RESUMO
DExD/H-box helicase (DDX) 5 belongs to the DExD/H-box helicase family. DDX family members play differential roles in the regulation of innate antiviral immune response. However, whether DDX5 is involved in antiviral immunity remains unclear. In this study, we found that DDX5 serves as a negative regulator of type I interferon (IFN) response. Overexpression of DDX5 inhibited IFN production induced by Spring viremia of carp virus (SVCV) and poly(I:C) and enhanced virus replication by targeting key elements of the RLR signaling pathway (MAVS, MITA, TBK1, IRF3 and IRF7). Mechanistically, DDX5 directly interacted with TBK1 to promote its autophagy-mediated degradation. Moreover, DDX5 was shown to block the interaction between TRAF3 and TBK1, hence preventing nuclear translocation of IRF3. Together, these data shed light on the roles of DDX5 in regulating IFN response.
Assuntos
Interferon Tipo I , Proteínas Serina-Treonina Quinases , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fosforilação , Diclorodifenil Dicloroetileno , Imunidade Inata , Interferon Tipo I/metabolismo , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , AntiviraisRESUMO
In this issue, we established rapid, cost-effective, and simple detection methods including recombines polymerase amplification with lateral flow dipstick (RPA-LFD) and real-time RPA for cyprinid herpesvirus 3(CyHV-3), and evaluated their sensitivity, specificity, and applicability, the real-time RPA method could achieve sensitive diagnosis of CyHV-3 within 1.3 copies per reaction, respectively. The real-time RPA method is 10-fold more sensitive than RPA-LFD method. The exact number of CyHV-3 can be calculated in each sample by real-time RPA. The sera from koi also can be tested in these methods. In addition, no cross-reaction was observed with other related pathogens, including carp oedema virus (CEV), spring viraemia of carp virus (SVCV), cyprinid herpesvirus 1(CyHV-1), cyprinid herpesvirus 2(CyHV-2), type I grass carp reovirus (GCRV-I), type II GCRV (GCRV-II), type III GCRV (GCRV-III), and Aeromonas hydrophila.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Herpesviridae , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Animais , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/virologia , Herpesviridae/isolamento & purificação , Herpesviridae/genética , Infecções por Herpesviridae/veterinária , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Carpas/virologia , Técnicas de Amplificação de Ácido Nucleico/veterinária , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/metabolismoRESUMO
Autophagy is a conservative and important process that exists in all eukaryotic cells in nature. Cyprinid herpesvirus 3 (CyHV-3), also known as KHV (Koi Herpesvirus), is a pathogen that mainly infecting common carp and koi. In the present study, we identified the CcLC3B gene, with a length of 379 bp and displaying a close evolutionary relationship with other sixteen different species, the tissue distribution and expression pattern of CcLC3 were also identified. We found that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage by using confocal fluorescence microscopy, transmission electron microscopy and western blotting. And we measured the protein levels associated with the Akt/mTOR signalling pathway, intracellular replication of CyHV-3 at the mRNA and protein levels as well as viral titters. Collectively, the results taken together suggested that CyHV-3 infection could promote autophagy in CCB cells at the early stage but inhibit autophagy at the late stage via mTOR and that promoting autophagy could facilitate CyHV-3 intracellular replication and extracellular viral yields in CCB cells. These findings revealed the relationship between CyHV-3 and autophagy and provided a novel treatment strategy targeting the autophagy signalling pathway against CyHV-3 infection.
