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1.
Arch Virol ; 165(1): 169-178, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31773326

RESUMO

Plant viruses can alter the behavior or performance of their arthropod vectors, either indirectly (through effects of virus infection on the host plant) or directly (from virus acquisition by the vector). Given the diversity of plant viruses and their arthropod vectors, the effects for any specific system are not possible to predict. Here, we present experimental evidence that acquisition of maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) modifies the biological traits of its insect vector, the small brown planthopper (SBPH) Laodelphax striatellus. MIMV is an economically important virus of maize and several other grass species. It is transmitted by SBPHs in a persistent-propagative manner. We evaluated the effects of MIMV acquisition by SBPH on its life history when reared on healthy barley plants (Hordeum vulgare). We conclude that 1) MIMV acquisition by SBPHs increases female fecundity, duration of the nymph stage, adult longevity, and survival of SBPHs, (2) the mortality rate and female-to-male sex ratio are reduced in MIMV-infected planthoppers, and (3) MIMV infection increases the concentration of some biochemical components of the infected plants, including carbohydrates, some amino acids, and total protein, which might influence the life traits of its insect vector. The results indicate the potential of MIMV to improve the ecological fitness of its vector, SBPH, through direct or indirect effects, with the potential to increase the spread of the virus.


Assuntos
Hemípteros/fisiologia , Rhabdoviridae/fisiologia , Zea mays/metabolismo , Zea mays/virologia , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos , Feminino , Fertilidade , Hemípteros/virologia , Insetos Vetores/fisiologia , Insetos Vetores/virologia , Longevidade , Masculino , Proteínas de Plantas/metabolismo , Vírus de Plantas/fisiologia
2.
Plant Dis ; 104(1): 222-226, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31660798

RESUMO

Wheat yellow striate virus (WYSV), which is found in wheat fields of Northwest China and transmitted by leafhopper vector Psammotettix alienus, is a tentative new species in the genus Nucleorhabdovirus. Although the insect vector and host range of WYSV have been characterized, many aspects of the acquisition and transmission processes by its insect vector have not been elucidated. Here, the transmission parameters of WYSV by P. alienus were determined using wheat cv. Yangmai 12 as the indicator plant under a controlled temperature (23 ± 1°C) and photoperiod (16 h of light). The results showed that the minimum periods for acquisition were 5 min and 10 min for inoculation access. The latent period for successful transmission was most commonly 16 to 20 days (minimum, 10 days; maximum, 22 days). The quantitative reverse-transcriptase PCR results indicated that the WYSV titer increased with time after acquisition, suggesting that WYSV can replicate in P. alienus. Notably, female P. alienus transovarially transmitted the virus to next generations at relatively high efficiency. Electron microscopy of the WYSV-infected leafhopper revealed bacilliform particles aggregated in the cytoplasm of the salivary gland and midgut tissues. Our present studies suggested that acquisition and transmission of WYSV by P. alienus is consistent with a propagative, circulative, and persistent mode of transmission. Details regarding transmission competencies and distribution of WYSV in P. alienus will provide a basis for designing preventive measures.


Assuntos
Hemípteros , Rhabdoviridae , Animais , China , Feminino , Hemípteros/ultraestrutura , Hemípteros/virologia , Insetos Vetores/virologia , Microscopia Eletrônica , Reação em Cadeia da Polimerase , Rhabdoviridae/genética , Rhabdoviridae/fisiologia , Triticum/virologia
3.
Fish Shellfish Immunol ; 94: 685-696, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31546038

