Functional and regulatory diversification of Period genes responsible for circadian rhythm in vertebrates.

The Period genes (Per) play essential roles in modulating the molecular circadian clock timing in a broad range of species, which regulates the physiological and cellular rhythms through the transcription-translation feedback loop. While the Period gene paralogs are widely observed among vertebrates, the evolutionary history and the functional diversification of Per genes across vertebrates are not well known. In this study, we comprehensively investigated the evolution of Per genes at the copy number and sequence levels, including de novo binding motif discovery by comparative genomics. We also determined the lineage-specific transcriptome landscape across tissues and developmental stages and phenotypic effects in public RNA-seq data sets of model species. We observed multiple lineage-specific gain and loss events of Per genes, though no simple association was observed between ecological factors and Per gene numbers in each species. Among salmonid fish species, the per3 gene has been lost in the majority, whereas those retaining the per3 gene exhibit not a signature of relaxed selective constraint but rather a signature of intensified selection. We also determined the signature of adaptive diversification of the CRY-binding region in Per1 and Per3, which modulates the circadian rhythm. We also discovered putative regulatory sequences, which are lineage-specific, suggesting that these cis-regulatory elements may have evolved rapidly and divergently across different lineages. Collectively, our findings revealed the evolution of Per genes and their fine-tuned contribution to the plastic and precise regulation of circadian rhythms in various vertebrate taxa.

Generation of Recombinant Snakehead Rhabdovirus (SHRV) Expressing Artificial MicroRNA Targeting Spring Viremia of Carp Virus (SVCV) P Gene and In Vivo Therapeutic Use Against SVCV Infection.

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates. To overcome in vivo limitation of miRNA mimics or plasmid-based miRNA expression systems, we rescued recombinant snakehead rhabdoviruses (SHRVs) expressing SVCV P gene-targeting AmiRNA (rSHRV-AmiR-P3) or control AmiRNA (rSHRV-AmiR-C) using reverse genetic technology. The successful expression of AmiR-P3 and AmiR-C in cells infected with the rescued viruses was verified by quantitative PCR. To evaluate the availability of rSHRV-AmiR-P3 for in vivo control of SVCV, zebrafish (Danio rerio) were (i) infected with either rSHRV-AmiR-C or rSHRV-AmiR-P3 followed by SVCV infection or (ii) infected with SVCV followed by either rSHRV-AmiR-C or rSHRV-AmiR-P3 infection. Fish infected with rSHRVs before and after SVCV infection showed significantly higher survival rates than fish infected with SVCV alone. There was no significant difference in survival rates between groups of fish infected with rSHRV-AmiR-C and rSHRV-AmiR-P3 before SVCV infection; however, fish infected with SVCV followed by infection with rSHRV-AmiR-P3 showed significantly higher survival rates than fish infected with rSHRV-AmiR-C. These results suggest that rSHRV-AmiR-P3 has therapeutic potential against SVCV in fish when administered after SVCV infection, and rSHRVs expressing artificial microRNAs targeting SVCV transcripts could be used as a tool to control SVCV infection in fish for a therapeutic purpose.

Compensatory mutations in the matrix protein of viral hemorrhagic septicemia virus (VHSV) genotype IVa in response to artificial mutation of two amino acids (D62A E181A).

The matrix (M) protein of rhabdoviruses locates between the inner line of the viral envelope and the nucleocapsids core and plays an important role in viral replication. In the present study, we aimed to rescue a mutant of VHSV genotype IVa that has artificial mutations in the M protein (M-D62A E181A). However, most rescued recombinant viruses unexpectedly showed non-targeted secondary mutations in the M protein. Therefore, this study was conducted to know whether the targeted artificial mutation can lead to specific non-targeted secondary mutations in the M protein and whether the secondary mutations are compensatory for the targeted artificial mutations. Experiments were conducted to rescue three kinds of M protein mutants (rVHSV-M-D62A, -E181A, and -D62A E181A), and rVHSV-M-E181A and rVHSV-M-D62A E181A without the secondary mutations were rescued only from IRF-9 gene-knockout EPC cells. Recombinant VHSVs having only targeted mutation(s) (rVHSV-M-D62A, -E181A, and -D62A E181A) showed slower CPE progression and retarded growth compared to rVHSV-wild. Although the sites of secondary mutations were changed in every transfection experiment to generate recombinant VHSVs, the positions of the secondary mutations were not random. Some amino acid residues in the M protein showed more frequent mutations than others, and the changed amino acid residues were always the same. EPC cells infected with rVHSV-M-D62A E181A showed significantly higher type I interferon response and NF-κB activity, and the inhibitory activity against type I interferon response and NF-κB activity in other recombinant VHSVs having secondary mutations in M gene were similar to those of rVHSV-wild. In conclusion, the present results showed that VHSV actively responded to the artificial mutation of M protein through the secondary mutations, and those secondary mutations occurred when the artificial mutations were deleterious to viral replication and protein stability. Furthermore, most secondary mutations in recombinant viruses compensated for the deleterious effect of the engineered mutations.

