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1.
Cells ; 10(9)2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34572091

RESUMO

The use of lumpfish (Cyclopterus lumpus) as a cleaner fish to fight sea lice infestation in farmed Atlantic salmon has become increasingly common. Still, tools to increase our knowledge about lumpfish biology are lacking. Here, we successfully established and characterized the first Lumpfish Gill cell line (LG-1). LG-1 are adherent, homogenous and have a flat, stretched-out and almost transparent appearance. Transmission electron microscopy revealed cellular protrusions and desmosome-like structures that, together with their ability to generate a transcellular epithelial/endothelial resistance, suggest an epithelial or endothelial cell type. Furthermore, the cells exert Cytochrome P450 1A activity. LG-1 supported the propagation of several viruses that may lead to severe infectious diseases with high mortalities in fish farming, including viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV). Altogether, our data indicate that the LG-1 cell line originates from an epithelial or endothelial cell type and will be a valuable in vitro research tool to study gill cell function as well as host-pathogen interactions in lumpfish.


Assuntos
Proliferação de Células , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Brânquias/citologia , Brânquias/fisiologia , Perciformes/fisiologia , Animais , Linhagem Celular , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Brânquias/virologia , Novirhabdovirus/fisiologia , Perciformes/classificação , Perciformes/virologia
2.
Mar Biotechnol (NY) ; 23(4): 546-559, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34268626

RESUMO

To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P. The replication of rVHSV-A-amiR-C in EPC-ΔDicer cells was not different from that in control EPC cells, while the copy number of rVHSV-A-amiR-P was increasingly risen up in EPC-ΔDicer cells compared to that in control EPC cells, and the final viral titer of rVHSV-A-amiR-P was enhanced by culture in EPC-ΔDicer cells. These results indicate that VHSV can be attenuated by the equipment of self-mRNA-targeting microRNA sequence in the genome, and the titer of artificial miRNA-expressing attenuated recombinant VHSVs can be enhanced by the knockout of Dicer gene in EPC cells.


Assuntos
Genoma Viral/genética , Novirhabdovirus/genética , Replicação Viral/genética , Animais , Sistemas CRISPR-Cas , Carpas , Linhagem Celular , Linhagem Celular Tumoral , Cricetinae , RNA Helicases DEAD-box/genética , Doenças dos Peixes/virologia , Técnicas de Inativação de Genes , Septicemia Hemorrágica Viral , MicroRNAs , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo
3.
Fish Shellfish Immunol ; 116: 84-90, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34214656

RESUMO

Viral hemorrhagic septicemia virus (VHSV) causes severe mortality among more than 90 fish species. The 11 kb viral genome encodes six proteins including nonvirion protein (NV). In previous study, we reported that NV gene variations of VHSV decrease cellular energy metabolism. Among several NV mutant proteins, NV-S56L showed the highest cellular energy deprivation. Based on this finding, we further examined a molecular mechanism of one amino acid (S56L) change on differential cellular dysregulation. In the fish cells, the NV-S56L protein showed an increased level of cellular expression than normal and other mutant NV proteins without change of mRNA expression. Using cycloheximide treatment for exclude de novo NV protein expression, NV-S56L had an extensive half-life of intracellular protein. The proteasome inhibitor, MG-132, treatment recovered the all NV protein levels. The ubiquitination of NV was increased in the treatment of MG132 via inhibition of the ubiquitin/proteasome system process. Finally, increased protein stability of NV-S56L led to downregulation of NF-κB response immune gene expression. These results indicate that the prolonged protein stabilization of NV protein variant (NV-S56L) increases its pathological duration and might eventually lead to high virulence activity in the host fish cell.


Assuntos
Septicemia Hemorrágica Viral , Novirhabdovirus/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Peixes , Expressão Gênica/imunologia , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/imunologia , Estabilidade Proteica
4.
J Fish Dis ; 44(10): 1553-1562, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34160839

RESUMO

T-helper cells express CD4 as a co-receptor that binds to major histocompatibility complex class II to synchronize the immune response against upcoming threats via mediating several cytokines. We have previously reported the presence of CD4 homologues in brown trout. The study of cellular immune responses in brown trout is limited by the availability of specific antibodies. We here describe the generation of a polyclonal antibody against CD4-1 that allows for the investigation of CD4+ cells. We used this novel tool to study CD4+ cells in different tissues during viral haemorrhagic septicaemia infection (VHSV) using flow cytometric technique. Flow cytometric analyses revealed an enhanced level of surface CD4-1 expression in the infected group in major lymphoid organs and in the intestine. These results suggest an important role for the T-helper cells within the immune response against viruses, comparable to the immune response in higher vertebrates.


