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Molecular biology of piscine herpesviruses
Project
Project code: FLI-IMVZ-08-DA_0002
Contract period: 01.01.2001
- 31.12.2020
Purpose of research: Applied research
Herpesvirus induced diseases can cause substantial economic losses in fish industry. The koi herpesvirus of (KHV) was first described in the late 1990ties, but in the mean time has spread over major parts of the world. KHV causes severe disease with high mortality rates not only in kois, but also in common carps. The DNA sequence of the virus genome has been completely determined, but up to now only few viral gene products were identified and structurally or functionally analyzed. Because of the high phylogenic distance to better characterized herpesviruses of mammals and birds nearly no sequence homologies can be found and conclusions by analogy are barely possible. On the other hand, there are several other poorly characterized, but economically important pathogenic herpesviruses of carps, eels and salmonides, which exhibit considerable genetic homology to KHV. Thus, the insights obtained by molecular characterization of KHV might be also helpful to combat of other fish diseases. Major aims of our current work are the identification of immunogenic envelope proteins of KHV, which, after expression in heterologous systems, might serve as diagnostic antigens, vectored or DNA vaccines, and directed deletion of nonessential genes from the KHV genome to generate attenuated live virus vaccines. Furthermore, KHV deletion mutants and corresponding trans-complementing cell lines shall be also used to elucidate the functions of individual, in particular membrane-associated virus proteins in receptor binding, virus entry, primary and secondary envelopment, and release of mature virus particles.
Up to now, we have investigated nine putative membrane proteins of KHV, and six of them (pORF25, pORF65, pORF81, pORF99, pORF136 und pORF149), as well as the major capsid protein (pORF92) could be detected in virus-infected cells using the prepared monospecific antisera. After plasmid cloning and transient expression in eukaryotic the gene products pORF25, pORF65 and pORF149 showed specific immunofluorescence reactions with serum antibodies of experimentally and naturally infected carps, indicating that these proteins might be suitable as diagnostic antigens. Furthermore, pORF149 was identified as the target protein of several KHV-specific monoclonal antibodies. The polyclonal antiserum against the abundant membrane protein pORF81 was most suitable for detection of KHV virions, and of infected cells in tissues of affected fish by Western blot, indirect immunofluorescence, immunhistochemistry and immunoelectron microscopy. Meanwhile we have generated KHV recombinants with deletions of the genes encoding the immunogenic envelope glycoproteins pORF25, pORF65, pORF148 and/or pORF149 demonstrating that they are dispensable for virus replication in cell culture. Regrettably in animal trials the ORF148 and ORF149 deletion mutants did not exhibit significantly reduced virulence, and, thus, are not suitable as live virus vaccines. However, by additional deletion of virulence factors serological DIVA (differentiation of infected from vaccinated animals) vaccines might be generated. Since for several mammalian herpesviruses genes encoding enzymes of nucleotide metabolism were identified as important virulence factors, we generated KHV recombinants possessing deletions of the genes of viral thymidine kinase (TK, pORF55), desoxyuridine triphosphate-hydrolase (DUT, pORF123), or the large subunit of ribonucleotide reductase (RNR, pORF141), as well as corresponding revertants. Because of the presence of functionally homologous cellular proteins, these deletion mutants were replication competent in carp cell cultures. However, compared to the parental virus and the revertant, mutant RNR-negative KHV exhibited severe replication defects, which might prevent its efficient production as a putative vaccine. In contrast, in vitro replication of the TK- and DUT-negative KHV mutants, and of corresponding double mutants, was not detectably affected. To further improve production capacities, the mutations have been introduced into a cell-culture adapted KHV strain (KHV-Taiwan), which grows to nearly 1000fold higher titers than the originally used field isolate from Israel. In experimentally infected carps, all mutants caused significantly less pronounced clinical symptoms, and much lower mortality rates than the highly pathogenic parental virus, and the TK- and DUT-negative double mutant proved to be almost avirulent. Nevertheless, after a single immunization by immersion the fish were protected against lethal challenge infections with virulent KHV. Furthermore, we have established TK-and DUT-gene-specific PCR techniques for rapid diagnostic discrimination of animals infected with the putative KHV vaccines or wild type viruses. Furthermore, a described KHV-specific real-time PCR (qPCR) was modified by additional TK gene-specific primers and probes to obtain a highly sensitive multiplex qPCR permitting differentiation of vaccinated and wild type virus-infected carp on the basis of gill swabs (genetic DIVA). Recently we have also generated KHV recombinants exhibiting combined deletions of TK, DUT, ORF148 und ORF149 genes, which will be now characterized in vitro und in vivo. Publications Fuchs W, Granzow H, Dauber M, Fichtner D, Mettenleiter TC. 2014. Identification of structural proteins of koi herpesvirus. Arch Virol 159, 3257-3268. Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. 2011. Generation and characterization of koi herpesvirus recombinants lacking viral enzymes of nucleotide metabolism.Arch Virol 156, 1059-1063. Rosenkranz D, Klupp BG, Teifke JP, Granzow H, Fichtner D, Mettenleiter TC, Fuchs W. 2008. Identification of envelope protein pORF81 of koi herpesvirus. J Gen Virol 89, 896-900.
Section overview
Subjects
- Animal health
- Freshwater fisheries
- Pond fisheries
- Biotechnology
Framework programme
Funding programme
Excutive institution
FLI - Institute of Molecular Virology and Cell Biology (FLI - IMVZ)