RESUMO

The S100 family proteins are a group of small acidic polypeptides and have diverse functions in regulating many aspects of physiological processes. They are structurally conserved and possess two EF-hands which are central for calcium-mediated functions. In this study, 14 S100 cDNA sequences were determined in zebrafish and their genomic organizations confirmed. Re-analyzing the gene synteny of the S100 loci identified two major S100 loci in Chr16 and Chr19 which share remarkable conservation with the S100 locus in human Chr1, suggesting they may have evolved from a single locus during the teleost specific whole genome duplication event. It appears that the homologues of human S100G and S100P have been lost in zebrafish. Expression analysis reveals that S100W, ICN1 and ICN2 are markedly expressed in embryos. Further, the transcripts of S100 genes are relatively abundant in mucosal tissues such as gills and gut. Intraperitoneal injection of poly(I:C) resulted in up-regulation of most S100 genes in the gut and spleen, with highest induction of S100V2 and S100Z detected. In fish challenged with spring viremia of carp virus (SVCV), expression of most S100 family genes was increased in the spleen between day 1 and 7 post infection, with consistent induction seen for the S100A1, S100A10b, S100B, S100ICN1, S100T, S100U, S100V1 and S100Z. Interestingly, intraperitoneal injection of Edwardsiella tarda down-regulated S100 expression in the gut but resulted in induction in the spleen. The results demonstrate that the S100 family genes are differentially modulated by bacterial and viral pathogens in zebrafish.


Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas S100/genética , Transcriptoma/imunologia , Peixe-Zebra/imunologia , Animais , Edwardsiella tarda/fisiologia , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Poli I-C/farmacologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Proteínas S100/química , Proteínas S100/metabolismo
4.
Fish Shellfish Immunol ; 93: 406-415, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31369857

RESUMO

Mandarin fish (Siniperca chuatsi) is a universally farmed fish species in China and has a large farming scale and economic value. With the high-density cultural mode in mandarin fish, viral diseases, such as infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV), have increased loss, which has seriously restricted the development of aquaculture. Y-Box binding protein 1 (YB-1) is a member of cold shock protein family that regulates multiple cellular processes. The roles of mammalian YB-1 protein in environmental stress and innate immunity have been studied well, but its roles in teleost fishes remain unknown. In the present study, the characteristic of S. chuatsi YB-1 (scYB-1) and its roles in cold stress and virus infection were investigated. The scYB-1 obtained an 1541 bp cDNA that contains a 903 bp open reading frame encoding a protein of 300 amino acids. Tissue distribution results showed that the scYB-1 is a ubiquitously expressed gene found among tissues from mandarin fish. Overexpression of scYB-1 can increase the expression levels of cold shock-responsive genes, such as scHsc70a, scHsc70b, and scp53. Furthermore, the role of scYB-1 in innate immunity was also investigated in mandarin fish fry (MFF-1) cells. The expression level of scYB-1 was significant change in response to poly (I:C), poly (dG:dC), PMA, ISKNV, or SCRV stimulation. The overexpression of scYB-1 can significantly increase the expression levels of NF-κB-responsive genes, including scIL-8, scTNF-α, and scIFN-h. The NF-κB-luciferase report assay results showed that the relative expression of luciferin was significantly increased in the cells overexpressed with scYB-1 compared with those in cells overexpressed with control plasmid. These results indicate that scYB-1 can induce the NF-κB signaling pathway in MFF-1 cells. Overexpressed scYB-1 can downregulate the expression of ISKNV viral major capsid protein (mcp) gene but upregulates the expression of SCRV mcp gene. Moreover, knockdown of scYB-1 using siRNA can upregulate the expression of ISKNV mcp gene but downregulates the expression of SCRV mcp gene. These results indicate that scYB-1 suppresses ISKNV infection while enhancing SCRV infection. The above observations suggest that scYB-1 is involved in cold stress and virus infection. Our study will provide an insight into the roles of teleost fish YB-1 protein in stress response and innate immunity.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Iridoviridae/fisiologia , Filogenia , Poli I-C/farmacologia , Polidesoxirribonucleotídeos/farmacologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Acetato de Tetradecanoilforbol/farmacologia , Proteína 1 de Ligação a Y-Box/química
5.
Fish Shellfish Immunol ; 93: 108-115, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31326582

