Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

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.

Self-assembling ferritin nanoplatform for the development of infectious hematopoietic necrosis virus vaccine.

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of infectious hematopoietic necrosis virus (IHNV).

Infectious hematopoietic necrosis virus (IHNV) is an economically important virus causing significant mortalities among wild and cultured salmonid fish worldwide. Rapid and sensitive diagnostic methods of IHNV are crucial for timely controlling infections. For better detection of IHNV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of IHNV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 41 °C and 35 min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15 min. The whole detection procedure including can be accomplished within one hour, with a detection sensitivity of about 9.5 copies/µL. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses HIRRV and VHSV, allowing naked-eye interpretation of the results through lateral flow or fluorescence under ultraviolet light. Overall, our results demonstrated that the developed RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for routine and on-site detection of IHNV, which shows a great application promise for the prevention of IHNV infections.

Traditional Chinese medicine bufalin inhibits infectious hematopoietic necrosis virus infection in vitro and in vivo.

Infectious hematopoietic necrosis virus (IHNV) causes infectious hematopoietic necrosis and severe economic losses to salmon and trout aquaculture worldwide. Currently, the only commercial vaccine against IHNV is a DNA vaccine with some biosafety concerns. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, 1,483 compounds were screened from a traditional Chinese medicine monomer library, and bufalin showed potential antiviral activity against IHNV. The 50% cytotoxic concentration of bufalin was >20 µM, and the 50% inhibitory concentration was 0.1223 µΜ against IHNV. Bufalin showed the inhibition of diverse IHNV strains in vitro, which confirmed that it had an inhibitory effect against all IHNV strains, rather than random activity against a single strain. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization. Bufalin also inhibited IHNV infection in vivo and significantly increased the survival of rainbow trout compared with the mock drug-treated group, and this was confirmed by in vivo viral load monitoring. Our data showed that the anti-IHNV activity of bufalin was proportional to extracellular Na+ concentration and inversely proportional to extracellular K+ concentration, and bufalin may inhibit IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout. IMPORTANCE: Infectious hematopoietic necrosis virus (IHNV) is the pathogen of infectious hematopoietic necrosis (IHN) which outbreak often causes huge economic losses and hampers the healthy development of salmon and trout farming. Currently, there is only one approved DNA vaccine for IHN worldwide, but it faces some biosafety problems. Hence, more effective vaccines and antiviral drugs are needed to prevent IHNV infection. In this study, we report that bufalin, a traditional Chinese medicine, shows potential antiviral activity against IHNV both in vitro and in vivo. The bufalin-mediated block of IHNV infection occurred at the viral attachment and RNA replication stages, but not internalization, and bufalin inhibited IHNV infection by targeting Na+/K+-ATPase. The in vitro and in vivo studies showed that bufalin significantly inhibited IHNV infection and may be a promising candidate drug against the disease in rainbow trout.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus.

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Differences between DNA vaccine and single-cycle viral vaccine in the ability of cross-protection against viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV).

Vaccination procedures can be stressful for fish and can bring severe side effects. Therefore, vaccines that can minimize the number of administrations and maximize cross-protection against multiple serotypes, genotypes, or even different species would be highly advantageous. In the present study, we investigated the cross-protective ability of two types of vaccines - viral hemorrhagic septicemia virus (VHSV) G protein-expressing DNA vaccine and G gene-deleted single-cycle VHSV genotype IVa (rVHSV-ΔG) vaccine - against both VHSV genotype Ia and infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). The results showed that rainbow trout immunized with VHSV genotype Ia G gene- or IVa G gene-expressing DNA vaccine were significantly protected against VHSV genotype Ia, but were not protected against IHNV. In contrast to the DNA vaccine, the single-cycle VHSV IVa vaccine induced significant protection against not only VHSV Ia but also IHNV. Considering no significant increase in ELISA titer and serum neutralization activity against IHNV in fish immunized with single-cycle VHSV IVa, the protection might be independent of humoral adaptive immunity. The scarcity of cytotoxic T cell epitopes between VHSV and IHNV suggested that the possibility of involvement of cytotoxic T cell-mediated cellular adaptive immunity would be low. The role of trained immunity (innate immune memory) in cross-protection should be further investigated.

In vitro propagation of infectious hypodermal hematopoietic necrosis virus (Penaeus stylirostris penstyldensovirus) in PmLyO-Sf9 cells.

