Functional characterization of irf3 against viral hemorrhagic septicemia virus infection using a CRISPR/Cas9-mediated zebrafish knockout model.

Interferon regulatory factors (IRFs) are transcription factors involved in immune responses, such as pathogen response regulation, immune cell growth, and differentiation. IRFs are necessary for the synthesis of type I interferons through a signaling cascade when pathogen recognition receptors identify viral DNA or RNA. We discovered that irf3 is expressed in the early embryonic stages and in all immune organs of adult zebrafish. We demonstrated the antiviral immune mechanism of Irf3 against viral hemorrhagic septicemia virus (VHSV) using CRISPR/Cas9-mediated knockout zebrafish (irf3-KO). In this study, we used a truncated Irf3 protein, encoded by irf3 with a 10 bp deletion, for further investigation. Upon VHSV injection, irf3-KO zebrafish showed dose-dependent high and early mortality compared with zebrafish with the wild-type Irf3 protein (WT), confirming the antiviral activity of Irf3. Based on the results of expression analysis of downstream genes upon VHSV challenge, we inferred that Irf3 deficiency substantially affects the expression of ifnphi1 and ifnphi2. However, after 5 days post infection (dpi), ifnphi3 expression was not significantly altered in irf3-KO compared to that in WT, and irf7 transcription showed a considerable increase in irf3-KO after 5 dpi, indicating irf7's control over ifnphi3 expression. The significantly reduced expression of isg15, viperin, mxa, and mxb at 3 dpi also supported the effect of Irf3 deficiency on the antiviral activity in the early stage of infection. The higher mortality in irf3-KO zebrafish than in WT might be due to an increased inflammation and tissue damage that occurs in irf3-KO because of delayed immune response. Our results suggest that Irf3 plays a role in antiviral immunity of zebrafish by modulating critical immune signaling molecules and regulating antiviral immune genes.

Genome-wide association study of VHSV-resistance trait in Paralichthys olivaceus.

In flounder aquaculture, selective breeding plays a vital role in the development of disease-resistant traits and animals with high growth rates. Moreover, superior animals are required to achieve high profits. Unlike growth-related traits, disease-resistant experiments need to be conducted in a controlled environment, as the improper measurement of traits often leads to low genetic correlation and incorrect estimation of breeding values. In this study, viral hemorrhagic septicemia virus (VHSV) resistance was studied using a genome-wide association study (GWAS), and the genetic parameters were estimated. Genotyping was performed using a high-quality 70 K single nucleotide polymorphism (SNP) Affymetrix® Axiom® myDesign™ Genotyping Array of olive flounder. A heritability of ∼0.18 for resistance to VHSV was estimated using genomic information of the fish. According to the GWAS, significant SNPs were detected in chromosomes 21, 24, and contig AGQT02032065.1. Three SNPs showed significance at the genome-wide level (p < 1 × 10-6), while others showed significance above the suggestive cutoff (p < 1 × 10-4). The 3% phenotypic variation was explained by the highest significant SNP, named AX-419319631. Of the important genes for disease resistance, SNPs were associated with plcg1, epha4, clstn2, pik3cb, hes6, meis3, prx6, cep164, siae, and kirrel3b. Most of the genes associated with these SNPs have been previously reported with respect to viral entry, propagation, and immune mechanisms. Therefore, our study provides helpful information regarding VHSV resistance in olive flounder, which can be used for breeding applications.

A single amino acid variation of NV protein of viral hemorrhagic septicemia virus increases protein stability and decreases immune gene expression.

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.

Genomic and immunogenic changes of Piscine novirhabdovirus (Viral Hemorrhagic Septicemia Virus) over its evolutionary history in the Laurentian Great Lakes.

A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically-restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv- (3.8%), and M- (2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHSV genogroups and subgenogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I, -II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an origin date). The 2016 isolates that we tested induced milder impacts on fish host cell innate antiviral responses, suggesting altered phenotypic effects. In conclusion, our overall findings indicate that VHSV-IVb has undergone continued sequence change and a trend to lower virulence over its evolutionary history (2003 through present-day), which may facilitate its long-term persistence in fish host populations.

Functional nutrition modulates the early immune response against viral haemorrhagic septicaemia virus (VHSV) in rainbow trout.

