ABSTRACT
A plethora of bat-associated lyssaviruses potentially capable of causing the fatal disease rabies are known today. Transmitted via infectious saliva, occasionally-reported spillover infections from bats to other mammals demonstrate the permeability of the species-barrier and highlight the zoonotic potential of bat-related lyssaviruses. However, it is still unknown whether and, if so, to what extent, viruses from different lyssavirus species vary in their pathogenic potential. In order to characterize and systematically compare a broader group of lyssavirus isolates for their viral replication kinetics, pathogenicity, and virus release through saliva-associated virus shedding, we used a mouse infection model comprising a low (102 TCID50) and a high (105 TCID50) inoculation dose as well as three different inoculation routes (intramuscular, intranasal, intracranial). Clinical signs, incubation periods, and survival were investigated. Based on the latter two parameters, a novel pathogenicity matrix was introduced to classify lyssavirus isolates. Using a total of 13 isolates from ten different virus species, this pathogenicity index varied within and between virus species. Interestingly, Irkut virus (IRKV) and Bokeloh bat lyssavirus (BBLV) obtained higher pathogenicity scores (1.14 for IRKV and 1.06 for BBLV) compared to rabies virus (RABV) isolates ranging between 0.19 and 0.85. Also, clinical signs differed significantly between RABV and other bat lyssaviruses. Altogether, our findings suggest a high diversity among lyssavirus isolates concerning survival, incubation period, and clinical signs. Virus shedding significantly differed between RABVs and other lyssaviruses. Our results demonstrated that active shedding of infectious virus was exclusively associated with two RABV isolates (92% for RABV-DogA and 67% for RABV-Insectbat), thus providing a potential explanation as to why sustained spillovers are solely attributed to RABVs. Interestingly, 3D imaging of a selected panel of brain samples from bat-associated lyssaviruses demonstrated a significantly increased percentage of infected astrocytes in mice inoculated with IRKV (10.03%; SD±7.39) compared to RABV-Vampbat (2.23%; SD±2.4), and BBLV (0.78%; SD±1.51), while only individual infected cells were identified in mice infected with Duvenhage virus (DUVV). These results corroborate previous studies on RABV that suggest a role of astrocyte infection in the pathogenicity of lyssaviruses.
Subject(s)
Chiroptera/virology , Lyssavirus/genetics , Lyssavirus/pathogenicity , Rhabdoviridae Infections/virology , Animals , Astrocytes/virology , Genome, Viral , Mice , Mice, Inbred BALB C , RNA, Viral , Random Allocation , Rhabdoviridae Infections/pathology , Virus Cultivation , Virus Replication , Virus SheddingABSTRACT
Due to the lack of relevant therapies for infectious haematopoietic necrosis virus (IHNV) infection, the viral outbreak invariably causes serious economic losses in salmonid species. In this study, we evaluated the anti-IHNV effects of 7-(6-benzimidazole) coumarin (C10) and 4-phenyl-2-thioxo-1,2,3,4-tetrahydro-5H-chromeno[4,3-d]pyrimidin-5-one (S5) in vitro and in vivo. The results revealed that C10 at 12.5 mg/L and S5 at 25 mg/L significantly inhibited IHNV replication in epithelioma papulosum cyprini (EPC) cells with a maximum inhibitory rate >90%, showing that IHNV-induced cytopathic effect (CPE) was alleviated by C10 and S5. There are two complementary effects on antiviral mechanism: 1. C10 completely inhibited IHNV infectivity when the virus was preincubated with C10 at 12.5 mg/L, determining that C10 may have a negative impact on IHNV binding to the cell; 2. C10 also up-regulated the gene expression of extracellular proto type galectin-1 (Gal1-L2) and a chimera galectin-3 (Gal3-L1) of EPC cells to inhibit IHNV adhesion. For the in vivo study, injection and immersion of the coumarins enhanced the survival rate of rainbow trout (Oncorhynchus mykiss) juveniles by 25% (at least) at 12 dpi. IHNV loads in the kidney and spleen were also obviously decreased at 96 h, and thus we considered that they had a delaying effect on IHNV replication in vivo. Meanwhile, C10 with a high stability in aquacultural water in immersion suppressed IHNV horizontal transmission by decreasing the viral loads in recipient fish. Overall, our data suggest that there is a positive effect of C10 and S5 against IHNV infection in aquaculture, and C10 had the potential to be a broad-spectrum antiviral against fish rhabdoviruses.