Effect of CRISPR/Cas9-mediated knockout of either IRF-3 or IRF-5 gene in Epithelioma papulosum cyprini cells on type I interferon response and NF-κB activity.

Transcription factors related to the activation of type I interferons (IFNs) and nuclear factor-kappa B (NF-κB) are known to be critical in innate immune responses. Interferon regulatory factors (IRFs) are a family of transcription factors. IRF-3 is known to act as the primary regulator in type I IFN signaling in response to viral infections, and the upregulation of IRF5 by virus infection has been reported in various fish species. One of the ways to know the functional role of certain genes is the production of target gene(s) knockout cells or organisms. In the present study, we produced either IRF3 or IRF5 gene knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 system, and investigated the effect of IRF3 gene and IRF5 gene knockout on polyinosinic:polycytidylic acid (ploly (I:C))-mediated and viral hemorrhagic septicemia virus (VHSV) infection-mediated type I IFN response and NF-κB activation. Both IRF3 knockout and IRF5 knockout EPC cells showed severely decreased type I IFN responses measured by ISRE activity and the expression of Mx1 and ISG15 genes when stimulated with poly (I:C), while the decreased level of type I IFN responses was not high as by poly (I:C) stimulation when infected with VHSV. Different from type I IFN response, NF-κB activities in IRF3 and IRF5 knockout cells were not highly different between poly (I:C) stimulated cells and VHSV-infected cells. Further studies are needed to elucidate pathways responsible for the type I IFN responses and NF-κB activation by VHSV infection.

CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus).

Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method. Under the optimal CRISPR engineering condition, we could efficiently produce either alr or asd gene knockout E. piscicida auxotrophic mutants, and this genome editing process was much simpler and faster than the allelic exchange method. The simultaneous knockout of double auxotrophic genes (alr and asd) and the insertion of a foreign gene expression cassette in E. piscicida chromosome were also successfully performed using the established CRISPR/Cas9 system. Furthermore, to enhance the possibility to get permission as a commercial vaccine, we produced an auxotrophic E. piscicida mutant having only one nucleotide-deleted alr gene (E. piscicida △alr-1). Olive flounder (Paralichthys olivaceus) fingerlings immunized with 1 × 106 and 1 × 105 CFU/fish of E. piscicida △alr-1 showed the superior ability in the induction of serum agglutination activity and in the protection against E. piscicida compared to killed E. piscicida. However, olive flounder immunized with 1 × 107 CFU/fish of E. piscicida △alr-1 showed high mortality far before the challenge, and the isolated E. piscicida from moribund and dead fish had the wild type alr gene, suggesting the reversion of one base-deleted alr gene to original form by a second mutation in olive flounder. Therefore, investigation on the minimum number of edited nucleotide for stable maintenance of E. piscicida mutants should be further conducted.

Effect of NV gene deletion in the genome of hirame rhabdovirus (HIRRV) on viral replication and the type I interferon response of the host cell.

Hirame rhabdovirus (HIRRV), a member of the genus Novirhabdovirus, causes morbidity and mortality in farmed olive flounder (Paralichthys olivaceus). As no information is available on the role of the NV gene of HIRRV, we produced a recombinant HIRRV with the NV gene deleted (rHIRRV-ΔNV) using reverse genetic technology and investigated whether the NV gene knockout affected HIRRV replication and the type I interferon response of the host cell. The rescue of rHIRRV-ΔNV was successful only when IRF9-gene-knockout Epithelioma papulosum cyprini (ΔIRF9-EPC) cells were used, suggesting that the NV protein of HIRRV might be involved in inhibition of the type I interferon response of the host cell. This conclusion was also supported by the significantly higher level of Mx gene induction in EPC cells infected with rHIRRV-ΔNV than in cells infected with recombinant HIRRV without the deletion. When cells were coinfected with rHIRRV-ΔNV and either wild-type HIRRV or wild-type viral hemorrhagic septicemia virus (VHSV), there was a decrease in the growth rate of not only wild-type HIRRV but also wild-type VHSV in a concentration-dependent manner. Further studies are required to investigate the role of HIRRV NV in virulence and its possible importance for the development of attenuated vaccines.