Although viruses represent a major threat for cultured fish worldwide, the commercialization of vaccines capable of providing effective and long-lasting protection is still lacking for most of these viral diseases. In this situation, the use of supplemented diets could be a suitable strategy to increase the immune status of the fish and reduce the impact of viral pathogens. Among possible immunostimulants that could be included in these functional feeds, some studies have previously shown that certain ß-glucans can significantly increase certain immune parameters of fish and reduce the impact of viral diseases. However, the mechanisms through which ß-glucans exert their activity have not been fully elucidated yet. In the current study, we have studied the immune response of different tissues to viral haemorrhagic septicaemia virus (VHSV) in rainbow trout fed with a non-supplemented control diet as well as in fish fed a commercial functional aquafeed (Protec™, Skretting) containing ß-glucans, vitamin C, vitamin E and zinc. For this, after 30 days of feeding the fish with one of the two diets, they were subsequently infected with VHSV by bath or mock-infected. After 2 or 6 days post-infection, fish were sacrificed and the levels of transcription of different immune genes such as IgM, IgT, IgD, Mx, interferon γ (IFN γ) and perforin studied in different tissues (kidney, gut and gills). Additionally, the levels of natural IgMs in serum were also determined. Our results demonstrate that fish fed the functional diet were capable of mounting an increased IgM, IgT, IgD and Mx transcriptional response to the virus. Additionally, these fish also showed increased levels of natural IgMs in serum. These results reveal a previously undescribed effect of functional diets on fish Ig production and point to Protec™ as an adequate diet to be incorporated in holistic programs aimed at mitigating the effect of viral diseases.

Effect of CRISPR/Cas9-mediated knockout of either Mx1 or ISG15 gene in EPC cells on resistance against VHSV infection.

Although the type I interferon-mediated increase of Mx1 and ISG15 gene expression in Epithelioma papulosum cyprini (EPC) cells has been reported, the antiviral role of Mx1 and ISG15 in EPC cells has not been investigated. In this study, to know the anti-viral hemorrhagic septicemia virus (VHSV) role of Mx1 and ISG15 of EPC cells, either Mx1 or ISG15 gene was knocked-out using a CRISPR/Cas9 system, and the progression of cytopathic effects (CPE) and viral growth were analyzed. Mx1 gene and ISG15 gene knockout EPC cells were successfully produced via CRISPR/Cas9 coupled with a single-cell cloning. Through the sequence analysis, one clone showing two heterozygous indel patterns in Mx1 gene and a clone showing three heterozygous indel patterns in ISG15 gene were selected for further analyses. Mx1 knockout EPC cells did not show any differences in VHSV-mediated CPE progression, even when pre-treated with polyinosinic:polycytidylic acid (poly I:C), compared to control EPC cells. These results suggest that Mx1 in EPC cells may be unfunctional to cytoplasmic RNA viruses. In contrast to Mx1, ISG15 knockout cells showed clearly hampered anti-VHSV activity even when pre-treated with poly I:C, indicating that ISG15 plays an important role in type I interferon-mediated anti-viral activity in EPC cells, which allowed VHSV to replicate more efficiently in ISG15 knockout cells than Mx1 knockout and control cells.

The Nucleoprotein and Phosphoprotein Are Major Determinants of the Virulence of Viral Hemorrhagic Septicemia Virus in Rainbow Trout.

Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus, infects several marine and freshwater fish species. There are many strains of VHSV that affect different fish, but some strains of one genetic subgroup have gained high virulence in rainbow trout (Oncorhynchus mykiss). To define the genetic basis of high virulence in trout, we used reverse genetics to create chimeric VHSVs in which viral nucleoprotein (N), P (phosphoprotein), or M (matrix protein) genes, or the N and P genes, were exchanged between a trout-virulent European VHSV strain (DK-3592B) and a trout-avirulent North American VHSV strain (MI03). Testing of the chimeric recombinant VHSV (rVHSV) by intraperitoneal injection in juvenile rainbow trout showed that exchanges of the viral P or M genes had no effect on the trout virulence phenotype of either parental strain. However, reciprocal exchanges of the viral N gene resulted in a partial gain of function in the chimeric trout-avirulent strain (22% mortality) and complete loss of virulence for the chimeric trout-virulent strain (2% mortality). Reciprocal exchanges of both the N and P genes together resulted in complete gain of function in the chimeric avirulent strain (82% mortality), again with complete loss of virulence in the chimeric trout-virulent strain (0% mortality). Thus, the VHSV N gene contains an essential determinant of trout virulence that is strongly enhanced by the viral P gene. We hypothesize that the host-specific virulence mechanism may involve increased efficiency of the viral polymerase complex when the N and P proteins have adapted to more efficient interaction with a host component from rainbow trout.IMPORTANCE Rainbow trout farming is a major food source industry worldwide that has suffered great economic losses due to host jumps of fish rhabdovirus pathogens, followed by evolution of dramatic increases in trout-specific virulence. However, the genetic determinants of host jumps and increased virulence in rainbow trout are unknown for any fish rhabdovirus. Previous attempts to identify the viral genes containing trout virulence determinants of viral hemorrhagic septicemia virus (VHSV) have not been successful. We show here that, somewhat surprisingly, the viral nucleocapsid (N) and phosphoprotein (P) genes together contain the determinants responsible for trout virulence in VHSV. This suggests a novel host-specific virulence mechanism involving the viral polymerase and a host component. This differs from the known virulence mechanisms of mammalian rhabdoviruses based on the viral P or M (matrix) protein.

