Production of monoclonal antibodies against yellowtail ascites virus

A total of 31 antibody-secreting hybridoma cells against the yellowtail ascites virus (YAV) were established. These monoclonal antibodies (MAbs) reacted with the cytoplasm of YAV-infected CHSE-214 cells, but not with uninfected CHSE-214 cells in an immunofluorescence test. Using these MAbs, two classes of polypeptides (VP2 and VP3) were characterized by immunoprecipitation followed by SDS-PAGE, although one MAb did not react with either polypeptide. Fifteen out of the 17 MAbs that were reactive with VP2 polypeptides neutralized virus infectivity, but all 13 MAbs that were reactive with VP3 did not neutralize infectivity. In the immunofluorescence test, 29 out of the 31 MAbs obtained showed the same reaction pattern to 12 YAV isolates from yellowtail, Seriola quinqueradiata, goldstriped amberjack, Seriola aureovittata, and threeline grunt, Parapristipoma trilineatum, from different geographical regions. The remaining two MAbs showed slightly different reaction patterns to the YAV isolates. The reaction patterns of the MAbs to the VR-299, Sp and Ab strains of IPNV were also investigated. Fourteen MAbs reacted to all three IPNV strains. The other 17 MAbs showed a negative reaction with at least one strain.     

Epitope analysis of infectious pancreatic necrosis virus (IPNV) isolated from lake trout, Salvelinus namaycush (Walbaum), by monoclonal antibodies

Abstract. A panel of 15 monoclonal antibodies (MAbs) were raised against infeetious panercretic mecrosis virus (IPNV) associated with lake trout. salvelinus namaycush (Walwaum). (LT-IPNV) in Cornwall Lake Alberta, for LT-IPNV epietope analysis and comparison with other Canadian IPNV isolates. All the MAbs reacted with IPNV VP2 polypeptide in western blot and 10 MAbs were neutralizing. Both conformation and sequence dependent epitopes were found to be present on the IPNV VP, protein. The antibodies reeognized different epitopes on VP, protein in reeiproeal bloeking ELISA. Twelve MAbs reeognized common epitopes present on LT-IPNV and IPNV from Aretic char. Salvelinus alpinus (L.), (AC-IPNV) in binding and neutralization assays. Three MAbs reacted only with LT-IPNV indicating that it has distinct epitopes, and thus clerly differentiaing it from AC-IPNV isolated from the adjacent Northwest Trritories.Only two MAbs bound to Ja and BCI-IPNV isolate and none of the MAbs neutralized these two IPNV isolates. LT-IPNV was found to be distinct isolate, more colosly related to AC-IPNV and Canda -2 than to Ja-IPNV from alberta or other isolates in Canda. Additionally, the panel of MAbs could differnciate all the propsed Canadian IPNV scrotypes, namely C1. C2. C3 and Ja.     

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.     

Use of cloned cDNA probes for diagnosis of infectious pancreatic necrosis virus infections

Abstract. A dot-blot hybridization test has been developed for the detection of infectious pancreatic necrosis virus (IPNV) in infected fish. For this purpose, cloning of the dsRNA of the West Buxton strain of IPNV was carried out. Two cDNA clones (WB and A4) were characterized for use as diagnostic probes and corresponded to IPNV genome segments A and B. respectively. Clone WB1, with an insert of 812 base pairs, showed an 87 and 77% nuclcotidc sequence homology with the corresponding sequences of Jasper and N1 strains, respectively. Clone A4, with an insert size of 596bp, presented a nuclcotidc sequence homology of 90 and 80% with the corresponding sequences of the Jasper and Sp strains, respectively. Both probes were able to detect 15 ng of purified dsRNA, and were highly efficient in detecting the RNA of American IPNV strains. However, the A4 probe was less effective than WB1 in hybridizing to RNA from European and Spanish strains of IPNV. Both probes detected IPNV RNA in cells 4–8h post-infection with the homologous West Buxton strain, 8–12h post-infection with other American strains and 24h post-infection with the European strains of IPNV. The method was less sensitive in detecting IPNV RNA directly in infected fish tissues. However, the present authors obtained a 100% effectiveness to detect viral RNA in cells inoculated with fish tissues confirmed by conventional diagnostic methods as being infected with IPNV. Therefore, the hybridization test is appropriate if combined with conventional diagnostic procedures, e.g. applying the dot blot hybridization test on tissue cultures 12–24 h after inoculation with infected fish tissue homogenates.     

