RESUMEN
The virus neutralization test (VNT) is a functional immunoassay which detects the presence and quantity of neutralizing antibodies. It is a highly sensitive and specific test. As with most neutralization assays, the EHDV VNT does not react with all virus-targeting antibodies, but specifically with those antibodies that bind to VP2, the outermost capsid structural protein of the virus. The interaction between VP2 and neutralizing antibodies can block EHDV cell binding, neutralizing its infectivity. The detection and quantification of neutralizing antibodies are indicative of how protected an animal is against reinfection. The EHD VNT can therefore be a useful tool to monitor the efficacy of a vaccination campaign. VP2 is also the main determinant of EHDV serotype specificity, and so EHDV-neutralizing antibodies which target VP2 are also serotype-specific. Throughdetecting and quantifying neutralizing antibodies, the VNT can discriminate the EHDV serotype responsible for an infection and provides insights into the time of infection. It is considered the gold standard test for identifying and quantifying antibodies against EHDV serotypes present in test serum samples. The assay is performed in vitro and is based on inhibition of virus infectivity in the presence of neutralizing antibodies. A neutralizing antibody titer is determined through the presence or absence of cytopathic effect in a cell monolayer. The VNT is a relatively inexpensive assay using standard laboratory equipment; however, to perform the assay, cell cultures, significant time, intensive labor, and technical skill are required.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Virus de la Enfermedad Hemorrágica Epizoótica , Pruebas de Neutralización , Pruebas de Neutralización/métodos , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Animales , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Virus de la Enfermedad Hemorrágica Epizoótica/inmunología , Serogrupo , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/diagnóstico , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/virologíaRESUMEN
Enzyme-linked immunosorbent assay (ELISA) is a relatively inexpensive, rapid, and high-throughput diagnostic tool to detect antibodies raised against epizootic hemorrhagic disease virus (EHDV) in ruminant serum. While the presence of EHDV antibodies only confirms prior exposure to the virus, it does not conclusively determine infection status. The c-ELISA can be used in conjunction with other diagnostic tests (e.g., real-time PCR) to reinforce diagnosis of infection or as a surveillance tool to support disease control. The EHDV competition ELISA (c-ELISA) described here is a commercial diagnostic assay, recommended by the World Organisation for Animal Health (WOAH), that detects ruminant antibodies against the highly conserved EHDV structural protein, VP7.
Asunto(s)
Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Virus de la Enfermedad Hemorrágica Epizoótica , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Virus de la Enfermedad Hemorrágica Epizoótica/inmunología , Infecciones por Reoviridae/diagnóstico , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/virología , OvinosRESUMEN
BF/C2 is a crucial molecule in the coagulation complement cascade pathway and plays a significant role in the immune response of grass carp through the classical, alternative, and lectin pathways during GCRV infection. In vivo experiments demonstrated that the mRNA expression levels of BF/C2 (A, B) in grass carp positively correlated with GCRV viral replication at various stages of infection. Excessive inflammation leading to death coincided with peak levels of BF/C2 (A, B) mRNA expression and GCRV viral replication. Correspondingly, BF/C2 (A, B) recombinant protein, CIK cells and GCRV co-incubation experiments yielded similar findings. Therefore, 3 h (incubation period) and 9 h (death period) were selected as critical points for this study. Transcriptome sequencing analysis revealed significant differences in the expression of BF/C2A and BF/C2B during different stages of CIK infection with GCRV and compared to the blank control group (PBS). Specifically, the BF/C2A_3 and BF/C2A_9 groups exhibited 2729 and 2228 differentially expressed genes (DEGs), respectively, with 1436 upregulated and 1293 downregulated in the former, and 1324 upregulated and 904 downregulated in the latter. The BF/C2B_3 and BF/C2B_9 groups showed 2303 and 1547 DEGs, respectively, with 1368 upregulated and 935 downregulated in the former, and 818 upregulated and 729 downregulated in the latter. KEGG functional enrichment analysis of these DEGs identified shared pathways between BF/C2A and PBS groups at 3 and 9 h, including the C-type lectin receptor signaling pathway, protein processing in the endoplasmic reticulum, Toll-like receptor signaling pathway, Salmonella infection, apoptosis, tight junction, and adipocytokine signaling pathway. Additionally, the BF/C2B groups at 3 and 9 h shared pathways related to protein processing in the endoplasmic reticulum, glycolysis/gluconeogenesis, and biosynthesis of amino acids. The mRNA levels of these DEGs were validated in cellular models, confirming consistency with the sequencing results. In addition, the mRNA expression levels of these candidate genes (mapk1, il1b, rela, nfkbiab, akt3a, hyou1, hsp90b1, dnajc3a et al.) in the head kidney, kidney, liver and spleen of grass carp immune tissue were significantly different from those of the control group by BF/C2 (A, B) protein injection in vivo. These candidate genes play an important role in the response of BF/C2 (A, B) to GCRV infection and it also further confirmed that BF/C2 (A, B) of grass carp plays an important role in coping with GCRV infection.