Harnessing snakehead rhabdovirus (SHRV) for gene editing by installment of CRISPR/Cas9 in viral genome.

As there is no risk of viral genome integration into host chromosome, cytoplasmic RNA viruses can be a safer vehicle to deliver CRISPR/Cas system. Snakehead rhabdovirus (SHRV) is a piscine RNA virus belonging to the family Rhabdoviridae, and, in the present study, we evaluated the availability of SHRV as a tool for CRISPR/Cas9 delivery in mammalian cells. SHRV was grown well in baby hamster kidney (BHK-21) cells at 28 °C, and the replication ability was greatly reduced by temperature up-shift to 37 °C. We rescued a recombinant SHRV that harboring not only the interferon regulatory factor 9 (IRF9) gene-targeting single-guide RNA (sgRNA) but also Cas9 gene in the genome using the reverse genetic technology. The IRF9 gene of BHK-21 cells was knocked-out by the infection with the IRF9 gene-targeting rSHRV. Moreover, the rSHRVs were sharply disappeared in the cells by elevating temperature to 37 °C, suggesting the possible regulation of knockout efficiency before virus infection-caused cell damage. Although further optimization researches are needed to enhance the editing efficiency using the recombinant SHRV, to our knowledge, this is the first report on the possible applicability of piscine RNA virus for the gene editing in mammalian cells.

Generation of Self-Inhibitory Recombinant Viral Hemorrhagic Septicemia Virus (VHSV) by Insertion of Viral P Gene-Targeting Artificial MicroRNA into Viral Genome and Effect of Dicer Gene Knockout on the Recombinant VHSV Replication.

To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P. The replication of rVHSV-A-amiR-C in EPC-ΔDicer cells was not different from that in control EPC cells, while the copy number of rVHSV-A-amiR-P was increasingly risen up in EPC-ΔDicer cells compared to that in control EPC cells, and the final viral titer of rVHSV-A-amiR-P was enhanced by culture in EPC-ΔDicer cells. These results indicate that VHSV can be attenuated by the equipment of self-mRNA-targeting microRNA sequence in the genome, and the titer of artificial miRNA-expressing attenuated recombinant VHSVs can be enhanced by the knockout of Dicer gene in EPC cells.

Effect of miR-155 on type I interferon response in Epithelioma papulosum cyprini cells.

MicroRNA-155 (miRNA-155) is known to play an important role in the regulation of innate and adaptive immune responses in mammals. However, no information is available on the role of miRNA-155 in relation to type I interferon (IFN) responses in fish cells. In the present study, we found that the protein inhibitor of activated STAT 4a (PIAS4a) gene of fathead minnow (Pimephales promelas) was a target of miR-155, which was verified by the inhibitory activity of miR-155 in the expression of reporter gene harboring 3'UTR of PIAS4a of EPC cells. Furthermore, cells over-expressing miR-155 showed a significantly higher type I IFN response after polyinosinic-polycytidylic acid (poly I:C) stimulation, suggesting the targeting of PIAS4a in EPC cells by miR-155 can be a cause of the up-regulation of type I IFN, and miR-155 can act as an antiviral factor. However, as the targeting PIAS4a might not be the sole cause of the type I IFN up-regulation by miR-155, further studies on the uncovering of miR-155 target genes that are involved in type I IFN responses in fish are required.

Enhancing immunogenicity of a reporter protein by fusion to glycoprotein and nucleoprotein of viral hemorrhagic septicemia virus (VHSV) particles.

The introduction of reverse genetic technology to generate recombinant VHSVs (rVHSVs) has contributed to the uncovering of functional roles of viral genes and to the development of attenuated prophylactic vaccines. In this study, to assess the possible use of rVHSVs as a tool of combined vaccines, we newly rescued rVHSVs that harbor viral envelop-studded eGFP (rVHSV-A-SGT) or nucleoprotein-fused eGFP (rVHSV-A-NLG), and the ability of these rVHSVs to induce adaptive humoral immunity in olive flounder (Paralichthys olivaceus) was compared with that of rVHSV-A-eGFP that expresses eGFP as a soluble form in the cytoplasm of infected cells. The results showed that antibodies against eGFP were efficiently induced by the immunization of olive flounder with rVHSV-A-SGT and rVHSV-A-NLG, while rVHSV-A-eGFP was poor in the ability to induce antibody response against eGFP. These results suggest that the display of heterologous antigens on VHSV envelop is a good way to develop efficient combined vaccines and the fusion of foreign antigen with N protein can also be a way to enhance immunogenicity of a foreign antigen. The present recombinant VHSVs - rVHSV-A-SGT and rVHSV-A-NLG - not only express foreign antigens in host cell cytoplasm but also display antigens in or on the virus particles. Further researches on the availability of recombinant VHSVs as combined vaccines against multiple fish pathogens are needed.

