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1.
Arch Virol ; 165(5): 1215-1218, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32140836

RESUMO

A novel lymphocystivirus causing typical signs of lymphocystis virus disease in whitemouth croaker (Micropogonias furnieri) on the coast of Uruguay was detected and described recently. Based on genetic analysis of some partially sequenced core genes, the virus seemed to differ from previously described members of the genus Lymphocystivirus. In this study, using next-generation sequencing, the whole genome of this virus was sequenced and analysed. The complete genome was found to be 211,086 bp in size, containing 148 predicted protein-coding regions, including the 26 core genes that seem to have a homologue in every iridovirus genome sequenced to date. Considering the current species demarcation criteria for the family Iridoviridae (genome organization, G+C content, amino acid sequence similarity, and phylogenetic relatedness of the core genes), the establishment of a novel species ("Lymphocystis disease virus 4") in the genus Lymphocystivirus is suggested.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Genoma Viral , Iridoviridae/classificação , Iridoviridae/isolamento & purificação , Perciformes/virologia , Análise de Sequência de DNA , Animais , Composição de Bases , Infecções por Vírus de DNA/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Iridoviridae/genética , Fases de Leitura Aberta , Filogenia , Homologia de Sequência de Aminoácidos , Uruguai
3.
Arch Virol ; 165(4): 973-976, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32060793

RESUMO

To separate and concentrate koi herpesvirus (KHV) from large-volume samples, a separation method based on immunomagnetic beads (IMBs) coated with polyclonal antibody directed against KHV was developed. After treatment with IMBs, viral DNA was extracted from samples and used as a template for quantitative PCR (qPCR). The results showed that the concentration of the template DNA extracted from the virus that had been separated using IMBs was 9.65-fold higher than that from virus not treated with IMBs. The detection limit of the IMBs/qPCR method was found to be at least 10 times lower than that of qPCR alone.


Assuntos
Carpas/virologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/veterinária , Herpesviridae/isolamento & purificação , Separação Imunomagnética/métodos , Animais , Anticorpos/análise , Herpesviridae/genética , Infecções por Herpesviridae/virologia , Separação Imunomagnética/instrumentação , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
4.
J Appl Microbiol ; 128(1): 41-53, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31529740

RESUMO

AIMS: To determine the immune gene expression response of gilthead seabream (Sparus aurata) that is experimentally infected with the lymphocystivirus LCDV-Sa. METHODS AND RESULTS: Viral DNA and transcripts were detected by qPCR in all samples from fish injected with LCDV-Sa, demonstrating that the virus establish a systemic and asymptomatic infection. The expression of 23 immune-related genes was also analysed by RT-qPCR in the head kidney (HK) and intestine at several times post-infection (dpi). In HK, the expression of five type I interferon (IFN)-related genes (ifn, irf3, mx2, mx3 and isg15), il10 and ck10 was upregulated at 1-3 dpi, while genes related to the inflammation process (tnfα, il1ß, il6, casp1) were not differentially expressed or even downregulated. The expression profile in the intestine was different regarding type I INF-related genes. An upregulated c3 and ighm expression was observed in both HK and intestine at 3-8 dpi. Finally, the transcription of nccrp1 and mhcIIα was induced in HK, whereas tcrß expression was downregulated in both organs. CONCLUSIONS: LCDV-Sa seems to trigger an immune response in gilthead seabream characterized by a partial activation of type I IFN system and a lack of systemic inflammatory response which may be related to viral persistence. SIGNIFICANCE AND IMPACT OF THE STUDY: The immune response observed in gilthead seabream infected by LCDV-Sa could be implicated in the establishment of an asymptomatic persistent infection.