RESUMO

Protein arginine methylation is a prevalent posttranslational modification and protein arginine methyltransferases 6 (PRMT6) has been identified as a suppressor of TBK1/IRF3 in human and mammals. To explore the role of PRMT6 in teleost fish, PRMT6 homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Black carp PRMT6 (bcPRMT6) transcription in host cells varies in response to different stimuli and bcPRMT6 migrates around 43 kDa in the immunoblot assay. Like its mammalian counterpart, bcPRMT6 has been identified to distribute majorly in the nucleus through the immunofluorescent staining assay. bcPRMT6 shows little interferon (IFN) promoter-inducing activity in the reporter assay and bcPRMT6 shows no antiviral activity against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV) in plaque assay. When co-expressed with bcPRMT6, the IFN promoter-inducing abilities of black carp TBK1 (bcTBK1) and IRF3/7 (bcIRF3/7) are fiercely attenuated. Accordingly, bcTBK1-mediated antiviral activity in EPC cells is obviously dampened by bcPRMT6. The interaction between bcPRMT6 and bcIRF3/7 has been identified by co-immunoprecipitation assay; however, no direct association between bcPRMT6 and bcTBK1 has been detected. Taken together, our data elucidates for the first time in teleost fish that PRMT6 suppresses TBK1-IRF3/7 signaling during host antiviral innate immune activation.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Perfilação da Expressão Gênica/veterinária , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Filogenia , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/imunologia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Transdução de Sinais
6.
Fish Shellfish Immunol ; 92: 224-229, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31200068

RESUMO

Fibroblast growth factor receptor (FGFR) 3 is one of the four distinct membrane-spanning tyrosine kinases required for proper skeletal development. In fish, the role of FGFR3 is still unclear. In this article, we reveal that zebrafish FGFR3 is a negative regulator of interferon (IFN) production in the innate immune response by suppressing the activity of TANK-binding kinase 1 (TBK1) in the process of virus infection. qPCR experiments demonstrate that the transcriptional level of cellular FGFR3 was upregulated by infection with spring viremia of carp virus (SVCV), indicating that FGFR3 might be involved in the process of host cell response to viral infection. Then, overexpression of FGFR3 significantly impeded the IFN promoter activity induced by a stimulator. In addition, the capabilities of a retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system to activate IFN promoter were decreased during the overexpression of FGFR3. Subsequently, FGFR3 decreased the phosphorylation of interferon regulatory factor 3 (IRF3) and mediator of IRF3 activation (MITA) by TBK1. These findings suggest that zebrafish FGFR3 is a negative regulator of IFN by attenuating the kinase activity of TBK1, leading to the suppression of IFN expression.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata/genética , Interferons/genética , Proteínas Serina-Treonina Quinases/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/imunologia , Animais , Interferons/metabolismo , Proteínas Serina-Treonina Quinases/imunologia , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/imunologia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Transdução de Sinais/imunologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia , Proteínas de Peixe-Zebra/fisiologia
7.
Fish Shellfish Immunol ; 92: 125-132, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31125665

RESUMO

Cytidine/uridine monophosphate kinase 2 (CMPK2) is known as a nucleoside monophosphate kinase in mitochondria to maintains intracellular UTP/CTP, and could be induced by immunostimulants LPS and Poly (I:C) in mammals, suggesting its potential antiviral and antibacterial role. In this study, CMPK2 was cloned and characterized in Fathead minnow (FHM) cells. In vivo analysis of tissue distribution revealed that CMPK2 transcript was detected in all the tissues of zebrafish (Danio rerio) examined in this study, particularly abundant in liver, spleen and kidney. In addition, indirect immunofluorescence showed that CMPK2 was localized in the cytoplasm of FHM cells. Expression of CMPK2 mRNA was significantly up-regulated following challenge with Spring viraemia of carp virus (SVCV), poly(I:C), or zebrafish IFN1 and IFN3 both in vitro and in vivo. Furthermore, overexpression and RNA interference of CMPK2 in SVCV-infected FHM cells showed significantly antiviral effect. In summary, this study for the first time shows the presence and distribution of CMPK2 in different tissues of zebrafish, but also demonstrates its antiviral potential against SVCV infection in vivo. These new findings could contribute to explain the molecular mechanism of the CMPK2 mediated antiviral function.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/imunologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Sequência de Aminoácidos , Animais , Perfilação da Expressão Gênica/veterinária , Interferons/metabolismo , Filogenia , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária
8.
New Phytol ; 223(4): 2120-2133, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31059138

