Search Results (27)

Infectious hematopoietic necrosis virus (IHNV) is a serious pathogen that causes great economic loss to the salmon and trout industry. Previous studies showed that IHNV alters the expression patterns of splenic microRNAs (miRNAs) in rainbow trout. Among the differentially expressed miRNAs, miRNA146a-3p was upregulated by IHNV. However, it is unclear how IHNV utilizes miRNA146a-3p to escape the immune response or promote viral replication. The present study suggested that one multiplicity of infection (MOI) of IHNV induced the most significant miR-146a-3p expression at 1 day post infection (dpi). The upregulation of miR-146a-3p by IHNV was due to viral N, P, M, and G proteins and relied on the interferon (IFN) signaling pathway. Further investigation revealed that Wingless-type MMTV integration site family 3a (WNT3a) and G1/S-specific cyclin-D1-like (CCND1) are the target genes of miRNA-146a-3p. The regulation of IHNV infection by miRNA-146a-3p is dependent on WNT3a and CCND1. MiRNA-146a-3p was required for the downregulation of WNT3a and CCND1 by IHNV. Moreover, we also found that WNT3a and CCND1 are novel proteins that induce the type-I IFN response in RTG-2 cells, and both of them could inhibit the replication of IHNV. Therefore, IHNV-induced upregulation of miRNA-146a-3p promotes early viral replication by suppressing the type-I IFN response by targeting WNT3a and CCND1. This work not only reveals the molecular mechanism of miRNA-146a-3p during IHNV infection but also provides new antiviral targets for IHNV. Full article

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins. Full article

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen that causes significant economic losses to salmon trout farming. Although vaccines have been invented for the treatment of IHNV, findings from our previous survey show that breeding enterprises and farmers require effective oral drugs or immune enhancers. However, studies on the development of oral drugs are limited. In the present study, we used bioinformatics methods to predict the protein targets of andrographolide (Andro) in IHNV. Cells were infected with IHNV, and the effect of andrographolide was explored by evaluating the expression levels of genes implicated in oxidative stress, activities of antioxidant enzymes, and the expression of genes implicated in apoptosis and necrosis. In the present study, cells were divided into NC, IHNV, IHNV+10 μM andrographolide, and IHNV+20 μM andrographolide groups. qRT-PCR was performed to determine the expression level of genes, and an antioxidant enzyme detection kit was used to evaluate the activities of antioxidant enzymes. Fluorescent staining was performed using a reactive oxygen species detection kit (ROS) and Hoechst 33342/PI double staining kit, and the mechanism of alleviation of apoptosis and oxidative stress andrographolide after IHNV infection was determined. The results indicated that andrographolide inhibits viral growth by binding to the NV protein of IHNV and increasing the antioxidant capacity of the body through the CTSK/BCL2/Cytc axis, thereby inhibiting the occurrence of IHNV-induced apoptosis. This is the first study to explore the antagonistic mechanism of action of andrographolide in alleviating IHNV infection. The results provide valuable information on alternative strategies for the treatment of IHNV infection during salmon family and provide a reference for the use of andrographolide as an antioxidant agent in agricultural settings. Full article

The ocular mucosa (OM) is an important and unique part of the vertebrate mucosal immune system. The OM plays an important role in maintaining visual function and defending against foreign antigens or microorganisms, while maintaining a balance between the two through complex regulatory mechanisms. However, the function of ocular mucosal defense against foreign pathogens and mucosal immune response in bony fish are still less studied. To acquire deeper understanding into the mucosal immunity of the OM in teleost fish, we established a study of the immune response of rainbow trout (Oncorhynchus mykiss) infected with the infectious hematopoietic necrosis virus (IHNV). Our findings revealed that IHNV could successfully infiltrate the trout’s OM, indicating that the OM could be an important portal for the IHNV. Furthermore, qPCR and RNA-Seq analysis results showed that a large number of immune-related genes were significantly upregulated in the OM of trout with IHNV infection. Critically, the results of our RNA-Seq analysis demonstrated that viral infection triggered a robust immune response, as evidenced by the substantial induction of antiviral, innate, and adaptive immune-related genes in the OM of infected fish, which underscored the essential role of the OM in viral infection. Overall, our findings revealed a previously unknown function of teleost OM in antiviral defense, and provided a theoretical basis for the study of the mucosal immunity of fish. Full article

