RESUMO
Infectious hematopoietic necrosis (IHN), caused by IHN virus, is a highly contagious and lethal disease that seriously hampers the development of rainbow trout (Oncorhynchus mykiss) aquaculture. However, the immune response mechanism of rainbow trout underlying IHNV infection remains largely unknown. MicroRNAs act as post-transcriptional regulators of gene expression and perform a crucial role in fish immune response. Herein, the regulatory mechanism and function of miR-206 in rainbow trout resistance to IHNV were investigated by overexpression and silencing. The expression analysis showed that miR-206 and its potential target receptor-interacting serine/threonine-protein kinase 2 (RIP2) exhibited significant time-dependent changes in headkidney, spleen and rainbow trout primary liver cells infected with IHNV and their expression displayed a negative correlation. In vitro, the interaction between miR-206 and RIP2 was verified by luciferase reporter assay, and miR-206 silencing in rainbow trout primary liver cells markedly increased RIP2 and interferon (IFN) expression but significantly decreased IHNV copies, and opposite results were obtained after miR-206 overexpression or RIP2 knockdown. In vivo, overexpressed miR-206 with agomiR resulted in a decrease in the expression of RIP2 and IFN in liver, headkidney and spleen. This study revealed the key role of miR-206 in anti-IHNV, which provided potential for anti-viral drug screening in rainbow trout.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Vírus da Necrose Hematopoética Infecciosa , MicroRNAs , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Oncorhynchus mykiss/imunologia , Oncorhynchus mykiss/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/imunologia , MicroRNAs/genética , MicroRNAs/imunologia , MicroRNAs/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genéticaRESUMO
Respiratory structures are crucial for vertebrate survival, as they serve not only to perform gas-exchange processes but also as entry points for opportunistic pathogens. Previous studies have demonstrated that fish contain gill mucosal-associated lymphoid tissue, and harbor a large number of commensal bacteria on their surface and contribute to maintaining fish health. However, by far, very limited information is known regarding the effects of viral infection on gill mucosal immunity and microbiota homeostasis. In this study, we conducted an infection model by bath with infectious hematopoietic necrosis virus (IHNV) and revealed a 27 % mortality rate among rainbow trout in the first two weeks after infection. Moreover, we found that diseased fish with the highest IHNV loads in gills exhibiting severe damage, as well as increased goblet cell counts in both primary lamellae (PL) and secondary lamellae (SL). Additionally, RT-qPCR and RNA-seq analyses revealed that IHNV infection induced a strong innate and adaptive antiviral immune responses. Interestingly, an antibacterial immune response was also observed, suggesting that a secondary bacterial infection occurred in trout gills after viral infection. Furthermore, 16S rRNA analysis of trout gills revealed a profound dysbiosis marked by a loss of beneficial taxa and expansion of pathobionts following IHNV infection. Overall, our finding demonstrates that IHNV infection induces significant changes of the microbial community in the fish respiratory surface, thus triggering local antiviral and bacterial mucosal immunity.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Microbiota , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Brânquias , Imunidade nas Mucosas , RNA Ribossômico 16SRESUMO
Rainbow trout (Oncorhynchus mykiss) is an important cold-water fish widely cultivated in China. The frequent occurrence of viral diseases caused by infectious hematopoietic necrosis virus (IHNV) seriously restricted the healthy development of the rainbow trout farming industry. However, the immune defense mechanism induced by IHNV in rainbow trout has not been fully elucidated. In the present study, we detected mRNA and miRNA expression profiles in rainbow trout head kidney after IHNV infection using RNA-seq and identified key immune-related genes and miRNAs. The results showed that a total of 7486 genes and 277 miRNAs were differentially expressed, and numerous differentially expressed genes (DEGs) enriched in the immune-related pathways such as Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathway were significantly up-regulated, including LGP2, MDA5, TRIM25, IRF3, IRF7, TLR3, TLR7, TLR8, MYD88, and IFN1. Integration analysis identified six miRNAs (miR-141-y, miR-200-y, miR-144-y, miR-2188-y, miR-725-y, and miR-203-y) that target at least six key immune-related genes (TRIM25, LGP2, TLR3, TLR7, IRF3, and IRF7). Further, we verified selected immune-related mRNAs and miRNAs through qRT-PCR and confirmed the reliability of the RNA-seq results. These findings improve our understanding of the immune mechanism of rainbow trout infected with IHNV and provide basic data for future breeding for disease resistance in rainbow trout.