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1.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638687

RESUMO

Exosomes are associated with cancer progression, pregnancy, cardiovascular diseases, central nervous system-related diseases, immune responses and viral pathogenicity. However, study on the role of exosomes in the immune response of teleost fish, especially antiviral immunity, is limited. Herein, serum-derived exosomes from mandarin fish were used to investigate the antiviral effect on the exosomes of teleost fish. Exosomes isolated from mandarin fish serum by ultra-centrifugation were internalized by mandarin fish fry cells and were able to inhibit Infectious spleen and kidney necrosis virus (ISKNV) infection. To further investigate the underlying mechanisms of exosomes in inhibiting ISKNV infection, the protein composition of serum-derived exosomes was analyzed by mass spectrometry. It was found that myxovirus resistance 1 (Mx1) was incorporated by exosomes. Furthermore, the mandarin fish Mx1 protein was proven to be transferred into the recipient cells though exosomes. Our results showed that the serum-derived exosomes from mandarin fish could inhibit ISKNV replication, which suggested an underlying mechanism of the exosome antivirus in that it incorporates Mx1 protein and delivery into recipient cells. This study provided evidence for the important antiviral role of exosomes in the immune system of teleost fish.


Assuntos
Infecções por Vírus de DNA , Exossomos , Doenças dos Peixes , Proteínas de Peixes , Peixes , Iridoviridae , Proteínas de Resistência a Myxovirus , Animais , Linhagem Celular , Infecções por Vírus de DNA/sangue , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Exossomos/imunologia , Exossomos/metabolismo , Doenças dos Peixes/sangue , Doenças dos Peixes/imunologia , Proteínas de Peixes/sangue , Proteínas de Peixes/imunologia , Peixes/sangue , Peixes/imunologia , Peixes/virologia , Iridoviridae/imunologia , Iridoviridae/metabolismo , Proteínas de Resistência a Myxovirus/sangue , Proteínas de Resistência a Myxovirus/imunologia
2.
Arch Virol ; 166(11): 3061-3074, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34462803

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV) is a fish-pathogenic virus belonging to the genus Megalocytivirus of the family Iridoviridae. In 2018, disease occurrences (40-50% cumulative mortality) associated with ISKNV infection were reported in grown-out Asian sea bass (Lates calcarifer) cultured in an inland freshwater system in Thailand. Clinical samples were collected from seven distinct farms located in the eastern and central regions of Thailand. The moribund fish showed various abnormal signs, including lethargy, pale gills, darkened body, and skin hemorrhage, while hypertrophied basophilic cells were observed microscopically in gill, liver, and kidney tissue. ISKNV infection was confirmed on six out of seven farms using virus-specific semi-nested PCR. The MCP and ATPase genes showed 100% sequence identity among the virus isolates, and the virus was found to belong to the ISKNV genotype I clade. Koch's postulates were later confirmed by challenge assay, and the mortality of the experimentally infected fish at 21 days post-challenge was 50-90%, depending on the challenge dose. The complete genome of two ISKNV isolates, namely KU1 and KU2, was recovered directly from the infected specimens using a shotgun metagenomics approach. The genome length of ISKNV KU1 and KU2 was 111,487 and 111,610 bp, respectively. In comparison to closely related ISKNV strains, KU1 and KU2 contained nine unique genes, including a caspase-recruitment-domain-containing protein that is potentially involved in inhibition of apoptosis. Collectively, this study indicated that inland cultured Asian sea bass are infected by homologous ISKNV strains. This indicates that ISKNV genotype I should be prioritized for future vaccine research.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Iridoviridae/genética , Perciformes/virologia , Adenosina Trifosfatases/genética , Animais , Aquicultura/estatística & dados numéricos , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/etiologia , Doenças dos Peixes/mortalidade , Água Doce , Genoma Viral , Genótipo , Iridoviridae/isolamento & purificação , Iridoviridae/patogenicidade , Filogenia , Reação em Cadeia da Polimerase , Tailândia/epidemiologia
3.
J Fish Dis ; 44(12): 2043-2053, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34449899