Infectious hypodermal hematopoietic necrosis virus (IHHNV/PstDVI) was isolated and propagated in the hybrid shrimp-insect cell line PmLyO-Sf9. A few hours after inoculation with an infected tissue extract or virus suspension, cytopathic changes could be observed in the cell line, including clustering, enlargement, syncytium formation, granulation, vacuole formation, tapering, irregularities in the plasma membrane with extended tails, detachment, cell death, and accumulation of cellular debris. Expression of viral genes, the presence of virions, and cytological changes observed using transmission electron microscopy suggested replication of the virus in these cells. The virus was purified by ultracentrifugation, negatively stained, and examined using an electron microscope, and the purified virus was found to be infectious both in vitro and in vivo. This development opens avenues for the study of the basic molecular mechanism of IHHNV infection, pathogenesis, and replication, which is much needed for developing an antiviral strategy in aquaculture.

Pathogenicity of two lineages of infectious hematopoietic necrosis virus (IHNV) to farmed rainbow trout (Oncorhynchus mykiss) in South Korea.

In May 2015, a high mortality event in farmed rainbow trout occurred in Jeollabuk-do province in Korea. Histopathological analysis revealed necrosis in the kidney, liver, branchial arch, and gills of moribund fish, and infectious hematopoietic necrosis virus (IHNV) was detected in the lesions by immunohistochemistry. Cytopathic effects were observed in EPC, FHM, and RTG-2 cell lines after inoculation with kidney and spleen tissues and IHNV was detected by reverse transcription polymerase chain reaction (PCR). The amplified PCR product was sequenced, and phylogenetic analysis placed IHNV in the JRt Nagano group. Both in vivo and in vitro trials were performed to compare the virulence properties between RtWanju15 isolate, which causes 100% mortality in imported fry, and a previous isolate RtWanju09 of the JRt Shizuoka group isolated from eggs of healthy broodfish. In vivo challenge with high dose on specific pathogen free (SPF) rainbow trout fry performed in Denmark with isolates RtWanju09, RtWanju15 and DF04/99 isolates showed a survival rates of 60%, 37.5% and 52.5% (average), respectively without statistical difference. The replication efficiency of the two isolates in the in vitro challenge was similar.

Comprehensive analysis of immune parameters, mRNA and miRNA profiles, and immune genes expression in the gill of rainbow trout infected with infectious hematopoietic necrosis virus (IHNV).

Rainbow trout (Oncorhynchus mykiss) is a species of cold-water fish with important economic values, widely cultivated worldwide. However, the outbreak of infectious hematopoietic necrosis virus (IHNV) caused the large-scale death of rainbow trout and seriously restricted the development of the trout farming industry. In this study, the changes of immune parameters in different periods (6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144 h post-infection (hpi)), transcriptome profiles of 48 hpi (T48G) compared to control (C48G), and key immune-related genes expression patterns were measured in rainbow trout gill following IHNV challenge through biochemical methods, RNA sequencing (RNA-seq), and quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that alkaline phosphatase (AKP), acid phosphatase (ACP), total superoxide dismutase (T-SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities, as well as lysozyme (LZM) and malonaldehyde (MDA) content decreased and then increased during infection, and remained at a high level after 48 hpi (P < 0.05), whereas catalase (CAT) activity showed a significant peak at 48 hpi (P < 0.05). The mRNA and miRNA analysis identified 4343 differentially expressed genes (DEGs) and 11 differentially expressed miRNAs (DEMs), and numerous immune-related DEGs involved in the Toll-like receptor signaling pathway, apoptosis, DNA replication, p53 signaling, RIG-I-like receptor signaling pathway, and NOD-like receptor signaling pathway and expression were significantly up-regulated in T48Gm group, including tlr3, tlr7, tlr8, traf3, ifih1, trim25, dhx58, ddh58, hsp90a.1, nlrc3, nlrc5, socs3, irf3, irf7, casp7, mx1, and vig2. The integrated analysis identified several important miRNAs (ola-miR-27d-3p_R+5, gmo-miR-124-3-5p, ssa-miR-301a-5p_L+2, and ssa-miR-146d-3p) that targeted key immune-related DEGs. Expression analysis showed that tlr3, tlr7, traf3, ifih1, dhx58, hap90a.1, irf3, irf7, and mx1 genes increased and then decreased during infection, and peaked at 72 hpi (P < 0.05). However, trim25 expression peaked at 96 hpi (P < 0.05). This study contributes to understanding immune response of rainbow trout against IHNV infection, and provides new insights into the immune regulation mechanisms and disease resistance breeding studies.

Protection of rainbow trout (Oncorhynchus mykiss) against VHSV genotype Ia and IHNV by immunization with VHSV genotype IVa backbone-based single-cycle viruses.

