Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 516
Filtrar
1.
Viruses ; 14(12)2022 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-36560639

RESUMO

Ranaviruses have been involved in amphibian mass mortality events worldwide. Effective screening to control this pathogen is essential; however, current sampling methods are unsuitable for the detection of subclinical infections. Non-lethal screening is needed to prevent both further spread of ranavirus and losses of at-risk species. To assess non-lethal sampling methods, we conducted two experiments: bath exposing common frogs to RUK13 ranavirus at three concentrations, and exposing common toads to RUK13 or PDE18. Non-lethal sampling included buccal, digit, body and tank swabs, along with toe clips and stool taken across three time-points post-exposure. The presence/load of ranavirus was examined using quantitative PCR in 11 different tissues obtained from the same euthanised animals (incl. liver, gastro-intestinal tract and kidney). Buccal swab screening had the highest virus detection rate in both species (62% frogs; 71% toads) and produced consistently high virus levels compared to other non-lethal assays. The buccal swab was effective across multiple stages of infection and differing infection intensities, though low levels of infection were more difficult to detect. Buccal swab assays competed with, and even outperformed, lethal sampling in frogs and toads, respectively. Successful virus detection in the absence of clinical signs was observed (33% frogs; 50% toads); we found no difference in detectability for RUK13 and PDE18. Our results suggest that buccal swabbing could replace lethal sampling for screening and be introduced as standard practice for ranavirus surveillance.


Assuntos
Infecções por Vírus de DNA , Ranavirus , Animais , Ranavirus/genética , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/epidemiologia , Anuros , Reino Unido
2.
Dis Aquat Organ ; 152: 127-138, 2022 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-36519684

RESUMO

In the early 2000s, numerous cases of European amphibian population declines and mass die-offs started to emerge. Investigating those events led to the discovery that wild European amphibians were confronted with grave disease threats caused by introduced pathogens, namely the amphibian and the salamander chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) and ranaviruses. In Greece, Bd was previously documented among wild amphibian populations in 2 different locations and 3 different species. However, no disease-related mass declines or mortality events have been reported. In this work, we build upon previous findings with new, subsequently obtained data, resulting in a 225-sample dataset of 14 species from 17 different locations throughout Greece, in order to examine the occurrence status of all 3 pathogens responsible for emerging infectious diseases in European amphibians. No positive samples for Bsal or ranavirus were recorded in any location. We confirmed the presence of Bd in 4 more localities and in 4 more species, including 1 urodelan (Macedonian crested newt Triturus macedonicus) and 1 introduced anuran (American bullfrog Lithobates catesbeianus). All insular localities were negative for Bd, except for Crete, where Bd was identified in 2 different locations. Again, no mass declines or die-offs were recorded in any Bd-positive area or elsewhere. However, given the persistence of Bd across Greece over the past ~20 yr, monitoring efforts should continue, and ideally be further expanded.


Assuntos
Quitridiomicetos , Doenças Transmissíveis Emergentes , Micoses , Ranavirus , Animais , Batrachochytrium , Grécia/epidemiologia , Micoses/epidemiologia , Micoses/veterinária , Micoses/microbiologia , Anfíbios/microbiologia , Doenças Transmissíveis Emergentes/veterinária , Rana catesbeiana
3.
Viruses ; 14(11)2022 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-36423155

RESUMO

Aquatic animal viruses infect and transmit in aquatic environments, causing serious harm to the aquaculture industry and a variety of wild aquatic animals. How are they affected by environmental factors and do they represent potential threat to mammalian heath or not? Here, the effects of environmental factors (ultraviolet radiation (UV), temperature, pH, and drying) and their threshold on five epidemic aquatic animal viruses infecting amphibians and bony fish, including Rana grylio virus (RGV), Andrias davidianus ranavirus (ADRV), Grass carp reovirus (GCRV), Paralichthys olivaceus rhabdovirus (PORV), and Scophthalmus maximus rhabdovirus (SMRV), were measured and compared in a fish cell line. The examination of virus titers after different treatment in fish cells showed that the two iridoviruses, RGV and ADRV, had a higher tolerance to all of the environmental factors, such as they only had a decay rate of 22-36% when incubated at 37 °C for 7 days. However, the rhabdovirus SMRV was sensitive to all of the factors, with a decay rate of more than 80% in most of the treatments; even a complete inactivation (100%) can be observed after drying treatment. To address the potential threat to mammals, infectivity and limitation factors of the five viruses in Baby hamster kidney fibroblast cells (BHK-21) were tested, which showed that three of the five viruses can replicate at a low temperature, but a high temperature strongly inhibited their infection and none of them could replicate at 37 °C. This study clarified the sensitivity or tolerance of several different types of aquatic animal viruses to the main environmental factors in the aquatic environment and proved that the viruses cannot replicate in mammalian cells at normal physiological temperature.


