Curt Endresen

Cloning of Two Distinct cDNAs Encoding Parvalbumin, the Major Allergen of Atlantic Salmon (Salmo salar)

Allergy to fish is common in Northern Europe. Variable reactions to different fish species are usually experienced among fish allergic patients. The allergens of cod fish and particularly the major allergen parvalbumin β (Gadus callarias) have been extensively studied in Norway. In the present communication, the white muscle parvalbumin was similarly found to be a major allergen in Atlantic salmon (Salmo salar, Sal s1 ). A purified salmon parvalbumin was obtained by anion exchange chromatography, gel filtration chromatography (GFC) and high‐performance liquid chromatography (HPLC) of the muscle extracts. The antigenicity and allergenicity of salmon parvalbumin were confirmed using various immunologic and electrophoretic techniques. The protein is representative for several isoallergens judged by the amino acid (AA) sequence variance at certain sites in the AA sequence of CNBr cleavage peptides. Using sera from patients with cod and salmon allergy Sal s1 was demonstrated to be the major allergen of Atlantic salmon, as judged by RAST‐ and ELISA‐inhibitions and crossed radioimmunoelectrophoresis (CRIE) techniques. The protein was also demonstrated to be antigenic by the use of polyclonal cod and salmon antibodies in IgG ELISA and immunoelectrophoretic methods. Cloning of parvalbumin cDNA from Atlantic salmon was performed based on an alignment of parvalbumin AA sequences from other species. A probe was generated by PCR and used for screening a salmon muscle cDNA‐library. Subcloning and sequencing of two hybridizing clones revealed transcripts from two different parvalbumin genes. The translated sequences of both clones belong to the β‐lineage of parvalbumins and include the entire coding region.

Cloning and sequence analysis of cDNAs encoding the MHC class II β chain in Atlantic salmon (Salmo salar)

Atlantic salmon (Salmo salar) cDNAs encoding the major histocompatibility complex (Mhc-Sasa) class II β chain were isolated from a leucocyte library by a polymerase chain reaction (PCR) approach. Three different cDNAs (c144, c22, and c157) encoding the entire mature β chain have been analyzed. Clone c144 differs from clone c157 in 12.6% of the nucleotides in the β1-encoding region. The corresponding differences between clones c144 and c22, and clones c22 and c157, are 10.3% and 5.2%, respectively. This variation is, at least in part, most likely attributable to allelism. The similarity indices between the highly conserved β2 domains from Atlantic salmon and corresponding sequences from humans (DQβ), chicken (BLβ), carp (TLAIIβ-1), and rainbow trout (O. M. No. 55) are 45%, 40%, 66%, and 97%, respectively. Variable residues in the β1 domains from Atlantic salmon correspond with polymorphic sites of β1 domains from higher vertebrates. The frequency of substitutions in the β1-encoding region exceeds that in the 3′-untranslated (UT) region with several folds, indicating extensive β1 polymorphism in Atlantic salmon.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers (C 144) X 70165, (C 157) X 70166, and (C 22) X 70167.

Molecular cloning of major histocompatibility complex class I cDNAs from Atlantic salmon (Salmo salar)

The major histocompatibility complex (Mhc) has attracted much attention because of its immense polymorphism, its importance in transplantation, and its indisputable role in disease susceptibility in humans (Chen and Parham 1989; Hill et al. 1991) and in animals (Lie 1990). Previously, typical Mhc features reflected in allograft rejection and mixed leucocyte reactivity were the only indications that an Mhc also existed in teleost fish (Stet and Egberts 1991). The use of polymerase chain reaction (PCR) with degenerate oligonucleotides from conserved Mhc regions provided the first direct evidence for Mhc class I and class II genes in a fish, the teleost carp (Hashimoto et al. 1990). The primary aim of our study was to isolate and characterize expressed Mhc molecules in Atlantic salmon, and thereby provide data for further studies on evolutionary and disease aspects of the Mhc and its polymorphism. An Atlantic salmon-specific Mhc probe from leucocyte RNA was generated by PCR based on primers from conserved regions of known Mhc genes. The oligonucleotides and detailed strategies are described in an accompanying paper by Hordvik and co-workers (this issue). This salmon-specific probe was employed to screen a leucocyte )~gtl0 cDNA library based on a few individuals, from which Mhc-positive cDNAs were derived. The cDNAs analyzed in this report were established as subclones in pGEM-7z(+)R (Promega, Madison, WI) and sequencing was performed on double-stranded DNA with SP6, T7, and internal primers, using the procedure supplied by Multi-Pol TM DNA

Identification and characterization of viral structural proteins of infectious salmon anemia virus.