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , MicroRNAs , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Vírus da Necrose Hematopoética Infecciosa/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , MicroRNAs/genética , Receptor 7 Toll-Like , Receptor 3 Toll-Like , Rim Cefálico/metabolismo , Reprodutibilidade dos Testes , Imunidade Inata/genéticaRESUMO
Antimicrobial peptides (AMPs) are considered a novel approach to stimulate fish antiviral mechanisms for defense against a broad range of viral infections by enhancing immunomodulatory activities. Octominin is an AMP derived from the defense proteins of Octopus minor. In this study, preliminary screening of octominin against viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and infectious pancreatic necrosis virus (IPNV) was carried out. Moreover, immune responses upon octominin treatment and IHNV challenge were investigated using fathead minnow (FHM) cells. The CC50s of octominin for FHM and Chinook salmon embryo-214 (CHSE-214) cells were 2146.2 and 1865.2 µg/mL, respectively. With octominin treatment, EC50 resulted in 732.8, 435.1, and 925.9 µg/mL for VHSV, IHNV, and IPNV, respectively. The selectivity indices were 2.9, 4.9, and 2.0, respectively. The transcriptional analysis results demonstrated the induced transcription factors (Irf3; 143-fold, Irf7; 105-fold, and NF-κB; 8-fold), stress response gene (HspB8; 2-fold), and apoptosis functional gene (p53; 3-fold) in octominin treated (500 µg/mL) FHM cells for 48 h. Moreover, IHNV viral copy number was slightly decreased with the octominin treatment (500 µg/mL) in FHM cells. Overall results suggest that octominin could be a potential antiviral agent, although further studies are necessary to understand its mode of action and the mechanism of its antiviral activity.
Assuntos
Cyprinidae , Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Vírus da Necrose Pancreática Infecciosa , Animais , Linhagem Celular , Peptídeos Antimicrobianos , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Antivirais/farmacologia , ImunidadeRESUMO
Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are typical pathogens of rainbow trout Oncorhynchus mykiss, and the concurrent infection of the two viruses is very common among modern trout hatcheries, which has caused huge economic losses to the rainbow trout farming industry. To prevent and control the spread of IHNV and IPNV in juvenile trout simultaneously, in this study a bivalent recombinant adenovirus vaccine with IHNV Glycoprotein (G) and IPNV VP2 genes was developed. After immunizing juvenile trout with this bivalent vaccine via the immersion route, the expression levels of IHNV G and IPNV VP2 and the representative immune genes in vaccinated and control rainbow trout were tested to evaluate the correlation of immune responses with the expression of viral genes. The neutralizing antibody level induced by this bivalent vaccine as well as the protection efficacy of the vaccine against IHNV and IPNV was also evaluated. The results showed that IHNV G and IPNV VP2 were successfully expressed in juvenile trout, and all the innate and adaptive immune genes were up-regulated. This indicated that the level of the innate and adaptive immune responses were significantly increased, which might be induced by the high expression of the two viral proteins. Compared with the controls, high levels of neutralizing antibodies against IHNV and IPNV were induced in the vaccinated trout. Besides, the bivalent recombinant adenovirus vaccine showed high protection rate against IHNV, with the relative percent survival (RPS) of 81.25%, as well as against IPNV, with the RPS of 78.95%. Taken together, our findings clearly demonstrated that replication-defective adenovirus can be developed as a qualified vector for fish vaccines and IHNV G and IPNV VP2 were two suitable antigenic genes that could induce effective immune protection against these two pathogens. This study provided new insights into developing bivalent vectored vaccines and controlling the spread of IHNV and IPNV simultaneously in juvenile trout.