RESUMO
Cyprinid herpesvirus 3 (CyHV-3) can induce up to 100% mortality among carp populations. To date, there has been no safe method to prevent the consequences of the activity of CyHV-3. Thyme is widely used in cooking due to its flavour. Both thyme and thyme essential oil (TEO) are used in traditional herbal medicine, mainly to treat respiratory system disorders. In this study, TEO containing predominantly cymene and thymol was applied to explore its antiviral effect. The toxicity of TEO was examined in MTT and crystal violet assays. The anti-CyHV-3 activity of TEO in the intracellular and extracellular stages of the viral replication cycle was explored in a plaque assay and TaqMan qPCR. TEO interfered with the intracellular stages of the CyHV-3 replication cycle with selectivity indexes (SI) of around 5. It also displayed virucidal activity in a dose- and time-dependent manner. Two-hour preincubation of CyHV-3 with TEO generated SI, ranging from 13.37 to 18.47 depending on cell line and method of examination. Preincubation of cells with TEO at a safe concentration did not decrease the intracellular viral DNA copy number, which suggests that TEO does not disturb the attachment of the virus to the cells. Further research regarding the antiviral activity of compounds of TEO is required in order to indicate the most potent molecules that could be considered candidates for application in aquaculture.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Herpesviridae , Óleos Voláteis , Thymus (Planta) , Animais , Óleos Voláteis/farmacologia , Doenças dos Peixes/tratamento farmacológico , Herpesviridae/fisiologia , Antivirais/farmacologia , Replicação ViralRESUMO
This study reports the occurrence of cyprinid herpesvirus 3 (CyHV-3) in koi carp (Cyprinus carpio koi) for the first time in India. The koi carp, with clinical signs of ulcer with haemorrhage on body surface, necrosis of fin and discolouration of gill associated with huge mortality, were observed in aquarium shops, rearing tanks and grow-out ponds located in Chennai, India. The PCR assay carried out on infected fish samples using different primer sets specific to CyHV-3 confirmed its presence in the infected fish. Sequence analysis of partial thymidine kinase gene revealed 100% similarity with the sequence of CyHV-3 available in GenBank. Cell lines of koi carp and catla were found to be susceptible to CyHV-3 and its replication was confirmed by viral-specific cytopathic effect, PCR and bioassay. The CyHV-3 infection was reproduced by intramuscular injection of inoculum prepared from CyHV-3-infected fish to satisfy Koch's postulates. Tissue tropism of CyHV-3 in infected fish by PCR assay revealed the presence of CyHV-3 in all vital organs with prominent band in gill and gut tissue.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Herpesviridae , Animais , Herpesviridae/genética , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/veterinária , Índia/epidemiologiaRESUMO
Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1ß, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.
Assuntos
Carpas , Doenças dos Peixes/prevenção & controle , Infecções por Herpesviridae/veterinária , Herpesviridae/imunologia , Vacinas contra Herpesvirus/imunologia , Fases de Leitura Aberta/imunologia , Vacinação/veterinária , Administração Oral , Animais , Anticorpos Antivirais/sangue , Carpas/imunologia , Carpas/virologia , Técnicas de Visualização da Superfície Celular , Epitopos de Linfócito B , Escherichia coli/genética , Doenças dos Peixes/virologia , Regulação da Expressão Gênica/imunologia , Infecções por Herpesviridae/prevenção & controle , Vacinas contra Herpesvirus/administração & dosagem , Esquemas de Imunização , Fases de Leitura Aberta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Análise de Sobrevida , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
Cyprinid herpesvirus 3, also known as koi herpesvirus (KHV), is a viral pathogen responsible for mass mortalities of carp worldwide. In this study, we compared the sensitivity and specificity of ELISA and quantitative PCR (qPCR) methods for the diagnosis of KHV in experimentally infected koi Cyprinus carpio over an 11 mo period. Koi were exposed to KHV at 18 ± 1°C (permissive temperatures for KHV disease) in laboratory-controlled conditions. At 21 d post challenge, the temperature in the system was decreased to <15°C (non-permissive temperature for KHV disease), and fish were monitored for the following 11 mo. At different time points throughout the study, samples of blood and gills were collected from exposed and control koi and subjected to qPCR and ELISA. Survival proportions of 53.3 and 98.8% in exposed and control treatments, respectively, were recorded at the end of the challenge. Traditional receiver-operating characteristic analysis was used to compare the sensitivity of the ELISA and blood and gill qPCR during permissive and non-permissive temperatures. ELISA was superior to qPCR of gills and whole-blood samples in detecting previous exposure to KHV. Similar results were obtained in a second experiment exposing koi to KHV and inducing persistent infection at >30°C (non-permissive temperature for KHV disease). Finally, KHV ELISA specificity was confirmed using cyprinid herpesvirus 1-exposed koi through a period of 3 mo. This study demonstrates that the combination of ELISA and gill qPCR should be recommended in the diagnosis of KHV exposure of suspected carrier-state fish.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Animais , HerpesviridaeRESUMO
