Study of the viral interference between infectious pancreatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)

The resistance of rainbow trout (Oncorhynchus mykiss) to an infectious haematopoietic necrosis virus (IHNV) challenge following a preceding non-lethal infection with infectious pancreatic necrosis virus (IPNV) was investigated through experimental dual infections. Trout initially infected with IPNV were inoculated 14 days later with IHNV. Single infections of trout with 1 of the 2 viruses or with cell culture supernatant were also carried out and constituted control groups. No mortality was noted in fish after a single infection with IPNV. This virus had no influence on the head kidney leucocyte phagocytic activity and plasma haemolytic complement activity. IHNV induced a high mortality (72%) and reduced the macrophage phagocytic activity and complement haemolytic activity. It also induced a late production of anti-IHNV antibodies which occurred after clearance of the virus in the fish. In trout co-infected with both viruses, a mortality rate of 2% occurred and the immune parameters were similar to those observed in the fish infected with IPNV only, demonstrating that in co-infected trout IPNV inhibits the effects of IHNV. The studied parameters did not allow us to define the mechanism of interference occurring between these 2 viruses, but some hypothesis are put forward to explain the interference between the 2 viruses.     

Inter-laboratory comparison of cell lines for susceptibility to three viruses: VHSV, IHNV and IPNV.

Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.     

Characteristics of inhibition of infectious pancreatic necrosis virus (IPNV) by normal rainbow trout Oncorhynchus mykiss serum

We studied the characteristics of rainbow trout serum (RTS) inhibitory activity against infectious pancreatic necrosis virus (IPNV). Serum inhibition was related to the serum source and host cell in which the virus had been propagated. IPNV was more efficiently inhibited by RTS in salmonid cell lines than in non-salmonid cell lines, with inhibition highest in rainbow trout gonad (RTG)-2 cells. The RTS sensitivity of the virus was modified by the cell line through which the virus passed, with multiple passages through Chinook salmon embryo (CHSE)-214 cells producing a virus that was less sensitive to RTS. The RTS inhibition level was dependent on cell density: at a cell density of < or = 2 x 10(5) cells ml(-1), inhibition was insignificant (tissue culture infective dose 50% = 10(-1.1) TCID50 ml(-1) reduction); however, above a density of 3 x 10(5) cells ml(-1), the inhibition level was very high (> or = 10(-6.3) TCID50 ml(-1) reduction). The salmonid sera tested showed high inhibition, except for brook trout serum (BTS), while non-salmonid sera did not inhibit IPNV, replication on RTG-2 cells. Pretreatment of cultured cells with RTS prior to exposure did not affect inhibition of IPNV and thus did not mask a viral receptor. The RTS inhibition level was dependent on the time of serum addition, with inhibition being maintained for at least 16 h postinfection. Pretreatment of IPNV revealed that the virus is directly inhibited by RTS, and more strongly so when RTS is present during viral replication.     

Molecular identification of a new strain of infectious pancreatic necrosis virus (IPNV) in a Croatian rainbow trout (Oncorhynchus mykiss) farm

Through the regular clinical control of a Croatian rainbow trout (Oncorhynchus mykiss) farm, aberrant fry behaviour was revealed whereby diagnostic protocols of sampled fish evidenced the presence of the infectious pancreatic necrosis virus (IPNV). Identification of an IPNV type isolated from the source farm was necessary to develop an adequate epizootiological survey at the country level. A previous IPNV outbreak and virus molecular characterization had been reported on only one previous occasion. Amplification of a 359‐bp fragment (GenBank HM036118 ) of the capsid protein VP2 variable region supported the hypothesis of the European origin of the isolate that clusters within genogroup III, belonging to A2 serotype, although showing a distinction from the previously reported strain.     

