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.

Systemic immune response of rainbow trout exposed to Flavobacterium psychrophilum infection.

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum is one of the most serious bacterial diseases leading to significant economic loss for rainbow trout (Oncorhynchus mykiss) aquaculture. However, little is known about the systemic immune response of rainbow trout against F. psychrophilum infection. This study investigated the immune response of rainbow trout to F. psychrophilum infection using multiple experiments, including bacterial load detection, phagocyte activity assessment, enzyme activity evaluation, and gene expression profiling. Results showed that the spleen index and intestinal pathogen load reached a peak at 3 days post-infection, with strong pro-inflammatory gene expression observed in rainbow trout. Leukocytes RBA and PKA were significantly elevated in the spleen, blood and intestine at 7 days post-infection. Heat map analysis demonstrated that the spleen had a more substantial pro-inflammatory response compared to the intestine post-infection and exhibited higher expression levels of immune-related genes, including IgM, il1ß, il6, cd4, cd8a, cd8b, c1q, chathelicidin, inos, and lysozyme. Both Th1 and Th2 polarized responses in the spleen were activated, with Th2 (il4/13a, gata3) (FC > 4) being more intense than Th1 (tnfα, t-bet) (FC > 2). Tight junction proteins exhibited down-regulation followed by up-regulation post-infection. Collectively, the results of this study expand our current understanding of the immune response of rainbow trout post F. psychrophilum infection but also provide new avenues for investigation in salmonid aquaculture.

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.

Identification of a novel type II-C Cas9 from the fish pathogen Flavobacterium psychrophilum.

Flavobacterium psychrophilum is the causative agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish worldwide. As an important fish pathogen, F. psychrophilum is frequently exposed to multiple invading genetic elements in natural environments. Endonuclease Cas9 provides bacteria with adaptive interference against invading genetic elements. Previous studies revealed that several F. psychrophilum strains harbored a type II-C Cas9 called Fp1Cas9, but little is known about the potential role of this endonuclease against invading genetic elements. In this work, we identified a gene encoding a novel type II-C Cas9 called Fp2Cas9 from F. psychrophilum strain CN46. Through bacterial RNA sequencing, we demonstrated active transcription of both Fp2Cas9 and pre-crRNAs in strain CN46. Bioinformatics analysis further revealed that the transcription of Fp2Cas9 and pre-crRNAs was driven by a newly integrated promoter sequence and a promoter element embedded within each CRISPR repeat, respectively. To formally demonstrate that Fp2Cas9 and associated crRNAs yielded functional interference in strain CN46, a plasmid interference assay was performed, resulting in adaptive immunity to target DNA sequences in Flavobacterium bacteriophages. Phylogenetic analysis demonstrated that Fp2Cas9 was present only in several F. psychrophilum isolates. Phylogenetic analysis revealed that this novel endonuclease was probably acquired through horizontal gene transfer from the CRISPR-Cas9 system in an unidentified Flavobacterium species. Comparative genomics analysis further showed that the Fp2Cas9 was integrated into the type II-C CRISPR-Cas locus in strain CN38 instead of the original Fp1Cas9. Taken together, our results shed light on the origin and evolution of Fp2Cas9 gene and demonstrated that this novel endonuclease provided adaptive interference against bacteriophage infections.

Supression Thioredoxin reductase 3 exacerbates the progression of liver cirrhosis via activation of ferroptosis pathway.