Carp from breeding strains with different genetic background present diverse levels of resistance to viral pathogens. Carp strains of Asian origin, currently being treated as Cyprinus rubrofuscus L., especially Amur wild carp (AS), were proven to be more resistant to koi herpesvirus disease (KHVD; caused by cyprinid herpesvirus 3, CyHV-3) than strains originating from Europe and belonging to Cyprinus carpio L., like the Prerov scale carp (PS) or koi carp from a breed in the Czech Republic. We hypothesised that it can be associated with a higher magnitude of type I interferon (IFN) response as a first line of innate defence mechanisms against viral infections. To evaluate this hypothesis, four strains of common carp (AS, Rop, PS and koi) were challenged using two viral infection models: Rhabdovirus SVCV (spring viremia of carp virus) and alloherpesvirus CyHV-3. The infection with SVCV induced a low mortality rates and the most resistant was the Rop strain (no mortalities), whereas the PS strain was the most susceptible (survival rate of 78%). During CyHV-3 infection, Rop and AS strains performed better (survival rates of 78% and 53%, respectively) than PS and koi strains (survival rates of 35% and 10%, respectively). The evaluation of virus loads and virus replication showed significant differences between the carp strains, which correlated with the mortality rate. The evaluation of type I IFN responses showed that there were fundamental differences between the virus infection models. While responses to the SVCV were high, the CyHV-3 generally induced low responses. Furthermore, the results demonstrated that the magnitude of type I IFN responses did not correlate with a higher resistance in infected carp. In the case of a CyHV-3 infection, reduced type I IFN responses could be related to the potential ability of the virus to interfere with cellular sensing of foreign nucleic acids. Taken together, the results broaden our understanding of how common carp from different genetic strains interact with various viral pathogens.
Assuntos
Carpas/genética , Carpas/imunologia , Resistência à Doença/genética , Doenças dos Peixes/imunologia , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Herpesviridae/fisiologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/veterinária , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterináriaRESUMO
Cyprinid herpesvirus 3 (CyHV-3), a virus that encodes an interleukin10 (IL-10) homologue, causes severe economic losses to the common carp and koi culture industry. The present study was devoted to this IL-10 homologue. Recombinant viral IL-10 (vIL-10) protein encoded by CyHV-3 ORF134 gene using prokaryotic expression system was obtained successfully. Bioinformatics analysis revealed that the amino acid sequence of CyHV-3 vIL-10 has low homology with other host IL-10 or viruses encoded IL-10s. However, their tertiary structure is quite similar, suggesting conservative biological functions between IL-10s and vIL-10s. The biological activity of CyHV-3 vIL-10 was detected by using CCK-8 kit and real time quantitative PCR. The results showed that CyHV-3 vIL-10 down regulate epithelioma papulosum cyprini (EPC) cellular activity at 72â¯h. Moreover, CyHV-3 vIL-10 inhibits the LPS-induced expression of proinflammatory genes, similar to common carp IL-10. Altogether, the results of this study demonstrate that a clear biological activity of CyHV-3 vIL-10 on its host cells and indicates CyHV-3 vIL-10 may play an important role in viral immune evasion.
Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Herpesviridae/genética , Herpesviridae/imunologia , Interleucina-10/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Carpas/microbiologia , Linhagem Celular , Evasão da Resposta Imune , Interleucina-10/química , Interleucina-10/genética , Macrófagos , Filogenia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência/veterinária , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
During a disease outbreak, affected fish exhibit particular clinical signs, and the task in veterinary diagnostics is to identify the causative agent(s) as a prerequisite for appropriate treatment measures. In this study, we present an outbreak of a multifactorial gill disease in a cohort of ornamental koi Cyprinus carpio with gill necrosis as the main exterior clinical sign. By means of pathogen identification and determining pathogen abundance in various tissues, mortality of individual fish was found to be caused by different agents. Three out of 5 diseased individuals suffered from koi herpesvirus disease (KHVD) associated with a systemic infection with cyprinid herpesvirus 3 (CyHV-3), 1 fish succumbed to koi sleepy disease (KSD) caused by a high carp edema virus (CEV) load in the gills co-infected with CyHV-3 and flavobacteria, and the last fish had low loads of both viruses but high flavobacteria and Ichthyobodo burdens and most likely died from an interaction of these bacterial and parasitic agents. The results indicated that correct identification of the agent responsible for the observed clinical signs or mortality during co-infection might require quantitative determination of the abundance of the pathogens as well as detailed knowledge of the infection biology of these pathogens.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Herpesviridae , Animais , Flavobacterium , Brânquias , Infecções por Herpesviridae/veterináriaRESUMO
In response to the constant challenge by potential pathogens, external surfaces of fish, their skin, gills and intestinal tract, are coated with mucus, a gel like substance which largely prevents the entry of pathogens. This mucus gel consists mainly of water and mucins, large O-glycosylated proteins, which are responsible for forming a gel like mixture. A modulation of the mRNA expression of mucins, was described in viral diseases in mammals however there is a knowledge gap about the regulation of mucins during viral infection in fish. Therefore, novel sequences for common carp mucins were located in an early version of the common carp genome and their mRNA expression measured in carp under infection with three different viral pathogens: (i) the alloherpesvirus cyprinid herpesvirus 3, (ii) the rhabdovirus spring viremia of carp virus and (iii) the poxvirus carp edema virus. The results showed a downregulation of mucin mRNA expression in gills and gut of common carp under infection with these pathogenic viruses. This could be a sign of a severe distress to the mucosal tissues in carp which occurs under viral infection. The reduced expression of mucins could help explaining the increased susceptibility of virus-infected carp to secondary bacterial infection.
Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Mucinas/genética , Mucinas/imunologia , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica , Herpesviridae/fisiologia , Infecções por Herpesviridae/imunologia , Mucosa/imunologia , Poxviridae/fisiologia , Infecções por Poxviridae/imunologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologiaRESUMO
Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of koi herpesvirus disease in koi and common carp. The disease is notifiable to the World Organisation for Animal Health. Three tests-quantitative polymerase chain reaction (qPCR), conventional PCR (cPCR) and virus isolation by cell culture (VI)-were validated to assess their fitness as diagnostic tools for detection of CyHV-3. Test performance metrics of diagnostic accuracy were sensitivity (DSe) and specificity (DSp). Repeatability and reproducibility were measured to assess diagnostic precision. Estimates of test accuracy, in the absence of a gold standard reference test, were generated using latent class models. Test samples originated from wild common carp naturally exposed to CyHV-3 or domesticated koi either virus free or experimentally infected with the virus. Three laboratories in Canada participated in the precision study. Moderate to high repeatability (81 to 99%) and reproducibility (72 to 97%) were observed for the qPCR and cPCR tests. The lack of agreement observed between some of the PCR test pair results was attributed to cross-contamination of samples with CyHV-3 nucleic acid. Accuracy estimates for the PCR tests were 99% for DSe and 93% for DSp. Poor precision was observed for the VI test (4 to 95%). Accuracy estimates for VI/qPCR were 90% for DSe and 88% for DSp. Collectively, the results show that the CyHV-3 qPCR test is a suitable tool for surveillance, presumptive diagnosis and certification of individuals or populations as CyHV-3 free.
Assuntos
Cyprinidae , Doenças dos Peixes/diagnóstico , Infecções por Herpesviridae/veterinária , Herpesviridae/classificação , Herpesviridae/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Animais , DNA Viral/genética , Doenças dos Peixes/virologia , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/virologia , Plasmídeos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a serious infective, notifiable disease affecting common carp and varieties. In survivors, infection is generally characterized by a subclinical latency phase with restricted viral replication. The CyHV-3 genome is difficult to detect in such carrier fish that represent a potential source of dissemination if viral reactivation occurs. In this study, the analytical and diagnostic performance of an alternative serum neutralization (SN) method based on the detection of CyHV-3-specific antibodies was assessed using 151 serum or plasma samples from healthy and naturally or experimentally CyHV-3-infected carp. French CyHV-3 isolate 07/108b was neutralized efficiently by sera from carp infected with European, American and Taiwanese CyHV-3 isolates, but no neutralization was observed using sera specific to other aquatic herpesviruses. Diagnostic sensitivity, diagnostic specificity and repeatability of 95.9%, 99.0% and 99.3%, respectively, were obtained, as well as a compliance rate of 89.9% in reproducibility testing. Neutralizing antibodies were steadily detected in infected carp subjected to restrictive or permissive temperature variations over more than 25 months post-infection. The results suggest that this non-lethal diagnostic test could be used in the future to improve the epidemiological surveillance and control of CyHV-3 disease.
