Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein.

Fourteen reptilian paramyxovirus isolates were chosen to represent the known extent of genetic diversity among this novel group of viruses. Selected regions of the fusion (F) gene were sequenced, analyzed and compared. The F gene of all isolates contained conserved motifs homologous to those described for other members of the family Paramyxoviridae including: signal peptide, transmembrane domain, furin cleavage site, fusion peptide, N-linked glycosylation sites, and two heptad repeats, the second of which (HRB-LZ) had the characteristics of a leucine zipper.

Complete genome sequence of Fer-de-Lance virus reveals a novel gene in reptilian paramyxoviruses.

The complete RNA genome sequence of the archetype reptilian paramyxovirus, Fer-de-Lance virus (FDLV), has been determined. The genome is 15,378 nucleotides in length and consists of seven nonoverlapping genes in the order 3' N-U-P-M-F-HN-L 5', coding for the nucleocapsid, unknown, phospho-, matrix, fusion, hemagglutinin-neuraminidase, and large polymerase proteins, respectively. The gene junctions contain highly conserved transcription start and stop signal sequences and tri-nucleotide intergenic regions similar to those of other Paramyxoviridae.

Investigations on the ORF 167L of lymphocystis disease virus (Iridoviridae).

The predicted open reading frame 167L (ORF 167L) of lymphocystis disease virus (LCDV, Iridoviridae ) isolated from plaice, dab and flounder was investigated. The ORF 167L corresponding genes of the three LCDV isolates were amplified, cloned and sequenced. A comparison of the LCDV strains showed that the nucleotide sequence of ORF 167L and its deduced amino acid sequence were highly conserved in the genus lymphocystivirus (a homology of 80% in dab and flounder/plaice, 97% in plaice and flounder).

Characterization of aquabirnaviruses from flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA.

Viruses were isolated in cell culture from tissue homogenates of flounder Pseudopleuronectes americanus and mummichog Fundulus heteroclitus in the Chesapeake Bay, Virginia, USA. Neutralization and immunofluorescence tests with aquabirnavirus (West Buxton strain)-specific polyclonal antisera indicated that both viruses were aquabirnaviruses belonging to Serogroup A, the most common aquabirnavirus serogroup in the United States. This was confirmed by RT-PCR, with primers targeting the VP3 and VP2 gene of aquabirnaviruses.

Nucleotide sequence analysis of the glycoprotein gene of putative spring viraemia of carp virus and pike fry rhabdovirus isolates reveals four genogroups.

RT-PCR methods have been applied to the detection and sequencing of the glycoprotein gene of putative spring viraemia of carp viruses (SVCV) and pike fry rhabdoviruses (PFRV), including isolates from tench, grass carp, roach, bream and false harlequin, sheatfish and orfe. Phylogenetic analysis of a 550 nucleotide (nt) region of the glycoprotein gene identified 4 groups, I to IV. Significantly, the majority of viruses previously identified as PFRV formed a distinct cluster (Genogroup IV) which shared <80% nucleotide identity with the PFRV reference strain (Genogroup III).

Spring viremia of carp (SVC).

Spring viremia of carp (SVC) is an important disease affecting cyprinids, mainly common carp Cyprinus carpio. The disease is widespread in European carp culture, where it causes significant morbidity and mortality. Designated a notifiable disease by the Office International des Epizooties, SVC is caused by a rhabdovirus, spring viremia of carp virus (SVCV).

Genomic and phylogenetic analyses of an adenovirus isolated from a corn snake (Elaphe guttata) imply a common origin with members of the proposed new genus Atadenovirus.

Approximately 60% of the genome of an adenovirus isolated from a corn snake (Elaphe guttata) was cloned and sequenced. The results of homology searches showed that the genes of the corn snake adenovirus (SnAdV-1) were closest to their counterparts in members of the recently proposed new genus ATADENOVIRUS: In phylogenetic analyses of the complete hexon and protease genes, SnAdV-1 indeed clustered together with the atadenoviruses. The characteristic features in the genome organization of SnAdV-1 included the presence of a gene homologous to that for protein p32K, the lack of structural proteins V and IX and the absence of homologues of the E1A and E3 regions.

First molecular evidence for the existence of distinct fish and snake adenoviruses.

From adenovirus-like viruses originating from a fish and a snake species, a conserved part of the adenoviral DNA polymerase gene was PCR amplified, cloned and sequenced. Phylogenetic analysis showed that the snake adenovirus is closely related to the members of the proposed genus Atadenovirus, whereas the fish isolate seems to represent a separate cluster, likely a new genus.