Assuntos
Ranavirus , Reoviridae , Rhabdoviridae , Animais , Raios Ultravioleta , Ranavirus/fisiologia , Urodelos , Mamíferos
4.
J Virol ; 96(20): e0068222, 2022 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-36190239

RESUMO

Iridoviruses are large DNA viruses which cause great economic losses to the aquaculture industry and serious threats to ecological diversity worldwide. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, causes high mortality in grouper aquaculture. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. Here, we reported that the protein encoded by SGIV ORF131R (VP131) was localized predominantly within the endoplasmic reticulum (ER). Ectopic expression of GFP-VP131 significantly enhanced SGIV replication, while VP131 knockdown decreased viral infection in vitro, suggesting that VP131 functioned as a proviral factor during SGIV infection. Overexpression of GFP-VP131 inhibited the interferon (IFN)-1 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), or melanoma differentiation-associated gene 5 (EcMDA5), whereas such activation induced by mitochondrial antiviral signaling protein (EcMAVS) was not affected. Moreover, VP131 interacted with EcSTING and degraded EcSTING through both the autophagy-lysosome pathway and ubiquitin-proteasome pathway, and targeted for the K63-linked ubiquitination. Of note, we also found that EcSTING significantly accelerated the formation of GFP-VP131 aggregates in co-transfected cells. Finally, GFP-VP131 inhibited EcSTING- or EcTBK1-induced antiviral activity upon red-spotted grouper nervous necrosis virus (RGNNV) infection. Together, our results demonstrated that the SGIV VP131 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion. IMPORTANCE STING has been identified as a critical factor participating in the innate immune response which recruits and phosphorylates TBK1 and IFN regulatory factor 3 (IRF3) to induce IFN production and defend against viral infection. However, viruses also distort the STING-TBK1 pathway to negatively regulate the IFN response and facilitate viral replication. Here, we reported that SGIV VP131 interacted with EcSTING within the ER and degraded EcSTING, leading to the suppression of IFN production and the promotion of SGIV infection. These results for the first time demonstrated that fish iridovirus evaded the host antiviral response via abrogating the STING-TBK1 signaling pathway.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Ranavirus , Animais , Ranavirus/genética , Bass/genética , Bass/metabolismo , Iridovirus/genética , Iridovirus/metabolismo , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/genética , Fator Regulador 3 de Interferon/metabolismo , Antivirais , Complexo de Endopeptidases do Proteassoma/metabolismo , Singapura , Proteínas de Peixes , Imunidade Inata/genética , Interferons/metabolismo , RNA Mensageiro/genética , GMP Cíclico , Ubiquitinas/metabolismo , Monofosfato de Adenosina
5.
Biol Lett ; 18(10): 20220359, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36259234

RESUMO

Hosts may limit exposure to pathogens through changes in behaviour, such as avoiding infected individuals or contaminated areas. Here, we tested for a behavioural response to ranavirus infection in juvenile wood frogs (Rana sylvatica) because the majority of dispersal between populations occurs during this life stage. We hypothesized that if infections are transmissible and detectable at this life stage, then susceptibles would display avoidance behaviours when introduced to an infected conspecific. Despite no apparent signs of infection, we observed a greater distance between susceptible-infected pairs, compared to pairs of either two infected or two susceptible animals. Further, distances between susceptible-infected pairs were positively related to the infection intensity of the focal exposed frog, suggesting the cue to avoid infected conspecifics may become more detectable with more intense infections. Although we did not quantify whether the transmission was affected by their distancing, our findings suggest that juvenile frogs have the potential to reduce terrestrial transmission of ranaviruses through avoidance behaviours.