AIMS: In the past decades, Txnrd3 as selenoprotein is considered to be highly expressed in testis and participate in sperm mature; however its role in liver diseases needs further study. Iron is essential for humans and animals, while its overload could damage to multiple organs. However, role of Txnrd3 and iron in cirrhosis is still unclear. MATERIALS AND METHODS: Forty 8-week-old wild-type and forty Txnrd3-/- mice were selected to build liver cirrhosis model using Thiacetamide solution, deposition of iron in liver was observed via Prussian blue staining. Txnrd3 overexpression/knockdown model in vitro was constructed based on cell transfection techniques in AML12 cells, expression abundance of ferroptosis pathway genes within cells and tissues were determined by qRT-PCR and Western Blot. KEY FINDINGS: Results showed that Txnrd3-/- mice developed more pronounced liver damage, accompanied by reduced GPX4 expression and iron deposition. A significant decrease in the expression abundance of GPX4 was also detected in Txnrd3 knock-down AML12 cells. In summary, Txnrd3 knockdown could result in iron overload and ferroptosis pathway activation within liver tissues and hepatocytes, ultimately lead to the occurrence of liver injury and cirrhosis. SIGNIFICANCE: These results will provide biological markers for early diagnosis during cirrhosis and lay a theoretical basis for clinical therapy.

Andrographolide Alleviates Oxidative Damage and Inhibits Apoptosis Induced by IHNV Infection via CTSK/BCL2/Cytc Axis.

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.

Dynamic Distribution of Infectious Pancreatic Necrosis Virus (IPNV) Strains of Genogroups 1, 5, and 7 after Intraperitoneal Administration in Rainbow Trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV) is the causative agent of rainbow trout (Oncorhynchus mykiss) IPN and causes significant loss of fingerlings. The currently prevalent IPNV genogroups in China are genogroups 1 and 5. However, in this study, we isolated and identified a novel IPNV, IPNV-P202019, which belonged to genogroup 7. Here, a total of 200 specific-pathogen-free rainbow trout (10 g average weight) were divided randomly into four groups to investigate the distribution of different IPNV strains (genogroups 1, 5, and 7) in 9 tissues of rainbow trout by means of intraperitoneal (ip) injection. Fish in each group were monitored after 3-, 7-, 14-, 21- and 28- days post-infection (dpi). The study showed no mortality in all groups. The distribution of IPNV genogroups 1 and 5 was similar in different tissues and had a higher number of viral loads after 3, 7, or 14 dpi. However, the distribution of IPNV genogroup 7 was detected particularly in the spleen, head kidney, and feces and had a lower number of viral loads. The results of this study provide valid data for the distribution of IPNV in rainbow trout tissues and showed that IPNV genogroups 1 and 5 were still the prevalent genogroups of IPNV in China. Although rainbow trout carried IPNV genogroup 7, the viral load was too low to be pathogenic.

Long-Term Protection Elicited by an Inactivated Vaccine Supplemented with a Water-Based Adjuvant against Infectious Hematopoietic Necrosis Virus in Rainbow Trout (Oncorhynchus mykiss).

Previous inactivated vaccines against infectious hematopoietic necrosis (IHN) usually had a strong early immune protective effect but failed to provide long-term protection in rainbow trout (Oncorhynchus mykiss). To find a method for stabilizing the desired protective effect of IHN vaccines, we assessed the immune enhancement effect of four adjuvants on formaldehyde inactivated vaccine for IHN at 60 days postvaccination (dpv). The efficacy of a two-dose vaccination with the candidate adjuvant-formaldehyde inactivated vaccine for IHN was evaluated in terms of early protection and long-term protection (30 to 285 dpv). Neutralizing antibody titers were also measured at each time point. The Montanide GEL 02 PR (Gel 02) adjuvant significantly enhanced the immune protection provided by the IHN inactivated vaccine, whereas the immune-boosting effect of the other tested adjuvants lacked statistical significance. Both tested Gel 02-adjuvanted IHN inactivated vaccine dosages had a strong immune protection effect within 2 months postvaccination, with a relative percent of survival (RPS) of 89.01% to 100%, and the higher dosage provided complete protection at 204 dpv and a RPS of 60.79% on 285 dpv by reducing viral titers in rainbow trout. The neutralizing antibodies were observed only in vaccinated fish on 30 and 60 dpv. Through compatibility with an appropriate adjuvant, the highly immune protective effect of an IHN inactivated vaccine was prolonged from 60 dpv to at least 284 dpv; this novel adjuvant-IHN inactivated vaccine has promise as a commercial vaccine that provides the best available and longest duration of protection against IHN to rainbow trout. IMPORTANCE Infectious hematopoietic necrosis virus (IHNV) is one of the most serious pathogens threatening the global salmon and trout industry. However, there is currently only one commercialized infectious hematopoietic necrosis (IHN) vaccine, and it is inadequate for solving the global IHN problem. In this study, a promising adjuvanted inactivated vaccine with long-term protection was developed and comprehensively studied. We confirmed the presence of a late antiviral response stage in vaccinated rainbow trout that lacked detectable neutralizing antibodies, which are commonly recognized to be responsible for long-term specific protection in mammals. These findings further our understanding of unique features of fish immune systems and could lead to improved prevention and control of fish diseases.