RESUMO

Plant viruses have been used as rapid and cost-effective expression vectors for heterologous protein expression in genomic studies. However, delivering large or multiple foreign proteins in monocots and insect pests is challenging. Here, we recovered a recombinant plant cytorhabdovirus, Barley yellow striate mosaic virus (BYSMV), for use as a versatile expression platform in cereals and the small brown planthopper (SBPH, Laodelphax striatellus) insect vector. We engineered BYSMV vectors to provide versatile expression platforms for simultaneous expression of three foreign proteins in barley plants and SBPHs. Moreover, BYSMV vectors could express the c. 600-amino-acid ß-glucuronidase (GUS) protein and a red fluorescent protein stably in systemically infected leaves and roots of cereals, including wheat, barley, foxtail millet, and maize plants. Moreover, we have demonstrated that BYSMV vectors can be used in barley to analyze biological functions of gibberellic acid (GA) biosynthesis genes. In a major technical advance, BYSMV vectors were developed for simultaneous delivery of CRISPR/Cas9 nuclease and single guide RNAs for genomic editing in Nicotiana benthamiana leaves. Taken together, our results provide considerable potential for rapid screening of functional proteins in cereals and planthoppers, and an efficient approach for developing other insect-transmitted negative-strand RNA viruses.


Assuntos
Grão Comestível/genética , Grão Comestível/virologia , Genoma de Planta , Genômica , Hemípteros/virologia , Vírus de Plantas/fisiologia , Rhabdoviridae/fisiologia , Animais , Sequência de Bases , DNA Complementar/genética , Edição de Genes , Vetores Genéticos/metabolismo , Glucuronidase/metabolismo , Hordeum/ultraestrutura , Hordeum/virologia , Folhas de Planta/virologia , Vírus de Plantas/ultraestrutura , RNA Guia/metabolismo , Rhabdoviridae/ultraestrutura , Tabaco/ultraestrutura , Tabaco/virologia
9.
Insect Biochem Mol Biol ; 109: 13-23, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959110

RESUMO

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.


Assuntos
Proteínas Argonauta/genética , Culex/imunologia , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , Vírus do Nilo Ocidental/fisiologia , Aedes/genética , Aedes/imunologia , Aedes/virologia , Animais , Proteínas Argonauta/imunologia , Linhagem Celular , Culex/genética , Culex/virologia , Feminino , Flavivirus/fisiologia , Proteínas de Insetos/imunologia , Intestinos/virologia , Ovário/metabolismo , Ovário/virologia , Interferência de RNA , RNA Interferente Pequeno/imunologia , Rhabdoviridae/fisiologia , Glândulas Salivares/metabolismo , Glândulas Salivares/virologia
10.
Virol Sin ; 34(4): 434-443, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30989427

RESUMO

Tripartite motif (TRIM) proteins were shown to play an important role in innate antiviral immunity. FinTRIM (ftr) is a new subset of TRIM genes that do not possess obvious orthologs in higher vertebrates. However, little is known about its function. In this study, we used bioinformatic analysis to examine the phylogenetic relationships and conserved domains of zebrafish (Danio rerio) ftr01, ftr42, and ftr58, as well as qualitative real-time PCR to examine their expression patterns in zebrafish embryonic fibroblast (ZF4) cells and zebrafish tissues. Sequence analysis showed that the three finTRIMs are highly conserved, and all contain a RING domain, B-box domain, and SPRY-PRY domain. In addition, ftr42 and ftr58 had one coiled-coil domain (CCD), whereas ftr01 had two CCDs. Tissue expression analysis revealed that the mRNA level of ftr01 was the highest in the liver, whereas those of ftr42 and ftr58 were the highest in the gill; the expression of these finTRIMs was clearly upregulated not in the eyes, but in the liver, spleen, kidney, gill, and brain of zebrafish following spring viremia of carp virus (SVCV) infection. Similarly, the expression of these three finTRIM genes also increased in ZF4 cells after SVCV infection. Our study revealed that ftr01, ftr42, and ftr58 may play an important role in antiviral immune responses, and these findings validate the need for more in-depth research on the finTRIM family in the future.