Assuntos
Infecções por Vírus de DNA , Ranavirus , Animais , Aprendizagem da Esquiva , Ranidae , Anfíbios , Anuros
6.
Front Immunol ; 13: 985291, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36203610

RESUMO

TRIM (tripartite motif) proteins have been demonstrated to exert critical roles in host defense against different microbial pathogens. Among them, TRIM23 acts as an important regulatory factor in antiviral immune and inflammatory responses, but the roles of fish TRIM23 against virus infection still remain largely unknown. Here, we investigated the characteristics of TRIM23 homolog from orange spotted grouper (Epinephelus coioides) (EcTRIM23). EcTRIM23 encoded a 580 amino acid peptide, which shared 93.1%, 89.73% and 86.36% identity with golden perch (Perca flavescens), zebrafish (Danio rerio) and human (Homo sapiens), respectively. The transcription levels of EcTRIM23 were significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection. EcTRIM23 overexpression in vitro significantly inhibited RGNNV and SGIV replication, evidenced by the delayed cytopathic effect (CPE) progression and the decreased expression of viral core genes. EcTRIM23 significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines, as well as the promoter activities of IFN and NF-κB, suggesting that EcTRIM23 exerted antiviral function by positively regulating host IFN response. Exogenous EcTRIM23 exhibited either diffuse or aggregated localization in grouper cells. After co-transfection, TANK binding kinase 1 (TBK1), TNF receptor associated factor (TRAF) 3 and TRAF4, TRAF5 and TRAF6 were found to interact with EcTRIM23 in grouper cells. Moreover, these proteins could be recruited and co-localized with EcTRIM23 in vitro. Together, our results demonstrated that fish TRIM23 exerted antiviral activity against fish viruses by interacting with multiple host proteins to regulate immune responses.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Nodaviridae , Ranavirus , Aminoácidos/genética , Animais , Antivirais/farmacologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/metabolismo , Proteínas de Ligação ao GTP , Humanos , Imunidade Inata/genética , Interferons/metabolismo , NF-kappa B/metabolismo , Nodaviridae/fisiologia , Ranavirus/fisiologia , Alinhamento de Sequência , Fator 4 Associado a Receptor de TNF/genética , Fator 4 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Peixe-Zebra/genética
7.
Fish Shellfish Immunol ; 130: 380-390, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36150412

RESUMO

Transcription factor ATF1 is a member of the ATF/CREB family of the CREB subfamily and is involved in physiological processes such as tumorigenesis, organ development, reproduction, cell survival, and apoptosis in mammals. However, studies on ATF1 in fish have been relatively poorly reported, especially on its role in antiviral immunity in fish. In this study, ATF1 from orange-spotted grouper (named EcATF1) were cloned and characterized. Molecular characterization analysis showed that EcATF1 encodes a 307-amino-acid protein, containing PKID and bZIP_CREB1 domains. Homology analysis showed that had the highest homology with E. lanceolatus(88.93%). Tissue expression pattern showed that EcATF1 was extensively distributed in twelve selected tissues, with higher expression in the skin, gill, liver and spleen. Subcellular localization analysis showed that EcATF1 was distributed in the nucleus of GS cells. EcATF1 overexpression inhibits Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) replication, as evidenced by a diminished degree of CPE induced by SGIV and RGNNV and a reduction in the level of viral gene transcription and viral capsid protein expression. Furthermore, EcATF1 overexpression upregulated interferon pathway-related genes and proinflammatory factors, and increased the promoter activities of IFN, IFN stimulated response element (ISRE), and nuclear factor κB(NFκB). Meanwhile, EcATF1 overexpression positive regulate the MHC-I signaling pathway, and upregulated the promoter activity of MHC-I. Collectively, these data demonstrate that EcATF1 plays an important role during the host antiviral immune response. This study provides insights into the function of ATF1 in the immune system of lower vertebrates.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Nodaviridae , Ranavirus , Sequência de Aminoácidos , Animais , Antivirais , Proteínas do Capsídeo/genética , Proteínas de Peixes , Imunidade Inata/genética , Interferons/genética , Mamíferos/genética , Mamíferos/metabolismo , NF-kappa B/metabolismo , Nodaviridae/fisiologia , Ranavirus/fisiologia , Alinhamento de Sequência
8.
Oecologia ; 200(3-4): 307-322, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35969272

