Inactivation of infectious pancreatic necrosis virus for vaccine use

Abstract. Formalin and β-propiolactone (BPL) were compared for efficacy in inactivating infectious pancreatic necrosis virus (IPNV) for vaccine use. Incubation with a 1:200 dilution of formalin at 20°C for four days or longer, or a 1:200 dilution of BPL at 4°C for six days or longer, completely inactivated the virus infectivity. However, whereas treatment with formalin caused only a slight reduction in anti-genicity as titrated by in vitro tests, treatment with BPL destroyed over 50% of the antigenicity of the virus. Virus inactivated with formalin also proved highly effective at inducing high titres of neutralizing antibody in trout.     

Persistent infections in salmonid fish cells with infectious pancreatic necrosis virus (IPNV)*

Abstract. Two cell lines that are persistently infected with IPN virus have been established. Viral persistence in the cultures was demonstrated by the detection of infectious virus (10²-10^5,5 TCID/ml₅₀) in the culture fluids, by specific immunofluorescence, and by their resistance to superinfection by homologous virus. The growth, rate of these cells was indistinguishable from normal, uninfected cells. Virus stocks harvested from persistently infected cultures contained a greater proportion of defective, interfering particles than those from lytically infected cells which suggests that these particles may be responsible for the maintenance of these carrier cultures.     

Genetic analysis of infectious pancreatic necrosis virus from Scotland

Infectious pancreatic necrosis (IPN) is a highly contagious disease of young salmonid fish, and is one of the most serious economic diseases in aquaculture. In Scotland, an increase in IPN virus (IPNV) outbreaks in seawater Atlantic salmon, Salmo salar, has been reported in recent years. The aim of this study was to analyse the VP2 gene from recent IPNV isolates from Scotland, to determine whether there are epidemiological links between IPNV isolates from farms (13), wild fish (17) and the environment (6) in order to investigate potential wild and farmed fish interactions. Comparison of the nucleotide sequence of the VP2 gene revealed that 34 of 36 isolates were 97.1-100% similar and the deduced amino acid sequences showed 97-100% identity. Two isolates from wild fish exhibited the most divergence at 85-87.3% similarity to the other isolates at the nucleotide level and 88.2-90.8% identity at the deduced amino acid level. Phylogenetic analyses revealed that 34 of 36 of the isolates from Scotland were genetically closely related to the A2 (Sp) serotype of IPNV. The two wild isolates from seatrout, Salmo trutta, and flounder, Platichthys flesus, were most closely related to the European A5 (Te) serotype. This study represents a comprehensive IPNV phylogenetic study that indicates that there are closely related or identical isolates in circulation in the marine environment, which adds evidence that disease interactions between wild and farmed fish may occur. This type of analysis is a useful tool in the management and control of fish diseases because it can assist in the identification of epidemiological links and highlight potential risks to aquaculture.     

Serological relationships among five strains of infectious pancreatic necrosis virus

Abstract. The serological relationships of five strains of infectious pancreatic necrosis virus (IPNV) were examined by cross-neutralization, cross-fluorescent antibody (FA) and cross-immunodiffusion (ID) tests. Few serological relationships among these strains were observed by the cross-neutralization test, which is consistent with previous studies. Some cross reactions were observed by the FA test when antisera were reacted with cells infected with heterologous strains of IPNV. However, close antigenic relationships were demonstrated among these strains of IPNV by using the ID test when antisera to each strain of IPNV were titrated with their respective or heterologous antigens. The results of the present study showed that substantial antigenic relationships exist among the strains of IPNV examined. It is suggested that strain specific antigens which can be detected by virus neutralization and FA tests might exist on the surface of the virion. The FA test proved to be a useful method for detecting viral antigens of several strains of IPNV in tissue culture cells since monovalent antiserum reacted with homologous and heterologous IPNV antigens.     

Antigenic diversity of eastern Canadian isolates of infectious pancreatic necrosis virus

A collection of infectious pancreatic necrosis virus (IPNV) isolated in five provinces of eastern Canada was analysed by an antigen-coated immunosorbent assay and by neutralization tests using selected monoclonal and polyclonal antibodies. Relevant antigenic sites of the two major capsid proteins, VP2 and VP3, were simultaneously compared. The A1 serotype was predominant and no significant variations of VP2 epitopes were observed. However, two subtypes could be distinguished on the basis of one or two epitopes on VP3. Other serotypes such as A6, A7 or A8 have been detected in piscicultures of New Brunswick and Nova Scotia. The antigenic characterization of IPNV strains appears of interest for epidemiological studies.     