Assuntos
Doenças dos Peixes/virologia , Imunidade Inata/genética , Filogenia , Proteínas com Motivo Tripartido/genética , Peixe-Zebra/genética , Animais , Biologia Computacional , Feminino , Doenças dos Peixes/imunologia , Expressão Gênica , Masculino , Rhabdoviridae/fisiologia , Análise de Sequência de DNA , Proteínas com Motivo Tripartido/imunologia , Peixe-Zebra/imunologia , Peixe-Zebra/virologia
11.
Fish Shellfish Immunol ; 89: 736-744, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31002927

RESUMO

TAK1-binding protein 1 (TAB1) forms the protein complex with TAK1 and enhances its kinase activity in human and mammals. To elucidate the role of TAB1 in the innate immunity of teleost sfih, the TAB1 homologue of black carp (Mylopharyngodon piceus) (bcTAB1) has been cloned and characterized in this paper. bcTAB1 is composed of 498 amino acids and contains a typical PP2Cc domain like its mammalian counterpart. The transcription of bcTAB1 gene in vivo and ex vivo varied in response to different stimuli; and the immunofluorescence staining showed that bcTAB1 was distributed in both cytoplasm and nucleus of host cell. The reporter assay showed that neither bcTAB1-expression alone nor co-expression of bcTAB1 and bcTAK1 could activate the transcription of IFN in EPC cells. Accordingly, EPC cells expressing bcTAB1 or co-expressing bcTAB1 and bcTAK1 showed no improved antiviral activity against grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). However, EPC cells co-expressing bcTAB1, bcTAK1 and bcIRF7 showed fiercely increased IFN-inducing ability in reporter assay and obviously improved antiviral activity in plaque assay compared with EPC cells co-expressing bcTAK1 and bcIRF7. The subsequent co-immunoprecipitation assay identified that bcTAB1 associated with bcTAK1 but not interacted with bcIRF7. Based on our previous finding that bcTAK1 up-regulates bcIRF7-mediated IFN signaling during host innate immune activation, the data generated in this study support the conclusion that bcTAB1 interacts with bcTAK1 and boosts bcTAK1-activated bcIRF7/IFN signaling during host antiviral innate immune response against GCRV and SVCV.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Peptídeos e Proteínas de Sinalização Intracelular/química , Filogenia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária
12.
Fish Shellfish Immunol ; 89: 18-26, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30905838

RESUMO

Triploid hybrid (3n = 150) of red crucian carp (♀, 2n = 100) and allotetraploid (♂, 4n = 200) presents the obviously stronger disease resistance than its parents. To elucidate the innate immunity of triploid hybrid, the MAVS homologues of triploid hybrid (3nMAVS), red crucian carp (2nMAVS) and allotetraploid (4nMAVS) have been identified and characterized separately in this study. 2nMAVS and 4nMAVS were evolutionarily conserved; however, 3nMAVS showed lower amino acid similarity and differently predicted structure to 2nMAVS or 4nMAVS. 3nMAVS transcription increase rate in host cells were obviously higher than 2nMAVS or 4nMAVS in response to different stimuli, which included spring viraemia of carp virus (SVCV), grass carp reovirus (GCRV) and poly (I:C). The reporter assay in EPC cells showed that 3nMAVS owned much stronger ability to induce the production of DrIFNφ1 and eIFN than either 2nMAVS or 4nMAVS. Accordingly, EPC cells transfected with 3nMAVS presented obviously stronger antiviral activity against both GCRV and SVCV than the cells expressing 2nMAVS or 4nMAVS. All the data support the conclusion that 3nMAVS-mediated antiviral signaling during innate immune activation was stronger than those of 2nMAVS and 4nMAVS, which provided us the new insight on the innate immune system of triploid hybrid.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Carpas/genética , Carpas/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Animais , Cruzamento , Doenças dos Peixes/imunologia , Poli I-C/farmacologia , Reoviridae/fisiologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/veterinária , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Tetraploidia , Triploidia
13.
J Fish Dis ; 42(5): 773-776, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30850994

RESUMO

This work reports a mortality outbreak, occurred in 2015 and affecting juveniles of European perch (Perca fluviatilis L.) farmed in Italy. Perch rhabdovirus (PRV) was detected by viral isolation and biomolecular investigations. Phylogenetic analysis clustered our isolate into genogroup B, which also includes PRV isolates from Perca fluviatilis identified in France (2004-2009); diagnostic investigations also revealed opportunistic bacteria (Aeromonas hydrophila) and parasites (Chilodonella piscicola). Since, occasionally, PRV has been reported in the natural environment, which is often a source of eggs and broodstock for farms, it could be possible that both similar France and Italian isolate were imported from a same place elsewhere and have a common origin. Improving biosecurity measures (batch control) and disinfection of egg strings with an iodine-based solution helps prevent apparent vertical transmission of PRV.