Distribution of marine birnavirus in cultured olive flounder Paralichthys olivaceus in Korea

Surveys of marine birnavirus (MABV) were undertaken in cultured olive flounder Paralichthys olivaceus from the south and west coastal areas and Jeju in Korea during the period January 1999 to April 2007. MABV was detected in all seasons from the fry, juveniles and adult fish from the areas examined. Evident cytopathic effects of the virus including rounding and cell lysis were observed in chinook salmon embryo (CHSE-214) and rainbow trout gonad (RTG-2) cells, but not in fathead minnow (FHM) and epithelial papilloma of carp (EPC) cells. Nucleotide sequences of the VP2/NS junction region of the Korean isolates showed 97.8% ~ 100% similarity, and they belonged to the same genogroup. Cross neutralization tests with serotype-specific rabbit antisera against MABV strains exhibited a close antigenic relationships between strains, and were distinct from infectious pancreatic necrosis virus (IPNV) strains. Coinfection of MABV with bacteria (Streptococcus iniae, Vibrio spp.) and viruses (nervous necrosis virus, lymphocystis disease virus, viral hemorrhagic septicemia virus) was observed.     

First isolation of an aquatic birnavirus from farmed and wild fish species in Australia

During routine sampling and testing, as part of a systematic surveillance program (the Tasmanian Salmonid Health Surveillance Program), an aquatic birnavirus was isolated from 'pin-head' (fish exhibiting deficient acclimatisation on transfer to saltwater) Atlantic salmon Salmo salar, approximately 18 mo old, farmed in net-pens located in Macquarie Harbour on the west coast of Tasmania, Australia. The isolate grows readily in a range of fish cell lines including CHSE-214, RTG-2 and BF-2 and is neutralised by a pan-specific rabbit antiserum raised against infectious pancreatic necrosis virus (IPNV) Ab strain and by a commercial pan-specific IPNV-neutralising monoclonal antibody. Presence of the virus was not associated with gross clinical signs. Histopathological examination revealed a range of lesions particularly in pancreatic tissue. The virus was localised in pancreas sections by immunoperoxidase staining using the polyclonal antiserum and by electron microscopy. Examination by electron microscopy demonstrated that the virus isolated in cell culture (1) belongs to the family Birnaviridae, genus Aquabirnaviridae; (2) was ultrastructurally and antigenically similar to virus identified in the index fish; (3) is related to IPNV. Western blot analysis using the polyclonal rabbit antiserum confirmed the cross-reactions between various aquatic birnavirus isolates. In addition, PCR analysis of isolated viral nucleic acid from the index case indicated that the virus is more closely related to IPNV fr21 and N1 isolates than to other birnavirus isolates available for comparison. Sampling of other fish species within Macquarie Harbour has demonstrated that the virus is present in several other species of fish including farmed rainbow trout Oncorhynchus mykiss, wild flounder Rhombosolea tapirina, cod Pseudophycis sp., spiked dogfish Squalus megalops and ling Genypterus blacodes.     

The Swiss/ICR (Ha) albino mouse as an experimental host for infectious pancreatic necrosis virus of trout

Intracranial inoculation of infectious pancreatic necrosis virus (IPNV), a pathogen of several species of trout, produced pancreatic necrosis in suckling Swiss albino mice. Peri-nuclear halos, cytoplasmic vacuoles, and necrosis were found in histologic sections of pancreas taken from mice killed 21 days post-inoculation. Virus was recovered from the pancreas of mice killed as early as 10 days post-inoculation. Rivers' postulates were fulfilled. Virus recovered from the infected mouse pancreas was neutralized by IPNV specific antiserum. The significance of the mouse as an experimental host is discussed.     