Assuntos
Vacinas contra Adenovirus , Infecções por Birnaviridae , Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Vírus da Necrose Pancreática Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Vacinas Virais , Animais , Vírus da Necrose Pancreática Infecciosa/fisiologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vacinas Sintéticas , Adenoviridae/genética , Infecções por Rhabdoviridae/prevenção & controle , Infecções por Rhabdoviridae/veterinária , Infecções por Birnaviridae/prevenção & controle , Infecções por Birnaviridae/veterináriaRESUMO
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.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Microbiota , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Viroses , Animais , Imunidade nas Mucosas , RNA Ribossômico 16S , Disbiose , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Antivirais , MamíferosRESUMO
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.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Antivirais , RNA Helicases DEAD-box/genética , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Mamíferos , Filogenia , Proteínas/genéticaRESUMO
Viral infection of the central nervous system (CNS) is often associated with blood-brain barrier (BBB) disruption. Mammals have developed complicated and efficient immune strategies to protect the BBB. However, the immune defense of brain and BBB permeability changes are not well-understood in teleost during virus invading. In this study, we constructed an infectious hematopoietic necrosis virus (IHNV) immersion infected rainbow trout model. After IHNV infection, pathological changes occurred in the brain, and MPO and ROS activities were significantly increased. In addition, the expression levels of BBB permeability-related genes were also changed. Transcriptome analysis showed that immune-related genes and signaling pathways in the brain were activated after IHNV infection. These results showed that the permeability of BBB increased significantly after IHNV infection, thus activating immune related factors and cells to enter the CNS through blood circulation to resist pathogenic infection.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Barreira Hematoencefálica , Imunidade , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Mamíferos , PermeabilidadeRESUMO
Utilizing RNA-seq, this study compared the transcriptomic responses of three improved strains (VSel, PSel, and CSel) of rainbow trout fry during acute stages of challenge with infectious hematopoietic necrosis virus (IHNV). The VSel strain has been selected for resistance against the specific strain of IHNV used in our challenge, PSel has undergone selection for utilization of plant-protein based feeds and previously has shown elevated non-specific disease resistance despite no disease related selection pressures, and the final strain, CSel, is a commercial strain that has been domesticated for several years but has not been selected for specific viral disease resistance. Following a 21-day IHNV challenge, Kaplan-Meier survival estimator curves and cumulative percent mortality (CPM) showed significant differences in IHNV resistance across strains: VSel - 19.3 ± 5.0%, PSel - 67. ± 3.03%, CSel - 94.6 ± 4.1% CPM. To evaluate acute responses to IHNV infection, whole blood, as well as samples from the kidney, liver, and intestine, were collected at 0, 4, 12, 24, and 48 h post infection (hpi). Serum lysozyme activity, a marker of non-specific innate immunity, showed strain and temporal effects during the acute infection phase with PSel showing the highest activity at 0 and 48 hpi. Differential gene expression responses were detected, with varying degrees, in all tissues, both between strains, as well as across acute timepoints within strains. The VSel strain showed upregulation for a particular subset of viral recognition genes during early infection timepoints and rather limited upregulation of immune genes later, while maintaining and reactivating metabolic pathways. The CSel strain showed a downregulation of metabolic related genes and a limited upregulation of immune genes, while the PSel strain showed similar downregulation of metabolic genes during acute infection, yet when compared to the CSel strain, showed a more robust innate immune response. Evaluation of upregulated immune response genes, as well as interferon-related genes showed the PSel strain to have the greatest number of uniquely upregulated immune genes in both the kidney and intestine, with CSel and PSel showing a similar number of such genes upregulated in liver. A moderate number of immune response genes were shared between PSel and CSel in all tissues, though both PSel and VSel showed a high number of uniquely overexpressed immune response genes in the kidney, and PSel showed the highest number of uniquely upregulated interferon related genes in the intestine. Overall, the VSel response was unique from the CSel with very little overlap in activated immune responses. Findings from this study highlight the disparity in IHNV resistance among genetic strains of rainbow trout, while identifying molecular mechanisms underlying differences in disease phenotypes. Furthermore, our results on trout strains with distinct selection backgrounds yields comparative insights into the adaptive gains brought about by selection programs for pathogen-specific disease resistance, as well as the non-specific immune enhancement associated with selection for utilization of plant-based diets.