Assuntos
Surtos de Doenças/veterinária , Doenças dos Peixes/mortalidade , Percas , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Animais , Aquicultura , Doenças dos Peixes/virologia , Itália/epidemiologia , Filogenia , Rhabdoviridae/classificação , Infecções por Rhabdoviridae/mortalidade , Infecções por Rhabdoviridae/virologia
14.
Fish Shellfish Immunol ; 87: 871-878, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30776542

RESUMO

Viral infection is often accompanied with alteration of intracellular redox state, especially an imbalance between reactive oxygen species (ROS) production and antioxidant cellular defenses. The previous studies showed that an antioxidant cellular defense system, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), played an important role against spring viraemia of carp virus (SVCV) infection in fish. To further reveal the mediated mechanism that Nrf2 active state was affected by protein kinase C (PKC), here we evaluated SVCV replication in host cells by treated with a strong activator of PKC phorbol-12-myristate-13-acetate (PMA) and an inhibitor staurosporine. Our results showed that PMA significantly repressed SVCV replication and viral-induced apoptosis in Epithelioma papulosum cyprini (EPC) cell, suggesting that PKC may exhibit an anti-SVCV effect. Likewise, PMA resulted in a higher phosphorylation levels of PKCε rather than PKCα/ß to participate in the activation of Nrf2, mainly involved in the activation of Nrf2 phosphorylation of Ser40 to favor Nrf2 translocation to nucleus. Furthermore, the data revealed that PMA up-regulated an antiviral response heme oxygenase-1 (HO1) gene expression that was confirmed as the key player against SVCV infection by HO1 specific siRNA. Overall, this study provided a new therapeutic target for the treatment of SVCV infection, and modulating PKC activity could be used for the prevention and treatment of SVCV.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Fator 2 Relacionado a NF-E2/imunologia , Proteína Quinase C-épsilon/imunologia , Rhabdoviridae/fisiologia , Acetato de Tetradecanoilforbol/análogos & derivados , Animais , Antioxidantes/metabolismo , Carpas/genética , Linhagem Celular , Proteínas de Peixes/genética , Fator 2 Relacionado a NF-E2/genética , Proteína Quinase C-épsilon/genética , Espécies Reativas de Oxigênio/metabolismo , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Acetato de Tetradecanoilforbol/farmacologia
15.
Fish Shellfish Immunol ; 87: 809-819, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30776543

RESUMO

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/veterinária
16.
Microb Pathog ; 129: 146-151, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30731189

RESUMO

To distinguish between three types of Siniperca chuatsi rhabdovirus (SCRV) viral RNA (vRNA, cRNA, and mRNA) and investigate SCRV transcription and replication dynamics in Chinese perch brain CPB cells, a novel, strand-specific, reverse transcriptase quantitative real-time PCR (RT-qPCR) assay was established. The method is based on strand-specific reverse transcription, using tagged primers to add a 'tag' sequence at the 5' end. We used the 'tag' sequence as the forward primer and a strand-specific reverse primer to quantify the three types of RNA. Three types of synthetic viral RNA were used as reference standards for validation and quantification. These assays were optimized to produce a standard curve from 102 to 107 copies/µL, with an efficiency of 91-101% and an R2 value of 0.9949-0.9999. The coefficients of variation for repeatability and reproducibility were less than 2.85% and 5.52%, respectively. Using this method, specific target RNA was detected at a 3500-70,000 fold higher level than other types of RNA. This method was also used to evaluate the dynamics of vRNA, cRNA and mRNA synthesis in CPB cells infected with SCRV. The results indicate that the intracellular dynamics of vRNA, cRNA and mRNA are different. In the earliest phase of SCRV infection, all three types of viral RNA increased very slowly. The copy number of vRNA and mRNA increased exponentially from 4 h post infection, while cRNA increased from 6 h post infection. The amount of cRNA was lower than vRNA and mRNA throughout the infection. The novel, strand-specific RT-qPCR method developed in this study provides critical data to aid the understanding of transcription and replication during SCRV infection.


