Infectious Pancreatic Necrosis Virus Infection Research Articles

Characterization of the activity of 2′-C- methylcytidine against infectious pancreatic necrosis virus replication

Infectious pancreatic necrosis virus (IPNV) is the pathogen of infectious pancreatic necrosis (IPN), which can cause high mortality in salmonids, harm the healthy development of salmon-trout aquaculture, and lead to huge economic losses. However, in China, there is currently neither a commercially available vaccine to prevent IPNV infection nor antiviral drugs to treat IPNV infection. The genome of IPNV consists of two segments of dsRNA named A and B. Segment B encodes the RNA-dependent RNA-polymerase (RdRp) VP1 which is essential for viral RNA replication and is therefore considered an important target for the development of antiviral drugs. In this study, we investigate whether 2′-C-methylcytidine (2CMC), a nucleoside analog which target viral polymerases, has an inhibitory effect on IPNV both in vitro and in vivo. The results show that 2CMC inhibits IPNV infection by inhibiting viral RNA replication rather than viral internalization or attachment. In vivo experiment results showed that 2CMC could inhibit viral RNA replication and reduce viral load in rainbow trout (Oncorhynchus mykiss). In our study, we have revealed that 2CMC has a potent inhibitory effect against IPNV infection. Our data suggest that 2CMC is an attractive anti-IPNV drug candidate which will be highly valuable for the development of potential therapeutics for IPNV.

Epigenetic reprogramming around IFN1 and IFNy2 promoters in rainbow trout cells inoculated with infectious pancreatic necrosis virus (IPNV).

Infectious pancreatic necrosis virus (IPNV) has proven to effectively evade the host antiviral responses. This study clarifies whether the modulation of the antiviral immune response exerted by IPNV involves epigenetic mechanisms. An in-silico characterization of the rainbow trout IFN1 and IFNγ2 promoters was performed, identifying the islands or sequences rich in CpG dinucleotides and the putative transcription factor binding sites (TBS) for both gene promoters. RTS11 cells (rainbow trout monocyte/macrophage) were infected with IPNV, and the course of viral infection was followed up to 48h post infection (hpi). Infected cells showed increased IFN1 and IFNγ2 transcriptional expression at 6 and 24 hpi, respectively. IPNV infection caused increases and decreases in global IFNγ2 promoter methylation at 6 and 24 hpi, respectively. The CpG dinucleotides at positions -392 and+38 of this promoter were the most sensitive to methylation changes. The IFN1 promoter remained fully unmethylated during the course of the infection, similar to the control. The changes in the methylation pattern observed for the IFNγ2 promoter were coincident with the changes in DNA methyltransferase (DNMT) expression levels, increasing at 6 hpi and decreasing below basal level at 24 hpi. Similarly, the H4 histones associated with the IFN1 and IFNγ2 promoters were hyperacetylated at 6 hpi, subsequently decreasing their acetylation below basal levels at 24 hpi, in both promoters. Coincidentally with the above, overexpression of histone acetyltransferase (HAT) was observed at 6 hpi and of histone deacetylase (HDAC) at 24 hpi, with return to baseline of HAT. These results suggest that IPNV would epigenetically modulate the expression of IFN1 by changing acetylation levels of the histones H4 associated with its promoter. Also, the modulation of the expression of IFNy2 would be by switching methylation/demethylation levels of its promoter, in addition to changes in acetylation levels of histones H4 associated with this promoter. This study is the first to demonstrate the effect of epigenetic reprogramming after IPNV infection in salmonid cells, demonstrating that promoter methylation/demethylation level and changes in the histone code associated with promoters may play a role in the modulation of the immune response induced by the virus.

