CD4 homologues in Atlantic salmon.

In mammals CD4 is a membrane glycoprotein on Th cells with four extracellular immunoglobulin-like (Ig-like) domains (D1-D4). It functions as a co-receptor during immune recognition between the TCR and the MHC II/peptide complex. The cytoplasmic domain binds p56lck, a protein kinase responsible for phosphorylating CD3 which is the first interaction in a cascade leading to T cell activation. We have previously reported a CD4-2 gene in rainbow trout (Oncorhynchus mykiss) which was found adjacent to the CD4-1 gene by synteny analysis. There are two subtypes (a and b) of CD4-2 in rainbow trout, with two Ig-like extracellular domains. Here we present the homologues of mammalian CD4 in Atlantic salmon (Salmo salar): CD4-1 with four extracellular domains and CD4-2a and CD4-2b with two extracellular domains. A Southern blot analysis shows two copies of the CD4-1 gene in the genomic DNA of the closely related rainbow trout. The genes for CD4-1 and CD4-2 have been sequenced and show typical traits for CD4 genes, such as the code for the first domain (D1) being divided between two exons and the other domains being largely coded for by single exons. The corresponding translated cDNAs show little (13-17%) identity to higher vertebrates and are approximately 37% similar to other translated, teleost sequences but are 89% identical to the closely related rainbow trout. However they exhibit conserved features such as the Lck binding motif in their cytoplasmic domains and the order of variable and constant type Ig-like domains. qRT-PCR data are presented describing the differential tissue expression of these genes together with other T cell markers (TCR and CD3) in several individuals.

Wavelength- and energy-dispersive Electron Probe Microanalysis (EPMA) measurements with non-perpendicular incidence of the electron beam.

A database of 416 wavelength- and energy-dispersive EPMA measurements on tilted specimens of NiAl, TiO(2) and Ti(3)Al is presented. The analyses were performed between 10 and 30 kV and the tilt angles were varied between 0 degrees and 60 degrees in seven steps. The necessary hardware modifications for the specimen holder are discussed, as well as the various focusing techniques used in the measurements. A comparison between the experimental data, the calculations of our proza96t program and the results of Monte Carlo simulations shows that up to 50 degrees tilt the predictions of our software are more than satisfactory. At larger tilt angles some deviations become noticeable. The Monte Carlo simulations appear to produce deviations at a somewhat earlier stage already, for reasons as yet unknown.

Characterisation of salmon and trout CD8alpha and CD8beta.

The genes and corresponding cDNAs of both alpha and beta chains of the Atlantic salmon (Salmo salar) CD8 molecule have been sequenced and characterized. In addition, the cDNAs for alpha and beta chains of brown trout (Salmo trutta) and for the beta chain in rainbow trout (Oncorhynchus mykiss) have been sequenced. The cDNAs code for signal sequences which are preceded by short 5' UTRs. These are followed by typical immunoglobulin superfamily variable sequences all of which contain two conserved cysteines for the intra-chain disulphide bond. The hinge regions display conserved cysteines for dimerisation and several O-glycosylation motifs for each predicted protein. The domain sharing the highest sequence identity with mammals is the single pass transmembrane domain for all sequences. In salmon, each domain is predominantly coded for by a single exon except the cytoplasmic/3' UTR domains, which are coded for by 3 and 2 exons for the alpha and beta genes, respectively. In the alpha gene, the second cytoplasmic exon may be spliced out to form an alternative shorter transcript which if expressed would exhibit a truncated cytoplasmic tail. A splice variant found for the salmon beta gene introduces a stop codon after only 40 amino acids. Overall amino acid identities between salmonid sequences were higher than 90%, whereas they shared only 15-20% identity with species such as, chicken and human. Analysis of the expression patterns of the two salmon genes using quantitative RT-PCR shows a very high expression in the thymus. This is mirrored by the expression of the TCRalpha gene, which is known to be co-expressed with CD8 on mammalian T cells. This is the first report of a sequence for CD8beta in a teleost and together with the CD8alpha sequence, it encodes the ortholog of the CD8 co-receptor molecule on mammalian T cells.

Cytotoxic T cell function in fish.