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Infecciones por Reoviridae , Reoviridae , Animales , Carpas/genética , Carpas/virología , Carpas/inmunología , Enfermedades de los Peces/virología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/genética , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/genética , Infecciones por Reoviridae/virología , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Reoviridae/fisiología , Perfilación de la Expresión Génica , Transcriptoma , Replicación Viral , Regulación de la Expresión GénicaRESUMEN
The grass carp (Ctenopharyngodon idella) constitutes a significant economic resource within the aquaculture sector of our nation, yet it has been chronically afflicted by the Grass Carp Reovirus (GCRV) disease. The complement system, a vital component of fish's innate immunity, plays a crucial role in combating viral infections. This research investigates the potential role of MASP1, a key molecule in the lectin pathway of the complement system, in the GCRV infection in grass carp. An analysis of the molecular characteristics of MASP1 in grass carp revealed that its identity and similarity percentages range from 35.10 to 91.00 % and 35.30-91.00 %, respectively, in comparison to other species. Phylogenetically, MASP1 in C. idella aligns closely with species such as Danio rerio, Cyprinus carpio, and Carassius carassius, exhibiting chromosomal collinearity with the zebrafish. Subsequent tissue analysis in both healthy and GCRV-infected grass carp indicated that MASP1's basal expression was predominantly in the liver. Post-GCRV infection, MASP1 expression in various tissues exhibited temporal variations: peaking in the liver on day 5, spleen on day 7, and kidney on day 14. Furthermore, employing Complement Component 3 (C3) as a benchmark for complement system activation, it was observed that MASP1 could activate and cleave C3 to C3b. MASP1 also demonstrated an inhibitory effect on GCRV replication (compared with the control group, VP2 and VP7 decreased by 6.82-fold and 4.37-fold) and enhanced the expression of antiviral genes, namely IRF3, IRF7 and IFN1 (compared with the control group, increased 2.25-fold, 45.38-fold and 22.37-fold, respectively). In vivo protein injection experiments substantiated MASP1's influence on the relative mRNA expression levels of C3 in various tissues and its protein expression in serum. This study also verified that C3 could modulate the expression of antiviral genes such as IFN1 and IRF3.
Asunto(s)
Carpas , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Filogenia , Infecciones por Reoviridae , Reoviridae , Animales , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Carpas/inmunología , Carpas/genética , Reoviridae/fisiología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/genética , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/inmunología , Inmunidad Innata/genética , Regulación de la Expresión Génica/inmunología , Perfilación de la Expresión Génica/veterinaria , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/genética , Secuencia de Aminoácidos , Alineación de Secuencia/veterinariaRESUMEN
RLR helicases RIG-I and MDA5, which are known as pattern recognition receptors to sense cytoplasmic viral RNAs and trigger antiviral immune responses, are DExD/H-box helicases. In teleost, whether and how non-RLR helicases regulate RLR helicases to affect viral infection remains unclear. Here, we report that the non-RLR helicase DHX40 from grass carp (namely gcDHX40) is a negative regulator of grass carp reovirus (GCRV) infection and RLR-mediated type I IFN production. GcDHX40 was a cytoplasmic protein. Ectopic expression of gcDHX40 facilitated GCRV replication and suppressed type I IFN production induced by GCRV infection and by those genes involved the RLR antiviral signaling pathway. Mechanistically, gcDHX40 promoted the generation of viral inclusion bodies (VIBs) by interacting with the NS38 protein of GCRV. Additionally, gcDHX40 interacted with RLR helicase, and impaired the formation of RLR-MAVS functional complexes. Taken together, our results indicate that gcDHX40 is a novel important proviral host factor involving in promoting the generation of GCRV VIBs and inhibiting the production of RLR-mediated type I IFNs.