Antiviral Activity of Crude Polysaccharide Derived from Seaweed against IHNV and IPNV In Vitro.

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.

Molecular characterization and expression analysis of claudin-4-like in rainbow trout involved in Flavobacterium psychrophilum infection.

The claudin family of proteins are pivotal components of tight junction (TJ) participating in the epithelial barrier function in fish. Our previous studies indicated that one of the claudins, claudin-4-like (OmCLDN4L) was differentially expressed in rainbow trout (Oncorhynchus mykiss) spleen post infection of Flavobacterium psychrophilum, which is the causative pathogen of bacterial coldwater disease (BCWD). However, little is known about the function of OmCLDN4L in rainbow trout against bacterial infection. In the present study, the OmCLDN4L was identified and functionally characterized from rainbow trout. The OmCLDN4L has an open reading frame (ORF) of 668 bp, encoding a 22.86 kDa four-transmembrane protein with function of bicellular tight junction and apical tight junction. OmCLDN4L has the highest similarity with CLDN28a, CLDN28b and CLDN30 in amino acid sequence. Phylogenetic analysis showed that all of CLDN4 and CLDN4-like from fish clustered together but diverged from their counterparts in mammals, with main differences lying in their N-terminus. RT-qPCR results indicated that OmCLDN4L was constitutively expressed in all tissues investigated under healthy conditions, primarily in mucus, liver, skin and intestine. The expression of OmCLDN4L in rainbow trout intestine was slightly down-regulated at day 1 while up-regulated at day 3 and day 7 post F. psychrophilum infection, with the similar profiling of CLDN30 and CLDN10e. The expression level of inflammatory cytokines TNF-α, IL4/13A, IL-6 and pattern recognition receptor TLR-2 showed the same trend with OmCLDN4L in the intestine at day 3 and day 7 post F. psychrophilum infection. Collectively, these findings demonstrate that OmCLDN4L participates in the immune response to bacterial infection, offering new insights into the molecular mechanism of intestinal barrier in rainbow trout against F. psychrophilum infection.

Early or Simultaneous Infection with Infectious Pancreatic Necrosis Virus Inhibits Infectious Hematopoietic Necrosis Virus Replication and Induces a Stronger Antiviral Response during Co-infection in Rainbow Trout (Oncorhynchus mykiss).

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.

Time-course transcriptome analyses of spleen in rainbow trout (Oncorhynchus mykiss) post-Flavobacterium psychrophilum infection.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease and rainbow trout fry syndrome, causes considerable losses in salmonid aquaculture globally. Systemic F. psychrophilum infections in rainbow trout (Oncorhynchus mykiss) lead to a range of clinical signs, including ulcerative lesions in the skin and muscle and splenitis. Previous studies offered an integrative analysis of the skeletal muscle response to F. psychrophilum infection in rainbow trout. However, little is known about the molecular mechanism of immune response in the spleen, which is an important immune organ of rainbow trout. Here, we investigated the time-course splenic transcriptome profiles in uninfected rainbow trout (CK) and F. psychrophilum-infected rainbow trout at day 3 and day 7 (D3, D7) by RNA-seq analyses. Among the 7,170 differentially expressed genes (DEGs) in the three comparisons (D3 vs. CK, D7 vs. CK, D3 vs. D7), 1,286 DEGs showed consistent upregulation or downregulation at D3 and D7 and were associated with pattern recognition, acute-phase response, complement cascade, chemokine and cytokine signaling, and apoptosis. The Real time quantitative PCR (RT-qPCR) analysis of eight DEGs confirmed the accuracy of the RNA-Sequencing (RNA-seq) data. Our results reflected a general process from pathogen recognition to inflammatory cytokine generation and delineated a putative Toll-like receptor signaling pathway in rainbow trout spleen, following F. psychrophilum infection. Taken together, these results provide new insights into the molecular mechanism of the immune response to F. psychrophilum infection and are a valuable resource for future research on the prevention and control of bacterial coldwater disease during salmon culture.