RESUMO

Natural enemy ecology strives to unify predator-prey and host-pathogen interactions under a common framework to gain insights into community- and ecosystem-level processes. To address this goal, ecologists need a greater emphasis on: (1) quantifying pathogen-mediated effects on community structure to enable comparisons with predator-mediated effects and (2) determining the interactive effects of combined natural enemies on communities. We conducted a mesocosm experiment to assess the individual and combined effects of predators (dragonfly larvae and adult water bugs) and a pathogen (ranavirus) on the abundance and composition of a larval amphibian assemblage. We found that our three natural enemies structured victim assemblages in unique ways, producing distinct assemblages. Additionally, we found that in combination treatments, predators mainly drove assemblage structure such that the assemblages most closely resembled their respective predator treatments. We also found that predators reduced infection prevalence in combination treatments, and that the magnitude of this effect was dependent on predator identity. Compared to virus-alone treatments, the presence of dragonflies and water bugs reduced infection prevalence by 79% and 63%, respectively. Additionally, the presence of dragonflies eliminated ranavirus infection in two species, which demonstrates the prominent role of predators in disease dynamics in this system. Overall, this work demonstrates the importance of considering natural enemies in community ecology, as each enemy can elicit a unique structural change. Additionally, this study provides a unique empirical test of the healthy herds hypothesis for multi-species assemblages and underscores the importance of advancing our understanding of multi-enemy interactions within communities.


Assuntos
Odonatos , Ranavirus , Animais , Ecossistema , Ecologia , Larva
9.
J Fish Dis ; 45(11): 1745-1756, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35989490

RESUMO

European North Atlantic ranavirus (ENARV, Iridoviridae), is a ranavirus species recently isolated from lumpfish (Cyclopterus lumpus, L.), which are used as cleaner fish in Atlantic salmon (Salmo salar) farming in Northern Europe. This study aimed to investigate (1) the virulence of ENARV isolates from Ireland, Iceland and the Faroe Islands to lumpfish; (2) horizontal transmission between lumpfish; and (3) virulence to Atlantic salmon parr. Lumpfish were challenged in a cohabitation model using intraperitoneally (IP) injected shedders, and naïve cohabitants. IP challenge with isolates from Iceland (1.9 × 107 TCID50  ml-1 ) and the Faroe Islands (5.9 × 107 TCID50  ml-1 ) reduced survival in lumpfish, associated with consistent pathological changes. IP challenge with the Irish strain (8.6 × 105 TCID50  ml-1 ) did not significantly reduce survival in lumpfish, but the lower challenge titre complicated interpretation. Horizontal transmission occurred in all strains tested, but no clinical impact was demonstrated in cohabitants. Salmon parr were challenged by IP injection with the Irish isolate, no virulence or virus replication were demonstrated. A ranavirus qPCR assay, previously validated for fish ranaviruses, was first used to detect ENARV in tissues of both in lumpfish and Atlantic salmon. This study provides the first data on the assessment of virulence of ENARV isolates to lumpfish and salmon, guidelines for the diagnosis of ENARV infection, and poses a basis for further investigations into virulence markers.


