GATA3 Inhibits Viral Infection by Promoting MicroRNA-155 Expression.

Recognition of viral RNAs by melanoma differentiation associated gene-5 (MDA5) initiates chicken antiviral response by producing type I interferons. Our previous studies showed that chicken microRNA-155-5p (gga-miR-155-5p) enhanced IFN-ß expression and suppressed the replication of infectious burse disease virus (IBDV), a double-stranded RNA (dsRNA) virus causing infectious burse disease in chickens. However, the mechanism underlying IBDV-induced gga-miR-155-5p expression in host cells remains elusive. Here, we show that IBDV infection or poly(I:C) treatment of DF-1 cells markedly increased the expression of GATA-binding protein 3 (GATA3), a master regulator for TH2 cell differentiation, and that GATA3 promoted gga-miR-155-5p expression in IBDV-infected or poly(I:C)-treated cells by directly binding to its promoter. Surprisingly, ectopic expression of GATA3 significantly reduced IBDV replication in DF-1 cells, and this reduction could be completely abolished by treatment with gga-miR-155-5p inhibitors, whereas knockdown of GATA3 by RNA interference enhanced IBDV growth, and this enhancement could be blocked with gga-miR-155-5p mimics, indicating that GATA3 suppressed IBDV replication by gga-miR-155-5p. Furthermore, our data show that MDA5 is required for GATA3 expression in host cells with poly(I:C) treatment, so are the adaptor protein TBK1 and transcription factor IRF7, suggesting that induction of GATA3 expression in IBDV-infected cells relies on MDA5-TBK1-IRF7 signaling pathway. These results uncover a novel role for GATA3 as an antivirus transcription factor in innate immune response by promoting miR-155 expression, further our understandings of host response against pathogenic infection, and provide valuable clues to the development of antiviral reagents for public health. IMPORTANCE Gga-miR-155-5p acts as an important antivirus factor against IBDV infection, which causes a severe immunosuppressive disease in chicken. Elucidation of the mechanism regulating gga-miR-155-5p expression in IBDV-infected cells is essential to our understandings of the host response against pathogenic infection. This study shows that transcription factor GATA3 initiated gga-miR-155-5p expression in IBDV-infected cells by directly binding to its promoter, suppressing viral replication. Furthermore, induction of GATA3 expression was attributable to the recognition of dsRNA by MDA5, which initiates signal transduction via TBK1 and IRF7. Thus, it is clear that IBDV induces GATA3 expression via MDA5-TBK1-IRF7 signaling pathway, thereby suppressing IBDV replication by GATA3-mediated gga-miR-155-5p expression. This information remarkably expands our knowledge of the roles for GATA3 as an antivirus transcription factor in host innate immune response particularly at an RNA level and may prove valuable in the development of antiviral drugs for public health.

Tween 80 improves the infectivity of BCL1 cell-adapted infectious bursal disease virus.

Non-ionic surfactants have the ability to alter the cell membrane's permeability for enhancing virus replication. The impact of non-ionic surfactant Tween 80 (TW80) on the infectivity of infectious bursal disease virus (IBDV) was studied in BCL1 cells. The toxicity of different concentrations of TW80 for BCL1 cells was determined for five-time passages. The confluent monolayer of BCL1 was infected by IBDV and subsequently passaged. The adaptation was confirmed by virus titration and RT-PCR assay. Replication kinetics of the cell-adapted IBDV was evaluated in pre-treatment and simultaneous treatment with TW80 at 0.01% concentration. The IBDV infectivity patterns were determined by virus titration, FRAP assay, and transmission electron microscopy. Sequence analysis, RNA secondary structure, and potential N-glycosylation site were conducted for IBDV VP2. Despite the similar cytopathic effects found in both TW80-treated cells and similar ROS levels, the IBDV titer was higher in TW80 pre-treated cells compared to the simultaneous treatment one. Such an increase in IBDV titer did not associate with changes in the VP2 sequence and RNA secondary structure. The possible antioxidant capacity of TW80 can attenuate the ROS damage and improve the cell viability, thereby improving IBDV infectivity.

Research Note: Anti-inflammatory effects and antiviral activities of baicalein and chlorogenic acid against infectious bursal disease virus in embryonic eggs.

