‐relationships Among Catalogued Alphaviruses) to be considered separate subtypes within an alphavirus species. Only when testing with an SAV subtype‐2‐specific monoclonal antibody was a major difference between homologous and heterologous neutralization capacity evident. These results provide new direct or indirect information in terms of SAV classification, vaccine efficacy and the selection and validation of reagents for serological and immunological diagnostic purposes.     

Quantitative expression (Walbaum) of immunological factors in rainbow trout, Oncorhynchus mykiss (Walbaum), after infection with either Flavobacterium psychrophilum, Aeromonas salmonicida, or infectious haematopoietic necrosis virus

To further enhance our understanding of immunological gene expression in rainbow trout, Oncorhynchus mykiss, after infection with naturally occurring pathogens, a series of probes and primers were developed for the quantification of immune factors. Separate groups of specific-pathogen-free rainbow trout were infected with either Flavobacterium psychrophilum, Aeromonas salmonicida or infectious haematopoietic necrosis virus (IHNV). Three different concentrations of each pathogen were used and samples from infected and mock-infected fish were taken at either 1 or 5 days after infection. Ten fish were sampled at each time point for individual sections of liver, spleen and head kidney. Organ specimens from five of the fish were used to re-isolate and quantify the pathogen at the time the samples were taken. Total RNA was extracted from the organs of the remaining five animals. Using real-time polymerase chain reaction with fluorescent-labelled probes, the RNA from these organs was examined for the level of expression of the following immunological factors; an interferon related protein (MX-1), interleukin-8 (IL-8), the cytotoxic T-cell marker CD-8 and complement factor C3 (C3). They were also measured for the level of beta-actin, which was used as a standardization control for cellular RNA expression. Infection with IHNV produced the greatest change in expression level for all the immunological related factors examined in this study. IHNV elicited the best dose response profile, which was typically seen at 5-days post-infection for MX-1, C3, IL-8 and CD-8. Infection with A. salmonicida and F. psychrophilum showed elevated, but variable expression levels for several of the genes tested.     

First detection and isolation of infectious haematopoietic necrosis virus from farmed rainbow trout in Nyeri County,Kenya

Infectious haematopoietic necrosis virus (IHNV) is the causative agent of infectious haematopoietic necrosis, a disease of salmonid responsible for great economic losses. The disease occurs in most parts of the world where rainbow trout is reared but has not been previously reported in Kenya. In this study, rainbow trout fry and growers from two farms in Nyeri County were screened for IHNV. Whole fry (n = 4 from each farm) and kidney samples from growers (n = 15 and n = 6 from the two farms, respectively) were collected and preserved for cell culture examination or PCR analysis. Screening of samples was done by PCR followed by sequencing of the glycoprotein gene of the virus. Demonstration of the virus was done by propagation in EPC cells followed by the indirect fluorescence antibody test (IFAT). The results revealed the presence of IHNV at low prevalence of 0.1 and 0.4 for the two farms. The virus was confirmed both by IFAT and by partial sequencing of the G gene. Phylogenetic analysis revealed that the Kenyan isolates were identical to those of the J genogroup found mostly in Asia. The findings have implications for biosecurity measures and import regulations for the Kenyan rainbow trout industry.     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers

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.     

Appearance of Infectious Hematopoietic Necrosis Virus in Rainbow Trout,Oncorhynchus mykiss,During Seawater Adaptation

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm in the South Sea of Korea during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo‐214 cells. The cell culture supernatant showing CPE contained specific 1527‐bp fragment for the infectious hematopoietic necrosis virus (IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results suggest that this IHNV isolate might have been introduced to rainbow trout farm (land‐based culture system) in Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.     

The route of entry and progression of infectious haematopoietic necrosis virus in Oncorhynchus mykiss (Walbaum): a sequential immunohistochemical study

Abstract. 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.     

Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool

Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.     

QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) × Yellowstone cutthroat (Oncorhynchus clarki bouvieri) trout cross

Infectious hematopoietic necrosis virus (IHNV) is a major constraint to rainbow trout culture. Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) have greater resistance to this virus than do rainbow trout (O. mykiss), but the genetic mechanism of this resistance is not understood. We conducted a genome scan using a backcross of cutthroat trout into a rainbow trout background to estimate the number and locations of quantitative trait loci (QTL) associated with IHNV resistance and growth in trout. IHNV resistance was considered in terms of both survival (binary trait) and days to death (quantitative trait). The genetic map was scanned using interval mapping via two different approaches: one model considered survival alone and a second two-part model combined both survival and days to death. Three QTL were significantly (P ≤ 0.05) associated with virus resistance genome-wide, explaining 32.5% of the phenotypic variation. Cutthroat alleles at two of these QTL resulted in increased resistance to the pathogen, as expected. No growth QTL were detected in this cross. We suggest that these traits are genetically independent.     

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)‐GN438A expresses VP2 gene‐encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.     

Disinfectants against cultured Infectious Salmon Anaemia (ISA) virus: the virucidal effect of three iodophors, chloramine T, chlorine dioxide and peracetic acid/hydrogen peroxide/acetic acid mixture

Following the infectious salmon anaemia outbreak in Scotland in May 1998, there was a strong interest in evaluating alternatives to sodium hypochlorite for the disinfection of fish farming equipment. Because no information was available, a programme of laboratory testing of several disinfectants against cultured ISA virus was commenced.

Cultured ISA virus was grown on permissive cell lines and a stock stored at −80 °C. Disinfectant assays were set up using a procedure similar to that recommended for viruses of higher animals under the UK Animal Health Act (1981). Dilutions of disinfectant at the manufacturer's recommended dose were made in W.H.O. hard water at 4 °C and ISA virus was added for 5 min exposure. Neutralizer was added to inactivate the disinfectant and the samples were dialysed. Residual and starting virus was titrated on SHK-1 cells using a focus-forming assay (FFA). Briefly, cell layers were fixed with acetone, incubated with diluted ISA virus monoclonal antibody 3H6F8, then incubated with diluted goat anti-mouse Ig phosphatase conjugate and bound conjugate visualised with fast red TR/napthol-AS MX stain. Stained foci were counted.

Virucidal reductions in titre of >4 log₁₀ focus forming units (ffu)/ml were obtained using the six types of virucide. A virus titre reduction of log₁₀ 4 is the approved disinfectant inactivation standard in the UK according to the current DEFRA guideline protocol for disinfectant testing for veterinary viruses. According to this definition six branded disinfectants were found effective for the disinfection of fish farming equipment or fish ova at the manufacturer's recommended dose for 5 min duration.