Rainbow Trout Red Blood Cells Exposed to Viral Hemorrhagic Septicemia Virus Up-Regulate Antigen-Processing Mechanisms and MHC I&II, CD86, and CD83 Antigen-presenting Cell Markers.

Nucleated teleost red blood cells (RBCs) are known to express molecules from the major histocompatibility complex and peptide-generating processes such as autophagy and proteasomes, but the role of RBCs in antigen presentation of viruses have not been studied yet. In this study, RBCs exposed ex vivo to viral hemorrhagic septicemia virus (VHSV) were evaluated by means of transcriptomic and proteomic approaches. Genes and proteins related to antigen presentation molecules, proteasome degradation, and autophagy were up-regulated. VHSV induced accumulation of ubiquitinated proteins in ex vivo VHSV-exposed RBCs and showed at the same time a decrease of proteasome activity. Furthermore, induction of autophagy was detected by evaluating LC3 protein levels. Sequestosome-1/p62 underwent degradation early after VHSV exposure, and it may be a link between ubiquitination and autophagy activation. Inhibition of autophagosome degradation with niclosamide resulted in intracellular detection of N protein of VHSV (NVHSV) and p62 accumulation. In addition, antigen presentation cell markers, such as major histocompatibility complex (MHC) class I & II, CD83, and CD86, increased at the transcriptional and translational level in rainbow trout RBCs exposed to VHSV. In summary, we show that nucleated rainbow trout RBCs can degrade VHSV while displaying an antigen-presenting cell (APC)-like profile.

Outbreak of viral haemorrhagic septicaemia (VHS) in lumpfish (Cyclopterus lumpus) in Iceland caused by VHS virus genotype IV.

A novel viral haemorrhagic septicaemia virus (VHSV) of genotype IV was isolated from wild lumpfish (Cyclopterus lumpus), brought to a land-based farm in Iceland, to serve as broodfish. Two groups of lumpfish juveniles, kept in tanks in the same facility, got infected. The virus isolated was identified as VHSV by ELISA and real-time RT-PCR. Phylogenetic analysis, based on the glycoprotein (G) gene sequences, may indicate a novel subgroup of VHSV genotype IV. In controlled laboratory exposure studies with this new isolate, there was 3% survival in the I.P. injection challenged group while there was 90% survival in the immersion group. VHSV was not re-isolated from fish challenged by immersion. In a cohabitation trial, lumpfish infected I.P. (shedders) were placed in tanks with naïve lumpfish as well as naïve Atlantic salmon (Salmo salar L.). 10% of the lumpfish shedders and 43%-50% of the cohabiting lumpfish survived after 4 weeks. 80%-92% of the Atlantic salmon survived, but no viral RNA was detected by real-time RT-PCR nor VHSV was isolated from Atlantic salmon. This is the first isolation of a notifiable virus in Iceland and the first report of VHSV of genotype IV in European waters.

Gene expression profiles alteration after infection of virus, bacteria, and parasite in the Olive flounder (Paralichthys olivaceus).

