Objective
The final goal of this proposal is to develop a vaccination vector useful in aquaculture, leading to protection of salmonids against viral diseases as those associated to rhabdoviruses (VHS: Viral Haemorrhagic Septicemia and IHN: Infectious Hematopoietic Necrosis) The fish model is the rainbow trout Oncorhynchus mykiss.
The initial proposal was a four-years program, including four main Tasks:
(1) to localize potential insertion sites in the Herpesvirus salmonids (SaHV-1) viral genome (use of reporter gene);
(2) to study the virulence of the corresponding SaHV-1 recombinants;
(3) to construct recombinants expressing heterologous viral antigens; and
(4) to perform in vivo assays of the recombinants.
According to comments from the Commission, the work program reduced to a period of two years, and the objectives were re-focused as follows:
1. the first one was to generate a viral replicating vector based on Herpesvirus salmonis;
2. the second one was to study the safety on this recombinant by evaluating its loss of virulence (in vivo assays in rainbow trout)
Conclusions
Characterization of the SaHV-1 genome structure has allowed to choose an attractive potential insertion site for foreign DNA, which is dUTPase coding gene. The choice of this site as a primary target for insertion of foreign genes opens the possibility of producing additional mutations in the TK gene (when it is identified), thus providing, a means of further attenuating the virus. The genome of SaHV-1 is similar in structure to those of several mammalian alphaherpesviruses, such as pseudorabies virus (of pigs), bovine herpesvirus 1 (of cattle), equine herpesvirus 1 (of horses) and varicella-zoster virus (of man), and different from that of channel catfish virus. However, SaHV-1 is much more closely related genetically to CCV than it is to mammalian herpesviruses. This supports the view that similar genome structures have arisen independently during herpesvirus evolution. Evidence that the genes in SaHV-1 and CCV are ordered differently mirrors the situation in the three subfamilies of mammalian herpesviruses, where conserved genes are present in several rearranged blocks, and thus provides important data for classification of herpesviruses of lower vertebrates.
Work aimed at determining the complete DNA sequence of SAHV- 1 was continued during the second year of the contract. A preliminary analysis of the SaHV-1 genomic sequence, including the sequence of the dUPase gene, was completed and accepted for publication (Journal of Virology, March 1998). Efforts to locate the TK gene by translational screening were be made while the sequencing is in progress. This gene is, to date, not yet identified.
There are three possible reasons why the TK gene has not been detected.
.Firstly, the genome may not encode a TK. This appears unlikely, since the virus is sensitive to nucleoside analogues that are activated by known herpesvirus thymidine kinases.
.Secondly, none of the random sequences analyzed to date may have originated from a conserved region of TK (about one third of the genome is not currently represented in the database).
.Thirdly, TK may be poorly conserved in SAHV- 1, so that even if some of the random sequences did originate from the TK gene, the gene would not be recognized.
TK is known as a rather poorly conserved protein; only a few residues are identical in all known herpesvirus TKs.
The final goal of this program is to use SaHV-1 as a vector for vaccinating salmonids against viral diseases as those associated to rhabdoviruses. The use of a reporter acne as the one encoding ?-galactosidase is an intermediate step that should facilitate selection of SaHV-1 recombinants. We constructed the pYub-gal plasmid, specifically designed to allow easy insertion of genes of interest in place of the reporter gene.
Transfections were initiated as soon as this plasmid was available. High difficulties appeared due to the very low transfection efficiency of RTG-2 cells. Other cell lines were tested in transient transfection experiments, and promising results were obtained with CHSE-214 cells. Optimal experimental conditions were determined to transfect CHSE-214 with this plasmid. However, cells were completely refractory to the entrance of SAHV- 1 viral DNA. Attempts to avoid this problem were done by using an alternative procedure consisting of transfecting cells previously infected with SAHV- 1. All attempts were unsuccessful. This lack of success in a crucial step of the program obviously led to a "bottle neck" in our initial plan. The initial purpose of testing in fish the virulence of at least one recombinant was thus seriously compromised.