Infectious pancreatic necrosis virus (IPNV) serotype Sp is prevalent in Turkish rainbow trout farms

Infectious pancreatic necrosis virus (IPNV) is a common pathogen of rainbow trout (Oncorhynchus mykiss) in Turkey. We found that 455 of 1,676 sample pools tested were IPNV positive. Positive samples were found in all geographical regions where sampling was conducted. Sequence and phylogenetic analyses of VP2 from 30 isolates representing all regions showed that the viruses were highly similar in sequence and grouped within Genogroup 5 (serotype Sp‐A2). No correlations between sequences, sampling sites or geographical origins were identified. Although clinical disease was evident in several farms, analyses of the amino acid sequence of VP2 showed that all virus strains harboured the P₂₁₇T₂₂₁ motif, assumed to be associated with low virulence. We conclude that IPNV is prevalent in Turkish rainbow trout farms and that the viruses are very homogenous and likely to be of European origin. Frequent exchange of eggs and live fish within the farming industry may explain the homogeneity of the IPNV.     

Study of virulence in field isolates of infectious pancreatic necrosis virus obtained from the northern part of Norway

In order to study the variety of infectious pancreatic necrosis virus (IPNV) strains involved in outbreaks of infectious pancreatic necrosis (IPN) in Atlantic salmon fish farms, samples were collected from 19 different outbreaks of IPN in the northern part of Norway. The main objective of this study was to examine whether IPNV isolates of different virulence were involved in the outbreaks and could explain the variable IPN protection observed in vaccinated post‐smolts in the field. Both the molecular basis of virulence of all field isolates and virulence expressed by mortality after bath challenge of unvaccinated post‐smolts with eight of the isolates were studied. Very little variation among the field isolates was detected when the 578‐bp variable region encoding the VP2 protein known to be involved in virulence was sequenced. The cumulative mortality after experimental challenge with field isolates genetically characterized as highly virulent was always high (40–56%), while the cumulative mortality of the same strains in vaccinated post‐smolts during the field outbreaks varied from 1 to 50%. Although the tested samples came from fish vaccinated with the same vaccine product, the protection against IPN varied. These results demonstrate that differences in virulence of the isolates were not the main reason for the variation in mortality in the field outbreaks. Most of the field isolates were of high virulence, which is shown in experimental challenges to be important for mortality, but clearly other factors that might affect the susceptibility of IPN also play an important role in the outcome of an IPNV infection.     

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.     

传染性胰腺坏死病病毒分离株VP2基因抗原表位区融合表达及免疫特性的分析

利用RT-PCR方法扩增出IPNV-ZYX分离株主要结构蛋白VP2的抗原表位区基因(616 bp), 命名为IPNV VP2 COE, 将其克隆到pCold TF表达载体中构建重组质粒pCold TF-VP2 COE, 在大肠杆菌BL21(DH5α)感受态表达, 经SDS-PAGE电泳分析, 表达蛋白约78 ku, 用镍离子亲和层析柱纯化该蛋白, 制备抗血清, 间接ELISA结果显示, IPNV (ATCC VR-1318)细胞培养物与鼠抗VP2 COE蛋白血清发生特异性反应, 效价为1∶12 800; 间接免疫荧光结果显示, 鼠抗VP2 COE血清可与黑龙江某渔场已知感染IPNV虹鳟肝组织产生特异性的荧光, 以上两项结果表明, 表达IPNV VP2 COE蛋白具有良好的免疫原性和免疫反应性, 为IPNV检测方法的建立及疫苗的制备提供理论依据     

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).     

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.     