Supplementation of Dietary Crude Lentinan Improves the Intestinal Microbiota and Immune Barrier in Rainbow Trout (Oncorhynchus mykiss) Infected by Infectious Hematopoietic Necrosis Virus.

The effects of crude lentinan (CLNT) on the intestinal microbiota and the immune barrier were evaluated in rainbow trout (Oncorhynchus mykiss) infected by infectious hematopoietic necrosis virus (IHNV). The results showed that supplementary CLNT declined the rainbow trout mortality caused by IHNV, which suggested that CLNT has preventive effects on IHNV infection. IHNV destroyed intestinal integrity, as well as caused the intestinal oxidative and damage in rainbow trout. Supplementary CLNT significantly strengthened the intestinal immune barrier by declining intestinal permeability, as well as enhancing intestinal antioxidant and anti-inflammatory abilities in IHNV-infected rainbow trout (P<0.05). In addition, CLNT modified the aberrant changes of intestinal microbiota induced by IHNV, mainly represented by promoting the growths of Carnobacterium and Deefgea and inhibiting Mycobacterium and Nannocystis. Especially, supplementing with CLNT significantly promoted the growth of short-chain fatty acid-producing bacteria (P<0.05) and consequently increased the production of acetic acid, butanoic acid, and hexanoic acid in the intestine of IHNV-infected rainbow trout. Furthermore, it was speculated that CLNT could regulate the self-serving metabolic pathways of intestinal microbiota induced by IHNV, such as fatty acid metabolism and amino acid metabolism. Together, CLNT played the antiviral effects on IHNV infection through strengthening the intestinal immune barrier, as well as regulating intestinal microbiota and SCFA metabolism in rainbow trout. The present data revealed that CLNT exerted a promising prebiotic role in preventing the rainbow trout from IHNV infection.

Identification and characterization of DEAD-box RNA helicase DDX3 in rainbow trout (Oncorhynchus mykiss) and its relationship with infectious hematopoietic necrosis virus infection.

DDX3, a member of the DEAD-box RNA helicase family and has highly conserved ATP-dependent RNA helicase activity, has important roles in RNA metabolism and innate anti-viral immune responses. In this study, five transcript variants of the DDX3 gene were cloned and characterized from rainbow trout (Oncorhynchus mykiss). These five transcript variants of DDX3 encoded proteins were 74.2 kDa (686 aa), 76.4 kDa (709 aa), 77.8 kDa (711 aa), 78.0 kDa (718 aa), and 78.8 kDa (729 aa) and the predicted isoelectric points were 6.91, 7.63, 7.63, 7.18, and 7.23, respectively. All rainbow trout DDX3 proteins contained two conserved RecA-like domains that were similar to the DDX3 protein reported in mammals. Phylogenetic analysis showed that the five cloned rainbow trout DDX3 were separate from mammals but clustered with fish, especially Northern pike (Esox lucius) and Nile tilapia (Oreochromis niloticus). RT-qPCR analysis showed that the DDX3 gene was broadly expressed in all tissues studied. The expression of DDX3 after infectious hematopoietic necrosis virus (IHNV) infection increased gradually after the early stage of IHNV infection, decreased gradually with the proliferation of IHNV in vivo (liver, spleen, and kidney), and was significantly decreased after the in vitro infection of epithelioma papulosum cyprini (EPC) and rainbow trout gonad cell line-2 (RTG-2) cell lines. We also found that rainbow trout DDX3 was significantly increased by a time-dependent mechanism after the poly I:C treatment of EPC and RTG cells; however no significant changes were observed with lipopolysaccharide (LPS) treatment. Knockdown of DDX3 by siRNA showed significantly increased IHNV replication in infected RTG cells. This study suggests that DDX3 has an important role in host defense against IHNV infection and these results may provide new insights into IHNV pathogenesis and antiviral drug research.