RESUMO

Megalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Iridoviridae/isolamento & purificação , Animais , Aquicultura , Proteínas do Capsídeo/genética , Linhagem Celular , Ciclídeos , Infecções por Vírus de DNA/mortalidade , Doenças dos Peixes/mortalidade , Peixes , Índia , Iridoviridae/genética , Iridoviridae/ultraestrutura , Rim/virologia , Baço/virologia
4.
Virus Genes ; 57(5): 448-452, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34272657

RESUMO

The genus Megalocytivirus includes viruses known to cause significant disease in aquacultured fish stocks. Herein, we report the complete genome sequences of two megalocytiviruses (MCVs) isolated from diseased albino rainbow sharks Epalzeorhynchos frenatum reared on farms in the United States in 2018 and 2019. Histopathological examination revealed typical megalocytivirus microscopic lesions (i.e., basophilic cytoplasmic inclusions) that were most commonly observed in the spleen and kidney. Transmission electron microscopic examination of spleen and kidney tissues from specimens of the 2018 case revealed hexagonally shaped virus particles with a mean diameter of 153 ± 6 nm (n = 20) from opposite vertices and 131 ± 5 nm (n = 20) from opposite faces. Two MCV-specific conventional PCR assays confirmed the presence of MCV DNA in the collected samples. Full genome sequencing of both 2018 and 2019 Epalzeorhynchos frenatus iridoviruses (EFIV) was accomplished using a next-generation sequencing approach. Phylogenomic analyses revealed that both EFIV isolates belong to the infectious spleen and kidney necrosis virus (ISKNV) genotype within the genus Megalocytivirus. This study is the first report of ISKNV in albino rainbow sharks.


Assuntos
Infecções por Vírus de DNA/genética , Genoma Viral/genética , Iridoviridae/genética , Tubarões/virologia , Animais , Infecções por Vírus de DNA/virologia , Fazendas , Doenças dos Peixes/genética , Doenças dos Peixes/virologia , Peixes/genética , Peixes/virologia , Humanos , Filogenia , Tubarões/genética , Estados Unidos , Sequenciamento Completo do Genoma
5.
Fish Shellfish Immunol ; 117: 17-23, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34280519

RESUMO

Iridovirus can cause a mass of death in grouper, leading to huge economic loss in recent years. At present, practical vaccine is still the best way to control the outbreak of this virus. Many researches had indicated that the major capsid protein (MCP) of grouper iridovirus of Taiwan (TGIV) is an effective antigen to induce a specific immune response in grouper. However, these traditional vaccines that based on large proteins or whole organisms are faced with challenges because of the unnecessary antigenic load. Thus, in this study, we screened the dominant linear epitope within the MCP of TGIV and then, a new peptide vaccine (P2) was developed via prokaryotic expression system. Furthermore, SWCNTs was used as a vaccine carrier to enhance the immunoprotective effect. To evaluate the immunoprotective effect of this vaccine, a total of 245 fish were vaccinated with P2 (5, 10, 20 mg L-1) and SWCNTs-P2 (5, 10, 20 mg L-1) via immersion before being challenged with live TGIV at 28 days post immunization (d.p.i.). Results showed that the serum antibody titer, enzymatic activity, expression level of some immune-related genes (CC chemokine, IgM and TNF-α) and survival rate were significantly increased (SWCNTs-P2, 20 mg L-1, 100%) compared to the control group (0%). These results indicated that this peptide vaccine could effectively induce specific immune response in vaccinated groupers. Functionalized SWCNTs could serve as a carrier of the peptide vaccine to enhance the immunoprotective effect via immersion. To sum up, epitope screening might be a potential way to develop an effective vaccine nowadays, and SWCNTs might provide a practical method that can be used in large-scale vaccination, especially for juvenile fish, to fight against diseases in aquaculture industry.


Assuntos
Proteínas do Capsídeo/imunologia , Infecções por Vírus de DNA/prevenção & controle , Portadores de Fármacos/administração & dosagem , Epitopos/imunologia , Doenças dos Peixes/prevenção & controle , Iridoviridae/imunologia , Nanotubos de Carbono , Perciformes , Vacinas de Subunidades/administração & dosagem , Vacinas Virais/administração & dosagem , Fosfatase Ácida/imunologia , Fosfatase Alcalina/imunologia , Animais , Antígenos Virais/imunologia , Infecções por Vírus de DNA/imunologia , Portadores de Fármacos/química , Doenças dos Peixes/imunologia , Expressão Gênica/efeitos dos fármacos , Nanotubos de Carbono/química , Perciformes/genética , Perciformes/imunologia , Perciformes/virologia , Superóxido Dismutase/imunologia , Vacinas de Subunidades/química , Vacinas Virais/química
6.
J Fish Dis ; 44(9): 1337-1342, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33966277