Asunto(s)
Carpas , ARN Helicasas DEAD-box , Enfermedades de los Peces , Proteínas de Peces , Inmunidad Innata , Infecciones por Reoviridae , Reoviridae , Proteínas no Estructurales Virales , Animales , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Carpas/inmunología , Carpas/genética , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Reoviridae/fisiología , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/inmunología , ARN Helicasas DEAD-box/metabolismo , Inmunidad Innata/genética , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/inmunología , Regulación de la Expresión Génica/inmunologíaRESUMEN
Epizootic hemorrhagic disease (EHD), caused by Epizootic hemorrhagic disease virus (EHDV), is an emerging and severe livestock disease. Recent incursion and distribution of EHDV in Europe have outlined the emerging character of EHD. Despite its worldwide impact, numerous knowledge gaps exist. A range of inconveniences restricts utilization of natural hosts of EHDV. Here, we show that adult mice deficient in type I IFN receptor (IFNAR(-/-)) are highly susceptible to EHDV-6 and EHDV-8 infection when the virus is administered subcutaneously. Disease was characterized by ruffled hair, reluctance to move, dehydration and conjunctivitis, with viraemia detected from day 5 post-infection. A deeper characterization of EHDV-8 infection showed viral replication in the lung, liver, spleen, kidney, testis and ovaries. Importantly, increased expression levels of pro-inflammatory cytokines IL-1ß, IL-6 and CXCL2 were observed in spleen after EHDV-8 infection. Furthermore, IFNAR(-/-) adult mice immunized with a EHDV-8 inactivated vaccine elicited neutralizing antibodies specific of EHDV-8 and full protection against challenge with a lethal dose of this virus. This study also explores the possibilities of this animal model for study of BTV and EHDV coinfection. In summary, the IFNAR(-/-) mouse model faithfully recapitulates EHD and can be applied for vaccine testing, which can facilitate progress in addressing the animal health challenge posed by this virus.
Asunto(s)
Modelos Animales de Enfermedad , Virus de la Enfermedad Hemorrágica Epizoótica , Receptor de Interferón alfa y beta , Vacunas Virales , Animales , Ratones , Receptor de Interferón alfa y beta/genética , Receptor de Interferón alfa y beta/metabolismo , Virus de la Enfermedad Hemorrágica Epizoótica/inmunología , Virus de la Enfermedad Hemorrágica Epizoótica/genética , Vacunas Virales/inmunología , Infecciones por Reoviridae/inmunología , Femenino , Ratones Noqueados , Anticuerpos Neutralizantes/inmunología , MasculinoRESUMEN
Grass Carp Reovirus (GCRV) and Aeromonas hydrophila (Ah) are the causative agents of haemorrhagic disease in grass carp. This study aimed to investigate the molecular mechanisms and immune responses at the miRNA, mRNA, and protein levels in grass carp kidney cells (CIK) infected by Grass Carp Reovirus (GCRV, NV) and Aeromonas hydrophilus (Bacteria, NB) to gain insight into their pathogenesis. Within 48 h of infection with Grass Carp Reovirus (GCRV), 99 differentially expressed microRNA (DEMs), 2132 differentially expressed genes (DEGs), and 627 differentially expressed proteins (DEPs) were identified by sequencing; a total of 92 DEMs, 3162 DEGs, and 712 DEPs were identified within 48 h of infection with Aeromonas hydrophila. It is worth noting that most of the DEGs in the NV group were primarily involved in cellular processes, while most of the DEGs in the NB group were associated with metabolic pathways based on KEGG enrichment analysis. This study revealed that the mechanism of a grass carp haemorrhage caused by GCRV infection differs from that caused by the Aeromonas hydrophila infection. An important miRNA-mRNA-protein regulatory network was established based on comprehensive transcriptome and proteome analysis. Furthermore, 14 DEGs and 6 DEMs were randomly selected for the verification of RNA/small RNA-seq data by RT-qPCR. Our study not only contributes to the understanding of the pathogenesis of grass carp CIK cells infected with GCRV and Aeromonas hydrophila, but also serves as a significant reference value for other aquatic animal haemorrhagic diseases.