Assuntos
Linfócitos T CD4-Positivos/fisiologia , Doenças dos Peixes/imunologia , Septicemia Hemorrágica Viral/imunologia , Novirhabdovirus/fisiologia , Truta , Animais , Fenômenos Biomecânicos , Linfócitos T CD4-Positivos/virologia , Doenças dos Peixes/virologia , Septicemia Hemorrágica Viral/virologia , Cinética
5.
J Fish Dis ; 44(9): 1369-1383, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34002876

RESUMO

Viral haemorrhagic septicaemia virus (VHSV) is the cause of an important listed disease in European rainbow trout (Oncorhynchus mykiss) aquaculture and can be present in a wide range of fish species, including marine fish, which can act as viral reservoir. Recent studies revealed putative genetic virulence markers of VHSV to rainbow trout highlighting the roles of the nucleoprotein, phosphoprotein and non-virion protein. Using reverse genetics, we produced recombinant viruses by introducing parts of or the entire nucleoprotein from a high-virulent isolate VHSV into a low-virulent backbone. Furthermore, we also made recombinant viruses by introducing residue modifications in the nucleoprotein that seem to play a role in virulence. Rainbow trout challenged with these recombinant viruses (rVHSVs) by intraperitoneal injection (IP) developed clinical signs and showed lower survival when compared to the parental rVHSV whereas fish challenged by immersion did not show clinical signs except for the high-virulent control. The mutations did not influence the viral growth in cell culture. The recombinant viruses and parental recombinant were unable to replicate and show cytopathic effect in EPC cells whereas the high-virulent control was well adapted in all the fish cell lines tested. We showed evidence that corroborates with the hypothesis that the nucleoprotein has virulence motifs associated with VHSV virulence in rainbow trout.


Assuntos
Septicemia Hemorrágica Viral/virologia , Novirhabdovirus/genética , Virulência/genética , Animais , Linhagem Celular , Doenças dos Peixes/virologia , Peixes , Injeções Intraperitoneais , Novirhabdovirus/patogenicidade , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Oncorhynchus mykiss/virologia
6.
PLoS One ; 16(5): e0232923, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34048438

RESUMO

A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically-restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv- (3.8%), and M- (2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHSV genogroups and subgenogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I, -II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an origin date). The 2016 isolates that we tested induced milder impacts on fish host cell innate antiviral responses, suggesting altered phenotypic effects. In conclusion, our overall findings indicate that VHSV-IVb has undergone continued sequence change and a trend to lower virulence over its evolutionary history (2003 through present-day), which may facilitate its long-term persistence in fish host populations.


Assuntos
Doenças dos Peixes/epidemiologia , Peixes/virologia , Septicemia Hemorrágica Viral/epidemiologia , Novirhabdovirus/genética , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Septicemia Hemorrágica Viral/genética , Septicemia Hemorrágica Viral/virologia , Humanos , Lagos/virologia , Novirhabdovirus/isolamento & purificação , Novirhabdovirus/patogenicidade , Filogenia
7.
Microb Pathog ; 157: 104993, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34044046

RESUMO

Hirame novirhabdovirus (HIRRV) is a severe viral pathogen of flounder resulting in significant losses to the aquaculture industry. However, the mortality due to the disease would be significantly reduced when the water temperature was increased from 10 to 20 °C. In this study, we examined the potentiality of vaccination with live HIRRV under a temperature-controlled culture condition for development of protective immunity in flounder. Flounders were infected with HIRRV at 10 °C and maintained for 2 days, and then the temperature was shift up to 20 °C. When the temperature was further shift down to 10 °C at 7 (S-7 group), 14 (S-14 group) or 21 (S-21 group) days post infection (dpi), mortality rates of 60%, 13.33% and 0 were observed, respectively. To investigate the development of protective immunity of survived flounder, a re-challenge was performed and a highest survival rate of 80% was found in S-21 group, which was significantly higher than S-14 group (65%) and S-7 group (45%). Moreover, it was found that a lower viral load was detected in the flounder maintained at 20 °C for a longer time, and a longer maintaining of survived flounder at 20 °C would also elicit higher percentages of IgM + B lymphocytes and specific antibodies levels. Notably, a significantly higher levels of specific antibodies were detected post re-challenge compared with the first peak level after initial infection. Therefore, these demonstrated that the initial infection with live HIRRV under a temperature-controlled condition elicited an effective protective immune response against HIRRV, and maintaining at 20 °C for a long enough time would allow the HIRRV-infected flounder to eliminate the virus completely and acquired a protective immunity against HIRRV infection. This is the first study showing the possibility of developing an effective preventive measure against HIRRV by vaccination with live virus under controlled water temperature.