Environmental toxicology: polychlorinated biphenyls impair TNF-alpha release in vitro.

Tumour necrosis factor (TNF-alpha) was induced by bacterial lipopolysaccharides (LPS)/phytohemagglutin (PHA) stimulated human whole blood in vitro. Levels of TNF-alpha were evaluated by enzyme-linked immunosorbent assay. Blood samples treated with polychlorinated biphenyls (PCB 77, PCB 126) exhibited impairment of TNF-alpha release: 50 pg/microl PCB reduced by up to 66% and 500 pg/microl PCB reduced by up to 93% the TNF-alpha release compared with the controls.

The epizootic haematopoietic necrosis virus (Iridoviridae) induces apoptosis in vitro.

The epizootic haematopoietic necrosis virus (EHNV) is an iridovirus causing severe disease in different fish species. We investigated the induction of apoptosis during EHNV infection of the epithelioma carp papulosum (EPC) cell line. Apoptosis reveals several characteristic morphological changes, such as chromatin condensation, nuclear fragmentation, cytoplasm membrane disorientation, or mitochondrial changes.

Comparison of the eIF-2alpha homologous proteins of seven ranaviruses (Iridoviridae).

The alpha-subunit of the eukaryotic initiation factor 2 (eIF-2alpha) is a key component of the translation machinery of the cell. In response to cellular stress such as viral infections, eIF-2alpha is phosphorylated by double-stranded RNA-dependent protein kinase (PKR) leading to the inhibition of cellular protein synthesis. The importance of eIF-2alpha as a regulatory mechanism for protein synthesis is illustrated by the wide variety of strategies employed by viruses to down-regulate PKR.

Occurrence of an invertebrate iridescent-like virus (Iridoviridae) in reptiles.

Viral isolates were obtained in 1998, 1999 and 2000 from the lung, liver and intestine of two bearded dragons (Pogona vitticeps) and a chameleon (Chamaeleo quadricornis) and from the skin of a frill-necked lizard (Chamydosaurus kingii) by using viper heart cells (VH2) at 28 degrees C. Electron microscopic examination of infected VH2 cells revealed the assembly of icosahedral iridovirus-like particles measuring 139 nm (side to side) and 151 nm (apex to apex). Negatively stained virus particles had dimensions of 149 nm (side to side) and 170 nm (apex to apex).

Identification and molecular characterization of 18 paramyxoviruses isolated from snakes.

Viral agents from 18 different snake species (families Colubridae, Viperidae, and Crotalidae) showing respiratory symptoms and neuronal disease were identified as paramyxoviruses by typical cytopathogenic effect (CPE), electron microscopy, and hemagglutination inhibition. Detailed molecular characterization of the viruses was performed by partial L- and F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) and sequencing, nucleotide and amino acid sequence alignment, and phylogenetic analysis (PHYLIP). RT-PCR of the partial L-gene (566 nt) was successful for all 18 viruses; amplicons of the partial F-gene (918 nt) could be obtained in 16 cases.

Viruses of lower vertebrates.

Viruses of lower vertebrates recently became a field of interest to the public due to increasing epizootics and economic losses of poikilothermic animals. These were reported worldwide from both wildlife and collections of aquatic poikilothermic animals. Several RNA and DNA viruses infecting fish, amphibians and reptiles have been studied intensively during the last 20 years.

[An in vitro test system for evaluation of human-immunotoxicological actions of polychlorinated biphenyls].

An in vitro test system using isolated human leukocytes for evaluation of immunotoxicological actions of polychlorinated biphenyls (PCB) is described. Immunotoxic effects of PCB 77, PCB 126 could be demonstrated by evaluation of the production of interleukin IL-1alpha, IL-2 and the tumor necrosis factor (TNF-alpha) of stimulated human leukocytes. The release of IL-1alpha, IL-2 and TNF-alpha was suppressed up to 60-80% by 0,05 ng of the PCB.

Adaptation of reptilian paramyxovirus to mammalian cells (Vero cells).

The reptilian paramyxovirus GOV was successfully adapted to Vero cells in 80 passages at 30 degrees C. The virus replicated with HA titres of 1:64 and 10(7.5) TCID50/ml in the heterologous host cells forming syncytia, giant cells and cytoplasmic inclusion bodies.

Reptilian paramyxoviruses induce cytokine production in human leukocytes.

The reptilian paramyxoviruses FDLV and GOV initiated the production and release of cytokines like IL-1alpha, IL-1beta, IL-2, TNF-alpha and IFN-alpha in human peripheral blood mononuclear cells (PBMC) at 37 degrees C. The target cells produced the cytokines without replication of virus.