Asunto(s)
Aeromonas hydrophila , Carpas , MicroARNs , ARN Mensajero , Reoviridae , Transcriptoma , Animales , Carpas/genética , Carpas/microbiología , Carpas/virología , Carpas/inmunología , MicroARNs/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reoviridae/fisiología , Proteómica/métodos , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Enfermedades de los Peces/genética , Perfilación de la Expresión Génica , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Bacterias Gramnegativas/microbiología , Infecciones por Bacterias Gramnegativas/genética , Línea Celular , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/genética , Redes Reguladoras de GenesRESUMEN
Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.
Asunto(s)
Células Dendríticas , Virus de la Enfermedad Hemorrágica del Conejo , Limosilactobacillus reuteri , Péptidos , Animales , Células Dendríticas/inmunología , Conejos , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Virus de la Enfermedad Hemorrágica del Conejo/genética , Limosilactobacillus reuteri/genética , Limosilactobacillus reuteri/inmunología , Péptidos/inmunología , Péptidos/genética , Infecciones por Caliciviridae/prevención & control , Infecciones por Caliciviridae/inmunología , Infecciones por Reoviridae/prevención & control , Infecciones por Reoviridae/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Vacunas Virales/inmunología , Vacunas Virales/genética , Lactobacillus/genética , Lactobacillus/inmunologíaRESUMEN
Epizootic haemorrhagic disease (EHD), caused by the EHD virus (EHDV), is a vector-borne viral disease transmitted through Culicoides biting midges. EHDV comprises seven serotypes (1, 2, and 4-8), with EHDV-8 having recently emerged and spread in Europe over the last two years. Such event has raised concerns about the significant threat posed by EHDV-8 to livestock industry. In this study, an inactivated vaccine against EHDV-8 (vEHDV8-IZSAM) was developed. Safety and efficacy of the vaccine were evaluated in calves through clinical, serological, and virological monitoring following experimental challenge. The vaccine was proven safe, with only transient fever and localized reactions observed in a few animals, consistent with adjuvanted vaccine side effects. vEHDV8-IZSAM elicited a robust humoral response, as evidenced by the presence of neutralizing antibodies. After challenge with a virulent isolate, viraemia and clinical signs were evidenced in control animals but in none of the vaccinated animals. This study highlights the potential of vEHDV8-IZSAM as a safe and highly effective vaccine against EHDV-8 in cattle. It offers protection from clinical disease and effectively prevents viraemia. With the recent spread of EHDV-8 in European livestock, the use of an inactivated vaccine could be key in protecting animals from clinical disease and thus to mitigate the economic impact of the disease. Further investigations are warranted to assess the duration of the induced immunity and the applicability of this vaccine in real-world settings. Accordingly, joint efforts between public veterinary institutions and pharmaceutical companies are recommended to scale up vaccine production.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , Enfermedades de los Bovinos , Virus de la Enfermedad Hemorrágica Epizoótica , Vacunas de Productos Inactivados , Vacunas Virales , Viremia , Animales , Bovinos , Vacunas de Productos Inactivados/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Viremia/prevención & control , Enfermedades de los Bovinos/prevención & control , Enfermedades de los Bovinos/virología , Enfermedades de los Bovinos/inmunología , Vacunas Virales/inmunología , Vacunas Virales/administración & dosificación , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Virus de la Enfermedad Hemorrágica Epizoótica/inmunología , Infecciones por Reoviridae/prevención & control , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/inmunología , Eficacia de las Vacunas , Vacunación/veterinariaRESUMEN
NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKß has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKß, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKß. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKß was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKß up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKß coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.