In terms of in vivo experiments, most of the work focused on the study of virulence of the wild-type SAHV- 1 for rainbow trout. Adult rainbow trout immunizations were also carried out in order to produce antisera. These antisera were tested in indirect immunofluorescence assays. After testing, any of the rainbow-trout antisera obtained could be considered as a good immunological tool. Alternatively, immunizations of rabbits and mice were conducted using a recombinant protein as immunization antigen. A part of the SaHV-1 gene corresponding, to CCV ORF46 was cloned and expressed in E. coli, and the purified protein was inoculated to mice and rabbits in order to produce specific antisera.
Virulence studies were conducted with wild-type SaHV-1 and the possibility of using bath delivery as administration route was evaluated as compared to individual fish injection. SaHV-1 virulence appeared as moderate but real in young fish (27% of mortality after 50 days), showing that infection by SaHV-1 is effective by bath delivery for juvenile rainbow-trout. Symptoms observed after infection were extremely crude, they died in less than 24 hours. Neither symptoms nor mortality appeared for older fish. To our knowledge we provide here the first demonstration of the SAHV- 1 Virulence for juvenile rainbow trout using this route of administration.
Downstream from these in vivo experiments, virological and histopathological analysis were conducted. Some of these studies should be confirmed and completed, in particular virolooical analysis, which was highly surprising no virus could be detected in cell culture in spite of the fish mortality observed after SaHV-1 administration by bath delivery. Histopathological studies indicated the appearance of some lesions in yours, fish, moderate and present in fish samples harvested late in the infection. None of the fish analysed within two weeks post-infection show a specific histopathological lesion. Lesions appeared the most often in the liver with a moderate hepatocyte necrosis, sometimes in gills with a widespread epithelial necrosis and pyknosis of the secondary lamellae, and once in the kidney with lesions showing scattered necrosis and pyknosis of epithelial cells of the renal tubules.
Pathogenicity of SaHV-1 was only described after inoculation by individual injection. Mortality rates observed in our challenge experiments demonstrates the viability of bath delivery as administration route of Herpesvirus salmonis (SaHV-1) to rainbow-trout. This obviously constitutes a crucial prerequisite for using SaHV-1 as a potential vector for vaccinating salmonids.
Work and achievements:
1. Preliminary steps
The achievement of the first objective of our program required several preliminary steps that were all initiated during, the first year of the program:
1. sequencing of the SAHV- 1 genome in order to identify potential insertion sites for foreign genes;
2. construction of an intermediate plasmid that will be used to generate SaHV-1 viral recombinants,
3. production and purification of viral DNA required for generating SaHV-1 recombinants
4. production of viral stocks required for fish immunizations
5. determination of the optimal experimental conditions for transfection experiments with the available plasmid.
The essential components of these different tasks foreseen for the year of the contract were accomplished, with a few modifications which were discussed during our coordination meetings. These meetings were organized every six months. Each meeting, was followed by a short report written by the coordinator and sent to each of the other partners. Results obtained during, the first reporting period can be summarized as follows:
1. Sequencing of the SaHV-1 genome allowed to identify a potential insertion site within the SaHV-1 genome. This target gene is the counterpart of CCV gene 49 encoding, deoxynucleoside triphosphatase (dUTPase). It constitutes an attractive insertion site for generating a viral recombinant since it is known to be non-essential in mammalian herpesviruses and to have a role in pathogenicity.
2. An intermediate plasmid was constructed by inserting a reporter gene within the dUTPase gene of the SaHV-1 genome, downstream of the CMV immediate early promoter. The reporter gene that we chose is the Boalactosidase coding gene.
3. SAHV- 1 DNA required for generating viral recombinants was purified from virus grown in mono-layer rainbow trout cells. Yield of DNA production was initially relatively low, but recent yields were significantly higher probably as a result of continued passage of the RTG-2 cell line and perhaps also due to a greater incubator temperature stability.
4. Viral stocks were produced for in vivo experiments, as wild-type virus is required as a positive control in virulence studies and for immunizations of adult rainbow trout in order to obtain specific antisera. These immunizations were not foreseen in the initial work-plan but were decided at our first coordination meeting
5. Transfections of RTG-2 with the available intermediate plasmid were initiated. Transfection efficiency was two low to envisage efficient co-transfections with both plasmid and viral DNA. It was decided to test other fish cell lines as EPC (Epithelioma papulosum cyprini) and CHSE214 (Chinook salmon embryo) cells. EPC cells are not susceptible to SaHV-1 infection but might be used in a first step of co-transfection with plasmid and viral DNAs followed by a second step of infection of susceptible cells. However, CHSE214 cells seemed to be the best candidates for obtaining, SAHV- 1 recombinants.