Assuntos
Anticorpos Antivirais/sangue , Carpas , Doenças dos Peixes/imunologia , Infecções por Herpesviridae/veterinária , Herpesviridae/imunologia , Testes de Neutralização/veterinária , Animais , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/virologia , Infecções por Herpesviridae/diagnóstico , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologiaRESUMO
Cyprinid herpesvirus 3 (CyHV3) is a large double-stranded DNA virus of Alloherpesviridae family in the order Herpesvirales. It causes significant morbidity and mortality in common carp and its ornamental koi variety, and threatens the aquaculture industries worldwide. Mimicry of cytokines and cytokine receptors is a particular strategy for large DNA viruses in modulating the host immune response. Here, we report the identification and characterization of two novel viral homologues of tumor necrosis factor receptor (TNFR) encoded by CyHV3-ORF4 and -ORF12, respectively. CyHV3-ORF4 was identified as a homologue of HVEM and CyHV3-ORF12 as a homologue of TNFRSF1. Overexpression of ORF4 and ORF12 in zebrafish embryos results in embryonic lethality, morphological defects and increased apoptosis. Although we failed to identify any interaction between the two vTNFRs and their potential ligands in zebrafish TNF superfamily by yeast two-hybrid system, the expression of some genes in TNF superfamily or TNFR superfamily were mis-regulated in ORF4 or ORF12-overexpressing embryos, especially the death receptor zHDR and its cognate ligand DL1b. Further studies showed that the apoptosis induced by the both CyHV3 vTNFRs is mainly activated through the intrinsic apoptotic pathway and requires the crosstalk between the intrinsic and extrinsic apoptotic pathway. Additionally, using RT-qPCR and Western blot assays, the expression patterns of the both vTNFRs were also analyzed during CyHV3 productive infection. Collectively, this is the first functional study of two unique vTNFRs encoded by a herpesvirus infecting non-mammalian vertebrates, which may provide novel insights into viral immune regulation mechanism and the pathogenesis of CyHV3 infection.
Assuntos
Doenças dos Peixes/genética , Infecções por Herpesviridae/veterinária , Herpesviridae/fisiologia , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas Virais/genética , Peixe-Zebra , Sequência de Aminoácidos , Animais , Carpas , Linhagem Celular , Feminino , Doenças dos Peixes/metabolismo , Doenças dos Peixes/virologia , Regulação da Expressão Gênica , Herpesviridae/genética , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/virologia , Masculino , Fases de Leitura Aberta , Membro 14 de Receptores do Fator de Necrose Tumoral/química , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/química , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Alinhamento de Sequência/veterinária , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The pathogenesis of cyprinid herpesvirus-3 (CyHV-3) was studied using different lineages of carp/koi. After exposure to the virus, infected cells were first found in the skin by histopathology and by in situ hybridization. The epidermis of the skin was most severely damaged and often sloughed off in the fish sampled on days 5 through 8, and the fish that were highly sensitive to the virus died within 8 or 10 days after infection. Serum osmolality of the infected fish, particularly just before death, was significantly lower, suggesting that the osmotic shock consequent on the damage to the skin was the direct cause of the acute deaths. On the other hand, clinical and histopathological observations indicate that the carp of a less sensitive lineage most probably died of viral encephalitis around 3 weeks after infection. For these fish, the largest number of infected cells was found in the central nervous system (CNS) sampled on day 12. A substantial amount of viral genome was found in the CNS of carp surviving more than 1 year after the infection. Thus, the CNS is probably a major target for CyHV-3, and the virus can persistently infect the CNS, presumably establishing latency.