RESUMO

Red sea bream iridovirus (RSIV) belonging to the genus Megalocytivirus of the family Iridoviridae is the cause of serious mass mortality of cultured marine fishes. RSIV-type megalocytiviruses show extremely high nucleotide sequence identities. Thus, epidemiological studies on this virus are limited. This study developed two primer sets amplifying the regions possessing single nucleotide polymorphism (SNP) to determine the relationships and divergence of RSIV-type megalocytiviruses isolated from cultured marine fishes in Japan. The two regions were designed according to the genome sequences of the representative RSIV genotype II of megalocytivirus members in GenBank. The SNP 1 and 2 regions have sequences homologous to hypothetical protein ORF 24 and ORF 31, respectively, of RSIV (accession no. AP017456.1). By sequencing the regions, 53 polymorphic sites were identified. The phylogenetic analysis of 25 RSIV-type megalocytivirus isolates, classified into RSIV cluster, was clustered into eight haplotypes (seven haplotypes from Oita, two haplotypes from Ehime, and one haplotype shared between Oita and Ehime). These findings suggested that SNP in the RSIV genome is a powerful application for the detection and identification of RSIV-type megalocytiviruses.


Assuntos
Doenças dos Peixes/virologia , Iridoviridae/genética , Polimorfismo de Nucleotídeo Único , Animais , Aquicultura , Peixes , Genótipo , Japão
7.
J Fish Dis ; 44(9): 1411-1422, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34041757

RESUMO

Scale drop disease virus (SDDV) is one of the most important pathogens that causes scale drop disease (SDD) in Asian sea bass (Lates calcarifer). The outbreaks of this disease are one of the factors causing substantial losses in Asian sea bass aquaculture. In this study, the uracil-DNA glycosylase (UDG)-supplemented cross-priming amplification (UCPA) combined with a colorimetric detection method using the hydroxynaphthol blue (HNB) and lateral flow dipstick (LFD) for detection of SDDV was developed. The UDG was utilized to prevent carryover contamination, and the CPA reactions can be readily observed by HNB and LFD. The CPA primers and probe were designed to target the major capsid protein (MCP) gene of the SDDV. The optimized UCPA conditions were performed at the temperature of 61°C for 60 min. The UCPA assays demonstrated specificity to SDDV without cross-reaction to other tested viruses including red-spotted grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV) and Lates calcarifer herpes virus (LCHV), and other bacterial species commonly found in aquatic animals. The sensitivity of the UCPA-HNB and UCPA-LFD was 100 viral copies/µl and 10 pg of extracted total DNA, which was 10-fold more sensitive than that of conventional PCR. The UCPA-HNB and UCPA-LFD assays could be used to detect the SDDV infection in all 25 confirmed SDDV-infected fish samples. Therefore, the UCPA coupled with HNB and LFD was rapid, simple and effective and might be applied for diagnosis of SDDV infection.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/diagnóstico , Iridoviridae/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Colorimetria , Apresentação Cruzada , Infecções por Vírus de DNA/diagnóstico , Doenças dos Peixes/virologia , Naftalenossulfonatos , Testes Sorológicos/métodos
8.
Int J Mol Sci ; 22(6)2021 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808870