ABSTRACT Infectious salmon anemia virus (ISAV) is an unclassified Orthomyxovirus that has been shown to contain a segmented genome with eight single-stranded RNA species coding for 10 viral proteins. Four major structural proteins were characterized in the present study: two glycosylated proteins with estimated molecular masses of 42 and 50 kDa, one 66-kDa phosphoprotein, and one 22-kDa protein. Examination of lysed virions revealed the two glycoproteins and the 22-kDa protein in the soluble fraction, while the 66-kDa phosphoprotein and a minor part of the 22-kDa protein were found in the pelleted fraction. Immunofluorescence staining of infected cells demonstrated that the 22-kDa protein was a late protein accumulating in the nucleus. We conclude that the 66-kDa protein is the nucleoprotein, the 22-kDa protein is the matrix protein, and the 42- and 50-kDa proteins are the surface proteins. Radioimmunoprecipitation analysis of the 42-kDa glycoprotein, which was previously shown to represent the ISAV hemagglutinin, indicated that this protein exists at least as dimers. Further, by labeling of purified ISAV with [1,3-3H]diisopropyl fluorophosphate, it was also demonstrated that the viral esterase is located with the hemagglutinin. This finding was confirmed by demonstration of acetylesterase activity in affinity-purified hemagglutinin preparations. Finally, the active-site serine residue could be tentatively identified at position 32 within the amino acid sequence of the hemagglutinin of ISAV strain Glesvaer/2/90. It is proposed that the ISAV vp66 protein be termed nucleoprotein, the gp42 protein be termed HE protein, and the vp22 protein be termed matrix protein.

Purification and partial characterization of an IgM-like serum immunoglobulin from Atlantic salmon (Salmo salar).

An IgM-like immunoglobulin was isolated from pooled sera collected from healthy Atlantic salmon. The immunoglobulin was purified by means of gel filtration followed by ion-exchange chromatography. It eluted from the ion-exchange column as two distinct peaks, but the two IgMs seems to be identical in their molecular natures except for net charge. The molecular weight of the unreduced (native) IgM was determined to be approximately 800 kilo Daltons (kD) when estimated by dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). However, when the molecular weight of the native IgM was estimated by gel filtration, a molecular weight of 1000 kD was obtained. Furthermore, the molecular weights of the heavy and the light chains were estimated by SDS-PAGE analysis to be about 72 and 27 kD respectively. The amount of IgM was found to range from 80 to 130 mg/100 ml serum. Isoelectric focusing demonstrated that the major part of the IgM molecules focused between pH 5 and pH 6.

Characterization of a new serotype of infectious pancreatic necrosis virus isolated from Atlantic salmon

SummaryVirus particles isolated from hatchery reared fish with infectious pancreatic necrosis (IPN) were neutralized by homologous immune sera but not by immune sera raised against IPN virus serotype 1, 2, and 3. This virus isolate, called the N 1 strain, was detected in one year old Atlantic salmon (Salmo salar) during an outbreak with histopathological lesions of IPN and slightly increased mortality. The polypeptide pattern of N 1 virus differed markedly from that of the three classical IPN virus serotypes.Double stranded RNA isolated from the N 1 virus particles, co-migrated during agarose gel electrophoresis with nucleic acid isolated from the IPN virus Jasper and Ab strains. Nucleic acid hybridizations using low stringency washing conditions and a synthetic DNA oligonucleotide probe (representing the 3′ end of the A segment of the Jasper strain) gave positive results with the IPN virus Jasper, Ab, Sp, and N 1 strains.The results presented in this paper show that the N 1 isolate differs immunologically and biochemically from the IPN virus serotypes 1, 2, and 3 and may represent a new serotype of IPNV.