Assuntos
Doenças dos Peixes , Iridoviridae , Perciformes , Ranavirus , Salmo salar , Animais , Peixes
10.
Fish Shellfish Immunol ; 128: 136-147, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35921938

RESUMO

Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) are important pathogens that cause high mortality and heavy economic losses in grouper aquaculture. Interestingly, SGIV infection in grouper cells induces paraptosis-like cell death, while RGNNV infection induces autophagy and necrosis characterized morphologically by vacuolation of lysosome. Here, a comparative transcriptomic analysis was carried out to identify the different molecular events during SGIV and RGNNV infection in grouper spleen (EAGS) cells. The functional enrichment analysis of DEGs suggested that several signaling pathways were involved in CPE progression and host immune response against SGIV or RGNNV. Most of DEGs featured in the KEGG "lysosome pathway" were up-regulated in RGNNV-infected cells, indicating that RGNNV induced lysosomal vacuolization and autophagy might be due to the disturbance of lysosomal function. More than 100 DEGs in cytoskeleton pathway and mitogen-activated protein kinase (MAPK) signal pathway were identified during SGIV infection, providing additional evidence for the roles of cytoskeleton remodeling in cell rounding during CPE progression and MAPK signaling in SGIV induced cell death. Of note, consistent with changes at the transcriptional levels, the post-translational modifications of MAPK signaling-related proteins were also detected during RGNNV infection, and the inhibitors of extracellular signal-regulated kinase (ERK) and p38 MAPK significantly suppressed viral replication and virus induced vacuoles formation. Moreover, the majority of DEGs in interferon and inflammation signaling were obviously up-regulated during RGNNV infection, but down-regulated during SGIV infection, suggesting that SGIV and RGNNV differently manipulated host immune response in vitro. In addition, purine and pyrimidine metabolism pathways were also differently regulated in SGIV and RGNNV-infection cells. Taken together, our data will provide new insights into understanding the potential mechanisms underlying different host cell responses against fish DNA and RNA virus.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Nodaviridae , Ranavirus , Animais , MAP Quinases Reguladas por Sinal Extracelular/genética , Proteínas de Peixes , Imunidade Inata/genética , Interferons/genética , Necrose , Nodaviridae/fisiologia , Purinas , Pirimidinas , Ranavirus/fisiologia , Singapura , Transcriptoma , Proteínas Quinases p38 Ativadas por Mitógeno/genética
11.
Viruses ; 14(7)2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35891548

RESUMO

Largemouth bass virus (LMBV), belonging to the genus Ranavirus, causes high mortality and heavy economic losses in largemouth bass aquaculture. In the present study, a novel cell line, designated as MsF, was established from the fin of largemouth bass (Micropterus salmoides), and applied to investigate the characteristics of cell death induced by LMBV. MsF cells showed susceptibility to LMBV, evidenced by the occurrence of a cytopathic effect (CPE), increased viral gene transcription, protein synthesis, and viral titers. In LMBV-infected MsF cells, two or more virus assembly sites were observed around the nucleus. Notably, no apoptotic bodies occurred in LMBV-infected MsF cells after nucleus staining, suggesting that cell death induced by LMBV in host cells was distinct from apoptosis. Consistently, DNA fragmentation was not detected in LMBV-infected MsF cells. Furthermore, only caspase-8 and caspase-3 were significantly activated in LMBV-infected MsF cells, suggesting that caspases were involved in non-apoptotic cell death induced by LMBV in host cells. In addition, the disruption of the mitochondrial membrane potential (ΔΨm) and reactive oxygen species (ROS) generation were detected in both LMBV-infected MsF cells and fathead minnow (FHM) cells. Combined with our previous study, we propose that cell death induced by LMBV infection was cell type dependent. Although LMBV-infected MsF cells showed the characteristics of non-apoptotic cell death, the signal pathways might crosstalk and interconnect between apoptosis and other PCD during LMBV infection. Together, our results not only established the in vitro LMBV infection model for the study of the interaction between LMBV and host cells but also shed new insights into the mechanisms of ranavirus pathogenesis.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Ranavirus , Viroses , Animais , Apoptose , Morte Celular , Infecções por Vírus de DNA/epidemiologia , Ranavirus/genética
12.
Viruses ; 14(6)2022 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-35746698

RESUMO

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 µg/mL, green tea polyphenols (TP) ≤ 10 µg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 µg/mL, (-)-epigallocatechin (EGC) ≤ 10 µg/mL, (-)-epicatechin gallate (EGC) ≤ 5 µg/mL, and (-)-epicatechin (EC) ≤ 50 µg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Ranavirus , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Vírus de DNA/veterinária , Iridovirus/genética , Ranavirus/fisiologia , Chá
13.
Viruses ; 14(6)2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35746713