Virion glycosylation governs integrity and infectivity of infectious pancreatic necrosis virus

The possible importance of the O-linked glycosylation in virion stability and infectivity of infectious pancreatic necrosis virus (IPNV) was analysed. Enzymatic treatment with O-glycosidase of radiolabelled virions under different ionic conditions, to allow for possible alternative exposure of glycosidic enzyme cleavage sites, did not alter the specific infectivity of virions re-isolated after rate-zonal centrifugation in glycerol gradients. As an alternative method to assess the significance of carbohydrates in IPNV integrity, periodate oxidation in the presence of an aldehyde quencher was chosen. Following re-isolation of viruses, a 3-5 (10)log-unit reduction in specific infectivity was revealed and, at higher concentrations, a total disruption or virion aggregation was observed. The loss of infectivity of intact virions was not because of a lack of attachment to cells. Additionally, re-evaluation of reading values from UV-spectra of purified IPNV yielded a specific infectivity of 3 × 10(11) TCID(50)-units mg(-1) of protein and a ratio of 40 virions per TCID(50)-unit in the CHSE-214 cell system.     

Studies on the carrier state of infectious pancreatic necrosis virus infections in rainbow trout, Salmo gairdneri Richardson

Abstract. A rainbow trout population of infectious pancreatic necrosis virus carriers was studied over a one-year period using both homogenization and co-cultivation for virus isolation. The percentage of virus-yielding fish was high between March and June, but declined during the latter part of the year. This was diametrically opposite to the trend in the serum antibody levels indicating that the marked humoral immune response resulted in a very significant reduction in the virus titres. The highest isolation rate was obtained from the kidneys after co-cultivation underlining the very high sensitivity of this method for virus detection. Virus was occasionally isolated from the faeces indicating that this may well be a possible avenue for horizontal transmission of the virus. No virus was ever detected in gonadal tissue; the mechanism of vertical transmission of the virus is still very poorly understood.     

Antigenic relationships of selected strains of infectious pancreatic necrosis virus and European eel virus

Abstract. An antigenic comparison of ten strains of infectious pancreatic necrosis virus (IPNV) and a single strain of European eel virus (EEV) revealed the presence of three distinct groups separable by cross-neutralization. The 1/ r value calculated from the formula r=√r₁× r₂ (Archetti & Horsfall 1950) was used to divide the virus strains into: group I, which included all six U.S.A. strains: Buhl, Idaho; Reno, Nevada; ATCC VR 299; Powder Mill, New Hampshire; West Buxton, Maine; and Cascade Locks, Oregon: group II, the European strains: ďHonnincthun, France; Sp Denmark; and Bonnamy, France: group III, Ab Denmark and EEV. The group II European strains of IPNV were more closely related to the group I U.S.A. strains than to those viruses in group III. European eel virus (EEV) was antigenically most closely related to the Ab strain of IPNV.     

Induction of apoptosis and secondary necrosis by infectious pancreatic necrosis virus in fish embryonic cells

Infectious pancreatic necrosis virus (IPNV) causes an acute, contagious disease in a number of economically important fish species (Pilcher & Fryer 1980). It is a member of the Birnaviridae (Brown 1986). In this study, the present authors first characterized the process of fish cell damage caused by IPNV infection in cultured cells, and found that it induced fragmentation of chromosomal DNA into oligonucleosomes and also caused nuclear fragmentation by secondary necrosis.     

Isolation of infectious pancreatic necrosis (IPN) virus from non-salmonid fish

Abstract. The isolation and characterization of infectious pancreatic necrosis (IPN) virus from a goldfish, discus fish and bream is described. The fish from which the isolates were recovered showed no pathological signs of IPN. All three virus isolates were neutralized by antiserum to IPN, strain Ab, but not by antiserum to the Sp or VR-299 strains. They were morphologically identical to IPN virus in negative stain electron microscopy, grew in the cytoplasm of BF-2 cells, as shown by immuno-fluorescence and, like IPNV, were stable to heating, lipid solvents and acid pH.     