To evaluate the protective effect of viral hemorrhagic septicemia virus genotype IVa (VHSV IVa) genome-based single-cycle viruses against VHSV genotype Ia (VHSV Ia) and infectious hematopoietic necrosis virus (IHNV) in rainbow trout, three kinds of single-cycle VHSVs were rescued using reverse genetic technology: i) rVHSV-IaGΔTM-IVaG containing the transmembrane and cytoplasmic region-deleted G protein (GΔTM) of VHSV Ia instead of VHSV IVa full G gene ORF and having VHSV IVa G proteins on the envelope; ii) rVHSV-IaGΔTM-IaG containing VHSV Ia GΔTM instead of VHSV IVa full G gene ORF and having VHSV Ia G proteins on the envelope; iii) rVHSV-IaGΔTM-ihnvGΔTM-IVaG containing not only VHSV Ia GΔTM instead of full G gene but also IHNV GΔTM instead of NV gene and having VHSV IVa G proteins on the envelope. Rainbow trout immunized with rVHSV-IaGΔTM-IaG and rVHSV-IaGΔTM-IVaG showed significantly higher serum antibody titers against both VHSV Ia and VHSV IVa, and showed no mortality against VHSV Ia infection, while fish in the control groups showed 100% mortalities. Fish immunized with rVHSV-IaGΔTM-ihnvGΔTM-IVaG showed significantly higher serum antibody titers against VHSV IVa, VHSV Ia, and IHNV compared to fish in the control group. Immunization with rVHSV-IaGΔTM-ihnvGΔTM-IVaG induced significantly higher protection against not only VHSV Ia but also IHNV. These results suggest that the present single-cycle rVHSV-based system can be used as a platform to produce combined vaccines that can protect fish from multiple pathogenic species. However, the mechanism of the high protection against IHNV despite comparatively low antibody titer remains to be investigated.

Comparative Study on Immune Function of the Head and Trunk Kidney in Rainbow Trout Responding to IHNV Infection.

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.

Specificity of DNA Vaccines against the Genogroup J and U Infectious Hematopoietic Necrosis Virus Strains Prevalent in China.

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.

Phylogenetic analysis and genotyping of Iranian infectious haematopoietic necrosis virus (IHNV) of rainbow trout (Oncorhynchus mykiss) based on the glycoprotein gene.

BACKGROUND: Infectious haematopoietic necrosis (IHN) is known as one of the most contagious systemic viral diseases in salmonids which can lead to significant mortality rates and negative impacts on the salmonid farming industry. Infectious haematopoietic necrosis virus (IHNV) was first detected in rainbow trout (Oncorhynchus mykiss) farms in Iran in 2003. OBJECTIVES: We conducted the present study to determine the detection of IHN genotypes in rainbow trout (O. mykiss) in farms in the central parts of Iran, using molecular and phylogenetic techniques. METHODS: Samples were collected from fries exhibiting clinical signs such as darkening of the skin, abdominal swelling, and loss of appetite. Phylogenetic analysis was performed by the neighbour-joining method, using MEGA 5.1 software. For phylogenetic analysis and genotyping of IHNV from central parts of Iran, the sequences of the glycoprotein gene were determined for two Iranian isolates (Jahad-UT1 and Jahad-UT2). RESULTS: Phylogenetic analysis revealed that the detected strains (Jahad-UT1 and Jahad-UT2 isolates) are closely related (97.23%-100%) to European isolates within genogroup 'E'. CONCLUSIONS: This finding indicates that Jahad-UT1 and Jahad-UT2 isolates have been widely transferred to Iran from European countries. Moreover, the nucleotide diversity of these Iranian isolates showed a close relationship with the North American and Asian isolates, although the Iranian isolates were collected from a smaller geographical area and within a shorter time period between 2014 and 2015.

Integrative analysis of miRNA and mRNA expression associated with the immune response in the intestine of rainbow trout (Oncorhynchus mykiss) infected with infectious hematopoietic necrosis virus.