Subject(s)
Antiviral Agents/pharmacology , Coumarins/pharmacology , Infectious hematopoietic necrosis virus/drug effects , Virus Attachment/drug effects , Virus Replication/drug effects , Animals , Aquaculture , Cell Line , Fish Diseases/drug therapy , Fish Diseases/mortality , Fish Diseases/pathology , Oncorhynchus mykiss/virology , Rhabdoviridae Infections/drug therapy , Rhabdoviridae Infections/mortality , Rhabdoviridae Infections/pathology , Survival Rate , Viral Load/drug effects , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
Autophagy and apoptosis are two key cell fate determination pathways, which play vital roles in the interaction between viruses and host cells. Previous research had confirmed that one strain of fish rhabdoviruses, Siniperca chuatsi rhabdovirus (SCRV), could induce apoptosis and autophagy after infection. In the current study, we continued to analyze the interaction of autophagy and apoptosis in SCRV-infected EPC cell lines after treatment with different autophagy or apoptosis inhibitors. We found that SCRV infection could activate the mitochondrial apoptotic pathway by the detection of the activities of the caspase-3 and caspase-9 and by flow cytometry analysis in JC-1-stained cells, respectively. Furthermore, no significant autophagy-related factors were disturbed in SCRV-infected cell after apoptosis inhibitor Z-VAD-FMK treatment, while autophagy inducer rapamycin could obviously delay the occurrence of CPE and cell death. Meanwhile, rapamycin was able to reduce the proportion of apoptotic cells. Besides that, rapamycin could disturb the expression of p62 and LC3B-II, and the transcription level of SCRV nucleoprotein mRNA. The progeny virus titers did not show a big difference between the rapamycin treatment or without it. Collectively, our data preliminarily confirmed that SCRV-activated autophagy could delay apoptosis in EPC cells and may not affect virus production. Further study may need to focus on the crosstalk regulation and its roles on the SCRV infection.
Subject(s)
Autophagy/physiology , Carcinoma/veterinary , Carcinoma/virology , Cyprinidae/virology , Rhabdoviridae Infections/veterinary , Rhabdoviridae/pathogenicity , Animals , Apoptosis/genetics , Apoptosis/physiology , Autophagy/genetics , Caspases/genetics , Cell Line , Fish Diseases/virology , Flow Cytometry , Rhabdoviridae Infections/pathology , Virus ReplicationABSTRACT
Diseases caused by rhabdoviruses have had a huge impact on the productive lives of the entire human population. The main problem is the lack of drugs for the treatment of this family of viruses. Infectious hematopoietic necrosis virus (IHNV), the causative agent of IHN, is a typical rhabdovirus which has caused huge losses to the salmonid industry. Therefore, in this study, IHNV was studied as a model to evaluate the antiviral activity of 35 novel coumarin derivatives. Coumarin A9 was specifically selected for further validation studies upon comparing the half maximum inhibitory concentration (IC50) of four screened candidate derivatives in epithelioma papulosum cyprinid (EPC) cells, as it exhibited an IC50 value of 2.96 µM against IHNV. The data revealed that A9 treatment significantly suppressed the virus-induced cytopathic effect (CPE) in EPC cells. In addition, A9 showed IC50 values of 1.68 and 2.12 µM for two other rhabdoviruses, spring viremia of carp virus and micropterus salmoides rhabdovirus, respectively. Furthermore, our results suggest that A9 exerts antiviral activity, but not by destroying the virus particles and interfering with the adsorption of IHNV. Moreover, we found that A9 had an inhibitory effect on IHNV-induced apoptosis in EPC cells, as reflected by the protection against cell swelling, formation of apoptotic bodies, and loss of cell morphology and nuclear division. There was a 19.05 % reduction in the number of apoptotic cells in the A9 treatment group compared with that in the IHNV group. In addition, enzyme activity assays proved that A9 suppressed the expression of caspase 3, 8 and 9. These results suggested that A9 inhibit viral replication, to some extent, by blocking IHNV-induced apoptosis. In an in vivo study, A9 exhibited an anti-rhabdovirus effect in virus-infected fish by substantially enhancing the survival rate. Consistent with the above results, A9 repressed IHNV gene expression in virus-sensitive tissues (brain, kidney and spleen) in the early stages of virus infection. Importantly, the data showed that horizontal transmission of IHNV was reduced by A9 in a static cohabitation challenge model, especially in fish that underwent bath treatment, suggesting that A9 might be a suitable therapeutic agent for IHNV in aquaculture. Therefore, coumarin derivatives can be developed as antiviral agents against rhabdoviruses.