Infectious hematopoietic necrosis virus (IHNV) is the most important pathogen threatening the aquaculture of salmonid fish in China. In addition to the common genogroup J IHNV, genogroup U has been newly discovered in China. However, there is no effective DNA vaccine to fight against this emerging genogroup U IHNV in China. In this study, DNA vaccines encoding the IHNV viral glycoprotein (G) gene of the GS2014 (genogroup J) and BjLL (genogroup U) strains isolated from northern China were successfully developed, which were identified by restriction analysis and IFA. The expression of the Mx-1 gene and G gene in the spleens and muscles of the injection site as well as the titers of the serum antibodies were measured to evaluate the vaccine efficacy by RT-qPCR and ELISA. We found that DNA vaccine immunization could activate Mx1 gene expression and upregulate G gene expression, and the mRNA levels of the Mx1 gene in the muscles were significantly higher than those in the spleens. Notably, DNA vaccine immunization might not promote the serum antibody in fish at the early stage of immunization. Furthermore, the efficacy of the constructed vaccines was tested in intra- and cross-genogroup challenges by a viral challenge in vivo. It seemed that the DNA vaccines were able to provide great immune protection against IHNV infection. In addition, the genogroup J IHNV-G DNA vaccine showed better immune efficacy than the genogroup U IHNV-G or divalent vaccine, which could provide cross-immune protection against the genogroup U IHNV challenge. Therefore, this is the first study to construct an IHNV DNA vaccine using the G gene from an emerging genogroup U IHNV strain in China. The results provide great insight into the advances of new prophylactic strategies to fight both the genogroup J and U IHNV in China. Full article

Despite the negative impact of viral hemorrhagic septicemia (VHS) and infectious hematopoietic necrosis (IHN) on European rainbow trout farming, no vaccines are commercially available in Europe. DNA vaccines are protective under experimental conditions, but testing under intensive farming conditions remains uninvestigated. Two DNA vaccines encoding the glycoproteins (G) of recent Italian VHSV and IHNV isolates were developed and tested for potency and safety under experimental conditions. Subsequently, a field vaccination trial was initiated at a disease-free hatchery. The fish were injected intramuscularly with either the VHS DNA vaccine or with a mix of VHS and IHN DNA vaccines at a dose of 1 µg/vaccine/fish, or with PBS. At 60 days post-vaccination, fish were moved to a VHSV and IHNV infected facility. Mortality started 7 days later, initially due to VHS. After 3 months, IHN became the dominant cause of disease. Accordingly, both DNA vaccinated groups displayed lower losses compared to the PBS group during the first three months, while the VHS/IHN vaccinated group subsequently had the lowest mortality. A later outbreak of ERM caused equal disease in all groups. The trial confirmed the DNA vaccines to be safe and efficient in reducing the impact of VHS and IHN in farmed rainbow trout. Full article

A teleost’s kidney was divided into head kidney and trunk kidney. The head kidney is an important lymphatic organ, while the trunk kidney mainly performs osmotic pressure regulation and excretion functions. Previous studies have shown that the teleost’s head kidney exerts a strong immune response against pathogen invasion, while the mechanism of immune response in the trunk kidney is still rarely reported. Therefore, in this study, we established an Infectious hematopoietic necrosis virus (IHNV) immersion infection model to compare the similarities and differences of immune response mechanisms between the head kidney and trunk kidney against viral infection. The results showed that IHNV infection causes severe tissue damage and inflammatory reaction in the head and trunk kidney, triggers a series of interferon cascade reactions, and produces strong immune response. In addition, the transcriptome data showed that the head kidney and trunk kidney had similar immune response mechanisms, which showed that the NOD-like receptor signaling pathway and Toll-like receptor signaling pathway were activated. In conclusion, despite functional differentiation, the teleost’s trunk kidney still has a strong immune response, especially the interferon-stimulated genes, which have stronger immune response in the trunk kidney than in the head kidney when responding to IHNV infection. This study contributes to a more comprehensive understanding of the teleost immune system and enriches the theory of kidney immunity in teleosts. Full article

A new cell line Japanese flounder spleen (JFSP) derived from the spleen of Japanese flounder (Paralichthys olivaceus) was established and characterized in this study. The JFSP cells grew rapidly at 29 °C, and the optimum fetal bovine serum concentration in the L-15 medium was 15%. Cells were subcultured for more than 80 passages. The JFSP cells have a diploid chromosome number of 2n = 68, which differs from the chromosome number of normal diploid Japanese flounder. The established cells were susceptible to Bohle virus (BIV), Viral hemorrhagic septicemia virus (VHSV), Hirame rhabdovirus (HIRRV), Infectious hematopoietic necrosis virus (IHNV), and Lymphocystis disease virus (LCDV), as evidenced by varying degrees of cytopathic effects (CPE). Replication of the virus in JFSP cells was confirmed by qRT-PCR and transmission electron microscopy. In addition, the expression of four immune-related genes, TRAF3, IL-1β, TNF-α, and TLR2, was differentially altered following viral infection. The results indicated that the cells underwent an antiviral immune response. JFSP cell line is an ideal tool in vitro for virology. The use of fish cell lines to study the immune genes and immune mechanism of fish and to clarify the immune mechanism of fish has important theoretical significance and practical application value for the fundamental prevention and treatment of fish diseases. Full article