Rainbow trout (Oncorhynchus mykiss), an economically important cold-water fish cultured worldwide, suffers from infectious hematopoietic necrosis virus (IHNV) infection, resulting in huge financial losses. In order to understand the immune response of rainbow trout during virus infection, we explored trout intestine transcriptome profiles following IHNV challenge, and identified 3355 differentially expressed genes (DEGs) and 80 differentially expressed miRNAs (DEMs). Transcriptome analysis revealed numerous DEGs involved in immune responses, such as toll-like receptor 3 (TLR3), toll-like receptor 7/8 (TLR7/8), tripartite motif-containing 25 (TRIM25), DExH-Box helicase 58 (DHX58), interferon-induced with helicase C domain 1 (IFIH1), interferon regulatory factor 3 (IRF3/7), signal transducer and activator of transcription 1 (STAT1) and heat shock protein 90-alpha 1 (HSP90A1). Integrated analysis identified five key miRNAs (miR-19-y, miR-181-z, miR-203-y, miR-143-z and miR-206-y) targeting at least two important immune genes (TRIM25, DHX58, STAT1, TLR7/8 and HSP90A1). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs and target genes were significantly enriched in various immune-related terms including immune system process, binding, cell part and pathways of Toll-like receptor signalling, RIG-I-like receptor signalling, NOD-like receptor signalling, JAK-STAT signalling, PI3K-Akt signalling, NF-kappa B signalling, IL-17 signalling and AGE-RAGE signalling. In addition, protein-protein interaction networks (PPI) was used to display highly interactive DEG networks involving eight immune-related pathways. The expression trends of 12 DEGs and 10 DEMs were further verified by quantitative real-time PCR, which confirmed the reliability of the transcriptome sequencing results. This study expands our understanding of the immune response of rainbow trout infected with IHNV, and provides valuable resources for future studies on the immune molecular mechanism and disease resistance breeding.

Infectious hematopoietic necrosis virus (IHNV) nucleoprotein amino acid residues affect viral virulence and immunogenicity in rainbow trout (Oncorhynchus mykiss).

This study compared the N protein sequences of genotype J with other genotypes of IHNV to select amino acid residues that may be related to the change in viral virulence. The recombinant viruses containing different mutation sites were rescued by alanine scanning mutagenesis and the reverse genetic system. The nine recombinant virus strains obtained in this work were named rIHNV-N85, rIHNV-N102, rIHNV-N146, rIHNV-N380, rIHNV-N85-102-146, rIHNV-N85-102-380, rIHNV-N85-146-380, rIHNV-N102-146-380, and rIHNV-N85-102-146-380. Pathogenicity and immunity assays were performed to determine the role of virulence sites. The result of the pathogenicity test showed that the survival rates of rIHNV-N85, rIHNV-N102, rIHNV-N85-102-146, and rIHNV-N85-102-380 groups were 52.5%, 55%, 67.5%, and 57.5%, while the survival rate of wild-type (wt) IHNV HLJ-09 group was only 10%. The replication ability of recombinant viruses with substitutions at positions 85 and 102 was significantly inhibited in vivo and in vitro. The qRT-PCR result indicated that the cytokines of IFN1, IL-8, and IL-1ß expression levels were increased in rIHNV-N85, rIHNV-N102, rIHNV-N85-102-146, and rIHNV-N85-102-380 groups. In addition, these four recombinant viruses could cause the rainbow trout to produce anti-IHNV-specific antibodies immunoglobulin M (IgM) earlier, confirming that 85 and 102 amino acid residues of N protein affected the virulence and immunogenicity of IHNV. All these results suggest that mutations of the N protein virulence sites reduce virulence while retaining immunogenicity. This also provides a new idea for studying the virulence mechanism of rhabdoviruses and preparing attenuated vaccines.

Pathogenic Mechanism of a Highly Virulent Infectious Hematopoietic Necrosis Virus in Head Kidney of Rainbow Trout (Oncorhynchusmykiss) Analyzed by RNA-Seq Transcriptome Profiling.

Infectious hematopoietic necrosis virus (IHNV) is a pathogen that causes high rates of mortality in salmonid fishes. Therefore, an RNA-seq-based transcriptome analysis was performed in the head kidney of rainbow trout infected with a highly virulent IHNV strain to understand the pathogenesis of and defense strategies for IHNV infection in rainbow trout. The results showed that the numbers of DEGs were 618, 2626, and 774 (control vs. IHNV) on days 1, 3, and 5, respectively. Furthermore, the enrichment analysis of gene ontology (GO) annotations to classify DEGs showed that GO terms considerably associated with DEGs were gluconeogenesis, inflammatory response, and cell adhesion in the Biological Process (BP) category, apical plasma membrane, extracellular matrix (ECM) in the Cellular Component category, and transporter activity, integrin binding, and protein homodimerization activity in the Molecular Function category, on days 1, 3, and 5, respectively. Notably, GO terms in the BP category, including the negative regulation of type I interferon production and positive regulation of interleukin-1ß secretion, were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, complement and coagulation cascades were commonly identified at all time points. Importantly, the widely recognized GO terms and KEGG pathways extensively linked to DEGs were related to energy metabolism on day 1, the immune response on day 3, and cell proliferation on day 5. Furthermore, protein-protein interaction networks and centrality analysis showed that the metabolism and signaling transduction pathways were majorly upregulated. Conclusively, the virulent IHNV infection drives pathogenesis by activating the metabolic energy pathway for energy use for viral replication, facilitating necrosis through autophagy, and causing a shutoff response of the host immune system through the downregulation of type I IFN at the initial stage of infection.