Assuntos
Doenças dos Peixes , Linguado , Novirhabdovirus , Infecções por Rhabdoviridae , Animais , Doenças dos Peixes/prevenção & controle , Temperatura , Vacinação
8.
Dis Aquat Organ ; 144: 245-252, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34042072

RESUMO

Processes that allow viral hemorrhagic septicemia (VHS) virus to persist in the marine environment remain enigmatic, owing largely to the presence of covert and cryptic infections in marine fishes during typical sub-epizootic periods. As such, marine host reservoirs for VHS virus have not been fully demonstrated, nor have the mechanism(s) by which infected hosts contribute to virus perpetuation and transmission. Here, we demonstrate that after surviving VHS, convalesced Pacific herring continue to shed virus at a low rate for extended periods. Further, exposure of previously naïve conspecific sentinels to this shed virus can result in infections for at least 6 mo after cessation of overt disease. This transmission mechanism was not necessarily dependent on the magnitude of the disease outbreak, as prolonged transmission occurred from 2 groups of donor herring that experienced cumulative mortalities of 4 and 29%. The results further suggest that the virus persists in association with the gills of fully recovered individuals, and long-term viral shedding or shedding relapses are related to cooler or decreasing water temperatures. These results provide support for a new VHS virus perpetuation paradigm in the marine environment, whereby the virus can be maintained in convalesced survivors and trafficked from these carriers to sympatric susceptible individuals.


Assuntos
Doenças dos Peixes , Septicemia Hemorrágica Viral , Novirhabdovirus , Animais , Surtos de Doenças , Doenças dos Peixes/epidemiologia , Peixes , Eliminação de Partículas Virais
9.
J Aquat Anim Health ; 33(1): 53-65, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33825241

RESUMO

Serological assays were conducted for anti-viral hemorrhagic septicemia virus (VHSV) antibodies in four species of fish in Wisconsin (Bluegill Lepomis macrochirus, Brown Trout Salmo trutta, Northern Pike Esox lucius, and Walleye Sander vitreus) to examine spatial and temporal distributions of exposure. Sera were tested for non-neutralizing anti-nucleocapsid antibodies to VHSV by blocking enzyme-linked immunosorbent assay (ELISA). Results (percent inhibition [%I]) were analyzed for differences among species, across geographic distance, and among water management units. Positive fish occurred in 37 of 46 inland water bodies tested, including in water bodies far from reported outbreak events. Using highly conservative species-specific thresholds (mean %I of presumptive uninfected fish + 2 SDs), 4.3% of Bluegill, 13.4% of Brown Trout, 19.3% of Northern Pike, and 18.3% of Walleye tested positive for VHSV antibodies by ELISA. Spatial patterns of seropositivity and changes in %I between sampling years were also analyzed. These analyses explore how serology might be used to understand VHSV distribution and dynamics and ultimately to inform fisheries management.


Assuntos
Esocidae , Doenças dos Peixes/epidemiologia , Septicemia Hemorrágica Viral/epidemiologia , Novirhabdovirus/isolamento & purificação , Percas , Perciformes , Animais , Doenças dos Peixes/virologia , Septicemia Hemorrágica Viral/virologia , Estudos Soroepidemiológicos , Truta , Wisconsin/epidemiologia
10.
Viruses ; 13(3)2021 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802100

RESUMO

Novirhabdoviruses cause large epizootics and economic losses of farmed trout. In this study, we surveyed Viral hemorrhagic septicemia virus and Infectious hematopoietic and necrosis virus (VHSV and IHNV) through both monitoring and investigation of clinical outbreaks reported by farmers in the regions with major rainbow trout production in Iran from 2015 to 2019. RT-PCR assays of the kidney samples and cell culture (EPC/FHM cells) samples confirmed the presence of the viruses, with 9 VHSV and 4 IHNV isolates, in both endemic and new areas of Iran. Sequence analysis of the G gene revealed that VHSV isolates belonged to genogroup Ia, and IHNV isolates were clustered into genogroup E, both typical for isolates from European countries. A haplotype analysis based on non-homologous amino acids of the G gene supports the emergence of two lineages of IHNV from clade 1 (E-1), as well as VHSV clade 2 (Ia-2) of the European genogroups, confirming that VHSV and IHNV isolates in Iran, have originated from Europe possibly via imported eggs.


