Emerging vesiculo-type virus infections of freshwater fishes in Europe.

Rhabdoviruses were isolated from perch Perca fluviatilis and largemouth bass Micropterus salmoides exhibiting clinical signs of disease. Preliminary studies indicated that these viruses could be neutralised by antisera to perch rhabdovirus (Dorson et al. 1984) and may be similar to those previously isolated from grayling Thymallus thymallus and pike-perch Stizostedion stizostedion.

Malacosporean-like spores in urine of rainbow trout react with antibody and DNA probes to Tetracapsuloides bryosalmonae.

Tetracapsuloides bryosalmonae is the myxozoan parasite causing proliferative kidney disease (PKD) of salmonid fishes in Europe and North America. The complete life cycle of the parasite remains unknown despite recent discoveries that the stages infectious for fish develop in freshwater bryozoans. During the course of examinations of the urine of rainbow trout (Oncorhynchus mykiss) with or recovering from PKD we identified spores with features similar to those of T.

Phylogenetic analysis of viral haemorrhagic septicaemia virus (VHSV) isolates from France (1971-1999).

The nucleotide sequences of a specific region of the glycoprotein gene were compared among 63 strains of viral haemorrhagic septicaemia virus (VHSV) isolated from fish in France between 1971 and 1999. The analysis was performed on a region corresponding to amino acids 238 to 331 of the glycoprotein gene, also designated the V2 region and previously shown to accumulate most of the mutations. The sequences of many VHSV isolates were found to be identical or very conserved.

Sea bream Sparus aurata, an asymptomatic contagious fish host for nodavirus.

During an epidemiological survey of viral encephalopathy and retinopathy (VER) in diseased sea bass Dicentrarchus labrax, a nodavirus isolate was recovered from net pen-reared sea bream Sparus aurata harboured in the same farming premises. After the virus was isolated and identified by immunofluorescence on SSN-1 cells, sequence analysis with a PCR product from the T4 region of the capsid protein gene indicated that the virus shared 100% identity with a pathogenic virus strain isolated from sea bass. Infection trials demonstrated the pathogenicity of the sea bream virus isolate for juvenile sea bass whereas sea bream infected with the same virus isolate remained asymptomatic even following intramuscular injection of virus.

Evidence that infectious stages of Tetracapsula bryosalmonae for rainbow trout Oncorhynchus mykiss are present throughout the year.

Proliferative kidney disease (PKD) is a hyperplastic condition of the lymphoid tissue of salmonids infected with the spores of Tetracapsula bryosalmonae, a myxozoan parasite formerly designated PKX, which has recently been described as a parasite of several species of bryozoans. The occurrence of PKD is generally associated with seasonal increase in water temperature, with research indicating that transmission of the disease does not occur below 12 to 13 degrees C. This suggested that the infectious stages are absent from about November to March/April.

Molecular evidence that the proliferative kidney disease organism unknown (PKX) is a myxosporean.

The proliferative kidney organism unknown (PKX), a serious salmonid fish pathogen, is considered to be a myxosporean on the basis of ultrastructural studies, but its real taxonomic position has never been confirmed. In order to ascertain its position, genomic DNA was extracted from PKX and small subunit (SSU) ribosomal DNA was amplified by PCR, cloned and sequenced. A phylogenetical analysis on SSU rDNA from 76 or 128 eucaryotic species was carried out.

Mutations in the glycoprotein of viral haemorrhagic septicaemia virus that affect virulence for fish and the pH threshold for membrane fusion.

To study the molecular basis of virulence of viral haemorrhagic septicaemia virus (VHSV), we used a cross-reactive neutralizing MAb to select MAb-resistant (MAR) mutants with reduced pathogenicity for fish. From sequence determination of the G gene of MAR mutants, attenuated laboratory variant and avirulent field strains, we identified two distant regions of the glycoprotein associated with virulence: region I (aa 135-161), homologous to the putative fusion peptide of both rabies virus (RV) and vesicular stomatitis virus (VSV), and region II (surrounding aa 431-433), homologous to RV and VSV domains controlling the conformational changes necessary for the fusion process to take place. Simultaneous mutations in both regions resulted in the most attenuated phenotype and we obtained genetic evidence that regions I and II may be structurally linked.