Assuntos
Doenças dos Peixes/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Transcrição Genética , Replicação Viral , Animais , Encéfalo/virologia , Percas , RNA Viral/genética , Reprodutibilidade dos Testes , Rhabdoviridae/genética , Infecções por Rhabdoviridae/virologia , Sensibilidade e Especificidade
17.
Mar Drugs ; 17(2)2019 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-30717094

RESUMO

Global health is under attack by increasingly-frequent pandemics of viral origin. Antimicrobial peptides are a valuable tool to combat pathogenic microorganisms. Previous studies from our group have shown that the membrane-lytic region of turbot (Scophthalmus maximus) NK-lysine short peptide (Nkl71⁻100) exerts an anti-protozoal activity, probably due to membrane rupture. In addition, NK-lysine protein is highly expressed in zebrafish in response to viral infections. In this work several biophysical methods, such as vesicle aggregation, leakage and fluorescence anisotropy, are employed to investigate the interaction of Nkl71⁻100 with different glycerophospholipid vesicles. At acidic pH, Nkl71⁻100 preferably interacts with phosphatidylserine (PS), disrupts PS membranes, and allows the content leakage from vesicles. Furthermore, Nkl71⁻100 exerts strong antiviral activity against spring viremia of carp virus (SVCV) by inhibiting not only the binding of viral particles to host cells, but also the fusion of virus and cell membranes, which requires a low pH context. Such antiviral activity seems to be related to the important role that PS plays in these steps of the replication cycle of SVCV, a feature that is shared by other families of virus-comprising members with health and veterinary relevance. Consequently, Nkl71⁻100 is shown as a promising broad-spectrum antiviral candidate.


Assuntos
Antivirais/farmacologia , Linguados , Fragmentos de Peptídeos/farmacologia , Proteolipídeos/química , Proteolipídeos/farmacologia , Rhabdoviridae/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antivirais/química , Linhagem Celular , Cyprinidae , Doenças dos Peixes/tratamento farmacológico , Doenças dos Peixes/virologia , Concentração de Íons de Hidrogênio , Fragmentos de Peptídeos/química , Fosfolipídeos/química , Fosfolipídeos/farmacologia , Rhabdoviridae/fisiologia , Viremia/tratamento farmacológico , Viremia/virologia , Replicação Viral/efeitos dos fármacos
18.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626676

RESUMO

The Sf9 and Sf21 cell lines derived from ovarian tissues of the wide-host-range phytophagous lepidopteran Spodoptera frugiperda are widely used for research and commercial-scale production of recombinant proteins. These cell lines are chronically infected with a rhabdovirus (Sf-RV) that does not cause any overt cytopathic effects. We demonstrate that wild populations of S. frugiperda in the eastern United States and Caribbean are infected with genetically diverse strains of Sf-RV and that this virus is also capable of infecting cells of Spodoptera exigua, Heliothis subflexa, and Bombyx mori Feeding studies demonstrated the ability of S. frugiperda larvae to deposit Sf-RV onto human-consumed vegetables during feeding. Although no evidence for replication in two species of plant cells was detected, subcellular localization studies demonstrated that the Sf-RV nucleocapsid was targeted to plasmodesmata, while two forms of the accessory protein were differentiated on the basis of their ability to localize to nuclei. Collectively, the results from this study suggest that environmental exposure of humans to Sf-RV is likely to be commonplace and frequent, but its inability to replicate in plant or human cells suggests that there is no substantial risk to human health.IMPORTANCE Insect-derived cell lines are widely used commercially for the production of vaccines and protein-based pharmaceuticals. After decades of safe and beneficial use, it was a surprise to the biotechnology industry to discover an endemic rhabdovirus in Sf9 cells. This discovery was made possible only by the substantial advancements in DNA sequencing technologies. Given the public health concerns associated with many rhabdovirus species, several initiatives were undertaken to establish that Spodoptera frugiperda rhabdovirus (Sf-RV) does not pose a threat to humans. Such actions include the generation of cell lines that have been cleared of Sf-RV. Given that Sf9 is derived from a moth whose larvae feed on human-edible foods, we explored the prevalence of Sf-RV in its wild and lab-grown populations, as well as its ability to be deposited on food items during feeding. Collectively, our data suggest that there is no overt risk from exposure to Sf-RV.