Impact of genotyping strategy on the accuracy of genomic prediction in simulated populations of purebred swine

Single-step genomic BLUP (ssGBLUP) has been widely used in genomic evaluation due to relatively higher prediction accuracy and simplicity of use. The prediction accuracy from ssGBLUP depends on the amount of information available concerning both genotype and phenotype. This study investigated how information on genotype and phenotype that had been acquired from previous generations influences the prediction accuracy of ssGBLUP, and thus we sought an optimal balance about genotypic and phenotypic information to achieve a cost-effective and computationally efficient genomic evaluation. We generated two genetically correlated traits (h² = 0.35 for trait A, h² = 0.10 for trait B and genetic correlation 0.20) as well as two distinct populations mimicking purebred swine. Phenotypic and genotypic information in different numbers of previous generations and different genotyping rates for each litter were set to generate different datasets. Prediction accuracy was evaluated by correlating genomic estimated breeding values with true breeding values for genotyped animals in the last generation. The results revealed a negligible impact of previous generations that lacked genotyped animals on the prediction accuracy. Phenotypic and genotypic data, including the most recent three to four generations with a genotyping rate of 40% or 50% for each litter, could lead to asymptotic maximum prediction accuracy for genotyped animals in the last generation. Single-step genomic best linear unbiased prediction yielded an optimal balance about genotypic and phenotypic information to ensure a cost-effective and computationally efficient genomic evaluation of populations of polytocous animals such as purebred pigs.     

Do fish lymphocytes secrete interferon-γ?

Macrophage activating factor (MAF)-containing supernatants, generated by mitogen (Con A/PMA) stimulation of rainbow trout leucocytes, were found to confer viral resistance (interferon, IFN, activity) on a rainbow trout epithelial cell line challenged with infectious pancreatic necrosis virus. Both the MAF and IFN activities co-eluted by HPLC size exclusion chromatography and showed similar sensitivities to acid (pH 2), temperature (60° C) and trypsin. The mode of induction of this IFN, its acid and temperature sensitivity and its possible MAF activity suggest that fish leucocytes can secrete an IFN-γ like molecule.     

Effects of salmonid fish viruses on Mx gene expression and resistance to single or dual viral infections

We examined the ability of several fish viruses to induce protection against homologous or heterologous viruses in single or double infections, and assessed whether such protection is correlated with innate immunity or expression of the Mx gene. Monolayers of BF2 cells pre-treated with supernatants of brown trout (Salmo trutta L.) macrophage cultures that had been stimulated with either polyinosinic polycytidylic acid (poly I:C) or viruses, such as infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) or a mixture of the two, showed varying degrees of protection against viral infections. The virus showing the strongest induction was IPNV, and the antiviral activity against IHNV was also high: around 6 log(10) reduction of virus yield. Consequently, the IPNV-IHNV co-infection yield was also reduced by varying amounts. In vivo, the cumulative mortality observed in the IPNV-IHNV co-infected fish was always less than that in those with a single infection. Stimulation with poly I:C for 7 days significantly reduced cumulative mortality in single-infected fish, but not in the double-infected, in which the IPNV was the only virus isolated from moribund animals. By RT-PCR, Mx was expressed in all the organ samples tested (kidney, liver and spleen) from virus-stimulated fish at 1, 2 and 3 days. By qRT-PCR the extent and timing of Mx expression was shown to differ in the poly I:C and the single or dual viral infections. The highest increase in Mx expression (21.6-fold above basal levels) occurred (after 24 h) in fish infected with the IHNV, and expression remained high until day 7. Mx expression in fish infected with IPNV peaked later, at 2 days post infection, and also remained high until day 7. The dual infection with IPNV-IHNV induced high Mx expression on day 1, which peaked on day 2 and remained high until day 7. These results indicate that activation of the immune system could explain the interference and loss of IHNV in the IPNV-IHNV co-infections.     

An antiviral state induced in Chinook salmon embryo cells (CHSE-214) by transfection with the double-stranded RNA poly I:C