2.In vivo experiments
The achievement of the second objective of the program required also a preliminary step of determining optimal experimental conditions for in vivo assays in rainbow-trout. Virulence studies were initiated in February 1997. The purpose was to evaluate the virulence of wild-type virus administered by bath delivery. Virulence appeared as moderate but real on young fish (27% of mortality after 50 days), showing that infection by SaHV-1 is effective by bath delivery for juvenile rainbow trout. For older fish, neither symptoms nor mortality was observed although mortality is effective when SaHV-1 is administered by injection instead of bath delivery (data not shown).
In terms of in vivo assays, immunizations of adult rainbow trout were also performed for producing antisera which were tested in neutralization assays and immunofluorescence. As few of these sera showed neutralizing activity and that titers of neutralizing antibodies were very low, several approaches were discussed to improve the immunization efficiency and it was decided to perform rabbits and mice immunization using a recombinant purified SaHV-1 protein. It was proposed to use as immunization antigen a recombinant protein expressed in E. coli and containing a part of the gp46 glycoprotein. A 0.7 Kb fragment containing a part of the CCV homologous-ORF46 sequence was isolated from a cosmid clone and inserted into an expression vector in order to produce a recombinant protein fused at its NH2-terminus to six histidine residues. Expressed protein was purified and inoculated to mice and rabbits to produce a specific antiserum.
3. Second period of the program
According to the results obtained in the preliminary steps described above, the following tasks were continued during the second period of the contract:
1°/ Sequencing the SaHV-1 genome was continued in order to identify another potential insertion site for foreign DNA, particularly the TK locus which was not yet identified at the end of the first year. Analysis of the genomic fragment previously identified indicated the presence of genes corresponding, in order, to open reading, frames (OR-Fs) 57 (DNA polymerase), 58, 49 (dUTPase) and 48 of channel catfish virus. This confirmed that the genomes of SAHV- 1 and CCV are collinear.
Sequences of 857 M13 templates were read and entered into the database. These cover about two-thirds of the SAHV- 1 genome. Sequences were analysed in order to identify the TK locus, their conceptual translation products being screened for similarity to TKs of other herpesviruses. The TK gene was still not detected at the end of this research program.
2°/ SaHV-1 DNA required for generating viral recombinants was purified regularly from batches of purified virions and for extracellular virus. DNA preparations were used in transfection experiments.
3°/ Stocks of wild-type SaHV-1 virus to be used in virulence studies were also produced regularly. A part of this virus was also used for infection of cells further transfected with the plasmid containing the sequence of interest.
4°/ Virological and histological studies were performed on fish harvested from in vivo experiments. Virolocical analyses were done on RTG-2 cell line but surprisingly any cytopathic effect was observed, in young as well as in older fish. Concerning histopathology, some moderate lesions were observed in fish samples hat-vested late in the infection, particularly in young fish. Lesions appeared the most often in the liver, sometimes in gills, and once in the kidney. In older fish, lesions were only observed in gills.
5°/ Many attempts of transfecting RTG-2 cells (Rainbow Trout Gonad cells) were done using several methods and numerous conditions, but without success. It was thus decided to try another cell line for transfections: CHSE-214 (Chinook Salmon Embryo) cells. CHSE-214 fish cells could be transfected with the plasmid DNA, however they were totally refractory to the entrance of SaHV-1 DNA. Any recombinant virus could be isolated. In fact, any cytopathic effect was observed after transfection with viral DNA, even when SaHV-1 DNA was used alone in simple transfection experiments instead of co-transfections. An alternative method was assayed that consisted of transfecting with the plasmid SaHV-1 infected cells, at successive post-infection times (24, 48 and 72 hours). In this case, infection was effective, however any transfected cell could be revealed further.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- social sciences sociology demography mortality
- medical and health sciences basic medicine immunology immunisation
- natural sciences biological sciences microbiology virology
- natural sciences biological sciences genetics DNA
- medical and health sciences clinical medicine embryology
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4000 Sart Tilman - Liege
Belgium
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