Asunto(s)
Secuencia de Aminoácidos , Carpas , Proteínas de Peces , Interleucina-8 , FN-kappa B , Proteínas Serina-Treonina Quinasas , Regulación hacia Arriba , Animales , Carpas/genética , Carpas/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Proteínas de Peces/química , FN-kappa B/genética , FN-kappa B/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Interleucina-8/inmunología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Enfermedades de los Peces/inmunología , Transducción de Señal , Reoviridae/fisiología , Filogenia , Quinasa de Factor Nuclear kappa B , Regulación de la Expresión Génica/inmunología , Poli I-C/farmacología , Lipopolisacáridos/farmacología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Alineación de Secuencia/veterinaria , Inmunidad Innata/genética , Secuencia de Bases , Perfilación de la Expresión Génica/veterinariaRESUMEN
Grass carp, one of the major freshwater aquaculture species in China, is susceptible to grass carp reovirus (GCRV). GCRV is a non-enveloped RNA virus and has a double-layered capsid, causing hemorrhagic disease and high mortalities in infected fish. However, the tropism of GCRV infection has not been investigated. In this study, monoclonal antibodies against recombinant VP35 protein were generated in mice and characterized. The antibodies exhibited specific binding to the N terminal region (1-155 aa) of the recombinant VP35 protein expressed in the HEK293 cells, and native VP35 protein in the GCRV-II infected CIK cells. Immunofluorescent staining revealed that viruses aggregated in the cytoplasm of infected cells. In vivo challenge experiments showed that high levels of GCRV-II viruses were present in the gills, intestine, spleen and liver, indicating that they are the major sites for virus infection. Our study showed that the VP35 antibodies generated in this study exhibited high specificity, and are valuable for the development of diagnostic tools for GCRV-II infection.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Carpas , Enfermedades de los Peces , Infecciones por Reoviridae , Reoviridae , Animales , Carpas/inmunología , Carpas/virología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/virología , Reoviridae/inmunología , Reoviridae/fisiología , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Ratones , Humanos , Células HEK293 , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Tropismo Viral , Proteínas de la Cápside/inmunología , Proteínas de la Cápside/metabolismo , Ratones Endogámicos BALB C , ChinaRESUMEN
Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type â IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.
Asunto(s)
Carpas , Enfermedades de los Peces , Interferón Tipo I , Interleucina-6 , Infecciones por Reoviridae , Reoviridae , Factor de Transcripción STAT3 , Transducción de Señal , Replicación Viral , Animales , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Interleucina-6/genética , Interleucina-6/inmunología , Interleucina-6/metabolismo , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Reoviridae/fisiología , Carpas/inmunología , Carpas/genética , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/inmunología , Transducción de Señal/inmunología , Interferón Tipo I/inmunología , Interferón Tipo I/genética , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Inmunidad Innata/genéticaRESUMEN
Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3'-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.
Asunto(s)
Autofagia , Carpas , Enfermedades de los Peces , MicroARNs , Proteínas Proto-Oncogénicas c-akt , Infecciones por Reoviridae , Reoviridae , Animales , MicroARNs/genética , MicroARNs/inmunología , Carpas/inmunología , Carpas/genética , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/veterinaria , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reoviridae/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Línea Celular , Regulación de la Expresión Génica/inmunologíaRESUMEN
Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous µ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV µ1 protein expression by shRNA could impair MRV proliferation. Specifically, µ1 protein inhibited MRV or poly(I:C)-induced IFN-ß expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that µ1 protein significantly decreased IFN-ß mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that µ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein µ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection.
Asunto(s)
Proteínas de la Cápside , Proteína 58 DEAD Box , Factor 3 Regulador del Interferón , Helicasa Inducida por Interferón IFIH1 , Orthoreovirus de los Mamíferos , Receptores Inmunológicos , Transducción de Señal , Animales , Humanos , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Proteína 58 DEAD Box/metabolismo , Células HEK293 , Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/metabolismo , Interferón beta/inmunología , Helicasa Inducida por Interferón IFIH1/metabolismo , Helicasa Inducida por Interferón IFIH1/genética , Orthoreovirus de los Mamíferos/inmunología , Orthoreovirus de los Mamíferos/fisiología , Fosforilación , Proteínas Serina-Treonina Quinasas , Infecciones por Reoviridae/inmunología , Transducción de Señal/inmunología , Proteínas Virales/metabolismo , Proteínas de la Cápside/metabolismoRESUMEN
Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.