Olive flounder (Paralichthys olivaceus) is one of economically valuable fish species in the East Asia. In comparison with its economic importance, available genomic information of the olive flounder is very limited. The mass mortality caused by variety of pathogens (virus, bacteria and parasites) is main problem in aquaculture industry, including in olive flounder culture. In this study, we carried out transcriptome analysis using the olive flounder gill tissues after infection of three types of pathogens (Virus; Viral hemorrhagic septicemia virus, Bacteria; Streptococcus parauberis, and Parasite; Miamiensis avidus), respectively. As a result, we identified total 12,415 differentially expressed genes (DEG) from viral infection, 1,754 from bacterial infection, and 795 from parasite infection, respectively. To investigate the effects of pathogenic infection on immune response, we analyzed Gene ontology (GO) enrichment analysis with DEGs and sorted immune-related GO terms per three pathogen groups. Especially, we verified various GO terms, and genes in these terms showed down-regulated expression pattern. In addition, we identified 67 common genes (10 up-regulated and 57 down-regulated) present in three pathogen infection groups. Our goals are to provide plenty of genomic knowledge about olive flounder transcripts for further research and report genes, which were changed in their expression after specific pathogen infection.

CRISPR/Cas9-mediated knockout of HIF-1α gene in epithelioma papulosum cyprini (EPC) cells inhibited apoptosis and viral hemorrhagic septicemia virus (VHSV) growth.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1ß, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection.

Interferon-induced protein 56 (IFI56) is induced by VHSV infection but not by bacterial infection in olive flounder (Paralichthys olivaceus).

Interferon-inducible protein 56 (IFI56, also known as ISG56/IFIT1, interferon-induced protein with tetratricopeptide repeats 1) is strongly induced in response to interferon and a potent inhibitor of viral replication and translational initiation. Here, we describe the identification of IFI56 (OfIFI56) in olive flounder, its characteristic features, and expression levels in various tissues before and after viral hemorrhagic septicemia virus (VHSV) infection. The full-length OfIFI56 sequence was identified from rapid amplification of cDNA ends PCR. The complete coding sequence of OfIFI56 is 1971 bp in length and encodes 431 amino acids. The putative OfIFI56 protein has multiple tetratricopeptide (TPR) motifs, which regulate diverse biological processes, such as organelle targeting, protein import, and vesicle fusion. Based on sequence analysis, the Larimichthys crocea IFI56 protein (61%) had the highest sequence homology to OfIFI56. In healthy olive flounder, OfIFI56 mRNA expression was detected in many tissues such as intestine, gill, head kidney, heart, spleen, and trunk kidney tissues. After VHSV challenge, OfIFI56 mRNA was significantly up-regulated in these tissues. Additionally, OfIFI56 expression was induced by poly I:C but not by Streptococcus parauberis and S. iniae infection or lipopolysaccharide injection in kidney and spleen tissues of olive flounder. These results demonstrate that piscine OfIFI56 expression is not induced by bacterial infection but is selectively induced by viral infection, especially VHSV, and that OfIFI56 may play an important role in the host response against VHSV infection.

Antiviral effects of extracts from Celosia cristata and Raphanus sativus roots against viral hemorrhagic septicemia virus.

The antiviral activity of an extract mixture from Celosia cristata and Raphanus sativus was tested against viral hemorrhagic septicemia virus (VHSV). Pretreatment of EPC cells with this extract up to 72 h before VHSV infection markedly reduced the virus titer, but it had no effect when added after virus inoculation. In olive flounder that received 5 µg of extract per fish, Mx expression peaked at 48 h after treatment. In contrast, ISG15 and TLR2 expression peaked at 72 h, and that of TLR7 peaked at 48 h, followed by a slight decrease at 72 h, indicating that the antiviral activity was mediated by induction of gene expression involved in the innate immune response.

Montanide IMS 1312 VG adjuvant enhances the efficacy of immersion vaccine of inactivated viral hemorrhagic septicemia virus (VHSV) in olive flounder, Paralichthys olivaceus.

Vaccination by immersion is suitable for mass vaccination of small size fish. However, no viral vaccine has been developed for immersion applications, because of low efficacy. In this study, we evaluated the efficacy and safety of immersion vaccine against viral hemorrhagic septicemia (VHS) containing Montanide IMS 1312 VG adjuvant in olive flounder (Paralichthys olivaceus). Healthy fish were vaccinated by an immersion method with a heat-inactivated FP-VHS2010-1 strain of VHS virus (VHSV) in combination with Montanide IMS 1312 VG for 5 min at 20 ± 2 °C. The control group was vaccinated with sterile PBS. No toxicity of immersion vaccine with Montanide IMS 1312 VG adjuvant was observed by hematological and histopathological analysis. Immersion vaccine with adjuvant enhanced gene expression of immune-associated genes, i.e., genes encoding interleukin (IL)-1ß, IL-6, IL-8, and Toll-like receptor (TLR) 3. Relative percent survival (RPS) of fish was measured on weeks 4 and 8 post vaccination. In fish vaccinated with adjuvant, RPS was significantly higher than that of fish vaccinated without adjuvant. The results of the present study provide evidence that the VHSV immersion vaccine with Montanide IMS 1312 VG induces protective immunity in olive flounder against VHS.

