Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan).

Pancreas disease is responsible for major economic losses in the European salmonid farming industry. It was previously believed that a single subtype (salmon pancreas disease virus) of the virus species Salmonid alphavirus (SAV) was responsible for all outbreaks of pancreas disease in the UK and Norway. However, the recent discovery that pancreas disease in Norway is caused by a new and distinct subtype of salmonid alphavirus, exclusively found in Norway, has advocated the need for better diagnostic tools. In the present paper, three real-time PCR assays for all known subtypes of salmonid alphavirus have been developed; the Q_nsP1 assay is a broad-spectrum one that detects RNA from all subtypes, the Q_SPDV assay specifically detects the salmon pancreas disease virus subtype, and the Q_NSAV assay only detects the new Norwegian salmonid alphavirus subtype. The results demonstrated the assays to be highly sensitive and specific, detecting <0.1TCID50 of virus stocks. Regression analysis and standard curves calculated from the Ct-values from 10-fold serial dilutions of virus stocks showed that the assays were highly reproducible over a wide range of RNA input. Thirty-nine field samples were tested in triplicate and compared with traditional RT-PCR. Overall, the real-time assays detected 35 positive compared to 29 positives in standard RT-PCR, and were thus shown to be more sensitive for detecting salmonid alphaviruses in field samples.

Characterization of parvalbumin, the major allergen in Alaska pollack, and comparison with codfish Allergen M.

Increased fish consumption has led to frequent reporting of fish allergy and adverse reactions. Alaska pollack (Theragra chalcogramma) is a globally important commercial fish species, belonging to the Gadidae family. This family of fish also includes cod whose parvalbumin, Allergen M (Gad c 1), has been thoroughly studied and considered as a reference to sensitization in fish allergy. In the present study, parvalbumin from Alaska pollack, designated The c 1, was purified by use of anion exchange chromatography. To demonstrate the homogeneity of the purified protein, reverse phase high performance liquid chromatography was performed and showed two distinct fractions which had similar IgG and IgE binding capacities. Accordingly, cDNA cloning revealed two isotypic parvalbumin transcripts in pollack muscle. Recombinant parvalbumins of pollack exhibited low IgG and IgE binding capacities, in contrast to the native counterparts, which were almost as potent as cod Gad c 1. The allergenicity of The c 1 was assayed by ELISA inhibition, and compared to cod, the concentration required for obtaining 50% ELISA inhibition (C 50%) was only 18% higher for The c 1.

Structure and organization of the T cell receptor alpha chain genes in Atlantic salmon.

A cDNA fragment of the T cell receptor (TCR) alpha chain mRNA in Atlantic salmon (Salmo salar) was amplified by PCR and used as a probe to isolate a full-length clone from a leukocyte cDNA library. Additionally, a genomic lambda clone comprising the TCR alpha chain constant region (Calpha) gene and flanking regions was isolated and partially sequenced. The Calpha gene consists of three exons corresponding to the immunoglobulin (Ig) domain, the hinge region and the transmembrane peptide/cytoplasmatic tail, and two exons corresponding to the untranslated tail of the mRNA. Remnants of a transposase gene and a partial duplication of the Calpha gene were found nearby the intact gene. One J segment was found 1.5kb upstream of the Calpha gene. Twenty-six other J elements were identified among cDNA fragments covering the V/J/Calpha junction. Representatives of five Valpha gene families were identified by PCR amplification of genomic DNA fragments. PCR amplification of Calpha fragments from another individual revealed a slightly different Calpha gene which most likely represents an allelic variant.

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.

Analysis of two IgM isotypes in Atlantic salmon and brown trout.

Atlantic salmon (Salmo salar) possesses two distinct subpopulations of polymeric IgM which are separable by anion exchange chromatography. Consistent with this finding there are two isotypic IgM heavy chain genes, CmuA and CmuB, in the genome of this species, presumably as a result of ancestral tetraploidy. In the present study it was shown that IgM of brown trout (Salmo trutta) is also separated into two subpopulations by anion exchange chromatography, while IgM of rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) are eluted in one peak. Molecular cloning of IgM heavy chain cDNAs from brown trout revealed messages of two distinct constant region genes, named CmuA and CmuB. As deduced from the translated cDNA sequences (and in agreement with isoelectric focusing of the corresponding proteins) the mean pI values of the heavy chains in brown trout differ with only 0.14 units, in comparison to a 0.67 unit difference in salmon. Based on the present sequence analysis we suggest that an additional cysteine near the C-terminus of CmuB is critical in relation to the fractionation of IgM by anion exchange chromatography, for example by altering the overall structure of the IgM polymer and the exposure of charged residues. Most likely, the Cmu subvariant with the characteristic extra cysteine residue arose in the ancestor of Atlantic salmon and brown trout, i.e. after the three genera Salmo, Oncorhynchus and Salvelinus radiated.

Infectious salmon anemia virus (ISAV) genomic segment 3 encodes the viral nucleoprotein (NP), an RNA-binding protein with two monopartite nuclear localization signals (NLS).