Assuntos
Doenças dos Peixes , Vírus da Necrose Hematopoética Infecciosa , Oncorhynchus mykiss , Infecções por Rhabdoviridae , Animais , Resistência à Doença/genética , Vírus da Necrose Hematopoética Infecciosa/fisiologia , InterferonsRESUMO
Tradeoff theory, which postulates that virulence provides both transmission costs and benefits for pathogens, has become widely adopted by the scientific community. Although theoretical literature exploring virulence-tradeoffs is vast, empirical studies validating various assumptions still remain sparse. In particular, truncation of transmission duration as a cost of virulence has been difficult to quantify with robust controlled in vivo studies. We sought to fill this knowledge gap by investigating how transmission rate and duration were associated with virulence for infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Using host mortality to quantify virulence and viral shedding to quantify transmission, we found that IHNV did not conform to classical tradeoff theory. More virulent genotypes of the virus were found to have longer transmission durations due to lower recovery rates of infected hosts, but the relationship was not saturating as assumed by tradeoff theory. Furthermore, the impact of host mortality on limiting transmission duration was minimal and greatly outweighed by recovery. Transmission rate differences between high and low virulence genotypes were also small and inconsistent. Ultimately, more virulent genotypes were found to have the overall fitness advantage, and there was no apparent constraint on the evolution of increased virulence for IHNV. However, using a mathematical model parameterized with experimental data, it was found that host culling resurrected the virulence tradeoff and provided low virulence genotypes with the advantage. Human-induced or natural culling, as well as host population fragmentation, may be some of the mechanisms by which virulence diversity is maintained in nature. This work highlights the importance of considering non-classical virulence tradeoffs.
Assuntos
Doenças dos Peixes/virologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/virologia , Virulência , Eliminação de Partículas Virais , Animais , Cinética , Oncorhynchus mykiss/crescimento & desenvolvimento , Carga ViralRESUMO
Infectious hematopoietic necrosis virus (IHNV) causes clinical diseases and mortality in a wide variety of salmonid species. Here, we studied transcriptional responses in rainbow trout infected by the IHNV-Nagano strain isolated in Korea. RNA-seq-based transcriptome analysis of head kidney tissues cataloged differentially expressed genes. Enrichment analysis of gene ontology annotations was performed, and a total of fifteen biological process terms were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes pathway analysis, pathogen recognition receptor (PRR) signaling pathways such as the retinoic-acid-inducible gene-I-like receptor signaling pathway and the Toll-like receptor signaling pathway were identified at all time points. The nucleotide-binding oligomerization-domain-like receptor signaling pathway and cytosolic DNA-sensing pathway were identified at days 1 and 3. Protein-protein interaction network and centrality analyses revealed that the immune system, signaling molecules, and interaction pathways were upregulated at days 1 and 3, with the highest centrality of tumor necrosis factor. Cancer, cellular community, and endocrine system pathways were downregulated, with the highest centrality of fibronectin 1 at day 5. STAT1 was upregulated from days 1 to 5 with a high centrality. The reproducibility and repeatability of the transcriptome analysis were validated by RT-qPCR. IHNV-Nagano infection dynamically changed the transcriptome profiles in the head kidney of rainbow trout and induced a defense mechanism by regulating the immune and inflammatory pathways through PRR signaling at an early stage. Downregulated pathways involved in extracellular matrix formation and focal adhesion at day 5 indicated the possible failure of wound healing, which is important in the pathogenesis of IHNV infection.