Assuntos
Infecções Assintomáticas , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Iridoviridae/fisiologia , Dourada/imunologia , Animais , Citocinas/genética , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Rim Cefálico/imunologia , Intestinos/imunologia , Dourada/genética , Dourada/virologia
5.
J Fish Dis ; 43(1): 139-146, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31724200

RESUMO

Infectious pancreatic necrosis virus (IPNV) is the aetiological agent of a highly contagious disease that affects farmed salmonids. IPNV isolates have been phylogenetically classified into eight genogroups, of which two are present in Chile, genogroups 1 and 5. Here, we compare the mortality rate caused by isolates from both genogroups in rainbow trout (Oncorhynchus mykiss) fry to determine if there is an association between host susceptibility and phylogenetic characterization of IPNV. Fish were challenged by immersion with one of four isolates (two for each genogroup), and mortality curves were assessed after 30 days. Viral load was measured in all mortalities and in live fish sampled at 1, 7 and 20 days post-infection. Although mortality was low throughout the challenge, differences were found between fish infected with different isolates. Both isolates from genogroup 1 caused greater cumulative mortalities than either of the isolates from genogroup 5. When combined, the overall mortality rate of fish challenged with genogroup 1 isolates was significantly higher than those infected with genogroup 5. However, viral load was lower on trout infected with genogroup 1 isolates. These results suggest that rainbow trout are more susceptible to IPNV isolates from genogroup 1 than genogroup 5.


Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/mortalidade , Vírus da Necrose Pancreática Infecciosa/fisiologia , Oncorhynchus mykiss , Carga Viral/veterinária , Animais , Infecções por Birnaviridae/mortalidade , Infecções por Birnaviridae/virologia , Chile/epidemiologia , Doenças dos Peixes/virologia , Genótipo , Vírus da Necrose Pancreática Infecciosa/genética , Filogenia
6.
Pol J Vet Sci ; 22(4): 785-787, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31867926

RESUMO

The European eel (Anguilla anguilla) is a catadromous fish with a complicated life cycle. The long-term impact of anthropopressure, environmental pollution and diseases have led to a risk of extinction. The aim of the present study was to determine the influence of Anguillid herpesvirus-1 infection on the innate immunity of European eel from natural conditions. Spleen phagocyte respiratory burst activity and potential killing activity, as well as pronephros lymphocyte proliferation stimulated by concanavalin A or lipopolysaccharide were measured. The analyses of the results showed that all studied parameters were significantly higher (P⟨0.05) in AngHV-1-negative fish compared to the ones where the presence of viral DNA was confirmed.


Assuntos
Anguilla/virologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/veterinária , Herpesviridae , Imunossupressão/veterinária , Animais , Proliferação de Células , Doenças dos Peixes/imunologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Linfócitos/fisiologia , Linfócitos/virologia , Explosão Respiratória
7.
BMC Genomics ; 20(1): 1019, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31878870

RESUMO

BACKGROUND: Infectious disease outbreaks form major setbacks to aquaculture production and to further development of this important sector. Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide. Genetically disease resistant strains are highly sought after as a sustainable solution to this problem. To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection. RESULTS: In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish. Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types. Susceptible fish elicited a typical anti-viral response, involving interferon and interferon responsive genes, earlier than resistant fish did. Furthermore, chemokine profiles indicated that the two fish types elicited different cellular immunity responses. A comparative phylogenetic approach assisted in chemokine copies annotation pointing to different orthologous copies common to bony-fishes and even carp-specific paralogs that were differentially regulated and contributed to the different response of these two fish types. Susceptible fish up-regulated more ccl19 chemokines, which attract T-cells and macrophages, the anti-viral role of which is established, whereas resistant fish up-regulated more cxcl8/il8 chemokines, which attract neutrophils, the antiviral role of which is unfamiliar. CONCLUSIONS: Taken together, by pointing out transcriptional differences between susceptible and resistant fish in response to CyHV-3 infection, this study unraveled possible genes and pathways that take part in disease resistance mechanisms in fish and thus, enhances our understanding of fish immunogenetics and supports the development of sustainable and safe aquaculture.


Assuntos
Carpas/genética , Carpas/virologia , Resistência à Doença/genética , Doenças dos Peixes/virologia , Predisposição Genética para Doença/genética , Herpesviridae/fisiologia , Transcrição Genética , Animais , Doenças dos Peixes/imunologia , Locos de Características Quantitativas/genética
8.
Environ Sci Pollut Res Int ; 26(34): 35266-35269, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31728943

RESUMO

In combination, pollution and pathogens represent a serious threat to the health of European eels that has been increasingly recognized. Thus, the impact of contaminants, cadmium, lead, mercury, and selenium, on anguillid herpesvirus 1 infection in wild European eels has been evaluated. Despite the small sample size, results indicate that selenium and mercury concentrations may compromise the European eel immune system as herpesvirus infection was more prevalent in specimens with higher Hg and Se hepatic concentrations.