Genetic stability within the Norwegian subtype of salmonid alphavirus (family Togaviridae)

Summary.Salmonid alphavirus (SAV) (family Togaviridae) causes mortality in Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss W.) in Norway, France, UK, and Ireland. At least three subtypes of SAV exist: SPDV in UK/Ireland, SDV in France/UK, and the recently reported Norwegian salmonid alphavirus (NSAV) in western Norway. During 2003 and 2004, disease caused by NSAV was reported for the first time in northern Norway, more than 800 km away from the enzootic area in western Norway. The present study has investigated the phylogenetic relationships among 20 NSAV isolates, based on a 1221-nt-long segment covering part of the capsid gene, E3, and part of the E2 gene, collected over a period of eight years. The results revealed genetic homogeneity among NSAV isolates, including those from northern Norway. The SDV or SPDV subtypes were not found in diseased Norwegian fish. A substitution rate of 1.70 (±1.03) × 10−4 nt subst/site/year was obtained for the NSAV subtype by maximum likelihood analysis. The second aim of this study was to clarify whether NSAV changes genotypically in cell culture by culturing a NSAV isolate through 20 passages in CHSE-214 cells. Sequencing of almost the entire genome (11530 nt) after 20 passages revealed four nucleotide substitutions, all resulting in amino acid substitutions. One of these substitutions, serine to proline in E2 position 206, was also found to have occurred in field isolates.

Mapping of neutralization epitopes on infectious pancreatic necrosis viruses

We have characterized and mapped variable and conserved neutralization epitopes of serogroup A strains of aquatic birnaviruses. Epitope mapping using monoclonal antibodies (MAbs) and Escherichia coli-expressed deletion fragments of VP2 of the N1 strain of infectious pancreatic necrosis virus (IPNV) demonstrated that two variable epitopes, H8 and B9, depend on the variable region between amino acid 204-330. A conserved neutralization epitope, F2, was shown to depend on the same region as epitopes H8 and B9 but was additionally dependent on amino acids between 153-203. The neutralization epitopes H8, B9 and F2 were also shown to overlap by a competitive binding assay. One conserved neutralization epitope, AS-1, was not exposed on any of the recombinant VP2 deletion fragments and was therefore not possible to map. However, the MAbs AS1 and F2 were partly competitive indicating that these epitopes are overlapping. All neutralization epitopes were independent of a conserved non-neutralization epitope, E4. Our results demonstrate that the central third of VP2 contains several partly overlapping neutralization epitopes, both variable and conserved among serogroup A strains of IPNV.

Molecular phylogeny of microsporidians with particular reference to species that infect the muscles of fish

Ribosomal DNA from eight species of microsporidians infecting fish have been sequenced. Seven of these species infect the skeletal muscle of fish (Pleistophora spp.) and one species infects migratory mesenchyma cells (Glugea anomala). These sequences, in addition to other available microsporidian rDNA sequences from a broad range of host taxa, have been used in phylogenetic analysis. This analysis revealed that muscle‐infecting microsporidians from fish are a polyphyletic group, indicating that characters supposed to be important in the classification of the genus Pleistophora have to be re‐evaluated. One character that probably has a polyphyletic origin is the amorphous coat, which has been extensively used in the definition of this genus. Furthermore, our results showed that the insect parasitizing Pleistophora spp. are not related to the true pleistophorans parasitic in skeletal muscle of fish. Phylogenetic analysis of small subunit rDNA sequences revealed disagreements between the molecular phylogeny and classifications based upon ultrastruclure. Many of the morphological characters claimed to be important in microsporidian classifications appeared to have arisen several times during evolution: for example, the diplokaryon and sporophorous vesicles.

The major allergen (parvalbumin) of codfish is encoded by at least two isotypic genes: cDNA cloning, expression and antibody binding of the recombinant allergens.

The major allergen (parvalbumin) from cod, designated Allergen M Gad c 1, has been intensively studied both from the structural and immunological sides. In the present study, transcripts of two isotypic parvalbumin genes in Atlantic cod were identified and characterized. Subsequently, subfragments were inserted into the expression vector pET-19b, generating plasmids with coding capacity for complete parvalbumin polypeptides fused to an N-terminal his(10) tag. Most of the recombinant products were found in the soluble fraction of the expression host Escherichia coli. The target proteins showed to react with polyclonal antibodies raised against Allergen M and demonstrated binding to specific IgE from 12 sera of patients allergic to cod in ELISA inhibition experiments. Sera with classes 4 and 5 CAP FEIA exhibited also strong binding to recombinant parvalbumins in immunoblots.