Assuntos
Doenças dos Peixes/virologia , Rim Cefálico/virologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/veterinária , Transcriptoma , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Genótipo , Rim Cefálico/imunologia , Rim Cefálico/metabolismo , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes , República da Coreia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/metabolismo , Infecções por Rhabdoviridae/virologia , Transdução de SinaisRESUMO
Salmonids can be co-infected by infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) under natural or experimental conditions. To reveal the influence of IPNV on IHNV in co-infections, CHSE-214 cells were inoculated with IPNV at different time intervals prior to or after IHNV infection. Propagation of IHNV was determined by an immunofluorescence antibody test, real-time quantitative polymerase chain reaction, flow cytometry, and virus titration. The results showed that when cells were inoculated with IPNV prior to IHNV, IHNV multiplication was inhibited. This inhibitory effect became stronger with increasing time intervals (P < 0.05). When cells were inoculated with IPNV after IHNV, the inhibitory effect became weaker with increasing time intervals (P < 0.05), and no significant inhibition was observed at 12 h (P > 0.05) compared with the single IHNV infection group. The findings suggest that IHNV is inhibited at the early stage of infection by IPNV and in a time dependent manner during co-infection. Furthermore, the effect of IPNV on IHNV entry and expression of IHNV entry-related genes clathrin, dynamin-2, adaptor protein 2, and vacuolar protein sorting 35 were also determined. The results showed that IPNV did not affect the amount of IHNV entering the cells. However, the expression levels of clathrin and dynamin-2 were significantly lower in co-infection than those in single IHNV infection, which suggests that IPNV likely inhibits IHNV by affecting IHNV invasion via downregulating IHNV entry-related genes clathrin and dynamin-2.
Assuntos
Infecções por Birnaviridae/veterinária , Coinfecção/veterinária , Doenças dos Peixes/imunologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Vírus da Necrose Pancreática Infecciosa/fisiologia , Infecções por Rhabdoviridae/veterinária , Salmão , Animais , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Linhagem Celular , Coinfecção/imunologia , Coinfecção/virologia , Regulação para Baixo , Embrião não Mamífero , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologiaRESUMO
Emerging evidence suggests that bitter and sweet Taste receptors (TRs) in the airway are important sentinels of innate immunity. TRs are G protein-coupled receptors that trigger downstream signaling cascades in response to activation of specific ligands. Among them, the T1R family consists of three genes: T1R1, T1R2, and T1R3, which function as heterodimers for sweet tastants and umami tastants. While the other TRs family components T2Rs function as bitter tastants. To understand the relationship between TRs and mucosal immunity in teleost, here, we firstly identified and analyzed the molecular characteristics of three TRs (T1R1, T1R3, and T2R4) in rainbow trout (Oncorhynchus mykiss). Secondly, by quantitative real-time PCR (qPCR), we detected the mRNA expression levels of T1R1, T1R3 and T2R4 and found that the three genes could be tested in all detected tissues (pharynx, buccal cavity, tongue, nose, gill, eye, gut, fin, skin) and the expression levels of T1R3 and T2R4 were higher in buccal mucosa (BM) and pharyngeal mucosa (PM) compare to other tissues. It may suggest that T1R3 and T2R4 play important roles in BM and PM. Then, to analyses the changes of expression levels of the three genes in rainbow trout infected with pathogens, we established three infection models Flavobacterium columnare (F. cloumnare), infectious hematopoietic necrosis virus (IHNV) and Ichthyophthirius multifiliis (Ich). Subsequently, by qPCR, we detected the expression profiles of TRs in the gustatory tissues (BM, PM and skin) of rainbow trout after infection with F. cloumnare, IHNV, and Ich, respectively. We found that under three different infection models, the expression of the T1R1, T1R3 and T2R4 showed their own changes in mRNA levels. And the expression levels of the T1R1, T1R3 and T2R4 changed significantly at different time points in response to three infection models, respectively, suggesting that TRs may be associated with mucosal immunity.
Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Imunidade nas Mucosas/genética , Oncorhynchus mykiss/genética , Sequência de Aminoácidos , Animais , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/parasitologia , Infecções por Cilióforos/veterinária , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Proteínas de Peixes/imunologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/fisiologia , Perfilação da Expressão Gênica/veterinária , Hymenostomatida/fisiologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss/imunologia , Filogenia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Infecções por Rhabdoviridae/virologia , Alinhamento de Sequência/veterináriaRESUMO
RNA viruses including many retroviruses encode "late-domain" motifs that can interact with host proteins to mediate viral assembly and affect viral budding and pathogenicity. For IHNV, our previous studies demonstrated that the respective interactions of the L domains of IHNV with host proteins could mediate viral assembly and budding. To our knowledge, the role of L domains of the IHNV in the budding and pathogenicity has not investigated yet. In this study, we generated two recombinant IHNV strains rIHNV-M(PH>A4) and rIHNV-G(PS>A4) with mutations in the L domains (PPPH to AAAA or PSAP to AARA) of IHNV by reverse genetics and explored the effect of the mutations on budding and pathogenicity of the two recombinant viruses. The RT-qPCR results showed that the production levels of the extracellular particles of rIHNV-M(PH>A4) or rIHNV-G(PS>A4) declined significantly, compared with those of wild-type (wt) IHNV HLJ-09. Furthermore, the challenge test showed that the survival rates of juvenile rainbow trout challenged with rIHNV-M(PH>A4) or rIHNV-G(PS>A4) were 90% or 87%, respectively; however, the survivability was zero in groups challenged with wtIHNV HLJ-09 or rIHNV HLJ-09 (recombinant IHNV). Additionally, the RT-qPCR results showed that the recombinant viruses induced higher expression levels of IFN1, IL-1ß, and IL-8 compared with those induced by wtIHNV HLJ-09 as well as the ELISA results showed that fish vaccinated with recombinant viruses produced high levels of specific IgM antibodies, demonstrating that the two recombinant viruses may induce immune responses to resist infection by IHNV. Also, these results demonstrated for the first time that the L domains of the M and G proteins of IHNV could affect the budding and pathogenicity of IHNV, which may be beneficial in the prevention and control of IHNV infections in fish. Taken together, our study as the first research provides the foundation for effect of rhabdovirus L domains on viral budding and pathogenicity.
Assuntos
Doenças dos Peixes/virologia , Proteínas de Ligação ao GTP/genética , Vírus da Necrose Hematopoética Infecciosa/patogenicidade , Oncorhynchus mykiss/virologia , Proteínas Virais/genética , Liberação de Vírus , Animais , Vírus da Necrose Hematopoética Infecciosa/genética , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss/imunologia , Proteínas da Matriz Viral/genética , Virulência , Montagem de VírusRESUMO
Infectious hematopoietic necrosis virus (IHNV) is a fish viral pathogen that causes severe disease and huge economic losses in the salmonid aquaculture industry. However, anti-IHNV drugs currently are scarce. For the purpose of seeking out anti-IHNV drugs, the anti-IHNV activities of 32 medicinal plants were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Prunella vulgaris L. (PVL) showed the strongest inhibition on IHNV replication with an inhibitory percentage of 99.3% at the concentration 100â¯mg/L. Further studies demonstrated that ursolic acid (UA), a major constituent of PVL, also showed a highly effective anti-IHNV activity. The half-maximal inhibitory concentration (IC50) at 72â¯h of UA on IHNV was 8.0⯵M. Besides, UA could significantly decrease cytopathic effect (CPE) and the viral titer induced by IHNV in EPC cells. More importantly, UA also showed a strong anti-IHNV activity in vivo, as indicated by increasing the survival rate of rainbow trout and inhibiting viral gene expression. Intraperitoneal injection of UA increased the relative percentage of survival of rainbow trout by 18.9% and inhibited IHNV glycoprotein mRNA expression by > 90.0% in the spleen at the 1st-day post-infection. Altogether, UA was expected to be a therapeutic agent against IHNV infection in aquaculture.