RESUMO

Megalocytivirus is an important viral pathogen to many farmed fishes, including Japanese flounder (Paralichthys olivaceus). In this study, we examined megalocytivirus-induced RNA responses in the spleen of flounder by high-throughput sequencing and integrative analysis of various RNA-seq data. A total of 1327 microRNAs (miRNAs), including 368 novel miRNAs, were identified, among which, 171 (named DEmiRs) exhibited significantly differential expressions during viral infection in a time-dependent manner. For these DEmiRs, 805 differentially expressed target mRNAs (DETmRs) were predicted, whose expressions not only significantly changed after megalocytivirus infection but were also negatively correlated with their paired DEmiRs. Integrative analysis of immune-related DETmRs and their target DEmiRs identified 12 hub DEmiRs, which, together with their corresponding DETmRs, formed an interaction network containing 84 pairs of DEmiR and DETmR. In addition to DETmRs, 19 DEmiRs were also found to regulate six key immune genes (mRNAs) differentially expressed during megalocytivirus infection, and together they formed a network consisting of 21 interactive miRNA-messenger RNA (mRNA) pairs. Further analysis identified 9434 circular RNAs (circRNAs), 169 of which (named DEcircRs) showed time-specific and significantly altered expressions during megalocytivirus infection. Integrated analysis of the DETmR-DEmiR and DEcircR-DEmiR interactions led to the identification of a group of competing endogenous RNAs (ceRNAs) constituted by interacting triplets of circRNA, miRNA, and mRNA involved in antiviral immunity. Together these results indicate that complicated regulatory networks of different types of non-coding RNAs and coding RNAs are involved in megalocytivirus infection.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Iridoviridae/fisiologia , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , RNA Circular , RNA Mensageiro/genética
9.
J Fish Dis ; 44(8): 1131-1145, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33835515

RESUMO

Whiteleg shrimp is a widely cultured crustacean, but frequent disease outbreaks have decreased production and caused significant losses. Toll-like receptors (TLRs) comprise a large innate immune family that is involved in the innate immune response. However, understanding of their regulatory mechanism is limited. In this study, PacBio sequencing and Illumina sequencing were applied to the gill and hepatopancreas tissues of whiteleg shrimp and an integrated transcript gene set was established. The upregulation of Toll1, Toll2 and Toll3 transcripts in the hepatopancreas tissue of whiteleg shrimp after iridescent virus infection implies that these proteins are involved in the immune response to the virus; simultaneously, the TRAF6 and relish transcripts in the Toll pathway were also upregulated, implying that the Toll pathway was activated. We predicted the three-dimensional structure of the five Toll proteins in whiteleg shrimp and humans and constructed a phylogenetic tree of the Toll protein family. In addition, there was a large discrepancy of Toll1 between invertebrates and vertebrates, presumably because of the loss of Toll1 protein sequence during the evolution process from invertebrates to vertebrates. Our research will improve the cognition of Toll protein family in invertebrates in terms of evolution, structure and function and provide theoretical guidance for researchers in this field.


Assuntos
Proteínas de Artrópodes/genética , Evolução Molecular , Iridoviridae/fisiologia , Penaeidae/genética , Receptores Toll-Like/genética , Animais , Proteínas de Artrópodes/metabolismo , Penaeidae/virologia , Análise de Sequência de DNA , Análise de Sequência de Proteína , Receptores Toll-Like/metabolismo , Transcrição Genética
10.
Fish Shellfish Immunol ; 113: 139-147, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33848638

RESUMO

In fish, interleukin-6 (IL-6) is a very important immune-regulatory cytokine that plays a polyfunctional role in inflammation, metabolism, regeneration, and neural processes. IL-6 signal transducer (IL-6ST) is a specific receptor for IL-6 and expressed mainly in immune cells and hepatocytes. In this study, the complete cDNA and genomic DNA sequences of mandarin fish (Siniperca chuatsi) IL-6 and IL-6ST genes were identified and analyzed. Quantitative real-time PCR showed that IL-6 and IL-6ST were chiefly expressed in the immune organs. After challenge with infectious spleen and kidney necrosis virus (ISKNV), the expression levels of IL-6 were significantly up-regulated after 6 h and 24 h in the head kidney and spleen, respectively (p < 0.01), the peak value for both reached at 72 h, IL-6ST increased significantly after 120 h with a peak at 168 h in the head kidney (p < 0.01) and improved markedly at 168 h in the spleen (p < 0.01). Besides, IL-6 and IL-6ST have been identified 3 and 8 single nucleotide polymorphisms (SNPs), respectively. Statistical analysis showed that one SNP locus (1625C/T) in the coding region of IL-6 was significantly related to the resistance of mandarin fish against ISKNV. The 1625C→T locus in the coding region of IL-6 is a synonymous mutation; compared with the susceptible group, the frequency of allele T in the disease resistance group was significantly higher, which may be due to the rare codon produced by the mutation affecting translation. The involvement of IL-6 and IL-6ST in response to ISKNV infection in mandarin fish clearly indicate that the role of SNP markers in IL-6 was associated with the ISKNV resistance, which was demonstrated for the first time in our results. Thus, the current study may provide fundamental information for further breeding of mandarin fish with resistance to ISKNV infection.