Subject(s)
Antiviral Agents/chemical synthesis , Coumarins/chemistry , Rhabdoviridae/drug effects , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Apoptosis/drug effects , Cell Line , Coumarins/pharmacology , Coumarins/therapeutic use , Down-Regulation/drug effects , Fish Diseases/drug therapy , Fish Diseases/mortality , Fish Diseases/pathology , Humans , Oncorhynchus mykiss/metabolism , Oncorhynchus mykiss/virology , Rhabdoviridae Infections/drug therapy , Rhabdoviridae Infections/mortality , Rhabdoviridae Infections/pathology , Structure-Activity Relationship , Survival Rate , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Internalization/drug effectsABSTRACT
Viral encephalitis initiates a series of immunological events in the brain that can lead to brain damage and death. Astrocytes express IFN-ß in response to neurotropic infection, whereas activated microglia produce proinflammatory cytokines and accumulate at sites of infection. Here, we observed that neurotropic vesicular stomatitis virus (VSV) infection causes recruitment of leukocytes into the central nervous system (CNS), which requires MyD88, an adaptor of Toll-like receptor and interleukin-1 receptor signaling. Infiltrating leukocytes, and in particular CD8+ T cells, protected against lethal VSV infection of the CNS. Reconstitution of MyD88, specifically in neurons, restored chemokine production in the olfactory bulb as well as leukocyte recruitment into the infected CNS and enhanced survival. Comparative analysis of the translatome of neurons and astrocytes verified neurons as the critical source of chemokines, which regulated leukocyte infiltration of the infected brain and affected survival.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Chemokines/metabolism , Encephalitis, Viral/immunology , Myeloid Differentiation Factor 88/metabolism , Rhabdoviridae Infections/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Disease Models, Animal , Encephalitis, Viral/pathology , Encephalitis, Viral/virology , Female , Humans , Male , Mice , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Neurons/metabolism , Olfactory Bulb/cytology , Olfactory Bulb/immunology , Olfactory Bulb/pathology , Olfactory Bulb/virology , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Signal Transduction/immunology , Vesiculovirus/immunologyABSTRACT
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).
Subject(s)
Herbicides/toxicity , Immunity, Cellular/drug effects , Immunity, Humoral/drug effects , Infectious hematopoietic necrosis virus/physiology , Oncorhynchus mykiss/genetics , Oncorhynchus mykiss/immunology , Rhabdoviridae Infections/immunology , Rhabdoviridae Infections/veterinary , Aniline Compounds/toxicity , Animals , Fish Diseases/genetics , Fish Diseases/immunology , Fish Diseases/virology , Gene Expression Regulation/drug effects , Oncorhynchus mykiss/virology , Rhabdoviridae Infections/genetics , Rhabdoviridae Infections/pathology , Water Pollutants, Chemical/toxicityABSTRACT
An adult male harbour porpoise (Phocoena phocoena) stranded off the coast of Alaska displaying poor body condition, scattered mild ulcerative dermatitis, and necrotizing balanoposthitis. Necropsy findings included severe verminous panniculitis, pneumonia, hepatitis, and enteritis. Histopathological examination of skin lesions revealed a pustular epidermitis and dermatitis, with ballooning degeneration of keratinocytes and occasional amphophilic intracytoplasmic inclusion bodies. A swab sample collected from the ulcerative penile lesions was processed for virus isolation resulting in cytopathic effects observed in primary beluga whale kidney (BWK) cells. Transmission electron microscopy revealed bullet-shaped virions budding from the cell surface of infected BWK cells consistent with a rhabdovirus. A cDNA library was prepared using RNA extracted from infected cell culture supernatant and sequenced on an Illumina MiSeq sequencer. The near-complete genome of a novel rhabdovirus was recovered. Genetic and phylogenetic analyses based on the complete L gene supported the harbour porpoise rhabdovirus (HPRV) as a new species. HPRV clustered phylogenetically with dolphin rhabdovirus (DRV) and this cetacean rhabdovirus clade was found to be the sister group to members of the genus Perhabdovirus that infect fish. A specific nested RT-PCR assay detected HPRV RNA in the epaxial musculature of the harbour porpoise. Our results are consistent with a previous hypothesis that cetacean rhabdoviruses may have arisen following a host jump from fish and suggest that DRV and HPRV represent separate species belonging in a new genus within the family Rhabdoviridae. Further research is needed to determine the health impact of HPRV in harbour porpoise populations, its prevalence, and route of transmission.