Fish possess immunoglobulins, major histocompatibility complex (MHC), T-cell receptors, and lymphocyte populations analogous to B and T cells and can evoke specific immune responses against a variety of antigens. However, T-cell subsets have yet to be demonstrated and the information on cell-mediated immunity is limited. Here we briefly review our recent studies on specific cell-mediated immunity, particularly on cytotoxic T-cell function employing isogeneic fish and cell lines. Analyses of the graft-versus host reaction (GVHR) and cell-mediated cytotoxicity (CMC) against allogeneic erythrocytes or cell lines show alloantigen-specific cytotoxicity in clonal ginbuna crucian carp. We also describe specific cytotoxicity against virus-infected cells using clonal ginbuna and a syngeneic cell line. Lastly, we report MHC-restriction in CMC against virus-infected cells using homozygous clonal rainbow trout and trout cell line which share the same MHC class I allele. These studies on CMC strongly suggest the presence of antigen specific cytotoxic T cells in teleosts and functional similarities between the immune systems of fish and higher vertebrates. Experimental model systems established in these studies can be applied to the investigation of protective antigens to induce cell-mediated immunity for the development of fish vaccines.

Exogenous antigens and the stimulation of MHC class I restricted cell-mediated cytotoxicity: possible strategies for fish vaccines.

An MHC class I restricted cytotoxic T lymphocyte (CTL) activity assay has recently been established for rainbow trout. MHC class I restricted cytotoxicity probably plays a critical role in immunity to most viral diseases in mammals and may play a similar role in fish. Therefore, it is very important to investigate what types of vaccines can stimulate this immune response. Although logical candidates for vaccine components that can stimulate an MHC class I restricted response are live attenuated viruses and DNA vaccines, these materials are generally not allowed in fish for commercial vaccine use due to potential safety issues. In mammals, however, a number of interesting vaccination strategies based on exogenous antigens that stimulate MHC class I restricted cytotoxicity have been described. Several of these strategies are discussed in this review in the context of fish vaccination.

Luciferase expression 2 years after DNA injection in glass catfish (Kryptopterus bicirrhus).

Glass catfish (Kryptopterus bicirrhus) have transparent muscles and skin. Intramuscular injection of DNA encoding luciferase into these fish induced luciferase expression that was measurable in vivo with a low light video image analyser. Expression could be detected up to at least 2 years after DNA injection. Although luciferase is not representative of all types of antigen, this study stresses the need for future studies directed to limit the period of antigen expression after DNA vaccination.

Specific cell-mediated immunity in fish.

This review describes the fish immune system, focusing on specific cell-mediated immunity. Specific in vivo cell-mediated immune responses have been shown by allograft rejection, graft-versus-host reaction (GVHR) and delayed hypersensitivity reaction (DTH). Recent in vitro studies also showed specific cell-mediated cytotoxicity against allogeneic target cells. These in vivo and in vitro experiments strongly suggest the presence of cytotoxic T cells in fishes. Also described are current studies on shark and trout MHC class I polymorphism and function that demonstrate strong similarities between fish and mammals.

Glycoproteins gM and gN of pseudorabies virus are dispensable for viral penetration and propagation in the nervous systems of adult mice.

Glycoproteins gM and gN are conserved throughout the herpesviruses but are dispensable for viral replication in cell cultures. To assay for a function of these proteins in infection of an animal, deletion mutants of pseudorabies virus lacking gM or gN and corresponding revertants were analyzed for the ability to penetrate and propagate in the nervous systems of adult mice after intranasal inoculation. We demonstrate that neither of the two glycoproteins is required for infection of the nervous systems of mice by pseudorabies virus.

Inhibition of virion maturation by simultaneous deletion of glycoproteins E, I, and M of pseudorabies virus.

Glycoprotein M (gM), the product of the UL10 gene of pseudorabies virus (PrV), is one of the few nonessential glycoproteins conserved throughout the Herpesviridae. In contrast to wild-type PrV strains, the UL10 gene product of the attenuated PrV vaccine strain Bartha (PrV-Ba) is not modified by N-glycans due to a mutation in the DNA sequence encoding the consensus N-glycosylation motif. To assay function of the UL10 protein in PrV-Ba, a UL10-deletion mutant (PrV-Ba-UL10(-)) was isolated. Surprisingly, in contrast to gM-deleted wild-type PrV, PrV-Ba-UL10(-) was severely impaired in plaque formation, inducing only foci of very few infected RK13, Vero, and PSEK cells and tiny plaques on MDBK cells. Since this effect was significantly more dramatic than in wild-type PrV, additional mutations known to be present in PrV-Ba were analyzed for their contribution to this phenotype. trans-complementation of the mutated PrV-Ba UL21 or gC protein by the wild-type version had no influence on the observed phenotype. In contrast, complementation of the gE/gI deletion rescued the phenotype. The synergistic effect of deletions in gE/gI and gM on plaque size was verified by construction of a gE/I/M triple mutant derived from wild-type PrV which exhibited the same phenotype. The dramatic effect of deletion of gM on plaque size in a gE/I- virus background was mainly attributable to a function of gM, and not of the gM/gN complex, as shown by analysis of a gE/I/N triple mutant. Interestingly, despite the strong effect on plaque size, penetration was not significantly impaired. In noncomplementing cells infected with the gE/I/M triple mutant, electron microscopy showed absence of secondary envelopment in the cytoplasm but occurrence of intracytoplasmic accumulations of nucleocapsids in association with electron dense material, presumably tegument proteins. These structures were not observed after infection of cells expressing either gE/I or gM. We suggest that gE/I and gM are required for late stages in virion morphogenesis prior to final envelopment in the cytoplasm.