RESUMO

Singapore grouper iridovirus (SGIV) causes high economic losses in mariculture. Effective drugs for managing SGIV infection are urgently required. Medicinal plant resources are rich in China. Medicinal plants have a long history and significant curative effects in the treatment of many diseases. Reverse-transcription quantitative real-time PCR is the most commonly used method for detecting virus infection and assessing antiviral efficacy with high accuracy. However, their applications are limited due to high reagent costs and complex time-consuming operations. Aptamers have been applied in some biosensors to achieve the accurate detection of pathogens or diseases through signal amplification. This study aimed to establish an aptamer-based high-throughput screening (AHTS) model for the efficient selection and evaluation of medicinal plants components against SGIV infection. Q2-AHTS is an expeditious, rapid method for selecting medicinal plant drugs against SGIV, which was characterized as being dram, high-speed, sensitive, and accurate. AHTS strategy reduced work intensity and experimental costs and shortened the whole screening cycle for effective ingredients. AHTS should be suitable for the rapid selection of effective components against other viruses, thus further promoting the development of high-throughput screening technology.


Assuntos
Aptâmeros de Nucleotídeos , Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Ranavirus , Animais , Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Ensaios de Triagem em Larga Escala
14.
Fish Shellfish Immunol ; 127: 956-964, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35764286

RESUMO

Growing evidences have demonstrated that multiple TRIM (tripartite motif) proteins exert critical roles in host defense against different microbial pathogens. Although mammalian TRIM21 has been reported to function as an important regulatory factor in antiviral immune and inflammatory response, the role of fish TRIM21 against virus infection still remains largely unknown. In the present study, we investigated the characteristics of TRIM21 gene (EcTRIM21) from orange spotted grouper (Epinephelus coioides). The full-length EcTRIM21 cDNA encoded a 557 amino acid peptide with 92.1% and 31.14% identity with giant grouper (Epinephelus lanceolatus) and human (Homo sapiens), respectively. EcTRIM21 contained four conserved domains, including RING, B-Box, PRY and SPRY domain. EcTRIM21 expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, suggesting that EcTRIM21 might be involved in host defense against fish virus infections. Subcellular localization showed that EcTRIM21 were distributed in the cytoplasm in a punctate manner. Overexpression of EcTRIM21 in vitro significantly inhibited RGNNV and SGIV replication, as evidenced by the decreased severity of cytopathic effect (CPE) and the reduced expression levels of viral core genes. Consistently, knockdown of EcTRIM21 by small interfering RNA (siRNA) promoted the replication of RGNNV and SGIV in vitro. Furthermore, EcTRIM21 overexpression increased both interferon (IFN) and interferon stimulated response element (ISRE) promoter activities. In addition, the transcription levels of IFN signaling related molecules were positively regulated by EcTRIM21 overexpression. Together, our data demonstrated that fish TRIM21 exerted antiviral activity against fish viruses through positive regulation of host interferon response.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Nodaviridae , Ranavirus , Sequência de Aminoácidos , Animais , Antivirais/farmacologia , Proteínas de Peixes/química , Humanos , Interferons/genética , Mamíferos/genética , Mamíferos/metabolismo , Nodaviridae/fisiologia , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência , Proteínas com Motivo Tripartido/química
15.
Viruses ; 14(5)2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35632650

RESUMO

The Andrias davidianus ranavirus (ADRV) is a member of the family Iridoviridae and belongs to the nucleocytoplasmic large DNA viruses. Based on genomic analysis, an ADRV-encoding protein, ADRV 12L, and its homologs from other iridoviruses were predicted as Rad2 family proteins based on the conserved amino acids, domains, and secondary structures. Expression analysis showed that the transcription of ADRV 12L started at 4 h post infection, and its expression was not inhibited by a DNA-replication inhibitor. Meanwhile, immunofluorescence localization showed that ADRV 12L mainly localized in viral factories and colocalized with the viral nascent DNA, which hinted at a possible role in DNA replication. Furthermore, a mutant ADRV lacking 12L (ADRV-Δ12L) was constructed. In both luciferase assays based on homologous recombination (HR) and double-strand break repair (DSBR) that followed, ADRV-Δ12L induced less luciferase activity than the wild-type ADRV, indicating that HR and DSBR were impaired in ADRV-Δ12L infected cells. In addition, infection with ADRV-Δ12L resulted in smaller plaque sizes and lower viral titers than that with wild-type ADRV, indicating an important role for 12L in efficient virus infection. Therefore, the results suggest that Rad2 homologs encoded by iridovirus have important roles in HR- and DSBR-process of the viral DNA and, thus, affect virus replication and the production of progeny virions.