Assuntos
Antivirais/farmacologia , Antivirais/uso terapêutico , Vírus da Necrose Hematopoética Infecciosa/efeitos dos fármacos , Prunella/química , Infecções por Rhabdoviridae/veterinária , Triterpenos/farmacologia , Triterpenos/uso terapêutico , Animais , Aquicultura , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/virologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Concentração Inibidora 50 , Oncorhynchus mykiss/virologia , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Infecções por Rhabdoviridae/tratamento farmacológico , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Ácido UrsólicoRESUMO
Aquatic ecosystems are now chronically polluted by a cocktail of many chemical substances. There is now clear evidence of associations between exposure to pollutants and greater susceptibility to pathogens. The aim of the present study was to characterize the defense capacities of rainbow trout (Oncorhynchus mykiss), chronically exposed to pendimethalin (PD), to subsequent experimental challenge with the infectious hematopoietic necrosis virus (IHNV). Immunological responses were examined at different organizational levels, from individuals to gene expression. No negative effects of PD were noted on the Fulton index nor on the liver or spleen somatic indices (LSI; SSI) before viral infection, but the infectious stress seems to generate a weak but significant decrease in Fulton and LSI values, which could be associated with consumption of energy reserves. During the viral challenges, the distribution of cumulative mortality was slightly different between infected groups. The impact of the virus on fish previously contaminated by PD started earlier and lasted longer than controls. The proportion of seropositive fish was lower in the fish group exposed to PD than in the control group, with similar quantities of anti-IHNV antibodies secreted in positive fish, regardless of the treatment. While no significant differences in C3-1 expression levels were detected throughout the experiment, TNF1&2, TLR3, Il-1ß and IFN expression levels were increased in all infected fish, but the difference was more significant in fish groups previously exposed to herbicide. On the other hand, ß-def expression was decreased in the pendimethalin-IHNV group compared to that in fish only infected by the virus (control-IHNV group).
Assuntos
Herbicidas/toxicidade , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Compostos de Anilina/toxicidade , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/patologia , Poluentes Químicos da Água/toxicidadeRESUMO
Aquatic rhabdoviruses are globally significant pathogens associated with disease in both wild and cultured fish. Infectious hematopoietic necrosis virus (IHNV) is a rhabdovirus that causes the internationally regulated disease infectious hematopoietic necrosis (IHN) in most species of salmon. Yet not all naïve salmon exposed to IHNV become diseased, and the mechanisms by which some individuals evade or rapidly clear infection following exposure are poorly understood. Here we used RNA-sequencing to evaluate transcriptomic changes in sockeye salmon, a keystone species in the North Pacific and natural host for IHNV, to evaluate the consequences of IHNV exposure and/or infection on host cell transcriptional pathways. Immersion challenge of sockeye salmon smolts with IHNV resulted in approximately 33% infection prevalence, where both prevalence and viral kidney load peaked at 7 days post challenge (dpc). De novo assembly of kidney transcriptomes at 7 dpc revealed that both infected and exposed but noninfected individuals experienced substantial transcriptomic modification; however, stark variation in gene expression patterns were observed between exposed but noninfected, infected, and unexposed populations. GO and KEGG pathway enrichment in concert with differential expression analysis identified that kidney responses in exposed but noninfected fish emphasised a global pattern of transcriptional down-regulation, particularly for pathways involved in DNA transcription, protein biosynthesis and macromolecule metabolism. In contrast, transcriptomes of infected fish demonstrated a global emphasis of transcriptional up-regulation highlighting pathways involved in antiviral response, inflammation, apoptosis, and RNA processing. Quantitative PCR was subsequently used to highlight differential and time-specific regulation of acute phase, antiviral, inflammatory, cell boundary, and metabolic responsive transcripts in both infected and exposed but noninfected groups. This data demonstrates that waterborne exposure with IHNV has a dramatic effect on the sockeye salmon kidney transcriptome that is discrete between resistant and acutely susceptible individuals. We identify that metabolic, acute phase and cell boundary pathways are transcriptionally affected by IHNV and kidney responses to local infection are highly divergent from those generated as part of a disseminated response. These data suggest that primary resistance of naïve fish to IHNV may involve global responses that encourage reduced cellular signaling rather than promoting classical innate antiviral responses.