The skin is the largest organ on the surface of vertebrates, which not only acts as the first line of defense against pathogens but also harbors diverse symbiotic microorganisms. The complex interaction between skin immunity, pathogens, and commensal bacteria has been extensively studied in mammals. However, little is known regarding the effects of viral infection on the skin immune response and microbial composition in teleost fish. In this study, we exposed rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus (IHNV) by immersion infection. Through pathogen load detection and pathological evaluation, we confirmed that IHNV successfully invaded the rainbow trout, causing severe damage to the epidermis of the skin. qPCR analyses revealed that IHNV invasion significantly upregulated antiviral genes and elicited strong innate immune responses. Transcriptome analyses indicated that IHNV challenge induced strong antiviral responses mediated by pattern recognition receptor (PRR) signaling pathways in the early stage of the infection (4 days post-infection (dpi)), and an extremely strong antibacterial immune response occurred at 14 dpi. Our 16S rRNA sequencing results indicated that the skin microbial community of IHNV-infected fish was significantly richer and more diverse. Particularly, the infected fish exhibited a decrease in Proteobacteria accompanied by an increase in Actinobacteria. Furthermore, IHNV invasion favored the colonization of opportunistic pathogens such as Rhodococcus and Vibrio on the skin, especially in the later stage of infection, leading to dysbiosis. Our findings suggest that IHNV invasion is associated with skin microbiota dysbiosis and could thus lead to secondary bacterial infection. Full article

Both infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are the causative agents of acute and highly contagious diseases of juvenile salmonids, resulting in severe economic losses to these cold-water fish globally. There is an urgent need to explore antiviral agents against IHNV and IPNV due to the lack of commercially available vaccines and antiviral drugs. More importantly, the co-infection of IHNV and IPNV is prevalent in nature, which not only aggravates extensive damage to the salmonids but also poses challenges to its prevention and control. The antiviral effects of a crude polysaccharide derived from seaweed (CSP) on IHNV and IPNV were evaluated in this study separately. Furthermore, the underlying antiviral mechanisms of CSP to IHNV and IPNV were analyzed, respectively. The results showed that CSP possessed excellent safety and good ability to inhibit IHNV, IPNV, and their co-infection. CSP preferred to act at the early stage of viral infection. The antiviral mechanism of CSP on IHNV is possibly involved in preventing viral attachment and release, while in IPNV, it is involved in suppressing viral attachment, entry, and release. Taken together, the results of this study shed new light on developing novel agents against viral infection in salmonid fish. Full article

Fish novirhabdoviruses, including viral hemorrhagic septicemia virus (VHSV), hirame rhabdovirus (HIRRV), and infectious hematopoietic necrosis virus (IHNV), harbor a unique non-virion (NV) gene that is crucial for efficient replication and pathogenicity. The effective levels and the function of the N-terminal region of the NV protein, however, remain poorly understood. In the present study, several recombinant VHSVs, which completely lack (rVHSV-ΔNV) or harbor an additional (rVHSV-dNV) NV gene, were generated using reverse genetics. To confirm the function of the N-terminal region of the NV protein, recombinant VHSVs with the NV gene that gradually mutated from the start codon (ATG) to the stop codon (TGA), expressed as N-terminally truncated NV proteins (rVHSV-NV1, -NV2, and -NV3), were generated. CPE progression and viral growth analyses showed that epithelioma papulosum cyprini (EPC) cells infected with rVHSV-ΔNV or rVHSV-NV3—which did not express NV protein—rarely showed CPE and viral replication as opposed to EPC cells infected with rVHSV-wild. Interestingly, regardless of the presence of two NV genes in the rVHSV-dNV genome, EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP (control) failed to induce CPE and viral replication. In EPC cells infected with rVHSV-dNV or rVHSV-A-EGFP, which harbored a longer VHSV genome than the wild-type, Mx gene expression levels, which were detected by luciferase activity assay, were particularly high; Mx gene expression levels were higher in EPC cells infected with rVHSV-ΔNV, -NV2, or -NV3 than in those infected with rVHSV-wild or rVHSV-NV1. The total amount of NV transcript produced in EPC cells infected with rVHSV-wild was much higher than that in EPC cells infected with rVHSV-dNV. However, the expression levels of the NV gene per viral particle were significantly higher in EPC cells infected with rVHSV-dNV than in cells infected with rVHSV-wild. These results suggest that the NV protein is an essential component in the inhibition of host type-I interferon (IFN) and the induction of viral replication. Most importantly, viral genome length might affect viral replication efficiency to a greater extent than does NV gene expression. In in vivo pathogenicity experiments, the cumulative mortality rates of olive flounder fingerlings infected with rVHSV-dNV or rVHSV-wild were similar (60–70%), while those of fingerlings infected with rVHSV-A-EGFP were lower. Moreover, the virulence of rVHSV-ΔNV and rVHSV, both harboring a truncated NV gene (rVHSV-NV1, -NV2, and -NV3), was completely attenuated in the olive flounder. These results suggest that viral pathogenicity is affected by the viral replication rate and NV gene expression. In conclusion, the genome length and NV gene (particularly the N-terminal region) expression of VHSVs are closely associated with viral replication in host type-I IFN response and the viral pathogenicity. Full article