The purpose of this study was to investigate if baicalein and chlorogenic acid could inhibit the inflammatory responses induced by and protect against infectious bursal disease virus (IBDV) in chicken embryonic eggs. Nine-day-old embryonated chicken eggs were randomly divided into 3 groups of 50 eggs per group: 1) treatment with varying concentrations of baicalein, 2) treatment with varying concentrations of chlorogenic acid, or 3) left untreated as a control. Forty-eight hours after hatching, each group was inoculated with a very virulent IBDV isolate, and the survival of the embryo was monitored daily until the embryonic livers were collected 72 h after inoculation. After IBDV infection, the viral loads in the embryonic livers were evaluated using qRT-PCR, and the hepatic content of inflammatory mediators, such as histamine, interleukin 1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and nuclear factor-kappa B (NF-κB), were examined. Significant antiviral potential was demonstrated at concentrations of 108 and 215 µg/egg of baicalein and chlorogenic acid, respectively. We observed a concentration-dependent response in the antiviral properties of these chemicals. Treating the embryos with baicalein and chlorogenic acid significantly reduced histamine production. Moreover, pretreatment with baicalein and chlorogenic acid significantly inhibited NF-κB activation, and this inhibited the subsequent production of the proinflammatory cytokines TNF-α and IL-1ß in the context of IBDV infection. These findings suggest that baicalein and chlorogenic acid have anti-IBDV properties, and they may be useful in the prevention of inflammation-related diseases.

Research Note: Evaluation of acute oral toxicity of povidone-iodine in cockerels using the up-and-down procedure.

Povidone-iodine (Polidine) is a synthetic broad-spectrum antiseptic and being applied topically to treat wounds and prevent their infection. It is however used by poultry farmers, field veterinarians, and other animal health workers with the claim that it is effective for treatment of infectious bursal disease when administered orally. Hence, an acute oral toxicity study was conducted to ascertain its safety profile. Ten cockerel chicks were randomly selected and divided into 2 groups of 5 chicks per group. One group served as the negative control, whereas the other group was administered povidone-iodine at a dose of 2,000 mg/kg of BW orally. The blood sample was collected at the end of the study to determine changes in hematological and biochemical parameters. In addition, vital organs were also harvested and preserved for histopathological examinations. The result showed that the median lethal dose (LD50) of the povidone-iodine is higher than 2,000 mg/kg of BW in cockerels. There were no significant changes in the hematological parameters measured. Biochemical evaluation (renal and liver function test) showed an increase in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels after administration of povidone-iodine. The study indicated that the LD50 of povidone-iodine is higher than 2,000 mg/kg of BW of cockerels, and there were increases in urinary and liver enzymes at this dose.

Dietary supplementation with Withania somnifera root powder ameliorates experimentally induced Infectious Bursal Disease in chicken.

Infectious Bursal Disease (IBD) is an economically important, immunosuppressive viral disease of chicken. Withania somnifera, a well-known Indian medicinal plant and functional food, finds extensive ethnomedicinal and ethnoveterinary use in the subcontinent. Root extracts of Withania somnifera have been shown to inhibit IBD virus (IBDV) in vitro. The effect of dietary supplementation with whole root powder of Withania somnifera was studied in chicken experimentally infected with IBDV. Dietary supplementation with the root powder improved erythrocytic indices, biochemical parameters, bursal weight index, and lymphocyte stimulation indices, and reduced histopathological insult in the infected birds. Viral load decreased to less than one-fourth in the birds receiving dietary supplementation with Withania somnifera root powder. It could be concluded that continued supplementation of IBDV-infected chicken with Withania somnifera root powder alleviated virus-induced stress and histological and immunological alterations and reduced viral persistence in the host.

Alleviation of infectious-bursal-disease-virus-induced bursal injury by betaine is associated with DNA methylation in IL-6 and interferon regulatory factor 7 promoter.