Subject(s)
Phocoena/virology , Phylogeny , Rhabdoviridae Infections/veterinary , Rhabdoviridae/classification , Rhabdoviridae/isolation & purification , Alaska , Animals , Autopsy , Cells, Cultured , Genome, Viral , Kidney/cytology , Male , Rhabdoviridae/pathogenicity , Rhabdoviridae Infections/pathology , WhalesABSTRACT
Neddylation is a type of post-translational protein modifications, in which neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) is covalently conjugated to the lysine residues of target substrates. The best characterized principal substrates of neddylation are the cullin-RING ligases (CRLs). In addition, neddylation also modifies non-cullin proteins to affect gene regulation, cell survival, organ development, and stress response. However, the role of neddylation in antiviral innate immunity remain largely unknown. Here, we found that when neddylation was blocked by the NEDD8 activating enzyme E1 (NAE) inhibitor, MLN4924, the cellular and organismal antiviral response was suppressed. Moreover, the disruption of nedd8 increased the sensitivity of zebrafish to SVCV infection. Further assays indicated that blocking or silencing neddylation significantly downregulated key antiviral genes after poly (I:C) stimulation or SVCV infection, but dramatically increased SVCV replication. Neddylation of Irf3 and Irf7 was readily detected, but not of Mda5, Mavs, and Tbk1. Thus, our results not only demonstrated that neddylation facilitated the antiviral response in vitro and in vivo, but also revealed a novel role of nedd8 in antiviral innate immunity.
Subject(s)
Fish Diseases/immunology , NEDD8 Protein/immunology , Rhabdoviridae Infections/immunology , Rhabdoviridae/immunology , Signal Transduction/immunology , Ubiquitination/immunology , Zebrafish Proteins/immunology , Zebrafish , Animals , Cell Line , Cyclopentanes/pharmacology , Fish Diseases/pathology , Pyrimidines/pharmacology , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/veterinary , Signal Transduction/drug effects , Ubiquitination/drug effects , Zebrafish/immunology , Zebrafish/virologyABSTRACT
Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-κB heterodimers, which induce genes encoding interferon beta (IFN-ß) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN-ß in response to virus infections. Unexpectedly, infection of Rela-/- fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes.IMPORTANCE RelA/NF-κB participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFN-ß, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-κB signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-κB activities while alleviating the viral load.
Subject(s)
Embryo, Mammalian/metabolism , Fibroblasts/metabolism , Host-Pathogen Interactions , Rhabdoviridae Infections/metabolism , Transcription Factor RelA/metabolism , Vesiculovirus/physiology , 3T3 Cells , Animals , Cell Line , Chlorocebus aethiops , Embryo, Mammalian/pathology , Embryo, Mammalian/virology , Fibroblasts/pathology , Fibroblasts/virology , Mice , Rhabdoviridae Infections/pathology , Vero CellsABSTRACT
Vesicular stomatitis virus (VSV) is an insect-transmitted rhabdovirus that is neurovirulent in mice. Upon peripheral VSV infection, CD169+ subcapsular sinus (SCS) macrophages capture VSV in the lymph, support viral replication, and prevent CNS neuroinvasion. To date, the precise mechanisms controlling VSV infection in SCS macrophages remain incompletely understood. Here, we show that Toll-like receptor-7 (TLR7), the main sensing receptor for VSV, is central in controlling lymph-borne VSV infection. Following VSV skin infection, TLR7-/- mice display significantly less VSV titers in the draining lymph nodes (dLN) and viral replication is attenuated in SCS macrophages. In contrast to effects of TLR7 in impeding VSV replication in the dLN, TLR7-/- mice present elevated viral load in the brain and spinal cord highlighting their susceptibility to VSV neuroinvasion. By generating novel TLR7 floxed mice, we interrogate the impact of cell-specific TLR7 function in anti-viral immunity after VSV skin infection. Our data suggests that TLR7 signaling in SCS macrophages supports VSV replication in these cells, increasing LN infection and may account for the delayed onset of VSV-induced neurovirulence observed in TLR7-/- mice. Overall, we identify TLR7 as a novel and essential host factor that critically controls anti-viral immunity to VSV. Furthermore, the novel mouse model generated in our study will be of valuable importance to shed light on cell-intrinsic TLR7 biology in future studies.