The use of the complement fixation test for rapid typing of infectious pancreatic necrosis virus

A method for the rapid serological identification of infectious pancreatic necrosis (IPN) virus using the complement fixation test, is described. The test uses a simple, easily prepared tissue culture antigen. Methods used for preparation of suitable antisera for the test and their inclusion in a polyvalent antiserum are described.     

抗传染性胰腺坏死病毒VP3蛋白单克隆抗体的制备

利用纯化后的传染性胰腺坏死病毒(IPNV VP3)重组蛋白免疫BALB/c小鼠,通过细胞融合技术,采用间接ELISA和有限稀释法筛选杂交瘤细胞,利用染色体鉴定、蛋白印迹和免疫荧光等方法对单克隆抗体进行鉴定,共得到2株能稳定分泌特异性抗体的阳性细胞株,分别命名为2F1、4A7,亚类鉴定2株单抗均为IgG1亚类。ELISA检测其腹水效价,蛋白印迹检测表明获得的2株单抗均能特异性识别IPNV VP3蛋白;间接免疫荧光鉴定表明2株单抗均与IPNV发生反应;间接ELISA检测结果表明2株单抗均不与HSV、SVCV、HRV等病毒反应,与IPNV具有较强的特异性反应。     

Salmon cells SHK‐1 internalize infectious pancreatic necrosis virus by macropinocytosis

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE‐214 by macropinocytosis. In this study, we have extended our investigation into SHK‐1 cells, a macrophage‐like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK‐1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK‐1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK‐1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis‐mediated entry of IPNV into its target cells.     

An indirect fluorescent antibody test for the rapid detection of infectious pancreatic necrosis virus in tissues

Abstract. An indirect fluorescent antibody (IFA) test was developed to detect viral antigen in tissue sections prepared from rainbow trout experimentally inoculated with infectious pancreatic necrosis virus (IPNV). Specific fluorescence was present in the pancreatic acinar tissue and occurred as brilliant fluorescence throughout the mesentery surrounding the pyloric caeca and intestines. In addition, multiple foci of fluorescent cells were seen occasionally in the kidneys and liver of infected fry. Fluorescence was not observed in tissues other than the pancreas, kidneys, and liver. The IFA test was found to be quite specific and offers a rapid means of diagnosing IPN during acute outbreaks.     

An antigenic comparison of three strains of infectious pancreatic necrosis virus of salmonid fishes

Three strains of infectious pancreatic necrosis (IPN) virus of salmonid fishes were found to be closely related antigenically when compared by cross plaque neutralization. They differed significantly, however, in stability during freezing and storage at ?60°C. The American reference strain VR-299, which was the most stable, required consistently less antibody for a given degree of neutralization than the other two, regardless of whether the antiserum was specific for that strain or for either of the others. The CTT strain was the most unstable of the three, and required the greatest amount of antibody for a comparable degree of neutralization. The COHO strain was intermediate both in stability and in antibody requirement.The results seem to indicate that the CTT virus required the most antibody because it contained the largest amount of non-infectious virus (to chinook salmon embryo cells), which would still bind antibody and render it unavailable for neutralizing activity. The VR-299 virus contained the least amount of non-infectious virus, and consequently required the smallest amount of antibody.It is suggested that the relative stability of viral strains and possible variation in the content of non-infectious virus should be taken into account or controlled when quantitative neutralization data are used to differentiate or identify viral serotypes.     

Replication of infectious pancreatic necrosis virus in trout leucocytes and detection of the carrier state

Abstract. The exact cellular site of replication of infectious pancreatic necrosis virus (IPNV) in carrier fish is unknown. In order to determine if IPNV replicates in trout leucocytes, we purified leucocytes from normal (non-carrier) trout and separated the cells into an adherent and a non-adherent population. IPNV replicated in less than 0-01 % of the adherent leucocytes with a yield of about 400 p.f.u./cell. IPNV also became associated with less than 0.07% of the non-adherent leucocytes; either IPNV did not replicate in these cells or the yield was, at best, only a few p.f.u./cell. Trout persistently infected with IPNV (carrier fish) were tested for the presence of IPNV in leucocytes by co-cultivating with a sensitive fish cell line; this same population of trout was also tested for IPNV by organ sampling using standard methods. Ninety-eight per cent of the trout were positive for IPNV by organ sampling, but only 75 % yielded IPNV from leucocytes. Thus a blood sample from a living fish can be used to detect the presence of IPNV.