Different point mutations within the conserved N-glycosylation motif of pseudorabies virus glycoprotein M result in expression of a nonglycosylated form of the protein.

Glycoprotein M (gM) constitutes one of the rare examples of a nonessential glycoprotein conserved throughout all herpesvirus subfamilies. Whereas gM in wild-type pseudorabies virus (PrV) strains carries an N-glycan, gM of the attenuated strain Bartha is not glycosylated due to a point mutation in the N-glycosylation motif. Since PrV Bartha lacks glycoproteins E and I and carries a mutated gC, we analysed glycosylation of gM in isogenic PrV glycoprotein deletion mutants. Whereas gM was glycosylated normally in most mutants, two independent gC deletion mutants and a gI mutant expressed a nonglycosylated form of gM. DNA sequence analyses revealed the presence of point mutations in the N-glycosylation consensus motif. Surprisingly, mutations in strain Bartha, the two gC-deletion mutants and the gI mutant proved to be different, although all affected the N-glycosylation motif. Thus, our data show that different, apparently independent point mutations cause expression of nonglycosylated gM.

Glycoproteins M and N of pseudorabies virus form a disulfide-linked complex.

Genes homologous to the herpes simplex virus UL49.5 open reading frame are conserved throughout the Herpesviridae. In the alphaherpesvirus pseudorabies virus (PrV), the UL49.5 product is an O-glycosylated structural protein of the viral envelope, glycoprotein N (gN) (A. Jöns, H. Granzow, R. Kuchling, and T. C. Mettenleiter, J. Virol. 70:1237-1241, 1996). For functional characterization of gN, a gN-negative PrV mutant, PrV-gNbeta, and the corresponding rescuant, PrV-gNbetaR, were constructed, gN-negative PrV was able to productively replicate on noncomplementing cells, and one-step growth in cell culture was only slightly reduced compared to that of wild-type PrV. However, penetration was significantly delayed. In indirect immunofluorescence assays with rabbit serum directed against baculovirus-expressed gN, specific staining of wild-type PrV-infected cells occurred only after permeabilization of cells, whereas live cells failed to react with the antiserum. This indicates the lack of surface accessibility of gN in the plasma membrane of a PrV-infected cell. Western blot analyses and radioimmunoprecipitation experiments under reducing and nonreducing conditions led to the discovery of a heteromeric complex composed of gM and gN. The complex was stable in the absence of 2-mercaptoethanol but dissociated after the addition of the reducing agent, indicating that the partners are linked by disulfide bonds. Finally, gN was absent from gM-negative PrV virions, whereas gM was readily detected in virions in the absence of gN. Thus, gM appears to be required for virion localization of gN.

Intracellular processing of pseudorabies virus glycoprotein M (gM): gM of strain Bartha lacks N-glycosylation.

Genes encoding homologs of the herpes simplex virus type 1 UL10 product, glycoprotein M, are conserved in all herpesviruses investigated so far. Recently, we identified pseudorabies virus (PrV) gM as a 45-kDa structural component of purified virions. A gM-PrV mutant could be propagated in cell culture, albeit at lower titers and with delayed penetration kinetics. Thus, gM has a nonessential but modulatory function in PrV infection. PrV gM is modified by addition of an N-linked glycan at a consensus sequence located between the predicted first and second hydrophobic region of the protein. This N-glycosylation site is conserved in all gM homologs sequenced so far, indicating an important functional role. To analyze intracellular processing of PrV gM, Western blot analyses were performed. In PrV-infected cells, mature 45-kDa gM as well as 33- and 35-kDa precursor forms were detectable. Presumably dimeric 90- and 70-kDa proteins were also observed. The 33- and 35-kDa proteins represent nonglycosylated and glycosylated precursors as shown by endoglycosidase digestions. Investigation of several PrV strains revealed that the UL10 product of PrV strain Bartha, an attenuated virus used as vaccine, was not modified by N-glycosylation. Sequence analysis showed that the N-glycosylation consensus sequence was altered from NDT to NDA, which resulted in loss of the N-glycosylation signal. To our knowledge, this is the only gM homolog identified so far which is not N-glycosylated. To investigate whether this form of the protein is functionally competent, the UL10 gene of strain Bartha was inserted into PrV strain Kaplan by substitution of the wild-type UL10 gene. The resulting recombinant expressed a UL10 protein lacking N-glycans. In vitro replication analyses did not reveal any difference in virus production, but plaque size and penetration kinetics were slightly reduced. In summary, we show that wild-type gM is modified by N-glycosylation at one conserved site. However, although this site is highly conserved throughout the herpesviruses, loss of N-glycans due to mutation of the consensus sequence had only a minor effect on propagation of PrV in cell culture.