Assuntos
Receptor gp130 de Citocina/imunologia , Resistência à Doença/genética , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Interleucina-6/imunologia , Iridoviridae/fisiologia , Perciformes/imunologia , Animais , Receptor gp130 de Citocina/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , DNA Complementar , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Interleucina-6/genética , Perciformes/genética , Polimorfismo de Nucleotídeo Único/imunologia , Distribuição Aleatória , Transcriptoma
11.
Front Immunol ; 12: 636806, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33767703

RESUMO

Interferon-induced transmembrane proteins (IFITMs) are novel viral restriction factors which inhibit numerous virus infections by impeding viral entry into target cells. To investigate the roles of IFITMs during fish virus infection, we cloned and characterized an IFITM1 homolog from orange spotted grouper (Epinephelus coioides) (EcIFITM1) in this study. EcIFITM1 encodes a 131-amino-acid polypeptide, which shares 64 and 43% identity with Seriola dumerili and Homo sapiens, respectively. The multiple sequence alignment showed that EcIFITM1 contained five domains, including NTD (aa 1-45), IMD (aa 46-67), CIL (aa 68-93), TMD (aa 94-119), and CTD (aa 120-131). In vitro, the level of EcIFITM1 mRNA expression was significantly up-regulated in response to Singapore grouper iridovirus (SGIV), or red-spotted grouper nervous necrosis virus (RGNNV) infection. EcIFITM1 encoded a cytoplasmic protein, which was partly colocalized with early endosomes, late endosomes, and lysosomes. The ectopic expression of EcIFITM1 significantly inhibited the replication of SGIV or RGNNV, which was demonstrated by the reduced virus production, as well as the levels of viral gene transcription and protein expression. In contrast, knockdown of EcIFITM1 using small interfering RNAs (siRNAs) promoted the replication of both viruses. Notably, EcIFITM1 exerted its antiviral activity in the step of viral entry into the host cells. Furthermore, the results of non-targeted lipometabolomics showed that EcIFITM1 overexpression induced lipid metabolism remodeling in vitro. All of the detected ceramides were significantly increased following EcIFITM1 overexpression, suggesting that EcIFITM1 may suppress SGIV entry by regulating the level of ceramide in the lysosomal system. In addition, EcIFITM1 overexpression positively regulated both interferon-related molecules and ceramide synthesis-related genes. Taken together, our results demonstrated that EcIFITM1 exerted a bi-functional role, including immune regulation and lipid metabolism in response to fish virus infections.


Assuntos
Antígenos de Diferenciação/metabolismo , Infecções por Vírus de DNA/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Iridoviridae/fisiologia , Nodaviridae/fisiologia , Perciformes/imunologia , Infecções por Vírus de RNA/imunologia , Animais , Antígenos de Diferenciação/genética , Células Cultivadas , Clonagem Molecular , Proteínas de Peixes/genética , Metabolismo dos Lipídeos , RNA Interferente Pequeno/genética , Alinhamento de Sequência , Regulação para Cima , Internalização do Vírus , Replicação Viral
12.
Artigo em Inglês | MEDLINE | ID: mdl-33609809

RESUMO

MiR-150 is a microRNA (miRNA) present in a number of teleost species, but its target and regulation mechanism are unknown. Similarly, lysosome membrane protein 2-like (LMP2L) is a gene identified in fish but with unknown function. In this study, we examined the regulation mechanism and function of flounder miR-150 (named pol-miR-150) and its target gene LMP2L (named PoLMP2L) in association with bacterial and viral infection. We found that pol-miR-150 expression was not only modulated by the bacterial pathogen Streptococcus iniae but also by the viral pathogen megalocytivirus. Pol-miR-150 targeted PoLMP2L by binding to the 3'-untranslated region (3'-UTR) of PoLMP2L and inhibited PoLMP2L expression in vitro and in vivo. PoLMP2L is a member of the CD36 superfamily of scavenger receptors and homologous to but phylogenetically distinct from lysosomal integral membrane protein type 2 (LIMP2). PoLMP2L was localized mainly in the lysosomes and expressed in multiple organs of flounder. In vivo knockdown and overexpression of PoLMP2L enhanced and suppressed, respectively, S. iniae dissemination in flounder tissues, whereas in vivo knockdown and overexpression of pol-miR-150 produced the opposite effects on S. iniae dissemination. In addition, pol-miR-150 knockdown also significantly inhibited the replication of megalocytivirus. The results of this study revealed the regulation mechanism and immune functions of fish miR-150 and LMP2L, and indicated that LMP2L and miR-150 play an important role in the antimicrobial immunity of fish.