Assuntos
Anguilla/virologia , Doenças dos Peixes/virologia , Infecções por Herpesviridae/veterinária , Compostos Inorgânicos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Cádmio , Enguias , Herpesviridae , Fígado , Mercúrio , Alimentos Marinhos , Selênio
9.
J Fish Dis ; 42(12): 1667-1676, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31612537

RESUMO

In recent years, the use of cleaner fish for biological control of sea lice has increased considerably. Along with this, a number of infectious diseases have emerged. The aim of this study was to investigate the susceptibility of lumpfish (Cyclopterus lumpus) to Betanodavirus since it was detected in asymptomatic wild wrasses in Norway and Sweden. Three betanodaviruses were used to challenge lumpfish: one RGNNV genotype and two BFNNV genotypes. Fish were injected and monitored for 4 weeks. Brain samples from clinically affected specimens, from weekly randomly selected fish and survivors were subjected to molecular testing, viral isolation, histopathology and immunohistochemistry. Reduced survival was observed but was attributed to tail-biting behaviour, since no nervous signs were observed throughout the study. Betanodavirus RNA was detected in all samples, additionally suggesting an active replication of the virus in the brain. Viral isolation confirmed molecular biology results and revealed a high viral titre in BFNNV-infected groups associated with typical lesions in brains and eyes of survivor fish. We concluded that lumpfish are susceptible to Betanodavirus, as proven by the high viral titre and brain lesions detected, but further studies are necessary to understand if Betanodavirus can cause clinical disease in this species.


Assuntos
Doenças dos Peixes/patologia , Nodaviridae/genética , Perciformes/virologia , Infecções por Vírus de RNA/veterinária , Animais , Suscetibilidade a Doenças , Doenças dos Peixes/virologia , Genótipo , Noruega , Infecções por Vírus de RNA/patologia
10.
Fish Shellfish Immunol ; 94: 800-809, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31580938

RESUMO

This review describes and summarizes the knowledge on established and experimental vaccines developed against viral and bacterial pathologies affecting the most important farmed marine finfish species present in the Mediterranean area, namely European seabass Dicentrarchus labrax, sea bream Sparus aurata, turbot Psetta maxima and meagre Argyrosomus regius. The diseases that have been recorded in seabass, sea bream and meagre are caused by bacteria Vibrio anguillarum, Photobacterium damselae, Tenacibaculum maritimum as well as by viruses such as Viral Encephalopathy and Retinopathy/Viral Nervous Necrosis and Lymphocystic disease. The main pathologies of turbot are instead bacteriosis provoked by Tenacibaculum maritimum, Aeromonas sp. and Vibrio anguillarum, and virosis by viral hemorrhagic septicaemia virus. Some vaccines have been optimized and are now regularly available for the majority of the above-mentioned pathogens. A measurable immune protection has been conferred principally against Vibrio anguillarum, Photobacterium damselae sub. piscicida and VER/VNN.


Assuntos
Vacinas Bacterianas/imunologia , Doenças dos Peixes/imunologia , Peixes/imunologia , Vacinas Virais/imunologia , Animais , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/veterinária , Doenças dos Peixes/microbiologia , Doenças dos Peixes/virologia , Mar Mediterrâneo , Viroses/imunologia , Viroses/veterinária , Viroses/virologia
11.
J Fish Dis ; 42(12): 1773-1783, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31637759