First isolation and pathogenicity analysis of a genogroup U strain of infectious hematopoietic necrosis virus from rainbow trout in China.

Infectious hematopoietic necrosis virus (IHNV) is a major fish viral pathogen causing acute clinical disease and death in a variety of salmonids. IHNV isolates have been classified into five major genogroups according to the phylogenetic analysis of partial G gene fragments or the complete G gene sequence: U, M, E, L and J. Genogroup U strains have been reported in North America and Japan prior to 1982, and genogroup J is the only genogroup that has been reported in China. Here, one of IHNV strain (BjLL) was isolated from a local farm in China and were characterized in this study. The homogenate tissues of infected fry induced IHNV-positive cytopathic effects in epithelioma papulosum cyprinid (EPC) cells that were confirmed by RT-PCR and sequencing. The complete genome sequence of BjLL comprised 11,129 nucleotides, which had been submitted to GenBank (accession no. MF509592). By the sequence comparison and phylogenetic analysis for the G gene sequence of BjLL with 51 reference sequences in GenBank, we confirmed that this Chinese isolate belonged to genogroup U. Furthermore, virus exposure experiments with juvenile rainbow trout were conducted to assess the virulence and pathogenicity of BjLL. Compared with GS-2014 of genogroup J, BjLL was an obviously less virulent strain that could result in lower mortality. Besides, typical clinical symptoms and pathological damages could be seen in fish following infection of BjLL. The present study is the first report of genogroup U IHNV infection in China and will provide essential information for future studies on pathogenesis of IHNV BjLL and development of efficient control strategies.

Virulence and Infectivity of UC, MD, and L Strains of Infectious Hematopoietic Necrosis Virus (IHNV) in Four Populations of Columbia River Basin Chinook Salmon.

Infectious Hematopoietic Necrosis Virus (IHNV) infects juvenile salmonid fish in conservation hatcheries and aquaculture facilities, and in some cases, causes lethal disease. This study assesses intra-specific variation in the IHNV susceptibility of Chinook salmon (Oncorhynchus tshawytscha) in the Columbia River Basin (CRB), in the northwestern United States. The virulence and infectivity of IHNV strains from three divergent virus genogroups are measured in four Chinook salmon populations, including spring-run and fall-run fish from the lower or upper regions of the CRB. Following controlled laboratory exposures, our results show that the positive control L strain had significantly higher virulence, and the UC and MD strains that predominate in the CRB had equivalently low virulence, consistent with field observations. By several experimental measures, there was little variation in host susceptibility to infection or disease. However, a small number of exceptions suggested that the lower CRB spring-run Chinook salmon population may be less susceptible than other populations tested. The UC and MD viruses did not differ in infectivity, indicating that the observed asymmetric field prevalence in which IHNV detected in CRB Chinook salmon is 83% UC and 17% MD is not due to the UC virus being more infectious. Overall, we report little intra-species variation in CRB Chinook salmon susceptibility to UC or MD IHNV infection or disease, and suggest that other factors may instead influence the ecology of IHNV in the CRB.

Molecular Epidemiology of Novirhabdoviruses Emerging in Iranian Trout Farms.

Novirhabdoviruses cause large epizootics and economic losses of farmed trout. In this study, we surveyed Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) through both monitoring and investigation of clinical outbreaks reported by farmers in the regions with major rainbow trout production in Iran from 2015 to 2019. RT-PCR assays of the kidney samples and cell culture (EPC/FHM cells) samples confirmed the presence of the viruses, with 9 VHSV and 4 IHNV isolates, in both endemic and new areas of Iran. Sequence analysis of the G gene revealed that VHSV isolates belonged to genogroup Ia, and IHNV isolates were clustered into genogroup E, both typical for isolates from European countries. A haplotype analysis based on non-homologous amino acids of the G gene supports the emergence of two lineages of IHNV from clade 1 (E-1), as well as VHSV clade 2 (Ia-2) of the European genogroups, confirming that VHSV and IHNV isolates in Iran, have originated from Europe possibly via imported eggs.

Transient expression of the full-length glycoprotein from infectious hematopoietic necrosis virus in bean (Phaseolus vulgaris) leaves via agroinfiltration.

The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO4  + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 µg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.