Infectious hematopoietic necrosis virus promoted infectious pancreatic necrosis virus multiplication during co-infection in rainbow trout (Oncorhynchus mykiss)

Because plenty of pathogenic microbe exist in natural aquatic environments, co-infection is common. Infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) are prevalent viral diseases in cultured salmonids, with infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) known to infect rainbow trout (Oncorhynchus mykiss). To understand the influence of IHNV on IPNV in co-infection in rainbow trout, we intraperitoneally (ip) inject rainbow trout with IHNV at different time intervals prior to or after IPNV injection, and determine propagation of IPNV with absolute quantitative analysis in brain, gill, heart, liver, spleen and head kidney tissues. Meanwhile, to prove the existence and replication of IHNV in co-infections, IHNV loads were also detected by real-time quantitative polymerase chain reaction (RT-qPCR). For rainbow trout ip-injected with IHNV simultaneously or after IPNV, IPNV multiplication increases (P < 0.05) in each tissue. For rainbow trout ip-injected with IHNV 2 d prior to IPNV (H2P), levels of IPNV are enhanced (P < 0.05) in all but brain and heart tissues on 2 d post IPNV infection. For rainbow trout ip-injected with IHNV 7 d prior to IPNV (H7P), IPNV multiplication increases (P < 0.05) only in gill, heart, liver, spleen and head kidney tissues on 7 d post IPNV infection. At the same time, antiviral responses also occurred in all challenge groups. Results reveal that ip injection of IHNV simultaneously or after IPNV can enhance IPNV multiplication in in vivo co-infection, and that when ip injection of IHNV occurs prior to IPNV injection, IPNV multiplication can also be enhanced, although premature injection of IHNV does not obviously affect IPNV proliferation.

RNA-seq analysis reveals the dynamic regulation of proteasomal and autophagic degradation systems of rainbow trout (Oncorhynchus mykiss) skeletal muscle challenged with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis virus (IPNV) is a critical pathogen causing high salmonid mortality worldwide. IPNV primarily replicates in internal fish organs such as the head kidney, spleen, and pancreas, triggering necrosis and other subclinical symptoms. To date, there are significant data regarding the molecular bases underlying IPNV infection in classic immune tissues (e.g., head kidney and spleen); however, the effects of IPNV in nonclassical immune tissues such skeletal muscle have not been established. In this work, RNA-seq analysis was performed to examine the transcriptional response of rainbow trout skeletal muscle challenged with IPNV. Twenty-eight juvenile rainbow trout were intraperitoneally administered vehicle or IPNV for five and ten days. Total RNA from the skeletal muscle of each fish was obtained and sequenced using NovaSeq Illumina technology. A total of 1,278,349,708 high-quality reads were mapped onto the reference rainbow trout genome enriched with a de novo assembled transcriptome. Transcriptome analysis revealed that 245 and 763 genes were differentially expressed after five and ten days of IPNV challenge, respectively. GO enrichment analyses revealed that the main biological processes modulated in rainbow trout skeletal muscle during IPNV challenge correspond to catabolism-related processes such proteolysis, the ubiquitin-proteasome system, and autophagy. In line with these results, key catabolism-related signaling pathways, such as proteasome and FoxO signaling, were greatly impacted in our in vivo assay. Interestingly, most of these catabolism-related processes and signaling pathways in skeletal muscle were first downregulated after five days of IPNV administration and then were upregulated after ten days. Considering the results obtained in this work, we propose that IPNV is involved in the physiological response of skeletal muscle in salmonids by modulating critical catabolism-related processes.

Non-muscle myosin heavy chain 9 is a critical factor for infectious pancreatic necrosis virus cellular entry

Infectious pancreatic necrosis virus (IPNV) is a highly pathogenic agent of salmonid. However, the critical factors responsible for IPNV binding largely remain unknown. Here, by using a combination of immunoprecipitation and mass spectrometry assays, we identified that non-muscle myosin heavy chain 9 (Myh9) played an important role in IPNV entry by interacting with viral outer capsid protein VP2 on cell surface. We also discovered that IPNV infection induced redistribution of Myh9 to the cell membrane at the initiation of viral entry and altered the expression level of total Myh9 during viral replication. Treatments with different Myh9 inhibitors, including anti-Myh9 antibody, ML-7 and siRNA, suppressed IPNV infection. On the contrary, overexpression of Myh9 promoted IPNV infection in both susceptible and less susceptible cells. By using quantum dots-labeling technology and confocal microscopy, we tracked the entry of IPNV in real-time and found the adsorbed virions increased significantly in Myh9 overexpressed cells. Additionally, we found IPNV infection induced an increase of intracellular nanotubes and the virus could move along nanotubes to neighboring cells. Overall, our results indicate that Myh9 plays a substantial role in the entry of IPNV.