Combined DNA immunization with the glycoprotein gene of viral hemorrhagic septicemia virus and infectious hematopoietic necrosis virus induces double-specific protective immunity and nonspecific response in rainbow trout.

Glycoprotein (G) of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) contains several neutralizing epitopes. However, recombinant G protein never matches intact viral particles for immunogenicity. DNA immunization offers the possibility to deliver the antigen through the cellular machinery, thus mimicking natural infection.

Sequence variation of the glycoprotein gene identifies three distinct lineages within field isolates of viral haemorrhagic septicaemia virus, a fish rhabdovirus.

To evaluate the genetic diversity of viral haemorrhagic septicaemia virus (VHSV), the sequence of the glycoprotein genes (G) of 11 North American and European isolates were determined. Comparison with the G protein of representative members of the family Rhabdoviridae suggested that VHSV was a different virus species from infectious haemorrhagic necrosis virus (IHNV) and Hirame rhabdovirus (HIRRV). At a higher taxonomic level, VHSV, IHNV and HIRRV formed a group which was genetically closest to the genus Lyssavirus.

Live fish vaccines: history and perspectives.

In the development of live vaccines against enzootic fish viral diseases, the conventional approaches, although somewhat successful, failed finally to deliver efficient, safe, and tagged strains for vaccine application. The genetically-engineered vaccine approach also gave similarly disappointing results. Faced with these realities during our work with VHS, we turned our research effort towards understanding the molecular basis of virulence and antigenicity of the virus.

Cloning, sequencing and expression of a cDNA encoding an antigen from the Myxosporean parasite causing the proliferative kidney disease of salmonid fish.

A pool of monoclonal antibodies (MAbs) raised against the unknown organism causing the proliferative kidney disease of salmonid fish (PKX) has been used to screen a cDNA expression library constructed from poly (A+) RNA extracted from PKX infected kidney. Four immunopositive lambda ZapII recombinant phages were selected. Sequencing of the cDNAs revealed identical 3' ends.

Serum neutralization test for epidemiological studies of salmonid rhabdoviroses in France.

Serological examination is not yet accepted as being a suitable diagnostic method for fish that are asymptomatic virus carriers. Nevertheless, encouraging preliminary results using an end-point serum neutralization test (SNT) in several French trout farm populations have demonstrated an excellent correlation between the SNT and the previously established virus histories of the tested populations. Following the isolation of infectious haematopoietic necrosis virus (IHNV) in France, serological screening of fish for a neutralizing antibody (NAb) to IHN was conducted on a national scale.

Genetic diversity and phylogenetic classification of viral hemorrhagic septicemia virus (VHSV).

The present study was undertaken to determine the genetic diversity of viral hemorrhagic septicemia virus (VHSV) and to gain insight into the molecular epidemiology of this fish rhabdovirus. The sequences of the nonstructural (NV) protein and the transmembrane (G) protein of sequential North American and European isolates of VHSV were determined and used to compute phylogenetic trees. According to the percentage of nucleotide or amino acid similarities, North American and European isolates formed 2 clearly distant genetic groups.

The glycoprotein of viral hemorrhagic septicemia virus (VHSV): antigenicity and role in virulence.

In order to study the antigenic structure of the G protein of VHSV, we produced several anti-G monoclonal antibodies (MAbs) and used 4 neutralizing MAbs (NMAbs) to select resistant (MAR) mutants. Each MAR mutant was confronted with the 4 NMAbs in a neutralization test, and also with our panel of MAbs in surface plasmon resonance (SPR) analysis to determine the extent of their relatedness. Determination of the sequence of the entire G gene of representative MAR mutants allowed us to map the mutations responsible for the resistant phenotypes.

Eighteen years of vaccination against viral haemorrhagic septicaemia in France.

Viral haemorrhagic septicaemia (VHS) has been considered for many years to be a major cause of loss in the French trout industry. The high prevalence of VHS in certain geographic areas made a control strategy based on control policy unfeasible. This provided the impetus for immunoprophylaxis development that resulted in 3 successive types of vaccines: inactivated, live attenuated and recombinant vaccines.