Assuntos
Doenças dos Peixes/patologia , Infecções por Herpesviridae/veterinária , Animais , Carpas , Sistema Nervoso Central/patologia , Sistema Nervoso Central/virologia , Doença Crônica , Epiderme/patologia , Doenças dos Peixes/mortalidade , Genoma Viral , Herpesviridae/genética , Herpesviridae/fisiologia , Infecções por Herpesviridae/mortalidade , Infecções por Herpesviridae/patologia , Rim/patologia , Rim/virologiaRESUMO
Koi herpesvirus (KHV, CyHV-3) causes severe economic losses in carp farms. Its eradication is challenging due to the establishment of latency in blood leukocytes and other tissues. To understand the molecular mechanisms leading to KHV infection in leukocytes, common carp were bath-exposed to KHV at 17 °C. After confirming the presence of viral transcripts in blood leukocytes at ten days post infection, RNA-Seq was performed on peripheral blood leukocytes on the Illumina NovaSeq. KHV infection triggered a robust immune response mediated by pattern recognition receptors, mainly toll-like receptors (tlr2, tlr5, tlr7, and tlr13), urokinase plasminogen activator surface receptor-like, galectin proteins, and lipid mediators such as leukotriene B4 receptor 1. Enriched pathways showed increased mitochondria oxidative phosphorylation and the activation of signalling pathways such as mitogen-activated protein kinases (MAPKs) and vascular endothelial growth factor (VEGF). KHV-infected leukocytes showed low production of reactive oxygen species (ROS) and glutathione metabolism, high iron export and phagocytosis activity, and low autophagy. Macrophage polarization was deduced from the up-regulation of genes such as arginase non-hepatic 1-like, macrophage mannose receptor-1, crem, il-10, and il-13 receptors, while markers for cytotoxic T cells were observed to be down-regulated. Further work is required to characterise these leukocyte subsets and the molecular events leading to KHV latency in blood leukocytes.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Herpesviridae , Animais , Infecções por Herpesviridae/veterinária , Fator A de Crescimento do Endotélio Vascular , Herpesviridae/genética , Perfilação da Expressão Gênica , LeucócitosRESUMO
Cyprinid herpesvirus 3 (CyHV-3) can cause severe disease in koi and common carp (Cyprinus carpio). Currently, no effective treatment is available against CyHV-3 infection in koi. Both LSD1 and JMJD2 are histone demethylases (HD) and are critical for immediate-early (IE) gene activation essential for lytic herpesvirus replication. OG-L002 and ML324 are newly discovered specific inhibitors of LSD1 and JMJD2, respectively. Here, HD inhibitors were compared with acyclovir (ACV) against CyHV-3 infection in vitro and in vivo. ML324, at 20-50 µM, can completely block ~1 × 103 PFU CyHV-3 replication in vitro, while OG-L002 at 20 µM and 50 µM can produce 96% and 98% inhibition, respectively. Only about 94% inhibition of ~1 × 103 PFU CyHV-3 replication was observed in cells treated with ACV at 50 µM. As expected, CyHV-3 IE gene transcription of ORF139 and ORF155 was blocked within 72 h post-infection (hpi) in the presence of 20 µM ML324. No detectable cytotoxicity was observed in KF-1 or CCB cells treated for 24 h with 1 to 50 µM ML324. A significant reduction of CyHV-3 replication was observed in ~6-month-old infected koi treated with 20 µM ML324 in an immersion bath for 3-4 h at 1-, 3-, and 5-days post-infection compared to the control and ACV treatments. Under heat stress, 50-70% of 3-4-month-old koi survived CyHV-3 infection when they were treated daily with 20 µM ML324 in an immersion bath for 3-4 h within the first 5 d post-infection (dpi), compared to 11-19% and 22-27% of koi in the control and ACV treatments, respectively. Our study demonstrates that ML324 has the potential to be used against CyHV-3 infection in koi.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Herpesviridae , Animais , Aciclovir/farmacologia , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/veterinária , Herpesviridae/genética , Doenças dos Peixes/tratamento farmacológicoRESUMO
Interferon regulatory factor 9 (IRF9) is an important transcriptional regulator involved in innate and adaptive immunity. Cyprinid herpesvirus-3 (CyHV-3) is a virus causing widespread death and great economic loss in farmed common carp (Cyprinus carpio). However, the effect of IRF9 on CyHV-3 infection in common carp has not been reported. In this study, during CyHV-3 infection, IRF9 overexpression in common carp fin epithelial (CCF) cells significantly reduced the expression of viral factor thymidine kinase (TK) and open reading frame 72 (ORF72), and knockdown of IRF9 produced the opposite results (p < 0.05). In CCF cells. The IRF9 protein was expression in the nucleus and was rapidly induced in CCF cells by CyHV-3 infection. In addition, several genes associated with virus infection, including type I interferon (IFNI), IFN-stimulated gene 15 (ISG15), myxovirus resistance 1 (Mx1) and Viperin were induced in CCF cells overexpressing IRF9 upon CyHV-3 infection. IRF9 overexpression induced by CyHV-3 infection significantly increased the gene expression of Mx1 and phosphoinositide 3-kinase (PI3K) and the protein expression of protein kinase B (AKT) (p < 0.01). Interestingly, IRF9 did not significantly affect Mx1 gene expression when AKT protein levels remained unchanged during CyHV-3 infection of CCF cells. Furthermore, a significant resistance-related locus was found in the IRF9 sequence in "Longke-11" mirror carp (M11) and Yellow River carp (p < 0.05). These results indicated that IRF9 inhibited viral replication by upregulating the expression of Mx1 via the PI3K-AKT signalling pathway during CyHV-3 infection in CCF cells and provide some basis for the study of the antiviral molecular mechanisms of common carp.