Viral hemorrhagic septicemia virus (VHSV) infection-mediated sequential changes in microRNAs profile of Epithelioma papulosum cyprini (EPC) cells.

MicroRNAs are small non-coding RNAs and are involved in the regulation of wide biological processes. Viral hemorrhagic septicemia virus (VHSV) is the causative agent of viral hemorrhagic septicemia (VHS) disease causing a heavy loss in aquaculture farms. In this study, we tried to explore the effect of VHSV infection on microRNAs profile of Epithelioma papulosum cyprini (EPC) cells at different points of time (0, 3, 12, 24, and 48 h post infection). A total of 355 conserved microRNAs and 3 novel microRNAs were identified, and among them, 103 microRNAs were differentially expressed. The number of differentially expressed microRNAs was highly increased at 24 h.p.i compared to 3 h.p.i and 12 h.p.i., suggesting that EPC cells might not actively respond to VHSV infection at an early infection period, which can allow viruses to transcript and translate their genes enough to produce viral particles that can infect to another cells. Among the differentially expressed microRNAs, 2 miRNAs (miR-735 and miR-738) that were reported only in fish species were highly upregulated, and based on the target prediction, they could regulate several immune pathways. Furthermore, the present results showed the upregulation of representative immune regulating microRNAs such as miR-146a, miR-155, and miR-99. The target prediction of differentially expressed miRNAs, GO, and KEGG pathways analysis revealed that several biological processes and different pathways were affected by the viral infection. The present dynamical changing patterns of differentially expressed microRNAs in response to the progression of VHSV infection suggest that microRNA profile that was analyzed at one time point cannot provide enough information for the interpretation of the disease mechanism. Considering the wide and complex interactions between microRNAs and genes expression, the present results provide the basis for the understanding of VHSV infection-mediated cellular responses and for future investigations on the development of possible control measures.

Differentially expressed genes after viral haemorrhagic septicaemia virus infection in olive flounder (Paralichthys olivaceus).

A strain of viral haemorrhagic septicaemia virus (VHSV) was isolated from cultured olive flounder (Paralichthys olivaceus) during epizootics in South Korean. This strain showed high mortality to olive flounder in in vivo challenge experiment. The complete genomic RNA sequences were determined and phylogenetic analysis of the amino acid sequences of glycoprotein revealed that this isolate was grouped into genotype IVa of genus Novirhabdovirus. Expression profile of genes in olive flounder was analyzed at day 1 and day3 after infection with this VHSV isolate by using cDNA microarray containing olive flounder 13K cDNA clones. Microarray analysis revealed 785 up-regulated genes and 641 down-regulated genes by at least two-fold in virus-infected fish compared to healthy control groups. Among 785 up-regulated genes, we identified seven immune response-associated genes, including the interferon (IFN)-induced 56-kDa protein (IFI56), suppressor of cytokine signaling 1 (SOCS1), interleukin 8 (IL-8), cluster of differentiation 83 (CD83), α-globin (HBA), VHSV-induced protein-6 (VHSV6), and cluster of differentiation antigen 9 (CD9). Our results confirm previous reports that even virulent strain of VHSV induces expression of genes involved in protective immunity against VHSV.

Changes in microRNAs expression profile of olive flounder (Paralichthys olivaceus) in response to viral hemorrhagic septicemia virus (VHSV) infection.

To know the effect of viral hemorrhagic septicemia virus (VHSV) infection on the cellular microRNA expression profile in olive flounder (Paralichthys olivaceus), fish were infected with VHSV, and cellular microRNAs expression was analyzed at 0 (control), 6, 12, 24, 48 and 72 h post-infection (h.p.i.) by the high-throughput sequencing. A total of 372 mature miRNAs were identified, and, among them, 63 miRNAs were differentially expressed during VHSV infection. The differentially expressed microRNAs number was greatly increased from 24 h.p.i. compared to the number at 6 and 12 h.p.i., suggesting that the alteration of microRNAs expression by VHSV infection may be related to the progression of VHSV disease. The target prediction analysis, the GO enrichment analysis, and the KEGG pathway analysis of the predicted target genes showed that various biological pathways could be affected by VHSV infection through the down-regulation or up-regulation of host miRNAs. The present results provide a basic information on the microRNAs related to VHSV infection in olive flounder. Considering broad effects of microRNAs on various biological pathways, data in this study can be used to interpret the mechanism of VHSV pathogenesis, which, vice versa, can be used to develop control measures against VHSV.