Assuntos
Doenças dos Peixes/epidemiologia , Vírus da Necrose Hematopoética Infecciosa/isolamento & purificação , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/epidemiologia , Infecções por Rhabdoviridae/veterinária , Animais , Sequência de Bases , Surtos de Doenças , Europa (Continente)/epidemiologia , Doenças dos Peixes/virologia , Pesqueiros , Genótipo , Haplótipos/genética , Vírus da Necrose Hematopoética Infecciosa/genética , Irã (Geográfico)/epidemiologia , Epidemiologia Molecular , Novirhabdovirus/genética , Novirhabdovirus/isolamento & purificação , Filogenia , Análise de Sequência de DNA
11.
Microb Pathog ; 154: 104868, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33771630

RESUMO

Hirame rhabdovirus (HIRRV) is one of the most important viruses of fish, posing a great threat to the fish industry in Asia and Europe. The glycoprotein (G) of HIRRV is known to play important roles in virus attachment and entry, making it an ideal target for both diagnosis and therapy. In this study, a truncated G of HIRRV was expressed as a fusion protein in Escherichia coli. Using the recombinant G protein (rG), monoclonal antibodies (mAbs) were prepared by the hybridoma technology. Subsequently, positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA) and further characterized by Western blot and immunofluorescence assay (IFA). ELISA results showed that two mAbs (3E5 and 4D10) could react with the rG, as well as the purified HIRRV. Western blot analysis showed that the mAbs belong to the IgG isotype and could recognize a 60 kDa viral protein, which is consistent with the molecular weight of G protein and determined to be the G protein of HIRRV by mass spectrometry. The virions in HIRRV-infected EPC could also be recognized by two mAbs in IFA. Moreover, neutralization assay showed that mAb 4D10 could significantly inhibit the proliferation of HIRRV and delay the development of cytopathic effect in viral-infected EPC cells, and in vivo neutralization assay also showed that mAb 4D10 could significantly reduce the mortality of HIRRV-infected flounder, indicating that mAb 4D10 can partially neutralize the HIRRV infection. Western blot analysis showed that mAb 4D10 could specifically bind the C-terminal domain of HIRRV-G protein. These results demonstrated that the produced mAbs could specifically recognize the G protein of HIRRV and displayed virus-neutralizing activity in vitro and in vivo, which could serve as effective detection probes and potential neutralizing antibodies for HIRRV.


Assuntos
Doenças dos Peixes , Novirhabdovirus , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Ásia , Ensaio de Imunoadsorção Enzimática , Europa (Continente) , Glicoproteínas
12.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572970

RESUMO

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.


Assuntos
Doenças dos Peixes/genética , Proteínas de Peixes/genética , Linguado/genética , Septicemia Hemorrágica/veterinária , Novirhabdovirus/fisiologia , Proteínas Repressoras/genética , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Linguado/imunologia , Linguado/fisiologia , Septicemia Hemorrágica/genética , Septicemia Hemorrágica/imunologia , Interações Hospedeiro-Patógeno , Novirhabdovirus/imunologia , Filogenia , Proteínas Repressoras/imunologia , Transcrição Genética
13.
Fish Shellfish Immunol ; 111: 152-159, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33556552

RESUMO

The tetraspanin superfamily proteins are transmembrane proteins identified in a diverse range of eukaryotic organisms. Tetraspanins are involved in a variety of essential biological functions, including cell differentiation, adhesion, migration, signal transduction, intracellular trafficking, and immune responses. For an infection to occur, viruses must interact with various cell surface components, including receptors and signaling molecules. Tetraspanin CD63 is involved in the organization of the cell membrane and trafficking of cellular transmembrane proteins that interact with many viruses. In this study, the cd63 gene was characterized by studying its expression and function in a zebrafish model. The functional domains and structural features of Cd63, such as the Cys-Cys-Gly (CCG) motif in the large extracellular loop and cysteine residues, are conserved in zebrafish. We confirmed that cd63 was expressed in immune system organs, such as the axial vein and pronephric duct, during the embryonic development of zebrafish. To better understand the role of cd63 in the zebrafish immune system, we established cd63-deficient zebrafish lines using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. A 19 bp insertion mutation was generated in single guide RNA (sgRNA) target sequence of exon 3 of the cd63 gene, to create a pre-mature stop codon. We then analyzed the expression of cd63-related genes cxcr4a and cxcr4b in wild type (WT) and cd63-deficient zebrafish. We believe our study provides an important model that could be used to investigate the roles of cd63 in viral infection in vivo.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade/genética , Tetraspanina 30/genética , Tetraspanina 30/imunologia , Peixe-Zebra/genética , Peixe-Zebra/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/deficiência , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Novirhabdovirus/fisiologia , Filogenia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Alinhamento de Sequência/veterinária , Tetraspanina 30/química , Tetraspanina 30/deficiência
14.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467734