The fish intestinal mucosa is among the main sites through which environmental microorganisms interact with the host. Therefore, this tissue not only constitutes the first line of defense against pathogenic microorganisms but also plays a crucial role in commensal colonization. The interaction between the mucosal immune system, commensal microbiota, and viral pathogens has been extensively described in the mammalian intestine. However, very few studies have characterized these interactions in early vertebrates such as teleosts. In this study, rainbow trout (Oncorhynchus mykiss) was infected with infectious hematopoietic necrosis virus (IHNV) via a recently developed immersion method to explore the effects of viral infection on gut immunity and microbial community structure. IHNV successfully invaded the gut mucosa of trout, resulting in severe tissue damage, inflammation, and an increase in gut mucus. Moreover, viral infection triggered a strong innate and adaptive immune response in the gut, and RNA−seq analysis indicated that both antiviral and antibacterial immune pathways were induced, suggesting that the viral infection was accompanied by secondary bacterial infection. Furthermore, 16S rRNA sequencing also revealed that IHNV infection induced severe dysbiosis, which was characterized by large increases in the abundance of Bacteroidetes and pathobiont proliferation. Moreover, the fish that survived viral infection exhibited a reversal of tissue damage and inflammation, and their microbiome was restored to its pre−infection state. Our findings thus demonstrated that the relationships between the microbiota and gut immune system are highly sensitive to the physiological changes triggered by viral infection. Therefore, opportunistic bacterial infection must also be considered when developing strategies to control viral infection. Full article

In the coastal region of Washington State, a major pathogen emergence event occurred between 2007 and 2011 in which steelhead trout (Oncorhynchus mykiss) experienced a high incidence of infection and disease outbreaks due to the rhabdovirus infectious hematopoietic necrosis virus (IHNV). Genetic typing showed that the introduced viruses were in the steelhead-specific MD subgroup of IHNV and indicated the most likely source was a virus from the nearby Columbia River Basin. In the current study, full-length viral glycoprotein (G) gene sequences were determined for 55 IHNV isolates from both coastal and Columbia fish populations to identify specific source populations and infer mechanisms of transmission to coastal steelhead. We identified three transmission links based on exact fullG genotype matches between Columbia and coastal fish. In all cases, the likely source population was infected juvenile fish, and sink populations were adult fish returning to coastal rivers to spawn. The time intervals between detection in source and sink populations varied from 6 months to nearly 4 years, suggesting different transmission pathways. Surprisingly, distances between source and sink populations varied between 140 and 1000 km. These results confirm repeated introductions of virus from Columbia River Basin fish as the cause of emergence of MD virus on the Washington coast from 2007 to 2011. Full article

Infectious hematopoietic necrosis (IHN) and infectious pancreatic necrosis (IPN) are the most common viral diseases of salmon in aquaculture worldwide. The co-infection of rainbow trout (Oncorhynchus mykiss) with IHN virus (IHNV) and IPN virus (IPNV) is known to occur. To determine the influence of IPNV on IHNV in co-infection, rainbow trout were intraperitoneally (i.p.) injected with IPNV at different time intervals prior to, simultaneously to, or after IHNV infection. The replication of IHNV in the brain, gill, heart, liver, spleen, and head kidney was detected by real-time quantitative polymerase chain reaction (qRT-PCR). The results showed that when rainbow trout were i.p. injected with IPNV prior to, simultaneously to, or after IHNV on 2 day (d), IHNV replication was inhibited (p < 0.05) in all collected tissues. Nevertheless, when rainbow trout were i.p. injected with IPNV after IHNV on 7 d (H7P), IHNV replication was only inhibited (p < 0.05) in the liver 14 d post-IHNV infection. Moreover, stronger antiviral responses occurred in all challenge groups. Our results suggest that IPNV can inhibit IHNV replication before or simultaneously with IHNV infection, and induce a stronger antiviral response, and that this inhibition is most sensitive in the liver. Early i.p. injection of IPNV can significantly reduce the mortality of rainbow trout, compared with the group only injected with IHNV. Full article