Comparative sequence analyses of sixteen reptilian paramyxoviruses.

Viral genomic RNA of Fer-de-Lance virus (FDLV), a paramyxovirus highly pathogenic for reptiles, was reverse transcribed and cloned. Plasmids with significant sequence similarities to the hemagglutinin-neuraminidase (HN) and polymerase (L) genes of mammalian paramyxoviruses were identified by BLAST search. Partial sequences of the FDLV genes were used to design primers for amplification by nested polymerase chain reaction (PCR) and sequencing of 518-bp L gene and 352-bp HN gene fragments from a collection of 15 previously uncharacterized reptilian paramyxoviruses.

Partial mapping and sequencing of a fish iridovirus genome reveals genes homologous to the frog virus 3 p31, p40 and human eIF2alpha.

Iridovirus-like pathogens have been recognized as a cause of serious systemic diseases among feral, cultured and ornamental fish in the recent years. Mortalities of fish due to systemic iridovirus infection reaching 30-100% were observed in Europe, Australia, Japan and Thailand. Up to now, the molecular biology of these important pathogens has been poorly documented.

Replication of reptilian paramyxovirus in avian host systems.

The reptilian paramyxovirus GOV replicated in chicken embryo fibroblasts, in embryonated chicken eggs and in explanted chorio-allantoic membrane with titres of up to 10(8.2) TCID50/ml at 28 degrees C. The virus did not multiply above 30 degrees C.

Comparison of European systemic piscine and amphibian iridoviruses with epizootic haematopoietic necrosis virus and frog virus 3.

Iridovirus-like agents isolated from systemic infected fish (Silurus glanis, SFIR; Ictalurus melas, CFIR I, CFIR II, CFIR III) and from frogs (Rana esculenta, REIR) in Europe, Epizootic Haematopoietic Necrosis Virus (EHNV) isolated in Australia from redfin perch (Perca fluviatilis), and Frog Virus 3 (FV 3) isolated from frogs (Rana pipiens) in the USA were investigated by electron microscopy, polypeptide composition, immunofluorescence, restriction endonuclease digestion, Southern-blot hybridization and polymerase chain reaction (PCR) amplification. All virus isolates proved to be similar in morphology and in size and reacted with EHNV polyclonal antiserum in the immunofluorescence. Whilst DNA restriction profiles of the European piscine isolates cleaved by BamH I were similar, they differed clearly from those of EHNV, REIR and FV 3.

Poxvirus preparation CONPIND initiates production of the major inflammatory mediators IL-1 alpha and TNF-alpha in human whole blood and in blood mononuclear cell cultures.

The poxvirus preparation CONPIND initiated the production of the two major inflammatory mediators interleukin-1 alpha (IL-1 alpha) and tumour necrosis factor-alpha (TNF-alpha) in whole blood and in blood mononuclear cell cultures of man at 37 degrees C within 12-48 h. The release of the cytokines followed a dose-efficacy curve. High levels of IL1-alpha (up to 810 pg/ml) and TNF-alpha (up to 533 pg/ml) were determined by sandwich enzyme-linked immunosorbent assay.

Immunoreactive cytokines within primates.

Peripheral blood mononuclear cells of primates (man, orang utan, gorilla, baboon), rodents (mouse, rat), carnivores (cat, dog), artiodactyls (cattle, goat, pig) and perissodactyls (horse) were isolated and stimulated with mitogens (5 micrograms/ml LPS, 5 micrograms/ml PHA) at 37 degrees C. Cytokines immunoreactive to monoclonal antibodies (mAb) directed to human cytokines (TNF-alpha, IL-1 alpha, IL-2, IL-6, IFN-gamma) could be detected by enzyme-linked immunosorbent assay (ELISA) in the case of primates only. The mAb used did not recognize cytokines of the other mammalian species investigated.

[Immune response of fishes with respect to phylogenetic aspects].

The immune response progressed by steps in the phylogeny of animals. The non-specific immune response has been improved by specific immune mechanisms during the early evolution of the organisms. Typical B- and T-cells as well as true antibodies originated first in fishes during the phylogenesis.

In vitro replication of a reptilian adenovirus.

The morphological alterations of IgH-2 cells infected with a reptilian adenovirus were investigated during 1 cycle of virus replication. The virus particles entering the cell 1 h post infection (pi) were present in cytoplasmic receptosomes few hours later. About 24 h pi nucleocapsids entered the nucleus.