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Encefalitis Viral , Neuronas , Infecciones por Reoviridae , Reoviridae , Animales , Ratones , Apoptosis/genética , Apoptosis/inmunología , Quimiocinas/inmunología , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Neuronas/inmunología , FN-kappa B/genética , FN-kappa B/metabolismo , Reoviridae/inmunología , Reoviridae/patogenicidad , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/virología , Interacciones Microbiota-Huesped/genética , Interacciones Microbiota-Huesped/inmunologíaRESUMEN
Mammalian orthoreovirus (reovirus) is a double-stranded RNA (dsRNA) virus which encapsidates its 10 genome segments within a double-layered viral particle. Reovirus infection triggers an antiviral response in host cells which serves to limit viral replication. This antiviral response is initiated by recognition of the incoming viral genome by host sensors present in the cytoplasm. However, how host sensors gain access to the reovirus genome is unclear, as this dsRNA is protected by the viral particle proteins throughout infection. To initiate infection, reovirus particles are endocytosed and the outer viral particle layer is disassembled through the action of host proteases. This disassembly event is required for viral escape into the cytoplasm to begin replication. We show that endosomal proteases are required even late in infection, when disassembly is complete, to induce an immune response to reovirus. Additionally, counter to dogma, our data demonstrate that at least some viral dsRNA genome is exposed and detectable during entry. We hypothesize that some proportion of reovirus particles remain trapped within endosomes, allowing for the breakdown of these particles and release of their genome. We show that rapidly uncoating mutants escape the endosome more rapidly and induce a diminished immune response. Further, we show that particles entering through dynamin-independent pathways evade detection by host sensors. Overall, our data provide new insight into how genomes from entering reovirus particles are detected by host cells. IMPORTANCE Viruses must infect host cells to replicate, often killing the host cell in the process. However, hosts can activate defenses to limit viral replication and protect the organism. To trigger these host defenses to viral infections, host cells must first recognize that they are infected. Mammalian orthoreovirus (reovirus) is a model system used to study host-virus interactions. This study identifies aspects of host and virus biology which determine the capacity of host cells to detect infection. Notably, entry of reovirus into host cells plays a critical role in determining the magnitude of immune response triggered during infection. Mutants of reovirus which can enter cells more rapidly are better at avoiding detection by the host. Additionally, reovirus can enter cells through multiple routes. Entry through some of these routes also helps reovirus evade detection.
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Inmunidad Innata , Infecciones por Reoviridae , Reoviridae , Animales , Factores de Restricción Antivirales/inmunología , Línea Celular , Orthoreovirus de los Mamíferos , Péptido Hidrolasas , ARN Bicatenario/genética , Reoviridae/fisiología , Infecciones por Reoviridae/inmunología , Proteínas Virales , Replicación ViralRESUMEN
Grass carp reovirus (GCRV) is a highly virulent RNA virus that mainly infects grass carp and causes hemorrhagic disease. The roles of nonstructural proteins NS38 and NS80 of GCRV-873 in the viral replication cycle and viral inclusion bodies have been established. However, the strategies that NS38 and NS80 used to avoid host antiviral immune response are still unknown. In this study, we report the negative regulations of NS38 and NS80 on the RIG-I-like receptors (RLRs) antiviral signaling pathway and the production of IFNs and IFN-stimulated genes. First, both in the case of overexpression and GCRV infection, NS38 and NS80 inhibited the IFN promoter activation induced by RIG-I, MDA5, MAVS, TBK1, IRF3, and IRF7 and mRNA abundance of key antiviral genes involved in the RLR-mediated signaling. Second, both in the case of overexpression and GCRV infection, NS38 interacted with piscine TBK1 and IRF3, but not with piscine RIG-I, MDA5, MAVS, and TNF receptor-associated factor (TRAF) 3. Whereas NS80 interacted with piscine MAVS, TRAF3, and TBK1, but not with piscine RIG-I, MDA5, and IRF3. Finally, both in the case of overexpression and GCRV infection, NS38 inhibited the formation of the TBK1-IRF3 complex, but NS80 inhibited the formation of the TBK1-TRAF3 complex. Most importantly, NS38 and NS80 could hijack piscine TBK1 and IRF3 into the cytoplasmic viral inclusion bodies and inhibit the translocation of IRF3 into the nucleus. Collectively, all of these data demonstrate that GCRV nonstructural proteins can avoid host antiviral immune response by targeting the RLR signaling pathway, which prevents IFN-stimulated gene production and facilitates GCRV replication.