Infectious bursal disease virus (IBDV) often infects young chickens and causes severe immunosuppression and inflammatory injury. Betaine is an antiviral and anti-inflammatory ingredient that may exert functions through epigenetic regulation. However, the effects of betaine on an IBDV-induced bursal injury and their underlying mechanisms have not been investigated. In this study, betaine was supplemented to the drinking water of newly hatched commercial broilers for 3 wk. Afterward, the chickens were infected with the IBDV. After 5 D of infection, the bursal lesions were examined. The mRNA expression levels of IBDV VP2 gene, pro-inflammatory cytokines, and interferons were detected. Furthermore, the 5-methylcytosine level of the CpG island in the promoter region of IL-6 and interferon regulatory factor 7 (IRF7) were determined. The IBDV induced the depletion of lymphocytes and inflammation in the bursal follicles. IBDV infection considerably elevated the mRNA levels of VP2, IL-6, types I (IFNα and IFNß) and II (IFNγ) interferons, and IRF7. The CpG island methylation in the promoter regions of IL-6 and IRF7 were substantially decreased after IBDV infection. Betaine administration attenuated the IBDV-induced bursal lesions. Meanwhile, the IBDV-induced mRNA expression levels of IL-6, IFNß, and IRF7 were suppressed by betaine consumption. Furthermore, the hypomethylation effects of IBDV infection to the promoter regions of IL-6 and IRF7 genes were eliminated and relieved by betaine administration. Our results indicated that the IBDV-induced expression levels of IL-6 and IRF7 genes are associated with the suppression of methylation in the promoter region. Betaine administration through drinking water may alleviate the IBDV-induced bursal injury via epigenetic regulation.

Immunomodulatory effects of heat stress and lipopolysaccharide on the bursal transcriptome in two distinct chicken lines.

BACKGROUND: Exposure to heat stress suppresses poultry immune responses, which can increase susceptibility to infectious diseases and, thereby, intensify the negative effects of heat on poultry welfare and performance. Identifying genes and pathways that are affected by high temperatures, especially heat-induced changes in immune responses, could provide targets to improve disease resistance in chickens. This study utilized RNA-sequencing (RNA-seq) to investigate transcriptome responses in the bursa of Fabricius, a primary immune tissue, after exposure to acute heat stress and/or subcutaneous immune stimulation with lipopolysaccharide (LPS) in a 2 × 2 factorial design: Thermoneutral + Saline, Heat + Saline, Thermoneutral + LPS and Heat + LPS. All treatments were investigated in two chicken lines: a relatively heat- and disease-resistant Fayoumi line and a more susceptible broiler line. RESULTS: Differential expression analysis determined that Heat + Saline had limited impact on gene expression (N = 1 or 63 genes) in broiler or Fayoumi bursa. However, Thermoneutral + LPS and Heat + LPS generated many expression changes in Fayoumi bursa (N = 368 and 804 genes). Thermoneutral + LPS was predicted to increase immune-related cell signaling and cell migration, while Heat + LPS would activate mortality-related functions and decrease expression in WNT signaling pathways. Further inter-treatment comparisons in the Fayoumi line revealed that heat stress prevented many of the expression changes caused by LPS. Although fewer significant expression changes were observed in the broiler bursa after exposure to Thermoneutral + LPS (N = 59 genes) or to Heat + LPS (N = 146 genes), both treatments were predicted to increase cell migration. Direct comparison between lines (broiler to Fayoumi) confirmed that each line had distinct responses to treatment. CONCLUSIONS: Transcriptome analysis identified genes and pathways involved in bursal responses to heat stress and LPS and elucidated that these effects were greatest in the combined treatment. The interaction between heat and LPS was line dependent, with suppressive expression changes primarily in the Fayoumi line. Potential target genes, especially those involved in cell migration and immune signaling, can inform future research on heat stress in poultry and could prove useful for improving disease resistance.

Pro-apoptosis effects of protocatechuic acid in the early stage of infectious bursal disease virus infection.

Infectious bursal disease virus (IBDV) is a very important small RNA virus in the family of Birnaviridae, which can cause severe immunosuppressive effects and pathological damages in young chickens. It can replicate in bursal lymphocytes and impede the growth and development of B cells, finally causing bursal lymphocytes apoptosis. Previous results have shown that protocatechuic acid (PCA) as an important phenolic compound could effectively improve the survival rate of chickens infected with IBDV. The current study aimed to explore how PCA influenced the pathogenesis of IBDV, especially lymphocyte apoptosis in the process of IBDV infection. The results showed that PCA could effectively alleviate bursal pathological changes at the early stage of IBDV invasion. Moreover, bursal lymphocyte apoptosis for tissue section samples was largely elevated by PCA by using the terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) method while the bursal lymphocyte apoptosis ratio was also increased by PCA by flow cytometry in the early stage of IBDV infection in vivo. Meanwhile, PCA could promote non-lymphocyte apoptosis in vitro. Further study displayed that the potential mechanisms mainly relied on regulation of the expressions of pro-apoptotic protein Bax and anti-apoptotic Bcl-2, thus speeding up the process of IBDV-infected cell apoptosis and preventing virus infection. Meanwhile, the results displayed that the PI3K/Akt and NF kappa B signal pathways might play an important role in promoting cell apoptosis after IBDV infection. Overall, PCA as a potent antiviral drug precursor is expected to be applied in the poultry industry as a substitute for clinical antiviral application.