Assuntos
Resistência à Doença/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Salmão/genética , Salmão/imunologia , Transcriptoma/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Rim/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Carga Viral/fisiologiaRESUMO
In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Salmão/imunologia , Proteínas Virais/metabolismo , Animais , Embrião não Mamífero/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Vírion/fisiologia , Liberação de VírusRESUMO
Infectious hematopoietic necrosis virus (IHNV) leads to serious disease and economic losses in the salmonid aquaculture industry. The present study aimed to develop an effective and efficient vaccine to protect rainbow trout (Oncorhynchus mykiss) against IHNV infection. Administered via the immersion route, a live vector vaccine containing the regions of the IHNV glycoprotein (G) induced immune responses in rainbow trout. Use of the immersion route induced more-efficient mucosal immunity than intramuscular injection vaccination. IHNV G gene expression was detected in the spleens of rainbow trout at 3, 7 and 15 days post-vaccination (dpv). The G gene expression continuously decreased between 3 and 15 dpv. In addition, the expression of TLR-3, TLR-7 and TLR-8 was upregulated after vaccination, and the highest expression levels of IFN-1, Mx-1, Mx-3, Vig-1 and Vig-2 were observed at 3 dpv. Four markers of the adaptive immune response (CD4, CD8, IgM and IgT) gradually increased. When experimental fish were challenged with IHNV by immersion, significant differences in cumulative percentage mortality were observed in the vaccinated fish and the unvaccinated (empty-plasmid-vaccinated) fish. The relative survival rate was 92% and 6% in the vaccinated group and empty-plasmid group, respectively. Serum antibody levels gradually increased in the vaccinated fish, unlike in the unvaccinated fish, after 7 dpv. Our results suggest there was a significant increase in fish immune responses and resistance to infection with IHNV following administration of the live vector vaccine. Therefore, this live vector vaccine is a promising vaccine that may be utilized to protect rainbow trout against IHNV.
Assuntos
Imunidade Adaptativa , Doenças dos Peixes/prevenção & controle , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss , Infecções por Rhabdoviridae/veterinária , Vacinas Virais/imunologia , Animais , Doenças dos Peixes/imunologia , Glicoproteínas/genética , Glicoproteínas/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/prevenção & controle , Baço/imunologia , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Our previous studies demonstrated that the nonstructural NV protein of infectious hematopoietic necrosis virus (IHNV) was essential for efficient viral replication and pathogenicity, and that the amino acid residues 32EGDL35 of the NV protein were responsible for nuclear localization, and played important roles in suppressing IFN and inhibiting NF-κB activity. However, little is known about the influence of 32EGDL35 on IHNV replication and pathogenicity. In the present study, two recombinant IHNV strains with deletions of NV 32EGDL35 were generated and the effect on IHNV replication and pathogenicity was explored. Our results showed that both mutants stably replicated in Chinook salmon embryo cells for 15 consecutive passages, and had similar host-tropism as wild-type (wt) IHNV; however, titers of the mutants were lower than those of wt IHNV in CHSE-214â¯cells. Infection of rainbow trout showed wt IHNV produced 90% cumulative mortality, while the mutants produced 55% and 60% cumulative mortality, respectively. Histopathological evaluation showed that tissues from the liver, brain, kidney, and heart of fish infected with wt IHNV exhibited pathological changes, but significant lesions were found only in the liver and heart of fish infected with the recombinant viruses. In addition, the recombinant viruses induced higher expression levels of IFN1, Mx-1, and IL-6 compared with those induced by wt IHNV. These results indicated that the 32EGDL35 residues were essential for the efficient anti-IFN and NF-κB-inhibiting activity of NV. Our results provide a basis for understanding the roles of 32EGDL35 in IHNV replication and pathogenicity, and may prove beneficial in the prevention and control of IHNV infections of fish.