Assuntos
Infecções por Vírus de DNA , Doenças dos Peixes , Proteínas de Peixes/imunologia , Linguado , Iridoviridae/imunologia , Lisossomos , MicroRNAs/imunologia , Infecções Estreptocócicas , Streptococcus iniae/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/microbiologia , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Linguado/imunologia , Linguado/microbiologia , Linguado/virologia , Lisossomos/imunologia , Lisossomos/microbiologia , Lisossomos/virologia , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Infecções Estreptocócicas/virologia
13.
Vet Res ; 52(1): 28, 2021 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597018

RESUMO

CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.


Assuntos
Linguado/genética , Linguado/imunologia , Regulação da Expressão Gênica/imunologia , Tetraspanina 29/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Edwardsiella , Escherichia coli , Brânquias/citologia , Rim Cefálico/metabolismo , Iridoviridae , Fígado/metabolismo , Modelos Moleculares , Conformação Proteica , Baço/metabolismo , Tetraspanina 29/metabolismo , Transcriptoma , Vibrio
14.
Transbound Emerg Dis ; 68(2): 964-972, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33448668

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV), a member of family iridoviridae, reported for the first time in a wide range of ornamental fish species in India. Significant mortalities during the year 2018-19 were reported from a number of retailers in the region with various clinical signs. The samples of moribund, dead and apparently healthy ornamental fishes were collected from retailers, located in three districts of Karnataka, India. Out of 140 fish samples, 16 samples (11.42%) representing 10 different fish species were found positive to ISKNV by OIE listed primers and same samples were reported to amplify the major capsid protein (MCP) gene of ISKNV. Further, sequence analysis of MCP gene showed that all strains detected in this study were closely related to other documented isolates from different countries with an identity ranging from 98.76% to 100%. Further, they clustered in the clade of ISKNV, during the phylogenetic analysis. The sequence similarity was high (99.94%) to ISKNV strains from Japan, Australia and Malaysia. This is the first report of an ISKNV infection in India. Moreover, out of 10 ISKNV-positive fish species, three species were reported positive to ISKNV for the first time in the world. Further, the in vitro experiment showed the growth of virus in Asian sea bass cell line, which is a natural host of ISKNV. Therefore, considering the lethal nature of megalocytiviruses to infect a vast range of species, proper biosecurity measures need to be taken to control these emerging pathogens.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Iridoviridae , Perciformes , Animais , Proteínas do Capsídeo/genética , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/epidemiologia , Índia/epidemiologia , Iridoviridae/isolamento & purificação , Filogenia
15.
Transbound Emerg Dis ; 68(3): 1550-1563, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32920975

RESUMO

In late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalization of chromatin and fine granulation. Transmission electron microscopy showed cells containing conspicuous virions with typical iridovirus morphology, that is enveloped, with icosahedral and/or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as infectious spleen and kidney necrosis virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow-up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR-positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.


Assuntos
Ciclídeos , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/diagnóstico , Iridoviridae/isolamento & purificação , Animais , Aquicultura , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Gana
16.
J Fish Dis ; 44(4): 461-467, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33118189

RESUMO

Non-destructive sampling methods offer practical advantages to detection and monitoring of viral pathogens in economically important farmed fish and broodstock. Here, we investigated whether blood, mucus and fin can be used as non-lethal sample sources for detection of scale drop disease virus (SDDV) in farmed Asian sea bass, Lates calcarifer. Detection of SDDV was performed in parallel from three non-destructive and seven destructive sample types, collected from both clinically sick fish and subclinical fish obtained from an affected farm. The results showed that SDDV was detectable in all 10 sample types with the percentage ranging from 20% to 100%. Blood was the best non-destructive sample source exhibited by the fact that it yielded 100% SDDV-positive tests from both sick (n = 12, 95% CI: 69.9-99.2) and clinically healthy fish (n = 4, 95% CI: 39.6%-97.4%) and is considered a "sterile" sample. This study also revealed concurrent infection of SDDV and two ectoparasites Lernanthropus sp. and Diplectanum sp., in all affected fish (n = 8, 95% CI: 46.7-99.3) during the disease outbreak. These ectoparasites also tested positive for SDDV by PCR, indicating that they were potential sample sources for PCR-based detection of SDDV and possibly other viruses infecting Asian sea bass.