Ginsenoside Rg1 enhanced immune responses to infectious bursal disease vaccine in chickens with oxidative stress induced by cyclophosphamide.

This study was designed to evaluate the effect of oral administration of ginsenoside Rg1 on oxidative stress induced by cyclophosphamide in chickens. Ninety-six chickens were randomly divided into 4 groups, each consisting of 24 birds. Groups 2 and 3 received intramuscular injection of cyclophosphamide at 100 mg/kg body weight for 3 d to induce oxidative stress and immune suppression. Groups 1 and 4 were injected with saline in the same way as groups 2 and 3. Then chickens in group 3 were orally administrated Rg1 of 1 mg/kg body weight in drinking water for 7 d. After that, groups 1 to 3 were orally vaccinated with attenuated infectious bursal disease vaccine (Strain B87). Blood samples were collected for determination of infectious bursal disease virus-specific antibodies, cytokines, and oxidative parameters. Splenocytes were prepared for lymphocyte proliferation assay. The results showed that oral administration of ginsenoside Rg1 significantly enhanced specific antibody, IFN-γ, and IL-6 responses, and lymphocyte proliferation induced by concanavalin A and lipopolysaccharide in chickens injected with cyclophosphamide. Antioxidant activity of ginsenoside Rg1 was also observed in chickens by increased total antioxidant capacity, total superoxide dismutase, catalase, glutathione peroxidase, glutathione, ascorbic acid, and α-tocopherol, as well as decreased malondialdehyde and protein carbonyl. Therefore, oral administration of Rg1 was shown to improve the immune responses to infectious bursal disease vaccine in chickens suffering from oxidative stress.

Molecular cloning of chicken IL-7 and characterization of its antiviral activity against IBDV in vivo.

Mammalian interleukin-7 (IL-7) is able to stimulate lymphocyte proliferation and maturation, and reverse immunosuppression. However, whether poultry IL-7 has similar functions remains unclear. Chicken infectious bursal disease virus (IBDV) causes serious immunosuppression in chicken due to virus-induced immune disorder. Whether chicken IL-7 (chIL-7) has the ability to restore the immunity during IBDV-induced immunosuppression is not clear. To test this, we amplified chIL-7 gene by RT-PCR, prepared recombinant chIL-7 using HEK293T cells and treated the chicken with the chIL-7 prior to IBDV infection. Our results indicate that chIL-7 promoted mouse B cell proliferation in vitro, and significantly reduced virus titer in bursal tissue and chicken morbidity of IBDV-infected chicken. Mechanically, chIL-7 induced chicken lymphocyte proliferation and interferon-γ production, but down-regulated TGF-ß expression, suggesting that chIL-7 has the ability to reverse IBDV-induced immunosuppression and might be a potential therapeutic agent for prevention and treatment of infectious bursal disease.

Antiviral Activity of Graphene-Silver Nanocomposites against Non-Enveloped and Enveloped Viruses.

The discovery of novel antiviral materials is important because many infectious diseases are caused by viruses. Silver nanoparticles have demonstrated strong antiviral activity, and graphene is a potential antimicrobial material due to its large surface area, high carrier mobility, and biocompatibility. No studies on the antiviral activity of nanomaterials on non-enveloped viruses have been reported. To investigate the antiviral activity of graphene oxide (GO) sheets and GO sheets with silver particles (GO-Ag) against enveloped and non-enveloped viruses, feline coronavirus (FCoV) with an envelope and infectious bursal disease virus (IBDV) without an envelope were chosen. The morphology and sizes of GO and GO-Ag were characterized by transmission, scanning electron microscopy, and X-ray diffraction. A virus inhibition assay was used to identify the antiviral activity of GO and GO-Ag. Go-Ag inhibited 25% of infection by FCoV and 23% by IBDV, whereas GO only inhibited 16% of infection by FCoV but showed no antiviral activity against the infection by IBDV. Further application of GO and GO-Ag can be considered for personal protection equipment to decrease the transmission of viruses.

Omega-3 polyunsaturated fatty acids enrichment alters performance and immune response in infectious bursal disease challenged broilers.