Type I interferons (IFN alpha and beta) convert vertebrate cells into an antiviral state by inducing expression of proteins that inhibit virus replication. In humans and mice, Mx proteins constitute one family of interferon-induced antiviral proteins. Mx genes have recently been cloned from Atlantic salmon and rainbow trout. Moreover, double-stranded RNA (dsRNA) and type I IFN-like activity have been shown to induce Mx protein in salmonid cells. Chinook salmon embryo cells (CHSE-214 cells) have been suggested to have a defect in the IFN-system because the dsRNA polyinosinic polycytidylic acid (poly I:C) failed to induce an antiviral state in the cells. We have studied this phenomenon more closely in the present work. CHSE-214 cells were either transfected with poly I:C or incubated with poly I:C without transfection reagent. The cells were then studied for Mx protein expression and protection against infectious pancreatic necrosis virus (IPNV) infection. The results showed that cells transfected with poly I:C were protected from IPNV infection, whilst cells incubated with poly I:C were not protected. Cells transfected with the double-stranded DNA poly dI:dC were also not protected against IPNV. Mx protein was expressed in CHSE-214 cells upon transfection with poly I:C, but not after incubation with poly I:C alone. Stimulation of CHSE-214 cells with supernatants from cells transfected with poly I:C, induced protection against IPNV, indicating production of type I IFN-like activity. These results suggest that CHSE-214 cells in fact are able to produce type I IFN, but may have defects in the mechanisms mediating uptake of poly I:C or may degrade unprotected poly I:C.     

Frequent occurrence of apoptosis is not associated with pathogenic infectious pancreatic necrosis virus (IPNV) during persistent infection

Infectious pancreatic necrosis virus (IPNV), a member of the genus Aquabirnavirus and family Birnaviridae, is an unenveloped icosahedral virus with two segments of double-stranded RNA. IPNV causes acute infection in salmonid fry and fingerlings with high mortality. However, this mortality is low as the age increases and survivors become IPNV-carrier fish. In this study, IPNV persistent infection was established in rainbow trout with no clinical signs or mortality. TUNEL staining and immunohistochemistry showed that IPNV antigen-positive cells did not have an apoptotic nucleus in almost all tissue sections and leucocyte smears, indicating that apoptosis was not induced in IPNV antigen-positive cells. The IPNV genome detected by in situ RT-PCR was more frequent than detection of the IPNV antigen by immunohistochemistry in the kidney, spleen, and liver. This result implies that the successive replication would not occur in many IPNV-infected cells. Further, apoptotic cells were predominant in the tissue sections where the signal-positive cells were frequently detected. Therefore, the presence of apoptosis in this study might be associated with host defense mechanisms, which eliminates IPNV-infected cells by the recognition of IPNV genome at the early stage of infection.     

Molecular filtration for recovery of waterborne viruses of fish

The effectiveness of tangential flow filtration (TFF) for the recovery of infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) from large volumes of water was evaluated. In laboratory studies, virus recovery from IHNV-seeded water following concentration by TFF was approximately 13%. However, the addition of 0.1 and 1% fetal bovine serum to deionized water stabilized the virus, increasing virus recoveries to 95%. The addition of 0.03 and 0.3% beef extract resulted in IHNV recoveries of 80 and 61%, respectively. Similar results were obtained with IPNV-seeded water. Field studies using the TFF procedure were conducted with water from areas where IHNV is endemic. IHNV was detected in effluent from an adult steelhead trout (Salmo gairdneri) holding pond at an estimated concentration of 1 PFU/5 ml of water. It was also detected at levels of 1 PFU/50 ml in water from a 2-m-diameter circular tank containing IHNV-infected steelhead trout fry. IHNV isolated in samples taken from the Metolius River was detected by TFF at estimated levels of 1 PFU/3 liters.     

Evaluation of the Best Linear Unbiased Prediction in Mixed Linear Models with Estimated Variance Components by Means of the MSE of Prediction and the Genetic Selection Differential

Prediction in mixed linear models by Henderson 's (1972) BLUP (Best Linear Unbiased Prediction) requires knowledge of the underlying variance/covariance components to have the property ‘best’. In breeding value prediction these parameters are not known, generally. They have to be replaced by estimations and BLUP becomes estimated BLUP (EBLUP). The aim of this investigation was the evaluation of EBLUP with help of a designed simulation experiment. Criteria used for the evaluation were the mean squared error (MSE) and the (genetic) selection differential (GSD). Besides, an idea of the overestimation of the accuracy of EBLUP by the naive MSE approximation based on the MSE formulas of BLUP with variance component estimations instead of unknown parameters is given.     