Subject(s)
Macrophages/immunology , Membrane Glycoproteins/immunology , Rhabdoviridae Infections/immunology , Sialic Acid Binding Ig-like Lectin 1/immunology , Toll-Like Receptor 7/immunology , Vesiculovirus/physiology , Virus Replication/immunology , Animals , Brain/immunology , Brain/virology , Macrophages/virology , Membrane Glycoproteins/genetics , Mice , Mice, Knockout , Rhabdoviridae Infections/genetics , Rhabdoviridae Infections/pathology , Sialic Acid Binding Ig-like Lectin 1/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Spinal Cord/immunology , Spinal Cord/virology , Toll-Like Receptor 7/genetics , Virus Replication/geneticsABSTRACT
Reverse genetics systems are powerful tools for understanding the virulence mechanisms and gene functions of negative-sense RNA viruses. The reverse genetics systems commonly used for recombinant infectious hematopoietic necrosis virus (IHNV) are based on vaccinia virus infection. To avoid the potential biological safety risks associated with vaccinia virus, a recombinant IHNV virus strain Sn1203 (rIHNV-Sn1203) was rescued in this study using a mammalian cell line, BHK-21. The genome sequence authenticity of rIHNV-Sn1203 was confirmed using two silent genetic tags introduced by site-directed mutagenesis. Indirect immunofluorescence assays and transmission electron microscopy revealed that rIHNV-Sn1203 and wild-type IHNV-Sn1203 (wtIHNV-Sn1203) had identical immunogenicity and virion morphology. The virulence and pathogenicity of rIHNV-Sn1203 were assessed in vitro and in vivo. Although rIHNV-Sn1203 displayed trends toward delayed intracellular viral replication and lower virion yields compared with wtIHNV-Sn1203, statistical analyses revealed no significant differences between these two viruses. Moreover, rainbow trout challenged with rIHNV-Sn1203 and wtIHNV-Sn1203 showed indistinguishable mortality. Together, these results show that IHNV was successfully rescued using BHK-21 cells. This method is very convenient and may also be suitable for use in the recovery of other Novirhabdoviruses.
Subject(s)
Infectious hematopoietic necrosis virus/growth & development , Reverse Genetics/methods , Virology/methods , Animals , Cell Line , Cricetinae , Fish Diseases/pathology , Fish Diseases/virology , Fluorescent Antibody Technique, Indirect , Infectious hematopoietic necrosis virus/genetics , Infectious hematopoietic necrosis virus/pathogenicity , Infectious hematopoietic necrosis virus/ultrastructure , Microscopy, Electron, Transmission , Oncorhynchus mykiss , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/veterinary , Rhabdoviridae Infections/virology , Survival Analysis , Vaccinia virus/genetics , Virion/ultrastructure , Virus ReplicationABSTRACT
Among the most important sensors of extracellular danger signals, purinergic receptors have been demonstrated to play crucial roles in host defense against infection. However, the function of P2 receptors in viral infection has been little explored. Here we demonstrated that P2Y13 and its ligand ADP play an important role in protecting hosts from viral infections. First, we demonstrate that P2Y13, as a typical interferon-stimulated gene, is induced together with extracellular ADP during viral infection. Most importantly, extracellular ADP restricts the replication of different kinds of viruses, including vesicular stomatitis virus, Newcastle disease virus, herpes simplex virus 1, and murine leukemia virus. This kind of protection is dependent on P2Y13 but not P2Y1 or P2Y12, which are also considered as receptors for ADP. Furthermore, cyclic adenosine monophosphate and EPAC1 are downregulated by extracellular ADP through the P2Y13-coupled Gi alpha subunit. Accordingly, inhibition or deletion of EPAC1 significantly eliminates ADP/P2Y13-mediated antiviral activities. Taken together, our results show that P2Y13 and ADP play pivotal roles in the clearance of invaded virus and have the potential as antiviral targets.