Microarray analysis of gene expression in olive flounder liver infected with viral haemorrhagic septicaemia virus (VHSV).

The most fatal viral pathogen in olive flounder Paralichthys olivaceus, is viral hemorrhagic septicemia virus, which afflicts over 48 species of freshwater and marine fish. Here, we performed gene expression profiling on transcripts isolated from VHSV-infected olive flounder livers using a 13 K cDNA microarray chip. A total of 1832 and 1647 genes were upregulated and down-regulated over two-fold, respectively, after infection. A variety of immune-related genes showing significant changes in gene expression were identified in upregulated genes through gene ontology annotation. These genes were grouped into categories such as antibacterial peptide, antigen-recognition and adhesion molecules, apoptosis, cytokine-related pathway, immune system, stress response, and transcription factor and regulatory factors. To verify the cDNA microarray data, we performed quantitative real-time PCR, and the results were similar to the microarray data. In conclusion, these results may be useful for the identification of specific genes or for the diagnosis of VHSV infection in flounder.

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.

A single amino acid mutation (I1012F) of the RNA polymerase of marine viral hemorrhagic septicemia virus changes in vitro virulence to rainbow trout gill epithelial cells.

UNLABELLED: Viral hemorrhagic septicemia virus (VHSV) is separated into four different genotypes (I to IV) with different sublineages (K. Einer-Jensen, P. Ahrens, R. Forsberg, and N. Lorenzen, J. Gen. Virol. 85:1167-1179, 2004; K. Einer-Jensen, J. Winton, and N. Lorenzen, Vet. Microbiol. 106:167-178, 2005). European marine VHSV strains (of genotypes I to III) are, in general, nonpathogenic or have very low pathogenicity to rainbow trout after a waterborne challenge, and here we also show that genotype IVa is nonpathogenic to trout. Despite several attempts, it has not been possible to link genomic variation to in vivo virulence. In vitro virulence to gill epithelial cells (GECs) has been used as a proxy for in vivo virulence, and here we extend these studies further with the purpose of identifying residues associated with in vitro virulence. Genotype Ia (DK-3592B) and III (NO/650/07) isolates, which are pathogenic to rainbow trout (O. B. Dale, I. Orpetveit, T. M. Lyngstad, S. Kahns, H. F. Skall, N. J. Olesen, and B. H. Dannevig, Dis. Aquat. Organ. 85:93-103, 2009), were compared to two marine strains that are nonpathogenic to trout, genotypes Ib (strain 1p8 [H. F. Mortensen, O. E. Heuer, N. Lorenzen, L. Otte, and N. J. Olesen, Virus Res. 63:95-106, 1999]) and IVa (JF-09). DK-3592 and NO/650/07 were pathogenic to GECs, while marine strains 1p8 and JF-09 were nonpathogenic to GECs. Eight conserved amino acid substitutions contrasting high- and low-virulence strains were identified, and reverse genetics was used in a gain-of-virulence approach based on the JF-09 backbone. Mutations were introduced into the G, NV, and L genes, and seven different virus clones were obtained. For the first time, we show that a single amino acid mutation in conserved region IV of the L protein, I1012F, rendered the virus able to replicate and induce a cytopathic effect in trout GECs. The other six mutated variants remained nonpathogenic. IMPORTANCE: This is the first study to clearly link in vitro virulence of viral hemorrhagic septicemia virus (VHSV) with an amino acid residue in the L protein, a site located in conserved region IV of the L protein. In vitro virulence is documented by induction of cytopathic effects and viability studies of gill epithelial cells, and the observed cellular responses to infection are associated with increased viral replication levels. There are no previous studies addressing the importance of the L protein or the RNA-dependent RNA polymerase for virus virulence in vitro or in vivo. Therefore, the findings reported here should broaden the search for pathogenicity traits in novirhabdoviruses, and there is a possibility that the polymerase participates in defining the host species virulence of various VHSV strains.