Polymorphisms in major histocompatibility complex class IIα genes are associated with resistance to infectious hematopoietic necrosis in rainbow trout,Oncorhynchus mykiss (Walbaum, 1792)

Infectious hematopoietic necrosis is a serious viral disease of salmonids, including rainbow trout Oncorhynchus mykiss, and causes tremendous economic losses to the rainbow trout farming industry. Major histocompatibility complex (MHC) genes are crucial elements of adaptive immunity in vertebrate organisms and have been linked with the resistance to numerous pathogenic diseases. In this study, polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP) followed by cloning and sequencing were used to examine polymorphisms in the DAA genes (specifically DAA exon 2 of MHC class IIα) of rainbow trout and investigate their association with the infectious hematopoietic necrosis virus (IHNV) resistance in rainbow trout. Seventeen alleles were resolved, including 13 novel alleles. Individuals possessed between two and five alleles, indicating that the genome harbours at least three closely‐related DAA exon 2 loci. The ratio of non‐synonymous to synonymous nucleotide substitutions suggested that DAA exon 2 is under positive selection. A greater variability of amino acids and non‐synonymous nucleotide substitution rate was evident in the peptide‐binding region (PBR) than in the non‐PBR (27.75%). Importantly, the analyses revealed that certain MHC class IIα alleles appear to confer resistance to IHNV in rainbow trout, while others confer susceptibility. The most common alleles in the resistant populations of rainbow trout, Onmy‐DAA*1301 and Onmy‐DAA*0304, confer resistance to IHNV and were not present in the susceptible population. Hence, these alleles may be ideal molecular markers that can assist the breeding of IHNV resistance in rainbow trout.     

Molecular filtration for recovery of waterborne viruses of fish.

The effectiveness of tangential flow filtration (TFF) for the recovery of infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) from large volumes of water was evaluated. In laboratory studies, virus recovery from IHNV-seeded water following concentration by TFF was approximately 13%. However, the addition of 0.1 and 1% fetal bovine serum to deionized water stabilized the virus, increasing virus recoveries to 95%. The addition of 0.03 and 0.3% beef extract resulted in IHNV recoveries of 80 and 61%, respectively. Similar results were obtained with IPNV-seeded water. Field studies using the TFF procedure were conducted with water from areas where IHNV is endemic. IHNV was detected in effluent from an adult steelhead trout (Salmo gairdneri) holding pond at an estimated concentration of 1 PFU/5 ml of water. It was also detected at levels of 1 PFU/50 ml in water from a 2-m-diameter circular tank containing IHNV-infected steelhead trout fry. IHNV isolated in samples taken from the Metolius River was detected by TFF at estimated levels of 1 PFU/3 liters.     

Comparison of two birnavirus-rhabdovirus coinfections in fish cell lines

Aquabirnaviruses, such as the infectious pancreatic necrosis virus (IPNV), Novirhabdoviruses, such as the infectious hematopoiteic necrosis virus (IHNV) and the viral hemorrhagic septicemia virus (VHSV), cause considerable losses to the salmonid industry worldwide. Coinfections of 2 viruses have been described, but the interactions between rhabdoviruses and birnaviruses have not been examined closely. Using virus titration, flow cytometry and RT-PCR assays, we compared the effect of IPNV on the replication of IHNV and VHSV in tissue culture cells. RT-PCR assays indicated that simultaneous infection of IPNV with VHSV does not affect the replication of the rhabdovirus either in the first or successive passages; the infective titers were similar in single and double infections. In contrast, coinfection of IPNV with IHNV induced a fall in infectivity, with reduced expression of IHNV viral antigens in BF-2 cells from Lepomis macrochirus and a loss of 4.5 log10 units of the infective titer after 3 successive passages. It was possible to stimulate BF-2 cells to produce significant interferon-like activity against IHNV but not against VHSV.