Assuntos
Bass , Copépodes/virologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/epidemiologia , Iridoviridae/isolamento & purificação , Trematódeos/virologia , Escamas de Animais/virologia , Animais , Infecções por Vírus de DNA/epidemiologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Prevalência , Tailândia/epidemiologia
17.
Ticks Tick Borne Dis ; 12(1): 101585, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33113476

RESUMO

Trombidiformes and Mesostigmata mites, as well as Ixodida ticks, infest ectothermic tetrapods worldwide, potentially acting as vectors of bacteria, viruses and protozoa. The relationship among ectoparasites, transmitted pathogenic agents (e.g., Borrelia spp., Coxiella spp., Hepatozoon spp., and Rickettsia spp.) and ectothermic hosts has been scarcely investigated. This research focuses on a large collection of Brazilian herpetofauna screened for the presence of arthropod ectoparasites and vector-borne microbial agents. Reptiles (n = 121) and amphibians (n = 49) from various locations were infested by ectoparasites. Following genomic extraction, microbial agents were detected in 81 % of the Acari (i.e. n = 113 mites and n = 26 ticks). None of the mites, ticks and tissues from amphibians yielded positive results for any of the screened agents. Blood was collected from reptiles and processed through blood cytology and molecular analyses (n = 48). Of those, six snakes (12.5 %) showed intraerythrocytic alterations compatible with Hepatozoon spp. gamonts and Iridovirus inclusions. Hepatozoon spp. similar to Hepatozoon ayorgbor and Hepatozoon musa were molecularly identified from seven hosts, two mite and two tick species. Rickettsia spp. (e.g., Rickettsia amblyommatis, Rickettsia bellii-like, Rickettsia sp.) were detected molecularly from four mite species and Amblyomma rotundatum ticks. Phylogenetic analyses confirmed the molecular identification of the above-mentioned microbial agents of mites and ticks related to snakes and lizards. Overall, our findings highlighted that the Brazilian herpetofauna and its ectoparasites harbour potentially pathogenic agents, particularly from the northern and south-eastern regions. The detection of several species of spotted fever group Rickettsia pointed out the potential role of ectothermic hosts and related arthropod ectoparasites in the epidemiological cycle of these bacteria in Brazil.


Assuntos
Eucoccidiida/isolamento & purificação , Iridoviridae/isolamento & purificação , Ixodidae , Ácaros , Répteis , Rickettsia/isolamento & purificação , Animais , Brasil , Reservatórios de Doenças , Eucoccidiida/classificação , Feminino , Iridoviridae/classificação , Ixodidae/crescimento & desenvolvimento , Ixodidae/microbiologia , Ixodidae/parasitologia , Ixodidae/virologia , Larva/crescimento & desenvolvimento , Larva/microbiologia , Larva/parasitologia , Larva/virologia , Masculino , Ácaros/crescimento & desenvolvimento , Ácaros/microbiologia , Ácaros/parasitologia , Ácaros/virologia , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Ninfa/parasitologia , Ninfa/virologia , Filogenia , Répteis/microbiologia , Répteis/parasitologia , Répteis/virologia , Rickettsia/classificação
18.
J Fish Dis ; 44(4): 401-413, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33340375

RESUMO

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.