Assuntos
Especificidade de Hospedeiro/fisiologia , Rhabdoviridae/fisiologia , Spodoptera/virologia , Animais , Linhagem Celular , Humanos , Insetos/virologia , Larva/metabolismo , Larva/virologia , Plantas/virologia , Proteínas Recombinantes/metabolismo , Rhabdoviridae/metabolismo , Células Sf9 , Spodoptera/metabolismo , Proteínas Virais/metabolismo
19.
J Immunol ; 202(1): 119-130, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30504422

RESUMO

Viral infection activates the transcription factor IFN regulatory factor 7 (IRF7), which plays a critical role in the induction of IFNs and innate antiviral immune response. How virus-induced IFN signaling is controlled in fish is not fully understood. In this study, we demonstrate that N-myc downstream-regulated gene 1a (NDRG1a) in zebrafish plays a role as a negative regulator for virus-triggered IFN induction. First, the activation of the IFN promoter stimulated by the polyinosinic-polycytidylic acid or spring viremia of carp virus was decreased by the overexpression of NDRG1a. Second, NDRG1a interacted with IRF7 and blocked the IFN transcription activated by IRF7. Furthermore, NDRG1a was phosphorylated by TANK-binding kinase 1 (TBK1) and promoted the K48-linked ubiquitination and degradation of IRF7. Finally, the overexpression of NDRG1a blunted the transcription of several IFN-stimulated genes, resulting in the host cells becoming susceptible to spring viremia of carp virus infection. Our findings suggest that fish NDRG1a negatively regulates the cellular antiviral response by targeting IRF7 for ubiquitination and degradation, providing insights into the novel role of NDRG1a on the innate antiviral immune response in fish.


Assuntos
Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Fatores Reguladores de Interferon/metabolismo , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/imunologia , Animais , Células Cultivadas , Suscetibilidade a Doenças , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Ubiquitinação , Proteínas de Peixe-Zebra/genética
20.
Dev Comp Immunol ; 90: 157-164, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30253130

RESUMO

RIG-I like receptor (RLR) signaling functions importantly in host innate immune response against RNA virus, which is tightly regulated by a number of mechanisms to prevent aberrant interferon production. The suppressor of IKKε (SIKE) has been identified as a suppressor of IKKε and TBK1, which are key components of RLR signaling. In this study, SIKE homologue (bcSIKE) of black carp (Mylopharyngodon piceus) has been cloned and characterized. The transcription of bcSIKE varied in host cells in response to the stimulation of LPS, poly (I:C) and viruses. bcSIKE migrated around 27 KDa in immunoblot assay and distributed in both cytoplasm and nucleus of host cell in immunofluorescent (IF) staining test. bcSIKE showed no IFN-inducing ability in reporter assay and EPC cells expressing bcSIKE showed no enhanced antiviral ability against either grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV). However, bcSIKE obviously dampened the IFN-inducing ability of RLR signaling members in reporter assay when bcSIKE was co-expressed with these molecules in EPC cells. The association between bcSIKE and bcTBK1 has been identified through IF and co-immunoprecipitation (co-IP) assay. The plaque assay demonstrated clearly that bcTBK1-mediated antiviral activity in EPC cells against both GCRV and SVCV was down regulated by bcSIKE. All the data generated in this paper support the conclusion that bcSIKE interacts with bcTBK1 and inhibits bcTBK1-mediated antiviral signaling during host innate immune activation, which is reported in teleost for the first time.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Vírus de RNA/fisiologia , Infecções por Reoviridae/imunologia , Reoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Rhabdoviridae/fisiologia , Animais , Células Cultivadas , Clonagem Molecular , Proteína DEAD-box 58/metabolismo , Proteínas de Peixes/metabolismo , Humanos , Imunidade Inata , Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais
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