RESUMO

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder's CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doenças dos Peixes/imunologia , Linguado/imunologia , Imunidade Celular , Imunidade Adaptativa , Animais , Anticorpos Monoclonais/química , Proliferação de Células , Doenças dos Peixes/virologia , Citometria de Fluxo , Perfilação da Expressão Gênica , Imunização , Nodaviridae , Novirhabdovirus , Oligonucleotídeos/química , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Citotóxicos/imunologia , Vacinação
15.
PLoS Pathog ; 17(1): e1009213, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33465148

RESUMO

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Naturally occurring VHSV strains vary greatly in virulence. Until now, little has been known about genetic alterations that affect the virulence of VHSV in flounder. We recently reported the full-genome sequences of 18 VHSV strains. In this study, we determined the virulence of these 18 VHSV strains in flounder and then the assessed relationships between differences in the amino acid sequences of the 18 VHSV strains and their virulence to flounder. We identified one amino acid substitution in the phosphoprotein (P) (Pro55-to-Leu substitution in the P protein; PP55L) that is specific to highly virulent strains. This PP55L substitution was maintained stably after 30 cell passages. To investigate the effects of the PP55L substitution on VHSV virulence in flounder, we generated a recombinant VHSV carrying PP55L (rVHSV-P) from rVHSV carrying P55 in the P protein (rVHSV-wild). The rVHSV-P produced high level of viral RNA in cells and showed increased growth in cultured cells and virulence in flounder compared to the rVHSV-wild. In addition, rVHSV-P significantly inhibited the induction of the IFN1 gene in both cells and fish at 6 h post-infection. An RNA-seq analysis confirmed that rVHSV-P infection blocked the induction of several IFN-related genes in virus-infected cells at 6 h post-infection compared to rVHSV-wild. Ectopic expression of PP55L protein resulted in a decrease in IFN induction and an increase in viral RNA synthesis in rVHSV-wild-infected cells. Taken together, our results are the first to identify that the P55L substitution in the P protein enhances VHSV virulence in flounder. The data from this study add to the knowledge of VHSV virulence in flounder and could benefit VHSV surveillance efforts and the generation of a VHSV vaccine.


Assuntos
Doenças dos Peixes/virologia , Linguado/virologia , Novirhabdovirus/genética , Fosfoproteínas/genética , Infecções por Rhabdoviridae/virologia , Proteínas Virais/genética , Virulência/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Genoma Viral , Novirhabdovirus/metabolismo , Novirhabdovirus/patogenicidade , Fosfoproteínas/metabolismo , RNA-Seq , Homologia de Sequência , Transcriptoma , Proteínas Virais/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
Arch Virol ; 166(1): 191-206, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33145636

RESUMO

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus that causes high mortality in cultured flounder. Viral growth and virulence rely on the ability to inhibit the cellular innate immune response. In this study, we investigated differences in the modulation of innate immune responses of HINAE flounder cells infected with low- and high-virulence VHSV strains at a multiplicity of infection of 1 for 12 h and 24 h and performed RNA sequencing (RNA-seq)-based transcriptome analysis. A total of 193 and 170 innate immune response genes were differentially expressed by the two VHSV strains at 12 and 24 h postinfection (hpi), respectively. Of these, 73 and 77 genes showed more than a twofold change in their expression at 12 and 24 hpi, respectively. Of the genes with more than twofold changes, 22 and 11 genes showed high-virulence VHSV specificity at 12 and 24 hpi, respectively. In particular, IL-16 levels were more than two time higher and CCL20a.3, CCR6b, CCL36.1, Casp8L2, CCR7, and Trim46 levels were more than two times lower in high-virulence-VHSV-infected cells than in low-virulence-VHSV-infected cells at both 12 and 24 hpi. Quantitative PCR (qRT-PCR) confirmed the changes in expression of the ten mRNAs with the most significantly altered expression. This is the first study describing the genome-wide analysis of the innate immune response in VHSV-infected flounder cells, and we have identified innate immune response genes that are specific to a high-virulence VHSV strain. The data from this study can contribute to a greater understanding of the molecular basis of VHSV virulence in flounder.