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。     

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.     

Antigenic analysis of Norwegian aquatic birnavirus isolates using monoclonal antibodies—with special reference to fish species, age and health status

Abstract. A panel of 11 monoclonal antibodies (MAbs) was used in an immunodot assay to investigate the antigenic relationship of a representative selection of aquatic biroaviruses isolated in Norway from different species of both carrier and diseased fish. All the 81 isolates shared a serogroup A-speeifie epitopc, and 67 of them had a reaction pattern identical to the Sp-type strain. Of the other 14 isolates, nine resembled the Sp-type strain, two resembled both the Sp- and He-type strains, two were closest to the Ab-type strain and one resembled the Te-type strain. Sp-related strains were predominant from all nine host groups, including from both Atlantic salmon, Salmo salar L., with infectious pancreatic necrosis (IPN) and healthy. Atlantic salmon. The N1 strain reacted identically to the Sp-type strain.     

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.     

Development of monoclonal antibodies against VP28 of WSSV and its application to detect WSSV using immunocomb

White spot disease (WSD) is an important viral disease of penaeid shrimp caused by white spot syndrome virus (WSSV). WSSV isolated from WSD outbreaks in commercial shrimp (Penaeus monodon) farms in India were propagated in the laboratory in healthy shrimp. The virus was purified from the infected tissues by sucrose gradient centrifugation. The VP28 was electroeluted from SDS-PAGE gels and was used to immunize Balb/c mice to produce hybridomas secreting monoclonal antibodies (MAb) against WSSV. A total of five hybridoma clones secreting MAbs to VP28 were produced. The MAbs were of the isotypes IgG1, IgG2b and IgM. The MAbs reacted with VP28 of WSSV and not with any other viral or shrimp protein in western blot. The MAbs were used to develop dot immunoblot assay using an immunocomb to detect WSSV from field samples. The test developed had an analytical sensitivity of 625 pg and a diagnostic sensitivity of 100% compared to single step polymerase chain reaction (PCR). The test can be used as an alternate for first step PCR to detect WSSV from field samples.     

Production of the lethal acetylcholinesterase toxin by different Aeromonas hydrophila strains

Abstract. Seven fish pathogenic isolates of Aeromonas hydrophila , one A. sobria and one A. caviae were investigated for production of the fish lethal acetylcholinesterase toxin (AcChE-toxin). Western blotting was used for screening the ECP of these strains with a rabbit antiserum prepared against the purified toxin of strain B₃₂ and all the isolates (except A. sobria ) gave positive results with different patterns of bands. The AcChE-toxin appears to be secreted as a protein of high molecular weight which is stable at −20°C, and in 90% of the strains tested, it appears to be split into lower molecular weight fragments by the action of other components present in the ECP. The smallest, stable and highly active fragment has a MW of 15kDa.     

Characterization and application of monoclonal antibodies against white spot syndrome virus

Three hybridoma clones secreting monoclonal antibodies (MAbs) were produced from mouse myeloma and spleen cells immunized with white spot syndrome virus (WSSV) isolated and purified from Penaeus monodon (Fabricius), collected from north-eastern Taiwan. By sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), the protein profile of this isolate contained four major proteins with sizes of approximately 35 (VP35), 28 (VP28), 24 (VP24), and 19 kDa (VP19). Western blot analysis revealed that two MAbs (1D7 and 6E1) recognized epitopes on VP28 and one MAb (3E8) recognized an epitope on VP19. The MAb 6E1 isotyped to the IgG₁ class was used in both an indirect immunofluorescence assay (IFA) and in an immunochemical staining protocol for successful identification and localization of WSSV in infected shrimp tissues. Antigenic similarity of isolates from Indonesia and Malaysia to the Taiwan isolate was illustrated by IFA with MAb 6E1. A MAb (2F6) which bound specifically to two shrimp proteins, 75 and 72 kDa, and reacted to the healthy and non-target tissues of WSSV in infected shrimp, such as hepatopancreas, is also described here and shows the necessity for specific identification of antibodies.