Subject(s)
Cyclic AMP/metabolism , Gene Expression/drug effects , Guanine Nucleotide Exchange Factors/metabolism , Interferons/pharmacology , Receptors, Purinergic P2/metabolism , Adenosine Diphosphate/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Guanine Nucleotide Exchange Factors/deficiency , Guanine Nucleotide Exchange Factors/genetics , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Purinergic P2/deficiency , Receptors, Purinergic P2/genetics , Rhabdoviridae Infections/mortality , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/prevention & control , Rhabdoviridae Infections/veterinary , Signal Transduction/drug effects , Survival Rate , Vesiculovirus/genetics , Vesiculovirus/pathogenicity , Virus Replication/drug effectsABSTRACT
The innate immune system plays a critical role in the initial antiviral response. However, the timing and duration of these responses must be tightly regulated during infection to ensure appropriate immune cell activation and anti-viral defenses. Here we demonstrate that during antiviral response, a negative regulator miR-221 was also induced in an ELF4-dependent manner. We further show that ELF4 promotes miR-221 expression through direct binding to its promoter. Overexpression and knockdown assay show that miR-221 can negatively regulate IFNß production in time of virus infection. RNA-seq analysis of miR-221 overexpressed cells revealed multiple candidate targets. Taken together, our study identified a novel negative microRNA regulator of innate antiviral response, which is dependent on ELF4.
Subject(s)
Immunity, Innate/genetics , MicroRNAs/metabolism , Rhabdoviridae Infections/immunology , Animals , Antagomirs/metabolism , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Down-Regulation , HEK293 Cells , Herpesvirus 1, Human/physiology , Humans , Interferon-beta/genetics , Interferon-beta/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/virology , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Promoter Regions, Genetic , Protein Serine-Threonine Kinases/genetics , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Transcription Factors/deficiency , Transcription Factors/genetics , Vesiculovirus/genetics , Vesiculovirus/immunologyABSTRACT
A batch of wild common carp and largemouth bass died in Andong, Gyeongsangbuk-do province, South Korea, in 2016. Moribund fish showed typical signs of spring viremia of carp (SVC) disease, which causes acute hemorrhage in the skin and ascites. Thus far, SVC disease has been detected in several regions of the world but never in South Korea. Suspecting the infectious agent to be the SCV virus (SVCV), the moribund fish were sampled and screened. The isolated virus developed a cytopathic effect in EPC cells. Both viral isolates from the common carp (ADC-SVC2016-1) and largemouth bass (ADC-SVC2016-3) were identical in terms of their genome sequence, which were 11,034 bp nucleotides in length. Genome comparison exhibited greater sequence similarity with the Asian SVCV sequences available at NCBI. Phylogenetic analysis revealed that the Korean SVCV isolates were clustered within the Asian clade. More specifically, evolutionary analysis by using the P gene sequences showed that the Korean isolates were sub-cladded within the Iai genogroup but diverged from Chinese strains of SH150514 and SH160901. The Korean isolates shared more than 98% sequence similarity with the two Chinese SVCV isolates, suggesting that the spread of SVCV originated from China. The isolated virus had cytopathic effects on EPC cells. Virus transmission studies showed that the virus exhibited the highest virulence at 15 °C, which was also dependent on the method used, with the injection method being better than the immersion and cohabitation methods. This is the first study to document that Korean SVCV isolates may be epizootic in wild common carp and other susceptible animal populations in South Korea.
Subject(s)
Bass/virology , Carps/virology , Fish Diseases/virology , Genome , Phylogeny , Rhabdoviridae Infections/veterinary , Rhabdoviridae , Animals , Base Sequence , Fish Diseases/epidemiology , Fish Diseases/pathology , Genotype , Molecular Sequence Data , Republic of Korea/epidemiology , Rhabdoviridae/classification , Rhabdoviridae/genetics , Rhabdoviridae/pathogenicity , Rhabdoviridae Infections/epidemiology , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Sequence Analysis, DNA , VirulenceABSTRACT
As one of nine piscine viruses recognized by the International Office of Epizootics, spring viraemia of carp virus (SVCV) is an important pathogen bringing high mortality to cyprinids. Up to now, there is no approved therapy on SVCV, making them strong public health threat in aquaculture. In this study, the anti-SVCV activities of 12 plant crude extracts were investigated by using epithelioma papulosum cyprini (EPC) cells. Among these plants, Psoralea corylifolia Linn. showed the highest inhibition on SVCV replication, with an inhibitory percentage of 67.98%. Further studies demonstrated that bavachin (BVN), one of the major constituents of Psoralea corylifolia Linn., was also highly effective to SVCV infection. The half maximal inhibitory concentrations (IC50) of BVN on SVCV glycoprotein and nucleoprotein expression were 0.46 (0.29-0.73) and 0.31 (0.13-0.55) mg/L, respectively. In addition, SVCV-induced apoptosis which may be negative to SVCV replication was inhibited by BVN. The apoptotic cells were decreased 21.42% for BVN compared with SVCV group. These results indicated that the inhibition of BVN on SVCV replication was, in some extent, via blocking SVCV induced apoptosis. Furthermore, cellular morphological damage induced by SVCV was also blocked by BVN treatment. Mechanistically, BVN did not affect SVCV infectivity and cannot be used for prevention of SVCV infection. Time-of-addition and viral binding assays revealed that BVN mainly inhibited the early events of SVCV replication but did not interfere with SVCV adsorption. In conclusion, BVN was considered to develop as a promising agent to treat SVCV infection.