Infectious hematopoietic necrosis virus enhanced infectious pancreatic necrosis virus multiplication during co-infection in Chinook salmon embryo cell lines

Complicated viral interactions make viral co-infections more difficult to treat. Co-infection of infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) causes huge economic losses to the salmonid industry in China. To reveal the impact of IHNV on IPNV during co-infection, Chinook salmon embryo (CHSE-214) cells were inoculated with IHNV prior to or after IPNV at different time intervals, and propagation of IPNV was determined at the RNA, protein, and virion levels by real-time quantitative polymerase chain reaction (RT-qPCR), an immunofluorescence antibody test (IFAT), flow cytometry, and virus titration. Taken together, the results showed that IPNV multiplication was enhanced in all of the co-infection groups compared with the single IPNV infection group. This enhancement became stronger with an increasing infection time interval irrespective of the infection order, and the highest IPNV multiplication level was observed when IHNV was inoculated 12 h after IPNV (P12H). Compared with the single IPNV infection group, the IPNV RNA level in the P12H group was at least 10-fold higher, the proportions of IPNV-infected cells determined by IFAT and flow cytometry were approximately 2-fold higher, and the viral titer was about 40-fold higher. A significant difference was detected between each group in which IHNV was inoculated after IPNV. These findings suggested that a synergistic effect was induced by IHNV over IPNV, and IPNV multiplication was enhanced during co-infection in CHSE-214 cells. This enhancement was dependent on the infection time interval but not the infection order and the synergistic effect became stronger with increasing infection time interval within the study period.

Necrosis Pancreatic Infectious Virus does not block 701-STAT1 (α/β) tyrosine in Oncorhynchus mykiss (‎Salmoniformes: ‎Salmonidae).

ABSTRACT: Necrosis Pancreatic Infectious Virus does not block 701-STAT1 (α/β) tyrosine in Oncorhynchus mykiss (‎Salmoniformes: ‎Salmonidae).&#x0D; Introduction: Infectious pancreatic necrosis virus (IPNV) is a pathogen important that affects predominantly salmonids. The type I interferon alpha system has a crucial role in the first line of defense against IPNV infection. IFN-I(α) activation triggers the signaling pathway JAK-STAT, binding to their receptors results in the rapid phosphorylation of STATs a critical step for the nuclear translocation to induce the interferon stimulated genes (ISGs). The relationship between infectivity level of IPNV strain and pathway signaling of IFN is yet poorly understood. Objective: our purpose was to investigate if the IFN-I(α) signaling pathway is affected by IPNV strains of different infectivity levels.&#x0D; Methods: We used two IPNV isolated (VR-299 and Sp) to infect RTG-2 cells. Total RNA was isolated using the commercial kit for determine to VP2 expression and ISGs using qRT-PCR. Western Immunoblotting analysis was carried out for determine the 701 STAT1(α/β) phosphorylation into infected cells.&#x0D; Results: Hence, a higher virulence strain is not associated with a greater blocking effect for interferon signaling. Furthermore, the activation of Y701-STAT1 (α/β) was significantly increased in serotype Sp virus infected cells compared with serotype VR-299 virus infected cells, indicating that IPNV inhibits IFN signaling pathway.&#x0D; Conclusions: As concluded, IPNV does not block the phosphorylation of 701-tyrosine STAT1 (α/β) stimulated by IFN-I(α), contrary to other RNA viruses.