Assuntos
Linguado/imunologia , Linguado/virologia , Septicemia Hemorrágica Viral/imunologia , Imunidade Inata/imunologia , Novirhabdovirus/genética , Novirhabdovirus/imunologia , Transcriptoma/genética , Virulência/genética , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Septicemia Hemorrágica Viral/virologia , RNA-Seq/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Transcriptoma/imunologia
17.
J Fish Dis ; 44(5): 563-571, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33170959

RESUMO

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96 hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.


Assuntos
Septicemia Hemorrágica Viral/virologia , Neutrófilos/metabolismo , Novirhabdovirus/fisiologia , Tropismo/fisiologia , Peixe-Zebra , Animais , Modelos Animais de Doenças
18.
J Fish Dis ; 44(2): 217-220, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33165930

RESUMO

Autophagy can markedly alter host response to infectious disease, and several studies have demonstrated that a restricted diet or deoxynivalenol modulates autophagy and reduces mortality of fish due to bacterial disease. The picture is less clear for viral diseases of fish. Duplicate tanks of fathead minnow, Pimephales promelas Rafinesque, were fed a replete diet (control), 100 µM chloroquine, 5 µM deoxynivalenol, 10% (fasted) or 40% of a replete diet (pair-fed) for 2 weeks and then experimentally infected by intraperitoneal injection with 2 × 105 viral haemorrhagic septicaemia virus IVb. Survival from highest to lowest for the different treatments was as follows: deoxynivalenol (average 43.3%); control (40.0%); pair-fed (35.0%); fasted (33.3%); and chloroquine (21.7%). No treatment significantly altered the survival rate of fathead minnow after VHSV IVb infection when compared to controls; however, the fish fed with chloroquine had significantly lower survival rate than the fish fed deoxynivalenol (p < .05).


Assuntos
Cloroquina/farmacologia , Doenças dos Peixes/virologia , Septicemia Hemorrágica Viral/patologia , Tricotecenos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Restrição Calórica , Cyprinidae , Novirhabdovirus/patogenicidade
19.
Fish Shellfish Immunol ; 109: 62-70, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33348035

RESUMO

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Glutationa Transferase/genética , Glutationa Transferase/imunologia , Imunidade Inata/genética , Smegmamorpha/genética , Smegmamorpha/imunologia , Sequência de Aminoácidos , Animais , Feminino , Doenças dos Peixes/virologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Glutationa Transferase/química , Masculino , Novirhabdovirus/fisiologia , Filogenia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Alinhamento de Sequência/veterinária
20.
J Fish Dis ; 44(4): 379-390, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33319917

RESUMO

Rainbow trout (Oncorhynchus mykiss) and common carp (Cyprinus carpio) are the two most common species in traditional fish farming in Germany. Their aquaculture is threatened upon others by viruses that can cause a high mortality. Therefore, this work focuses on three viruses-viral haemorrhagic septicaemia virus, infectious hematopoietic necrosis virus and cyprinid herpesvirus 3 (CyHV-3)-that endanger these species. To prevent their spread and contain further outbreaks, it is essential to know how long they can outlast in environmental waters and what affects their infectivity outside the host. Hence, the stability of the target viruses in various water matrices was examined and compared in this work. In general, all three viruses were quite stable within sterile water samples (showing mostly ≤1 log reduction after 96 hr) but were inactivated faster and to a higher extent (up to five log steps within 96 hr) in unsterile environmental water samples. The inactivation of the viruses correlated well with the increasing bacterial load of the samples, suggesting that bacteria had the greatest effect on their stability in the examined samples. In comparison, CyHV-3 seemed to be the most sensitive and maintained its infectivity for the shortest period.


Assuntos
Aquicultura , Herpesviridae/isolamento & purificação , Vírus da Necrose Hematopoética Infecciosa/isolamento & purificação , Novirhabdovirus/isolamento & purificação , Águas Residuárias/virologia , Animais , Carpas , Alemanha , Oncorhynchus mykiss , Águas Residuárias/análise
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