Genetically engineered virus-based prophylactic and therapeutic approaches against Spring Viremia of Carp Virus (SVCV) and Viral Hemorrhagic Septicemia Virus

Abstract

Fish rhabdoviruses are considered as one of most devastating viruses for the worldwide fish rearing field. Up to date, there is still no effective control strategy to be used for treating those viral diseases or to prevent their infection. During our study, we focused on two important rhabdoviruses, spring viremia of carp virus (SVCV) and viral hemorrhagic septicemia virus (VHSV) which are infamous cause of high mortalities in freshwater and marine fish, and we attempted the development of therapeutic and prophylactic approaches using reverse genetics in order to control those two viruses’ propagation and to understand more about their genes function and interaction with their host. In chapter 1, we have studied one of SVCV gene’s function, the phosphoprotein, since it is an essential component for virus replication, and it has a role in the suppression of type I interferon of the host cells. For that reason, we have generated recombinant snakehead expressing SVCV P gene by inserting the P gene into SHRV genome between its N and P gene. The in vitro viral growth test showed a significantly higher titer of rSHRV-A-Psvcv compared to the control virus rSHRV-A-eGFP. In addition, the result of the luciferase assay that was conducted to verify the effect of SVCV P gene when it is inserted into SHRV genome on type I interferon, showed that comparing to the control virus, rSHRV-A-Psvcv seemed to suppress more the interferon response. After confirming that in vitro, the role of SVCV P gene was investigated in vivo through conducting a virulence test by infecting zebrafish with either rSHRV-A-Psvcv or rSHRV-A-eGFP. As a result. high mortality occurred from the fish infected with rSHRV-A-Psvcv compared to the control virus group with a significantly higher viral growth (at 7d at 15°C and at 3d at 28°C). Through this findings, SVCV P gene could reverse the virulence of SHRV in vivo and it may have an effect on the replication of the virus by delaying the immune response which can be explained by the late appearance of the immune relevant pathways enriched through RNA-seq analysis. In the second part of this thesis, the development of a therapeutic tool has been carried out to restrain SVCV infection in vivo, using artificial microRNA (AmiRNA) targeting SVCV P gene transcript. synthesized AmiRNA mimics and AmiRNA-expressing vector system were used to determine the downregulation ability of the three candidates of AmiRNAs (AmiR-P1, -P2, and –P3) against SVCV P gene transcript, among which AmiR-P3 was chosen since it showed a higher inhibitory activity. Based on the in vitro results, we rescued (SHRVs) expressing the chosen SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) in order to overcome any limitation of AmiRNA mimics or the AmiRNA-expressing vector systems in in vivo. After verifying in vitro that the expression of AmiR-P3 and AmiR-C was successful through rSHRVs, we evaluated the availability of rSHRV-AmiR-P3 for in vivo control of SVCV. For that, zebrafish were infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. as a result, there was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C or rSHRV-AmiR-P3 before SVCV infection, however, the survival rate in the group of fish infected with SVCV followed by infection with rSHRV-AmiR-P3 was significantly higher than in the group of fish infected with rSHRV-AmiR-C. Based on the present study results, we could verify that rSHRV could be a suitable system for a successful expression of AmiRNA, and rSHRV expressing AmiR targeting SVCV P gene could be used as an alternative to control SVCV infection in fish for therapeutic purpose. In chapter 3, prophylaxis was developed based on SVCV glycoprotein in order to provide protection of fish against SVCV infection. we have used chimeric rSHRV-Gsvcv as a live attenuated vaccine for the protection of zebrafish against SVCV. At first two experiments were conducted where zebrafish were immunized with two different doses of chimeric rSHRV-Gsvcv (1×104 pfu or 1×103 pfu) and after SVCV challenge, both doses showed significant high survival rate comparing to the control group which lead to the choice of the lowest dose to be used in the third experiment where a third control group wad added (rSHRV). After immunization, the immunized fish were challenged with SVCV to evaluate the protectivity of rSHRV-Gsvcv. Consequently, chimeric rSHRV-Gsvcv showed a significantly higher survival rate compared to the control groups. Based on this, chimeric rSHRV-Gsvcv can be considered as a successful tool to be used in vivo to protect fish against SVCV infection. In chapter 4, we have focused on VHSV’s G protein (glycoprotein) which has always been an interesting element to study its function and role related to the viral replication and general mechanism. The manipulation of glycoprotein by exchanging its signal peptide with a high secreted antimicrobial peptide gene “piscidin” was performed in order to increase recombinant VHSV titer. rVHSV expressing piscidin signal peptide exchanged-glycoprotein (rVHSV-PspvG) was produced the in vitro comparison of its titer showed a lower titer comparing with rVHSV-wild titer. This could be related to the fact that overexpression of the protein can lead to its accumulation into the ER causing ER stress which results in the initiation of the unfolded protein response (UPR). This UPR activates protein kinase R-like ER kinase (PERK) that phosphorylates eukaryotic initiation factor 2α (eIF2 α), attenuating global translation. To investigate this fact, antioxidants (ER stress reliver; BHA and SA), PERK inhibitor (GSK) or ER stress inducer (Tunicamycin) were used to determine the relation between ER stress and PERK pathway on VHSV growth. The result of the viral growth showed that the highest titer of both rVHSV-PspvG and rVHSV-wild reached from the cells treated with PERK inhibitor and the lowest one was shown from the ER stress inducer treated cells. Through this result, VHSV replication seems to be related to the ER stress and influenced by the activation of PERK pathway. The effect of piscidin signal peptide on the virus replication and on the immunogenicity of glycoprotein was determined by conducting virulence test of rVHSV-PspvG and DNA vaccine based on G gene in vivo. The virulence test result showed that a late and lower mortality was triggered by rVHSV-PspvG at both injected doses (1×105 pfu or 1×103 pfu) in comparison with rVHSV-wild injected fish. The DNA vaccine data revealed that PspvG-immunized fish showed significantly lower survival rate compared to the original vG. These findings can interpret that the secretion of the glycoprotein influences the virus replication and the protective ability of glycoprotein in vivo. In the present study, we could prove that recombinant viruses generated through reverse genetics can serve for several purposes such as the study of a heterologous gene such as in our case the usage of rSHRV-A-Psvcv, for the delivery of therapeutic tools or for prophylactic reasons. In addition, viral replication depends on the ER stress and the pathways related to that and the secretion of the glycoprotein could impact the virus growth and its protection ability.