Development of a live vector vaccine against infectious pancreatic necrosis virus in rainbow trout

Infectious pancreatic necrosis virus (IPNV) causes severe viral disease in salmonids, with major economic losses in the global rainbow trout aquaculture industry following from a high mortality rate. In the present study, we aimed to develop a safe and efficient vaccine to protect rainbow trout against IPNV infection. Administered via the immersion route, a live vector vaccine containing the coding region of the IPNV VP2 induced protective immune responses in rainbow trout. For best protection, juvenile rainbow trout should be immunized with a 1:100 dilution of the titer with 1 × 1010.0 ml−1 TCID50 of the live vector vaccine, via 10-min immersion. Expression of the IPNV VP2 gene was confirmed in spleens of vaccinated rainbow trout, and reached the highest level at 3 days post-vaccination (dpv) and thereafter gradually decreased between 3 and 15 dpv. The expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were detected at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) were continuously increased in the spleens of vaccinated fish as compared with controls, between 3 and 15 dpv. The cumulative percentage mortality differed significantly between the vaccinated fish and the controls (empty-plasmid-vaccinated). The results of the IPNV challenge showed that the live vector vaccine had a high protection rate against IPNV (relative percent survival value of 88.24), and the vaccinated rainbow trout displayed high levels of serum antibodies against IPNV infection. Taken together, our results demonstrate that this is a promising live vector vaccine that could be used to protect rainbow trout against IPNV infection.

Oral immunization with recombinant Lactobacillus casei displayed AHA1-CK6 and VP2 induces protection against infectious pancreatic necrosis in rainbow trout (Oncorhynchus mykiss)

Infectious pancreatic necrosis virus (IPNV) primarily infects larvae and young salmonid with serious economic losses, which causes haemorrhage and putrescence of hepatopancreas. To develop a more effective oral vaccine against IPNV infection, the aeromonas hydrophila adhesion (AHA1) gene was used as a targeting molecule for intestinal epithelial cells. A genetically engineered Lactobacillus casei (pPG-612-AHA1-CK6-VP2/L. casei 393) was constructed to express the AHA1-CK6-VP2 fusion protein. The expression of interest protein was confirmed by western blotting and the immunogenicity of pPG-612-AHA1-CK6-VP2/L. casei 393 was evaluated. And the results showed that more pPG-612-AHA1-CK6-VP2/L. casei 393 were found in the intestinal mucosal surface of the immunized group. The Lactobacillus-derived AHA1-CK6-VP2 fusion protein could induce the production of serum IgM and skin mucus IgT specific for IPNV with neutralizing activity in rainbow trouts. The levels of IL-1β, IL-8 and TNF-α isolated from the lymphocytes stimulated by AHA1-CK6-EGFP produced were significantly higher than EGFP group. For transcription levels of IL-1β, IL-8, CK6, MHC-II, Mx and TNF-1α in the spleen, the result indicated that the adhesion and target chemokine recruit more immune cells to induce cellular immunity. The level of IPNV in the immunized group of pPG-612-AHA1-CK6-VP2/L. casei 393 was significantly lower than that in the control groups. These data indicated that the adhesion and target chemokine could enhance antigen delivery efficiency, which provides a valuable strategy for the development of IPNV recombination Lactobacillus casei oral vaccine in the future.

Autophagy induced by infectious pancreatic necrosis virus promotes its multiplication in the Chinook salmon embryo cell line CHSE-214