Assuntos
Ranavirus , Animais , Reparo do DNA , DNA Viral/genética , DNA Viral/metabolismo , Ranavirus/genética , Ranavirus/metabolismo , Urodelos , Replicação Viral
16.
Viruses ; 14(5)2022 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-35632694

RESUMO

As nucleocytoplasmic large DNA viruses, replication of ranaviruses (genus Ranavirus, family Iridoviridae) involves a series of viral and host proteins. We have described that the replication and transcription machinery of Andrias davidianus ranavirus (ADRV) which was isolated from the Chinese giant salamander contained host factors. Here, a new host factor, the MutS homolog 2 (MSH2), was proved as an important protein that participated in ADRV infection. Expression of MSH2 was stable during ADRV infection in cultured cells and it localized at the cytoplasmic viral factories and colocalized with virus nascent DNA, indicating its possible role in virus genome replication. Investigation of the viral proteins that interacted with MSH2 by co-immunoprecipitation showed that A. davidianus MSH2 can interact with ADRV-35L (possible components associated with virus transcription), ADRV-47L (virus DNA polymerase), and ADRV-98R. Further knockdown MSH2 expression by RNAi significantly reduced the late gene expression of ADRV. Additionally, MSH2 knockout by CRISPR/Cas9 significantly reduced viral titers, genome replication, and late gene transcription of ADRV. Thus, the current study proved that ADRV can engage cellular MSH2 for its efficient genome replication and late gene transcription, which provided new information for understanding the roles of host factors in ranavirus replication and transcription.


Assuntos
Infecções por Vírus de DNA , Ranavirus , Animais , Reparo de Erro de Pareamento de DNA , DNA Viral/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Ranavirus/genética , Ranavirus/metabolismo , Urodelos
17.
J Virol ; 96(11): e0063422, 2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35575553

RESUMO

The global amphibian declines are compounded by infections with members of the Ranavirus genus such as Frog Virus 3 (FV3). Premetamorphic anuran amphibians are believed to be significantly more susceptible to FV3 while this pathogen targets the kidneys of both pre- and postmetamorphic animals. Paradoxically, FV3-challenged Xenopus laevis tadpoles exhibit lower kidney viral loads than adult frogs. Presently, we demonstrate that X. laevis tadpoles are intrinsically more resistant to FV3 kidney infections than cohort-matched metamorphic and postmetamorphic froglets and that this resistance appears to be epigenetically conferred by endogenous retroviruses (ERVs). Using a X. laevis kidney-derived cell line, we show that enhancing ERV gene expression activates cellular double-stranded RNA-sensing pathways, resulting in elevated mRNA levels of antiviral interferon (IFN) cytokines and thus greater anti-FV3 protection. Finally, our results indicate that large esterase-positive myeloid-lineage cells, rather than renal cells, are responsible for the elevated ERV/IFN axis seen in the tadpole kidneys. This conclusion is supported by our observation that CRISPR-Cas9 ablation of colony-stimulating factor-3 results in abolished homing of these myeloid cells to tadpole kidneys, concurrent with significantly abolished tadpole kidney expression of both ERVs and IFNs. We believe that the manuscript marks an important step forward in understanding the mechanisms controlling amphibian antiviral defenses and thus susceptibility and resistance to pathogens like FV3. IMPORTANCE Global amphibian biodiversity is being challenged by pathogens like the Frog Virus 3 (FV3) ranavirus, underlining the need to gain a greater understanding of amphibian antiviral defenses. While it was previously believed that anuran (frog/toad) amphibian tadpoles are more susceptible to FV3, we demonstrated that tadpoles are in fact more resistant to this virus than metamorphic and postmetamorphic froglets. We showed that this resistance is conferred by large myeloid cells within the tadpole kidneys (central FV3 target), which possess an elevated expression of endogenous retroviruses (ERVs). In turn, these ERVs activate cellular double-stranded RNA-sensing pathways, resulting in a greater expression of antiviral interferon cytokines, thereby offering the observed anti-FV3 protection.