An inactivated vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss).

Infectious pancreatic necrosis virus (IPNV), belonging to the genus Aquabirnavirus within the family Birnaviridae, causes huge economic loss to the global salmonid industry every year. Recently, outbreaks of disease caused by genogroup I IPNV were found in many rainbow trout (Oncorhynchus mykiss) farms worldwide. An inactivated vaccine was prepared using a genogroup I IPNV isolate with an optimized procedure as incubation with ß-propanolactone (BPL) at the final concentration of 0.5% at room temperature for 48 h. The inactivated vaccine was used to immunize rainbow trout, and the protection efficiency was evaluated by viral loads determination, immune-related genes quantification, and neutralizing antibody tests. The viral loads in immunized rainbow trout were significantly decreased and the strongest antiviral effect was observed on 30 days post-immunization (d.p.i). The expression of innate immune-related genes IFN-1, and Mx-1 genes were significantly up-regulated on 3, 7, and 15 d.p.i (p < 0.05), and adaptive immune-related genes CD4, CD8, and IgM genes were significantly up-regulated on 15 and 30 d.p.i (p < 0.05). Neutralizing antibodies were firstly detected on 30 d.p.i and the highest titer was observed on 45 d.p.i, which began to decrease on 60 d.p.i, but was still significantly higher than that in negative control fish. The results indicated that the vaccine prepared in this study could stimulate the non-specific and specific immune response and provide significant immune protection to the vaccinated rainbow trout.

Comparative transcriptome analysis of long non coding RNA (lncRNA) in RTG-2 cells infected by infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is the vital pathogen that has caused the great economic loss in salmonid fisheries. To date, there is limited information concerning the changes of lncRNAs in RTG-2 cells infected by IHNV. In this study, a comparative transcriptome analysis of lncRNAs was performed in RTG-2 cells with and without IHNV infection to determine their changes and the effects on IHNV infection. The results showed that IHNV infection significantly changed the expression levels of lncRNAs and mRNAs, including 3693 differentially expressed lncRNAs (DE-lncRNAs) and 3503 differentially expressed mRNAs (DE-mRNAs) respectively. These DE-lncRNAs and DE-mRNAs induced by IHNV were mostly associated with immune response, RNA processing, and viral diseases related pathways. Further analysis found that some DE-lncRNAs might participate in the regulation of extracellular matrix metabolism, apoptosis, lipid synthesis, autophagy, and immune responses referring to the functions of their target genes. Afterwards, 349 co-expression relationships were constructed by 223 DE-lncRNAs and 271 DE-mRNAs, of which LTCONS_00146935 was the pivotal node in the interaction networks, and was together with its target genes modulated the immune responses under the IHNV infection. RT-qPCR results showed that the changes of the selected immune-related DEGs were in consistent with the RNA-seq data, suggesting that the sequencing data was relatively reliable. In summary, this is the first study to determine the changes and interactions of lncRNA-mRNA in RTG-2 cells under the IHNV infection. The results provided the valuable information concerning the lncRNAs in salmonid fish, which will benefit for future study on uncovering the roles of lncRNAs-mRNAs during the viral infection.