[Epidemiology of infectious hematopoietic necrosis (IHN) of salmonid fish in France: study of the course of natural infection by combined use of viral and seroneutralization test and eradication attempts].

Infectious haematopoietic necrosis (IHN), a rhabdoviral infection of salmonid fish, was considered to be an exotic disease in Europe until it was recognized in France and Italy in 1987. In France, the existence of this new condition led the authorities in charge of animal health to order epidemiological studies to be undertaken. These studies were based upon virological, serological and experimental diagnostic methods and also encompassed disease eradication attempts.

A recombinant viral haemorrhagic septicaemia virus glycoprotein expressed in insect cells induces protective immunity in rainbow trout.

Viral haemorrhagic septicaemia (VHS) is a fish rhabdovirus infection of world-wide importance. Control policies have been established but the disease still causes heavy losses in fish farming. The development of a recombinant subunit vaccine was initiated to produce a safe and effective vaccine to protect fish against VHS.

The polymerase-associated protein (M1) and the matrix protein (M2) from a virulent and an avirulent strain of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus.

We have cloned and sequenced the M1 and M2 genes of both a European (virulent) and a North American (avirulent) strains of viral hemorrhagic septicemia virus, an important fish pathogen. We also compared the transcription of the two genes following infection of cells and determined the phosphorylation status and detergent solubility of the two proteins. Despite a total lack of homology with any available rhabdoviral sequence, the two VHSV proteins share comparable structural features with their respective VSV and RV equivalents.

Highly sensitive immunoassay for direct diagnosis of viral hemorrhagic septicemia which uses antinucleocapsid monoclonal antibodies.

An antigen capture enzyme-linked immunosorbent assay (ELISA) based on the detection of the viral nucleocapsid (anti-N system) was developed for the diagnosis of viral hemorrhagic septicemia. Four monoclonal antibodies directed against the viral nucleocapsid were produced; they all recognized the four viral hemorrhagic septicemia virus (VHSV) serotypes. Three of these monoclonal antibodies were used in a new antigen capture ELISA.

Sequence of a cDNA carrying the glycoprotein gene and part of the matrix protein M2 gene of viral haemorrhagic scepticaemia virus, a fish rhabdovirus.

A cDNA clone encoding for the glycoprotein of the viral haemorrhagic scepticaemia virus, a fish rhabdovirus, has been sequenced. The cDNA was 2035 bp long and contained two open reading frames (ORF). A 1523 bp ORF corresponded to the glycoprotein and was adjacent, on its 5' side, to an incomplete 372 bp ORF.

Antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I.

An antigen-capture ELISA for viral haemorrhagic septicaemia virus serotype I (VHSV I) was developed. The assay employs two monoclonal antibodies (mAb) directed against distinct epitopes of the viral envelope glycoprotein (Gp). The antigen bound by the capture mAb (A17) was detected by addition of a second mAb (L7) conjugated to horseradish peroxidase, followed by addition of the enzyme substrate.

Cloning and sequencing the messenger RNA of the N gene of viral haemorrhagic septicaemia virus.

The mRNA transcribed from the N gene of viral haemorrhagic septicaemia virus (VHSV) of salmonids has been cloned in Escherichia coli and expressed. Fusion proteins were recognized by monoclonal antibody directed against the N protein from the viral particle. A 1212 bp long open reading frame (ORF) coding for 404 amino acids with a calculated Mr of 44590 was deduced from the nucleotide sequence.

Molecular cloning of the mRNA coding for the G protein of the viral haemorrhagic septicaemia (VHS) of salmonids.

Viral haemorrhagic septicaemia virus (VHSV), a rhabdovirus, is a major threat for continental European trout fish farming. The development of a recombinant subunit vaccine could solve that problem. The neutralizing epitopes are located on the glycoprotein or G protein, the surface antigen.

Natural anti-TNP antibodies from rainbow trout interfere with viral infection in vitro.

Normal and viral-infected rainbow trout (RT) were tested for serum antibody activity against self and nonself antigens. Particularly high titres of anti-trinitrophenyl (TNP) antibodies were noted, as in other fish species. To analyse this, the anti-TNP antibodies were isolated by affinity chromatography and their capacity to interfere with viral infection in vitro was studied.