Infectious pancreatic necrosis virus (IPNV) is a common pathogen that causes huge economic losses for the salmonid aquaculture industry. Autophagy plays an important regulatory role in the invasion of pathogenic microorganisms. In this study, we explored the relationship between IPNV infection and autophagy in Chinook salmon embryo (CHSE-214) cells using standard methods. Transmission electron microscopy showed that IPNV infection produced typical structures of autophagosomes in CHSE-214 cells. Transformation of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II protein, a marker of autophagy, was observed in IPNV-infected cells using confocal fluorescence microscopy and western blot analysis. Western blotting also showed that expression of the autophagy substrate p62 was significantly decreased in IPNV-infected cells. The influence of autophagy on IPNV multiplication was further clarified with cell culture experiments using autophagy inducer rapamycin and autophagy inhibitor 3-methyladenine. Rapamycin promoted IPNV multiplication at both the nucleic acid and protein levels, which led to higher IPNV yields; 3-methyladenine treatment had the opposite effect. This study has demonstrated that IPNV can induce autophagy, and that autophagy promotes the multiplication of IPNV in CHSE-214 cells.

Salmon cells SHK-1 internalize infectious pancreatic necrosis virus by macropinocytosis.

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE-214 by macropinocytosis. In this study, we have extended our investigation into SHK-1 cells, a macrophage-like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK-1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK-1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK-1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis-mediated entry of IPNV into its target cells.

Iron Overload Is Associated With Oxidative Stress and Nutritional Immunity During Viral Infection in Fish.

Iron is a trace element, essential to support life due to its inherent ability to exchange electrons with a variety of molecules. The use of iron as a cofactor in basic metabolic pathways is essential to both pathogenic microorganisms and their hosts. During evolution, the shared requirement of micro- and macro-organisms for this important nutrient has shaped the pathogen–host relationship. Infectious pancreatic necrosis virus (IPNv) affects salmonids constituting a sanitary problem for this industry as it has an important impact on post-smolt survival. While immune modulation induced by IPNv infection has been widely characterized on Salmo salar, viral impact on iron host metabolism has not yet been elucidated. In the present work, we evaluate short-term effect of IPNv on several infected tissues from Salmo salar. We observed that IPNv displayed high tropism to headkidney, which directly correlates with a rise in oxidative stress and antiviral responses. Transcriptional profiling on headkidney showed a massive modulation of gene expression, from which biological pathways involved with iron metabolism were remarkable. Our findings suggest that IPNv infection increase oxidative stress on headkidney as a consequence of iron overload induced by a massive upregulation of genes involved in iron metabolism.

Infectious pancreatic necrosis virus isolated from farmed rainbow trout and tilapia in Kenya is identical to European isolates.

Infectious pancreatic necrosis virus (IPNV) is an aquabirnavirus that causes serious diseases in a variety of fish species worldwide. It has been isolated from a large number of healthy fresh and marine water fish. Prior to this study, there was no record of the presence of IPNV infection in Kenya. Here, the presence of IPNV in farmed rainbow trout and tilapia was examined in Nyeri County of central Kenya. Head kidney samples taken from five rainbow trout and three tilapia farms and stored in RNALater® were processed by PCR followed by sequencing of a segment A fragment covering nucleotide positions 2,120-2,343bp. IPNV was detected in all the farms sampled with infection ratios ranging from 0.3 to 0.78 although the infections were not associated with any specific clinical signs of disease. These findings were supported by immunohistochemistry staining of the virus in the kidney and exocrine pancreas of rainbow trout. Sequence alignment and phylogenetic analysis revealed that the Kenyan isolates were identical to European isolates, suggesting a common origin. These findings highlight the need for better biosecurity procedures with more stringent surveillance programmes and control for fish diseases, especially focusing on imported breeding materials to Kenya.

Recombinant infectious hematopoietic necrosis virus expressing infectious pancreatic necrosis virus VP2 protein induces immunity against both pathogens

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout. Their co-infection is also common, which causes great economic loss in juvenile salmon species. Although vaccines against IHNV and IPNV have been commercialized in many countries, the prevalence of IHNV and IPNV is still widespread in modern aquaculture. In the present study, two IHNV recombinant viruses displaying IPNV VP2 protein (rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE) were generated using the RNA polymerase Ⅱ system to explore the immunogenicity of IHNV and IPNV. The recombinant IHNV viruses were stable, which was confirmed by sequencing, indirect immunofluorescence assay, western blotting, transmission electron microscopy and viral growth curve assay. IHNV and IPNV challenge showed that the recombinant viruses had high protection rates against IHNV and IPNV with approximately 65% relative percent survival rates. Rainbow trout (mean weight 20 g) vaccinated with these two recombinant viruses showed a high level of antibodies against IHNV and IPNV infection. Taken together, our findings demonstrate that rIHNV-IPNV VP2 and rIHNV-IPNV VP2COE might be promising vaccine candidates against IHNV and IPNV.