BACKGROUND: Infectious bursal disease (IBD) results in economic loss due to mortality, reduction in production efficiency and increasing the usage of antibiotics. This study was carried out to investigate the modulatory roles of dietary n-3 polyunsaturated fatty acids (PUFA) enrichment in immune response and performance of IBD challenged broiler chickens. METHODS: A total of 300 day old male broiler chicks were assigned to four dietary n-3 PUFA ascending levels as the treatment groups (T1: 0.5; T2: 8.0; T3: 11.5; T4: 16.5) using combinations of tuna oil and sunflower oil. All diets were isocaloric and isonitrogenous. On day 28, all birds were challenged with IBD virus. Antibody titer, cytokine production, bursa lesion pre and post-challenge and lymphoid organ weight were recorded. RESULTS: On d 42 the highest body weight was observed in the T2 and T3 and the lowest in T4 chickens. Feed conversion ratio of the T2 broilers was significantly better than the other groups. Although productive parameters were not responded to the dietary n-3 PUFA in a dose-dependent manner, spleen weight, IBD and Newcastle disease antibody titers and IL-2 and IFN-γ concentrations were constantly elevated by n-3 PUFA enrichment. CONCLUSIONS: Dietary n-3 PUFA enrichment may improve the immune response and IBD resistance, but the optimum performance does not coincide with the optimum immune response. It seems that dietary n-3 PUFA modulates the broiler chicken performance and immune response in a dose-dependent manner. Thus, a moderate level of dietary n-3 PUFA enrichment may help to put together the efficiency of performance and relative immune response enhancement in broiler chickens.

Hypericum perforatum extract therapy for chickens experimentally infected with infectious bursal disease virus and its influence on immunity.

Hypericum perforatum extract (HPE) has been proved a drug effective to many viral diseases. The purpose of this paper was to investigate the therapeutic efficacy and immuno-enhancement of HPE for chickens which were already challenged with infectious bursal disease virus (IBDV BC-6/85). Chickens infected with IBDV were treated with HPE for 5 consecutive days, the observation of immune organ indexes and pathological changes index, determination of IFN-α and detection of IBDV with RT-PCR were employed to assess in vivo whether or not HPE had the certain therapeutic efficacy on infectious bursal disease (IBD), and if HPE was able to improve the immunologic function. The results showed that 1330 and 667.9 mg/kg body weight (BW) per day of HPE had significant therapeutic efficacy and improvement immunologic functions for chickens infected experimentally with IBDV.

The interplay between infectious pancreatic necrosis virus (IPNV) and the IFN system: IFN signaling is inhibited by IPNV infection.

Infectious pancreatic necrosis virus (IPNV) is a major pathogen in the aquaculture industry worldwide. Factors contributing to IPNV pathogenicity are yet poorly understood. Indications of IPNV being able to evade or counteract innate host defense come from its lack of ability to induce strong type I interferon (IFN) responses in cell culture. We show here that addition of salmon rIFN-alpha1 to cells prior to IPNV infection halts the viral protein synthesis and prevents processing of pVP2 into mature VP2. Furthermore, compared to pre-treatment with IFN-alpha1 the antiviral state in cells infected with IPNV prior to IFN-treatment, was antagonized by IPNV, as detected by higher viral titers, faster viral protein synthesis and also by reduced Mx expression. The longer headstart the virus gets, the more prominent is the weakening of IFN signaling. IPNV VP4 and VP5 inhibit IFN-induced expression from the Mx promoter, indicating that these proteins contribute to the antagonistic effect.

Inhibitory effect of mycophenolic acid on the replication of infectious pancreatic necrosis virus and viral hemorrhagic septicemia virus.

Infectious pancreatic necrosis virus (IPNV) and viral hemorrhagic septicemia virus (VHSV) remain two of the most important pathogens of farmed trout worldwide. Mycophenolic acid (MPA) is an inhibitor of cellular inosine monophosphate dehydrogenase (IMPDH), an enzyme that catalyzes an essential step in the biosynthesis of GTP. In this report, the antiviral activity of MPA against IPNV and VHSV in cell culture was assessed. Cell viability, virus yield, protein and RNA synthesis determinations were used to evaluate the inhibitory effect of MPA. MPA caused a dose-dependent inhibition of IPNV and VHSV replication. It was found that MPA had a particularly potent effect against IPNV, inhibiting the production of infectious virus more than 10(5)-fold. MPA was also highly effective in preventing viral protein synthesis. Quantitative real-time RT-PCR was used to measure viral RNA in cells infected by IPNV or VHSV to evaluate the inhibitory capacity of MPA as well as to compare MPA to the established antiviral drug ribavirin. MPA showed a good efficacy in decreasing accumulation of viral RNA at low concentrations. Finally, time of addition and wash out experiments suggested that MPA may have a dual mechanism of action, targeting both a cell and a viral function. This study provides evidence that MPA can function as a broad-spectrum antiviral drug for use in therapy of rainbow trout diseases.