Infectious salmon anemia virus (ISAV) is the type species of the genus Isavirus belonging to the Orthomyxoviridae, and causes serious disease in Atlantic salmon (Salmo salar). This study presents the expression and functional analysis of the ISAV genome segment 3, and provides further evidence that it encodes the viral nucleoprotein (NP). The encoded protein was expressed in a baculovirus system, and Western blot analysis showed that it corresponds to the 66-71 kDa structural protein previously found in purified ISAV preparations. RNA-binding activity was established by the interaction of viral and recombinant NP with single-stranded RNA transcribed in vitro. Immunofluorescence studies of infected cells showed the ISAV NP to be an early protein. It locates to the nucleus of infected cells before it is transported to the cytoplasm prior to virus assembly. A similar localization pattern was observed in cells transfected with the NP gene, confirming that the encoded protein has an intrinsic ability to be imported into the nucleus. Two monopartite nuclear localization signals (NLS) at amino acids (230)RPKR(233) and (473)KPKK(476) were identified by computer analysis, and validated by site-directed mutagenesis. In contrast to other orthomyxovirus-NPs, that have several NLSs that function independent of each other, both NLSs had to be present for the ISAV NP protein to be transported into the nucleus, indicating that these motifs cooperate to target the protein to the nucleus.

Segment 8 encodes a structural protein of infectious salmon anaemia virus (ISAV); the co-linear transcript from Segment 7 probably encodes a non-structural or minor structural protein.

In this study we present the cloning, expression and partial identification of Genomic Segment 7 of infectious salmon anaemia virus (ISAV). The nucleotide sequence corresponding to Segment 7 was isolated from a bacteriophage lambda cDNA library and contained 2 overlapping open reading frames (ORFs) of 903 and 522 bases respectively. It also contained an ISAV-specific conserved nucleotide motif in the mRNA 5' region. The co-linear transcript representing the large ORF undergoes a splicing event that removes a 526 nucleotide intron to form a mRNA corresponding to the smaller reading frame. Thus, ISAV Genomic Segment 7 has a similar coding strategy as influenza A virus Segments 7 and 8. The largest ORF of Segment 7 and the first ORF of Segment 8 was expressed in E. coli as fusion proteins and rabbit antiserum was raised against the recombinant protein from Segment 8. Immunoblot studies using this antiserum and a serum against purified virus, show that Segment 8 encodes one of the major structural proteins of the virus whereas the co-linear ORF of Segment 7 probably encodes a non- or minor structural protein

Allergy to fish parvalbumins: studies on the cross-reactivity of allergens from 9 commonly consumed fish.

BACKGROUND: Fish-hypersensitive patients can probably tolerate some fish species while being allergic to others. OBJECTIVE: To determine the allergenic cross-reactivity between 9 commonly edible fish: cod, salmon, pollack, mackerel, tuna, herring, wolffish, halibut, and flounder. METHODS: Sera from 10 patients allergic to fish and rabbit antisera against 3 parvalbumins (Gad c 1, Sal s 1, and The c 1) were used. Cross-reactivity was investigated by SDS/PAGE and IgE immunoblotting, IgG ELISA, IgE ELISA inhibition, and skin prick test (SPT). RESULTS: Cod (Gad c 1), salmon (Sal s 1), pollack (The c 1), herring, and wolffish share antigenic and allergenic determinants as shown by immunoblots and IgE ELISA, whereas halibut, flounder, tuna, and mackerel displayed lowest cross-reactivities. The highest mean IgE ELISA inhibition percent of 10 sera was obtained by Gad c 1, followed by The c 1, herring, Sal s 1, wolffish, halibut, flounder, tuna, and mackerel with the least inhibition. Nine of the 10 patients showed positive SPT to cod, salmon, and pollack; 8 patients reacted to recombinant (r) Sal s 1. Positive SPTs to rGad c 1 and rThe c 1 were demonstrated in 1 patient. CONCLUSION: Gad c 1, Sal s 1, The c 1, herring, and wolffish contained the most potent cross-reacting allergens, whereas halibut, flounder, tuna, and mackerel were the least allergenic in the current study. The latter could probably be tolerated by some of the tested patients.

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

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-(3)H]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.

Cloning and identification of the infectious salmon anaemia virus haemagglutinin.

Infectious salmon anaemia virus (ISAV) is an orthomyxo-like virus that causes serious disease in Atlantic salmon (Salmo salar). Like the orthomyxoviruses, ISAV has been shown to possess haemagglutinin (HA) activity. This study presents the cloning, expression and identification of the ISAV HA gene, which was isolated from a cDNA library by immunoscreening. The HA gene contained an ISAV-specific conserved nucleotide motif in the 5' region and a 1167 bp open reading frame encoding a protein with a predicted molecular mass of 42.4 kDa. The HA gene was expressed in a baculovirus system. A monoclonal antibody (MAb) shown previously to be directed against the ISAV HA reacted with insect cells infected with recombinant baculovirus. Salmon erythrocytes also adsorbed to these cells and adsorption was inhibited by the addition of either the ISAV-specific MAb or a polyclonal rabbit serum prepared against purified virus, confirming the virus specificity of the reaction. Immunoblot analyses indicated that ISAV HA, in contrast to influenza virus HA, is not posttranslationally cleaved. Sequence comparisons of the HA gene from five Norwegian, one Scottish and one Canadian isolate revealed a highly polymorphic region that may be useful in epidemiological studies.