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection have not been studied yet.Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques.Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genesifn-1,pkrandmxgenes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line.Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

Infectious pancreatic necrosis virus triggers antiviral immune response in rainbow trout red blood cells, despite not being infective

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), replicate inside them and induce an immune response. However, the roles of RBCs in the context of infectious pancreatic necrosis virus (IPNV) infection have not been studied yet. Methods: Ex vivo rainbow trout RBCs were obtained from peripheral blood, Ficoll purified and exposed to IPNV in order to analyze infectivity and immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Despite not being infected, rainbow trout RBCs could respond to IPNV with increased expression of antiviral genes. Fish RBCs could be considered as mediators of the antiviral response and therefore targets of new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this antiviral response in rainbow trout RBCs.

Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective.

Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), highly replicate inside them and induce an immune response. However, the implications of RBCs in the context of birnavirus infection (i.e, infectious pancreatic necrosis virus (IPNV)) have not yet been studied. Methods:Ex vivo trout RBCs were obtained from peripheral blood, ficoll purified and exposed to IPNV in order to analyze infectivity and induced immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV-exposed RBCs increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Trout RBCs could trigger an antiviral immune response against IPNV infection despite not being infected. Fish RBCs could be considered mediators of the antiviral response and therefore targets of novel DNA vaccines and new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this immune response in trout RBCs.

Infectious pancreatic necrosis virus enters CHSE-214 cells via macropinocytosis

Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.

PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells.

The double-stranded RNA-activated protein kinase R (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV) infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α) phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells.

Gilthead seabream (Sparus aurata) Mx gene promoters respond differentially to IPNV and VHSV infections in RTG-2 cells

The understanding of virus–host interactions relies on the knowledge of the regulatory mechanisms of the type I interferon (IFN I)-stimulated genes (ISGs). Among ISGs, those coding Mx proteins play a main role due to their direct antiviral activity. The study of these genes in gilthead seabream is interesting, since this species displays high natural resistance to viral diseases, being asymptomatic carrier of infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV). Gilthead seabream has three Mx genes (Mx1, Mx2, and Mx3), encoding proteins with a wide spectrum of antiviral activity. The structure of the three promoters (pMx1, pMx2 and pMx3) has been previously disclosed, and their response to poly I:C in RTG-2 cells characterized. To further analyze these promoters, their response to two viral infections has been evaluated in the present study. For that purpose, RTG-2 cells transiently transfected with the luciferase gene under the control of each promoter were inoculated with either IPNV or VHSV at two different doses. The highest and lowest fold induction values were recorded for pMx2 and pMx3, respectively. The promoter induction was always stronger after VHSV inoculation than in IPNV-inoculated cells. In addition, the higher dose of VHSV tested induced higher response of the three promoters, whereas in IPNV-infected cells the highest induction was recorded after inoculation with the lower viral dose. To further study the response of the Mx2 promoter, RTG-2 cells stably transfected with the luciferase gene under the control of pMx2 were stimulated with poly I:C and subsequently infected with IPNV or VHSV. Interestingly, IPNV infection inhibited the induction caused by poly I:C, suggesting an antagonistic activity of IPNV on Mx2 transcription. In contrast, VHSV infection did not alter the response triggered by poly I:C. These results highlight the specific regulation that controls the activity of each promoter, and support the existence of complex interactions between host cells, specific Mx promoters, and viruses, which are responsible for the final outcome of a viral infection.