Subject(s)
Carps/virology , Fish Diseases/virology , Flavonoids/pharmacology , Psoralea/chemistry , Rhabdoviridae Infections/veterinary , Rhabdoviridae/drug effects , Animals , Apoptosis/drug effects , Cell Line , Cell Survival/drug effects , Down-Regulation/drug effects , Down-Regulation/genetics , Fish Diseases/pathology , Flavonoids/isolation & purification , Inhibitory Concentration 50 , Plants, Medicinal/chemistry , Rhabdoviridae/physiology , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Viral Load/drug effects , Viral Proteins/genetics , Virus Replication/drug effectsABSTRACT
Spring viremia of carp virus (SVCV) is a notifiable pathogen of the World Organization of Animal Health. Since SVCV was isolated in Lake Ontario in 2007, concern has grown about its spread in the Great Lakes basin and its potential negative impacts on fish species of importance in stock enhancement programs basinwide. The susceptibility of representative fish species from the families Cyprinidae (Fathead Minnow Pimephales promelas, Golden Shiner Notemigonus crysoleucas, Spotfin Shiner Cyprinella spiloptera, and Creek Chub Semotilus atromaculatus), Centrarchidae (Largemouth Bass Micropterus salmoides), Percidae (Walleye Sander vitreus), Salmonidae (Rainbow Trout Oncorhynchus mykiss), and Esocidae (Muskellunge Esox masquinongy) to SVCV was evaluated by experimental infection under laboratory conditions. Morbidity and mortality were recorded, and virus re-isolation, seminested reverse transcription PCR, and histopathological assessments were performed. Using intraperitoneal (i.p.) injection, Fathead Minnows and Golden Shiners were highly susceptible to SVCV (40-70% mortality). All dead or moribund and apparently healthy surviving Fathead Minnows and Golden Shiners were SVCV positive. The SVCV was also detected in challenged but healthy Spotfin Shiners (30%) and Creek Chub (5%). However, noncyprinid species exhibited no morbidity or mortality and were free of SVCV following an observation period of 30 d. In a follow-up experimental challenge, Fathead Minnows and Golden Shiners were SVCV challenged at 103 and 105 PFU/mL by means of waterborne immersion. After immersion, Fathead Minnows and Golden Shiners exhibited characteristic SVCV disease signs, but mortality was less (30% and 10% mortality, respectively) than that in fish with i.p. injections. The SVCV was detected in all mortalities and a subset of healthy Fathead Minnows and Golden Shiners. Necrotic changes were observed in the kidneys, liver, spleen, ovaries, and heart, and other histopathological lesions also occurred. These findings suggest that two of the four cyprinids tested are susceptible to SVCV-induced disease and that all four can act as potential carriers of SVCV in the Laurentian Great Lakes. Received January 11, 2017; accepted July 17, 2017.
Subject(s)
Fish Diseases/mortality , Fish Diseases/virology , Fishes , Rhabdoviridae Infections/veterinary , Rhabdoviridae/physiology , Animals , Fish Diseases/pathology , Great Lakes Region , Rhabdoviridae/classification , Rhabdoviridae Infections/mortality , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Species SpecificityABSTRACT
Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.