Assuntos
Infecções por Vírus de DNA , Retrovirus Endógenos , Ranavirus , Xenopus laevis , Animais , Linhagem Celular , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Resistência à Doença , Retrovirus Endógenos/imunologia , Interferons/imunologia , Rim/virologia , Larva/imunologia , Larva/virologia , RNA de Cadeia Dupla , Ranavirus/patogenicidade , Xenopus laevis/virologia
18.
Viruses ; 14(5)2022 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-35632814

RESUMO

To cope with amphibian die-offs caused by ranavirus, it is important to know the underlying ranavirus prevalence in a region. We studied the ranavirus prevalence in tadpoles of two native and one introduced anuran species inhabiting agricultural and surrounding areas at 49 locations across eight provinces of South Korea by applying qPCR. The local ranavirus prevalence and the individual infection rates at infected locations were 32.6% and 16.1%, respectively, for Dryophytes japonicus (Japanese tree frog); 25.6% and 26.1% for Pelophylax nigromaculatus (Black-spotted pond frog); and 30.5% and 50.0% for Lithobates catesbeianus (American bullfrog). The individual infection rate of L. catesbeianus was significantly greater than that of D. japonicus. The individual infection rate of P. nigromaculatus was related to the site-specific precipitation and air temperature. The individual infection rate gradually increased from Gosner development stage 39, and intermittent infection was confirmed in the early and middle developmental stages. Our results show that ranavirus is widespread among wild amphibians living in agricultural areas of South Korea, and mass die-offs by ranavirus could occur at any time.


Assuntos
Anuros , Infecções por Vírus de DNA , Ranavirus , Animais , Anuros/virologia , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/veterinária , Prevalência , Rana catesbeiana/virologia , Ranavirus/isolamento & purificação , Ranidae/virologia , República da Coreia/epidemiologia
19.
Fish Shellfish Immunol ; 126: 113-121, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35609761

RESUMO

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1ß, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Ranavirus , Animais , Clonagem Molecular , Proteínas de Peixes/química , Imunidade Inata/genética , Iridovirus/fisiologia , Mamíferos/genética , Mamíferos/metabolismo , Pró-Proteína Convertase 9/genética , Ranavirus/fisiologia
20.
Fish Shellfish Immunol ; 124: 372-379, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35430348

RESUMO

Grouper iridovirus is a devastating pathogen that belongs to the genus Ranavirus. Based on the previous results that natural ingredient quercetin isolated from Illicium verum Hook. f. could effectively inhibit Singapore grouper iridovirus (SGIV) replication, suggesting that quercetin could serve as potential antiviral agent against grouper iridovirus. To know about whether quercetin has indirect antiviral activity against SGIV, this study made the investigation in vitro and in vivo, and the potential mechanism was also explored. Pretreating the cells with quercetin (12.5 µg/mL) significantly inhibited the replication of SGIV, similar results were also confirmed in vivo. Importantly, quercetin pretreatment could induce the expression of genes involved in type I interferon (IFN) system (IFN, STAT1, PKR, MxI and ISG15) and TLR9. It suggested that quercetin exerted the indirect antiviral activity against SGIV infection through promoting the recognition of SGIV and activating the IFN pathway to establish the antiviral status of host cell. Taken together, our results shedded light on the indirect antiviral function of natural ingredient quercetin, and clearly demonstrated that natural ingredient quercetin will be an excellent potential agent against SGIV infection in grouper aquaculture.


Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Plantas Medicinais , Ranavirus , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Bass/genética , Infecções por Vírus de DNA/veterinária , Quercetina/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...