Deletion of glycoprotein gM of pseudorabies virus results in attenuation for the natural host.

Glycoprotein M (gM) is one of the very few non-essential glycoproteins conserved throughout the herpesvirus family. Despite this conservation little is known about its function in virus replication. To test for the importance of gM in vivo in a natural virus-host system, 6-week-old piglets were intranasally infected with a gM- mutant of the alphaherpesvirus pseudorabies virus (PrV). Following infection virus excretion from the nasal mucosa was decreased ca. 100-fold compared to wild-type or revertant virus. Clinical signs were limited to transiently elevated temperature. In contrast, animals infected by wild-type or revertant virus exhibited high fever, severe respiratory symptoms and affliction of the central nervous system. Prior infection with gM-PrV conferred protection against challenge infection and animals mounted an antibody response against gM after wild-type virus infection. Thus, gM is important for efficient virus replication in vivo and deletion of gM may contribute to development of live attenuated, genetically marked vaccines.

Effects of malnutrition on the erythrocyte fatty acid composition and plasma vitamin E levels of Pakistani children.

Erythrocyte fatty acids and plasma vitamin E concentrations were determined in 47 grade 2 and 21 grade 3 malnourished Pakistani children (ages 4-56 months). Data were compared with those of 26 age- and sex-matched apparently healthy controls. Evaluation with three statistical approaches revealed that both grade 2 and grade 3 malnourished children had decreased erythrocyte omega6 fatty acids and to a lesser extent decreased omega3 fatty acids. These decreases were compensated for by increased omega9 fatty acids. The patients tended to have lower plasma vitamin E concentrations. We conclude that malnourished Pakistani children have low essential fatty acid status, notably those of the omega6 series. The combination of low erythrocyte 22:6omega3 and a low 22:5omega6/22:4omega6 ratio in grade 2 patients suggests low delta4-desaturation activity, which may be due to impaired peroxisomal beta-oxidation.

Identification and transcriptional analysis of pseudorabies virus UL6 to UL12 genes.

We determined the nucleotide sequence of an 11059 bp fragment of the pseudorabies virus genome located in the right part of genomic BamHI fragment 3 and the adjacent part of BamHI fragment 6. Within this region eight open reading frames were identified whose deduced amino acid sequences exhibited homology to the UL6, UL7, UL8, UL8.5, UL9, UL10, UL11, and UL12 protein products of herpes simplex virus type 1. Transcriptional analyses indicated presence of 3'-coterminal mRNAs for genes UL8, UL8.5, and UL9 as well as for genes UL6 and UL7, respectively, while UL10 was represented by a very abundant unique transcript. Both gene arrangement and transcriptional organization within this region of the pseudorabies virus genome thus parallels the situation found in other alphaherpesviruses.

Identification and characterization of pseudorabies virus glycoprotein gM as a nonessential virion component.

Sequence analysis within BamHI fragment 3 of the pseudorabies virus (PrV) genome revealed an open reading frame homologous to the UL10 gene of herpes simplex virus. A rabbit antiserum directed against a synthetic oligopeptide representing the carboxy-terminal 18 amino acids of the predicted UL10 product recognized a major 45-kDa protein in lysates of purified Pr virions. In addition, a second protein of 90 kDa which could represent a dimeric form was observed. Enzymatic deglycosylation showed that the PrV UL10 protein is N glycosylated. Therefore, it was designated PrV gM according to its homolog in herpes simplex virus. A PrV mutant lacking ca. 60% of UL10 coding sequences was able to productively replicate on noncomplementing cells, demonstrating that PrV gM is not required for viral replication in cell culture. However, infectivity of the mutant virus was reduced and penetration was delayed, indicating a modulatory role of PrV gM in the initiation of infection.