Expression of immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana.

VP2 protein is the major host-protective immunogen of infectious bursal disease virus (IBDV) of chickens. Transgenic lines of Arabidopsis thaliana expressing recombinant VP2 were developed. The VP2 gene of an IBDV antigenic variant E strain was isolated, amplified by RT-PCR and introduced into a plant expression vector, pE1857, having a strong promoter for plant expression. A resulting construct with a Bar gene cassette for bialaphos selection in plant (rpE-VP2) was introduced into Agrobacterium tumefaciens by electroporation. Agrobacterium containing the rpE-VP2 construct was used to transform Ar. thaliana and transgenic plants were selected using bialaphos. The presence of VP2 transgene in plants was confirmed by PCR and Southern blot analysis and its expression was confirmed by RT-PCR. Western blot analysis and antigen-capture ELISA assay using monoclonal anti-VP2 were used to determine the expression of VP2 protein in transgenic plants. The level of VP2 protein in the leaf extracts of selected transgenic plants varied from 0.5% to 4.8% of the total soluble protein. Recombinant VP2 protein produced in plants induced antibody response against IBDV in orally-fed chickens.

The in vivo and in vitro effects of chicken interferon alpha on infectious bursal disease virus and Newcastle disease virus infection.

The in vitro and in vivo effects of chicken interferon alpha on infectious bursal disease virus (IBDV) infection were investigated in this study. A cDNA of interferon alpha was first cloned from a Chinese strain chicken Shiqi by reverse transcription-polymerase chain reaction. The deduced amino acid sequence has one amino acid substitution with chicken interferon alpha 1 at residue 65 (N to S) and two amino acid substitutions with chicken interferon alpha 2 at residues 50 (N to S) and 58 (P to L), respectively. A prokaryotic expression system was employed to produce a large quantity of recombinant protein. Recombinant interferon was purified in a one-step process, and an optimal refolding process was devised. About 51% recombinant protein from inclusion bodies was refolded, and the final yield of the recombinant interferon reached 24.66 mg/liter culture. The recombinant interferon suppressed IBDV plaque formation in a dose-dependent manner and ameliorated IBDV and Newcastle disease virus infection in both specific-pathogen-free (SPF) and commercial chickens. The antiviral effect of interferon alpha is more significant in commercial chickens than in SPF chickens, and the route of administration affects the efficacy of interferon therapy. This is the first reported study of the effects of interferon alpha on IBDV infection.

In vivo effect of EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide) on experimental infected rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) fry with infectious pancreatic necrosis virus.

The in vivo antiviral effect of 5-ethynyl-1-beta-D-ribofuranosylimidazole-carboxamide (EICAR) was evaluated in coho salmon and rainbow trout fry, experimentally infected with infectious pancreatic necrosis virus (IPNV). Treatment consisted of a daily bath of 2 h in 0.4 microg ml(-1) or 0.8 microg ml(-1) of EICAR, for approximately 20 days. The behavior of the fish was studied for 45 days post-infection. The survival of the infected treated groups was compared with the survival of non-infected and infected untreated control groups. The results showed that the survival of coho salmon and rainbow trout fry in the infected group treated with both doses of EICAR was similar to the survival observed in the healthy control group (approximately 94%). While, the survival of the infected and untreated control fish was 56% for salmon and 28% for trout, there were no significant difference in the weight of coho salmon fry between those treated with EICAR and non-infected and infected untreated control groups. However, in rainbow trout there was a statistically significant weight decrease in infected untreated group. Finally, the analysis of tissue samples of the fish by reverse transcription associated with the polymerase chain reaction (RT-PCR) suggest that EICAR have decreased the viral load in infected treated fry. Consequently, the results indicate that EICAR is an effective inhibitor of IPNV replication in vivo and could be a promissory antiviral compound for the treatment of IPNV disease.