Assuntos
Carpas , Doenças dos Peixes , Infecções por Herpesviridae , Animais , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Fator Gênico 3 Estimulado por Interferon, Subunidade gama , Fosfatidilinositol 3-Quinase , Células EpiteliaisRESUMO
Since emerged in the late 1990s, cyprinid herpesvirus 3 (CyHV-3) has caused huge economic losses in common and koi carp culture worldwide. Accumulating evidences suggest that teleost fish microRNA (miRNA), a class of non-coding RNA of â¼22 nucleotides, can participate in many cellular processes, especially in host antiviral defenses. However, the roles of miRNAs in CyHV-3 infection are still unclear. Here, using high-throughput miRNA sequencing and quantitative real-time PCR (qRT-PCR) verification, we found that miR-155 was significantly upregulated in common carp brain (CCB) cells upon CyHV-3 infection. Overexpression of miR-155 effectively inhibited CyHV-3 replication in CCB cells and promoted type I interferon (IFN-I) expression. Further study revealed that miR-155 targeted the 3' untranslated region (UTR) of the mRNA of 5'AMP-activated protein kinase (AMPK), and that AMPK could interact with and degrade the mitochondrial antiviral signaling protein (MAVS), resulting in the reduction of interferon (IFN) expression. Collectively, our results show that miR-155, induced by CyHV-3 infection, exhibits anti-CyHV-3 activity via regulating AMPK-MAVS-IFN axis, which will help design anti-CyHV-3 drugs.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Regiões 3' não Traduzidas , Proteínas Quinases Ativadas por AMP/farmacologia , Animais , Antivirais/farmacologia , Carpas , Linhagem Celular , Doenças dos Peixes/virologia , Herpesviridae , Infecções por Herpesviridae/genética , Interferon Tipo I/genética , MicroRNAs/genética , RNA Mensageiro/análise , Replicação Viral/efeitos dos fármacosRESUMO
Vaccination is the best form of protecting fish against viral diseases when the pathogen cannot be contained by biosecurity measures. Vaccines based on live attenuated viruses seem to be most effective for vaccination against challenging pathogens like Cyprinid herpesvirus 3. However, there are still knowledge gaps how these vaccines effectively protect fish from the deadly disease caused by the epitheliotropic CyHV-3, and which aspects of non-direct protection of skin or gill integrity and function are important in the aquatic environment. To elucidate some elements of protection, common carp were vaccinated against CyHV-3 using a double deletion vaccine virus KHV-T ΔDUT/TK in the absence or presence of a mix of common carp beta-defensins 1, 2 and 3 as adjuvants. Vaccination induced marginal clinical signs, low virus load and a minor upregulation of cd4, cd8 and igm gene expression in vaccinated fish, while neutralisation activity of blood serum rose from 14 days post vaccination (dpv). A challenge infection with CyHV-3 induced a severe disease with 80-100% mortality in non-vaccinated carp, while in vaccinated carp, no mortality was recorded and the virus load was >1,000-fold lower in the skin, gill and kidney. Histological analysis showed strongest pathological changes in the skin, with a complete destruction of the epidermis in non-vaccinated carp. In the skin of non-vaccinated fish, T and B cell responses were severely downregulated, inflammation and stress responses were increased upon challenge, whereas vaccinated fish had boosted neutrophil, T and B cell responses. A disruption of skin barrier elements (tight and adherence junction, desmosomes, mucins) led to an uncontrolled increase in skin bacteria load which most likely exacerbated the inflammation and the pathology. Using a live attenuated virus vaccine, we were able to show that increased neutrophil, T and B cell responses provide protection from CyHV-3 infection and lead to preservation of skin integrity, which supports successful protection against additional pathogens in the aquatic environment which foster disease development in non-vaccinated carp.