Subject(s)
Eels/virology , Fish Diseases/pathology , Fish Diseases/virology , Rhabdoviridae Infections/veterinary , Vesiculovirus/pathogenicity , Animal Structures/pathology , Animal Structures/virology , Animals , China , Computational Biology , Histocytochemistry , Immunohistochemistry , In Situ Hybridization , RNA, Viral/analysis , Reverse Transcriptase Polymerase Chain Reaction , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Sequence Homology, Nucleic Acid , Vesiculovirus/genetics , Vesiculovirus/isolation & purification , Viral Proteins/analysis , Whole Genome SequencingABSTRACT
Spring viraemia of carp virus (SVCV) is an OIE-listed notifiable pathogen, which has brought huge economic loss to the aquaculture industry. Outbreaks of SVC mostly occur in spring with water temperature 11-17°C. Presently, there is an increase in detection during import quarantine testing and associated with outbreaks of SVCV outside of China, yet China is regarded as the origin of SVCV Asian clade. However, recent isolates from the Shanghai area all showed to be low pathogenic to their original hosts. In this study, we isolated a new SVCV strain (nominated as SH160901) from grass carp in late summer in Shanghai, 2016. Phylogenetic analysis showed this strain formed a distinct new lineage in the Asian clade along with our isolate SH150514 in 2015, and was divergent from all other identified Asian isolates. Cell infection test demonstrated that this strain replicated most efficiently at 25°C and 28°C, and could induce obvious cytopathic effect in infected cells. In vivo infection test revealed this strain could cause severe symptoms in experimentally infected fish at 16-20°C. Inoculated fish died at 100% in grass carp and common carp (Cyprinus carpio carpio) within 13days, and at 100% in goldfish (Carassius auratus) and 90% in koi (Cyprinus carpio koi) within 40days. Experimental infections at 26°C also induced moderate mortalities in grass carp (25%) and common carp (20%). The biological changes characterized for SVCV isolate SH160901 warrant changes to surveillance plans, specifically there is a need to broaden the testing parameters previously associated with SVCV.
Subject(s)
Carps/virology , Rhabdoviridae Infections/veterinary , Rhabdoviridae/isolation & purification , Rhabdoviridae/pathogenicity , Animals , China , Cytopathogenic Effect, Viral , Genotype , Phylogeny , Rhabdoviridae/classification , Rhabdoviridae/genetics , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Seasons , Survival Analysis , Temperature , Virus Cultivation , Virus ReplicationABSTRACT
Novirhabdoviruses like the Viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses infecting fish. In the current study, RNA genomes of different VHSV field isolates classified as high, medium or low virulent phenotypes have been sequenced by next-generation sequencing and compared. Various amino acid changes, depending on the VHSV phenotype, have been identified in all the VHSV proteins. As a starting point, we focused our study on the non-virion (NV) non-structural protein in which an arginine residue (R116) is present in all the virulent isolates and replaced by a serine/asparagine residue S/N116 in the attenuated isolates. A recombinant virus derived from a virulent VHSV strain in which the NV R116 residue has been replaced by a serine, rVHSVNVR116S, was generated by reverse genetics and used to infect juvenile trout. We showed that rVHSVNVR116S was highly attenuated and that surviving fish were almost completely protected from a challenge with the wild-type VHSV.
Subject(s)
Amino Acid Substitution , Fish Diseases/pathology , Fish Diseases/virology , Novirhabdovirus/pathogenicity , Rhabdoviridae Infections/veterinary , Viral Proteins/genetics , Virulence Factors/genetics , Animals , Genome, Viral , Novirhabdovirus/genetics , Novirhabdovirus/isolation & purification , Phenotype , Reverse Genetics , Rhabdoviridae Infections/pathology , Rhabdoviridae Infections/virology , Sequence Analysis, DNA , Trout , VirulenceABSTRACT
The transcription regulator YAP controls organ size by regulating cell growth, proliferation and apoptosis. However, whether YAP has a role in innate antiviral immunity is largely unknown. Here we found that YAP negatively regulated an antiviral immune response. YAP deficiency resulted in enhanced innate immunity, a diminished viral load, and morbidity in vivo. YAP blocked dimerization of the transcription factor IRF3 and impeded translocation of IRF3 to the nucleus after viral infection. Notably, virus-activated kinase IKKÉ phosphorylated YAP at Ser403 and thereby triggered degradation of YAP in lysosomes and, consequently, relief of YAP-mediated inhibition of the cellular antiviral response. These findings not only establish YAP as a modulator of the activation of IRF3 but also identify a previously unknown regulatory mechanism independent of the kinases Hippo and LATS via which YAP is controlled by the innate immune pathway.