Comparative transcriptome analysis of rainbow trout gonadal cells (RTG-2) infected with U and J genogroup infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is the causative pathogen of infectious hematopoietic necrosis, outbreaks of which are responsible for significant losses in rainbow trout aquaculture. Strains of IHNV isolated worldwide have been classified into five major genogroups, J, E, L, M, and U. To date, comparative transcriptomic analysis has only been conducted individually for the J and M genogroups. In this study, we compared the transcriptome profiles in U genogroup and J genogroup IHNV-infected RTG-2 cells with mock-infected RTG-2 cells. The RNA-seq results revealed 17,064 new genes, of which 7,390 genes were functionally annotated. Differentially expressed gene (DEG) analysis between U and J IHNV-infected cells revealed 2,238 DEGs, including 1,011 downregulated genes and 1,227 upregulated genes. Among the 2,238 DEGs, 345 new genes were discovered. The DEGs related to immune responses, cellular signal transduction, and viral diseases were further analyzed. RT-qPCR validation confirmed that the changes in expression of the immune response-related genes trpm2, sting, itgb7, ripk2, and irf1, cellular signal transduction-related genes irl, cacnb2, bmp2l, gadd45α, and plk2, and viral disease-related genes mlf1, mtor, armc5, pik3r1, and c-myc were consistent with the results of transcriptome analysis. Taken together, our findings provide a comprehensive transcriptional analysis of the differential virulence of the U and J genogroups of IHNV, and shed new light on the pathogenic mechanisms of IHNV strains.

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.

Molecular Evolution of Infectious Pancreatic Necrosis Virus in China.

Passive virus surveillance was performed in twenty-nine salmon and trout farms from seven provinces and districts in China during the period 2017-2020. A total of 25 infectious pancreatic necrosis virus (IPNV) isolates were obtained, mainly from rainbow trout (Oncorhynchus mykiss). The molecular evolution of these Chinese IPNV isolates and the previously reported Chinese IPNV strains ChRtm213 and WZ2016 was analyzed, based on their VP2 gene coding region sequences (CDS). All 27 Chinese IPNV isolates clustered within genogroups I and V, with 24 of the IPNV isolates belonging to genogroup I (including ChRtm213 and WZ2016), and only three isolates clustering in genogroup V. The Chinese genogroup I IPNV isolates lacked diversity, composing six haplotypes with 41 polymorphic sites, and the identity of nucleotide and amino acid sequences among the entire VP2 gene CDS from these isolates was 97.44%-100% and 98.19%-100%, respectively. Divergence time analyses revealed that the Chinese genogroup I IPNV isolates likely diverged from Japanese IPNV isolates in 1985 (95% highest posterior density (HPD), 1965-1997), and diverged again in 2006 (95% HPD, 1996-2013) in China. Each of the three Chinese genogroup V IPNV isolates has a unique VP2 gene CDS, with a total of 21 polymorphic sites; the identity of nucleotide and amino acid sequences among all VP2 gene CDS from these isolates was 98.5%-99.5% and 98.6%-99.0%, respectively. The data demonstrate that genogroups I and V are more likely the currently prevalent Chinese IPNV genotypes.

Transcriptome profiling of immune response to Yersinia ruckeri in spleen of rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Yersinia ruckeri is a pathogen that can cause enteric redmouth disease in salmonid species, damaging global production of economically important fish including rainbow trout (Oncorhynchus mykiss). Herein, we conducted the transcriptomic profiling of spleen samples from rainbow trout at 24 h post-Y. ruckeri infection via RNA-seq in an effort to more fully understand their immunological responses. RESULTS: We identified 2498 differentially expressed genes (DEGs), of which 2083 and 415 were up- and down-regulated, respectively. We then conducted a more in-depth assessment of 78 DEGs associated with the immune system including CCR9, CXCL11, IL-1ß, CARD9, IFN, TNF, CASP8, NF-κB, NOD1, TLR8α2, HSP90, and MAPK11, revealing these genes to be associated with 20 different immunological KEGG pathways including the Cytokine-cytokine receptor interaction, Toll-like receptor signaling, RIG-I-like receptor signaling, NOD-like receptor signaling, and MAPK signaling pathways. Additionally, the differential expression of 8 of these DEGs was validated by a qRT-PCR approach and their immunological importance was then discussed. CONCLUSIONS: Our findings provide preliminary insight on molecular mechanism underlying the immune responses of rainbow trout following Y. ruckeri infection and the base for future studies of host-pathogen interactions in rainbow trout.