Generation of microRNA-30e-producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect on in vitro immune responses.

MicroRNAs (miRNAs) are non-coding small RNAs involved in the regulation of gene expression. In the present study, we firstly reported the use of a fish RNA virus, viral hemorrhagic septicemia virus (VHSV), as a delivery vehicle of a miRNA-30e, and the effect of miR-30e produced by the recombinant VHSV on the immune responses of Epithelioma papulosum cyprini (EPC) cells was investigated. The expression of functional miR-30e using a CMV promoter-driven vector was verified by the significantly lower eGFP expression in cells transfected with a vector containing miR-30e sponge sequence than that in cells transfected with a control vector that had mutated miR-30e sponge sequence. Furthermore, the down-regulation of reporter gene containing 3'-UTR of NF-κb inhibitor α-like protein B (NFκbiαb) by miR-30e was demonstrated, suggesting that miR-30e overexpression can increase immune responses related to NF-κB activation through inhibition of IκB. A miR-30e-expressing recombinant VHSV (rVHSV-A-miR30e) that had primary microRNA-30e sequence between N and P genes was rescued using the reverse genetic method, and the successful expression of miR-30e in the cells infected with rVHSV-A-miR30e was demonstrated using Northern blot and qRT-PCR. Cells infected with rVHSV-A-miR30e showed the increase of NF-κB activation and type I interferon induced genes expression, suggesting that rVHSV-A-miR30e can produce functional miR-30e in fish cells, and VHSV can be used as a vehicle to deliver functional microRNAs in fish.

Generation of a recombinant viral hemorrhagic septicemia virus (VHSV) expressing olive flounder (Paralichthys olivaceus) interferon-γ and its effects on type I interferon response and virulence.

Rhabdoviruses including viral hemorrhagic septicemia virus (VHSV) are highly susceptible to type I interferon (IFN) responses, and IFN-γ that is belonging to the type II IFN has been known to enhance type I IFN responses in mammals. In this study, we generated a recombinant VHSV that can express olive flounder IFN-γ (rVHSV-A-IFNγ) using reverse genetics technology, and analyzed the effect of rVHSV-A-IFNγ infection on type I IFN response in Epithelioma papulosum cyprini (EPC) cells. Furthermore, the virulence of rVHSV-A-IFNγ was evaluated by infection to olive flounder (Paralichthys olivaceus). Using a recombinant VHSV full genome vector in which the olive flounder IFN-γ ORF was inserted between N and P genes, rVHSV-A-IFNγ was successfully rescued, and the recombinant virus was grown well in EPC cells. On the other hand, the growth of rVHSV-A-IFNγ rescued from EPC cells was severely retarded when infected into hirame natural embryo (HINAE) cells that were originated from olive flounder. These results indicate that the EPC cell's IFN-γ receptor could not bind to olive flounder IFN-γ, but the species-specific binding of IFN-γ in HINAE cells induced antiviral responses. The expression of Mx1 gene in EPC cells infected with rVHSV-A-IFNγ was not greatly different from cells infected with rVHSV-Arfp (a recombinant VHSV harboring red fluorescent protein gene between N and P genes of the genome), however, in HINAE cells, rVHSV-A-IFNγ infection induced distinctively higher Mx1 gene expression compared to other recombinant viruses. These results suggest that olive flounder IFN-γ produced from rVHSV-A-IFNγ effectively enhanced type I IFN response in HINAE cells. In the present study, the lowest mortality of olive flounder fingerlings was recorded in the group of fish challenged with rVHSV-A-IFNγ, suggesting that the recombinant VHSV was attenuated by production of IFN-γ by itself. However, although rVHSV-A-IFNγ induced significantly lower mortality, the mortality still reached to 40%. Therefore, to be safely used in the aquaculture farms as prophylactic vaccines or immunostmulators, further manipulations that can guarantee safety are needed.