Assuntos
Aeromonas hydrophila/isolamento & purificação , Densovirinae/isolamento & purificação , Edwardsiella tarda/isolamento & purificação , Iridoviridae/isolamento & purificação , Microfluídica/métodos , Técnicas de Diagnóstico Molecular/veterinária , Técnicas de Amplificação de Ácido Nucleico/veterinária , Vibrio/isolamento & purificação , Vírus da Síndrome da Mancha Branca 1/isolamento & purificação , Animais , Crustáceos/microbiologia , Crustáceos/virologia , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/diagnóstico , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Peixes/microbiologia , Peixes/virologia , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Moluscos/microbiologia , Moluscos/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
19.
Vaccine ; 38(51): 8107-8115, 2020 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-33189430

RESUMO

A formalin-inactivated red sea bream iridovirus (RSIV) vaccine was prepared using the culture supernatant of a persistently infected Pagrus major fin cell line (PI-PMF) with IVS-1 strain (RSIV subtype II Meglaocytivirus). Rock bream (Oplegnathus fasciatus) were injected with a high-dose, ultracentrifuged megalocytivirus vaccine (Ultra HSCMV, 7.0 × 1010 copies/mL), a high-dose supernatant of cultured megalocytivirus vaccine (HSCMV, 1.0 × 1010 copies/mL), a supernatant of cultured megalocytivirus vaccine (SCMV, 1.0 × 109 copies/mL), and a low-dose of cultured megalocytivirus vaccine (LSCMV, 1.0 × 108 copies/mL). The vaccine efficacies for the various vaccine formulations were determined done following injection challenge with IVS-1 (1.0 × 104 copies/0.1 mL/fish), and the four different vaccines exhibited cumulative mortalities of 10.0 ± 0.0%, 48.3 ± 7.6%, 75.0 ± 5.0%, and 100.0 ± 0.0%, respectively. Additionally, the dose-dependent vaccine efficacy was also confirmed using two different cohabitation methods that included challenges G (general) and I (individual). When squalene + aluminum hydroxide (SqAl) was used as an adjuvant for the HSCMV or SCMV vaccine, cumulative mortalities of 30.0 ± 5.0% and 48.3 ± 7.6%, respectively, were obtained; moreover, these two adjuvants exhibited the highest efficacy in this study. The observed difference in survival post-challenge for the different vaccine concentrations was not reflected in the differences in neutralizing antibody titers. It was found that the water temperature during immune induction plays a less important a role than the water temperature during the challenge test, in which lowering the water temperature from 25 °C to 21 °C during a challenge improved the level of protection from cumulative mortalities from 35% to 10%. This study demonstrated that protection against mortality using inactivated vaccines against RSIVD in rock bream, which are known to be the most susceptible species to RSIV infection, is dependent upon antigen dose and temperature during the challenge.


Assuntos
Infecções por Vírus de DNA , Doenças dos Peixes , Iridoviridae , Perciformes , Vacinas , Animais , Linhagem Celular , Infecções por Vírus de DNA/prevenção & controle , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/prevenção & controle
20.
Fish Shellfish Immunol ; 107(Pt A): 269-276, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33068760

RESUMO

To reduce the largemouth bass ulcer syndrome (LBUSV) aquatic economic losses, it must take effective preventive measures and coping strategies should be urgently investigated. In this research, the effects of a functionalized single-walled carbon nanotubes (SWCNTs) applied as a delivery vehicle for DNA vaccine administration in largemouth bass (Micropterus Salmoides) against LBUSV were studied. Our results showed that SWCNTs loaded with DNA vaccine induced a better protection to largemouth bass against LBUSV. We found more than 10 times increase in serum antibody levels, enzyme activities and immune-related genes (IL-6, IL-8, IFN-γ, IgM and TNF-α) expression, in the SWCNTs-pcDNA-MCP immunized groups compared with PBS group and the pure SWCNTs group. The survival rates for control group (PBS), pure SWCNTs groups (40 mg L-1), four pcDNA-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) and four SWCNTs-pcDNA-MCP groups (5 mg L-1, 10 mg L-1, 20 mg L-1 and 40 mg L-1) were 0%, 0%, 0%, 2.77%, 11.11%, 19.44%, 27.78%, 38.89%, 52.78% and 61.11%, respectively. Our results demonstrate that the SWCNTs-DNA vaccine can be used as a new method against LBUSV showing protection following challenge with LBUSV.


Assuntos
Bass/imunologia , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/prevenção & controle , Iridoviridae/imunologia , Vacinação/veterinária , Vacinas Virais/administração & dosagem , Animais , Infecções por Vírus de DNA/prevenção & controle , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Imunidade Inata , Imunização/veterinária , Nanotubos de Carbono/análise , Vacinas de DNA/administração & dosagem
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