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Carpas/virología , ARN Helicasas DEAD-box/metabolismo , Evasión Inmune/inmunología , Infecciones por Reoviridae/veterinaria , Reoviridae/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Células Cultivadas , Enfermedades de los Peces/inmunología , Enfermedades de los Peces/virología , Factores Reguladores del Interferón/metabolismo , Interferones/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/patología , Factor 3 Asociado a Receptor de TNF/metabolismo , Replicación Viral/fisiologíaRESUMEN
It has been shown that Muscovy duck reovirus (MDRV) infection causes severe intestinal barrier damage and intestinal mucosal immune suppression. The health and balance of gut microbes is essential for the progression of intestinal infectious diseases. To investigate the interaction of MDRV, intestinal bacteria with host intestinal innate immunity, an MDRV contact-infection model was established in this study. High-throughput sequencing technology was used to sequence 16S rDNA and transcripts in ileal samples from experimental Muscovy ducklings. Our results suggest that intestinal opportunistic pathogens such as Streptococcus and Corynebacterium proliferated massively in MDRV-infected Muscovy ducklings. The body initiates antiviral and antibacterial immunity and actively fights the infection of gut microbes. The synthesis of peptidoglycan, lipopolysaccharide, and flagellin by intestinal bacteria activates the Toll-like receptor signaling pathway resulting in increased secretion of IFN-ß, IL-1ß, and IL-8. The RIG-I-like receptor signaling pathway is an important signaling pathway for the interaction between MDRV and the host. At the same time, we also observed that multiple genes in the JAK-STAT signaling pathway were significantly different. These genes are important targets for studying the immunosuppression caused by MDRV. In conclusion, we analyzed the interaction of MDRV, intestinal flora and host immune system during MDRV infection, which provides a basis for the further study on the mechanism of intestinal immunosuppression caused by MDRV.
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Patos , Microbioma Gastrointestinal , Interacciones Microbiota-Huesped , Inmunidad Innata , Infecciones por Reoviridae , Animales , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/inmunología , Interacciones Microbiota-Huesped/inmunología , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Reoviridae/fisiología , Infecciones por Reoviridae/inmunología , Infecciones por Reoviridae/microbiología , Infecciones por Reoviridae/veterinaria , Infecciones por Reoviridae/virología , TranscriptomaRESUMEN
BACKGROUND: Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. METHODS: Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. RESULTS: Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoides jacobsoni was identified as TIBOV, while the other two viruses from C. orientalis and C. tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. CONCLUSIONS: The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known.
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Ceratopogonidae/virología , Insectos Vectores/virología , Orbivirus/genética , Orbivirus/aislamiento & purificación , Infecciones por Reoviridae/transmisión , Animales , Anticuerpos Antivirales/sangre , Bovinos , Ceratopogonidae/clasificación , China , Femenino , Orbivirus/inmunología , Filogenia , ARN Viral/genética , Infecciones por Reoviridae/inmunología , TibetRESUMEN
Cyclic GMP-AMP synthase (cGAS) is known as a DNA sensor for the initiation of innate immune responses in human and other mammals. However, the knowledge about fish cGAS is limited. In this study, we identified two paralogs of cGAS genes from grass carp (Ctenopharyngodon idellus), namely, CicGASa and CicGASb. Grass carp cGASa and cGASb share some conservative domains with mammalian cGASs; however, cGASb contains a unique transmembrane domain. Grass carp cGASa and cGASb responded to GCRV and poly (dA:dT) infection, but they played opposite roles in the regulation of type I IFN response, i.e. cGASa served as an activator for ISGs and NF-κB in a dose-dependent manner, while cGASb acted as an inhibitor. We found that cGASa and cGASb interacted with STING. Similarly, cGASa is an activator for IRF7, but cGASb inhibited IRF7 expression. Both cGASa and STING can protect cells from GCRV infection. Grass carp cGASb inhibited cGASa-induced type I IFN response by the competitive interaction with STING, suggesting that cGASb may be a negative regulator of cGASa-STING-IRF7 axis.