RESUMO

Betanodaviruses are small ssRNA viruses that cause viral encephalopathy and retinopathy, a severe neuropathological infectious disease in marine fish species worldwide. In the present study, the occurrence of betanodaviruses was investigated in wild and cultured populations of fishes and invertebrates of the Greek territorial waters. Betanodaviruses were detected in 35 species belonging to 21 families and 12 orders. To our knowledge, 23 of those are reported for the first time in Greek waters, while 11 of them are reported for the first time globally. The positive samples were subjected to sequencing and phylogenetic analysis of partial segments of RNA1 and RNA2 genes. Almost all the viruses circulating in Greece fell within RGNNV genotype, while reassortant viruses were detected in three samples, namely two inter-RGNNV and one RGNNV/SJNNV. A novel unclassified Betanodavirus sequence was also identified. Most of the Greek sequence types have a restricted geographic distribution except for two RNA1 and one RNA2 sequence types that are widespread throughout the Mediterranean basin. The results of this study indicate the range of reservoirs/hosts of betanodaviruses and also their wide spread in the Greek territorial waters and reinforce the hypothesis that wild fish species transmit the virus to cultured ones and vice versa.


Assuntos
Peixes/virologia , Invertebrados/virologia , Nodaviridae/classificação , Animais , Doenças dos Peixes/virologia , Genótipo , Grécia , Filogenia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia , RNA Viral/genética , Vírus Reordenados
12.
J Fish Dis ; 42(11): 1609-1621, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637761

RESUMO

Koi herpesvirus (KHV; cyprinid herpesvirus-3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real-time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.


Assuntos
Carpas , Coinfecção/veterinária , Doenças dos Peixes/mortalidade , Infecções por Herpesviridae/mortalidade , Infecções por Poxviridae/mortalidade , Animais , Coinfecção/mortalidade , Coinfecção/virologia , Doenças dos Peixes/virologia , Herpesviridae/fisiologia , Infecções por Herpesviridae/virologia , Poxviridae/fisiologia , Infecções por Poxviridae/virologia , Estados Unidos/epidemiologia
13.
Elife ; 82019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31478480

RESUMO

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.


Assuntos
Arenavirus/isolamento & purificação , Doenças dos Peixes/virologia , Nidovirales/isolamento & purificação , Reoviridae/isolamento & purificação , Salmão/virologia , Viroses/veterinária , Animais , Arenavirus/classificação , Arenavirus/genética , Células Sanguíneas/virologia , Hibridização In Situ , Metagenômica , Nidovirales/classificação , Nidovirales/genética , Oceano Pacífico , Reoviridae/classificação , Reoviridae/genética , Análise de Sequência de DNA , Transcrição Genética , Viroses/virologia
14.
Aquat Toxicol ; 215: 105282, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31509759

RESUMO

Aquatic ecosystems are now chronically polluted by a cocktail of many chemical substances. There is now clear evidence of associations between exposure to pollutants and greater susceptibility to pathogens. The aim of the present study was to characterize the defense capacities of rainbow trout (Oncorhynchus mykiss), chronically exposed to pendimethalin (PD), to subsequent experimental challenge with the infectious hematopoietic necrosis virus (IHNV). Immunological responses were examined at different organizational levels, from individuals to gene expression. No negative effects of PD were noted on the Fulton index nor on the liver or spleen somatic indices (LSI; SSI) before viral infection, but the infectious stress seems to generate a weak but significant decrease in Fulton and LSI values, which could be associated with consumption of energy reserves. During the viral challenges, the distribution of cumulative mortality was slightly different between infected groups. The impact of the virus on fish previously contaminated by PD started earlier and lasted longer than controls. The proportion of seropositive fish was lower in the fish group exposed to PD than in the control group, with similar quantities of anti-IHNV antibodies secreted in positive fish, regardless of the treatment. While no significant differences in C3-1 expression levels were detected throughout the experiment, TNF1&2, TLR3, Il-1ß and IFN expression levels were increased in all infected fish, but the difference was more significant in fish groups previously exposed to herbicide. On the other hand, ß-def expression was decreased in the pendimethalin-IHNV group compared to that in fish only infected by the virus (control-IHNV group).


Assuntos
Herbicidas/toxicidade , Imunidade Celular/efeitos dos fármacos , Imunidade Humoral/efeitos dos fármacos , Vírus da Necrose Hematopoética Infecciosa/fisiologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/imunologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/veterinária , Compostos de Anilina/toxicidade , Animais , Doenças dos Peixes/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Oncorhynchus mykiss/virologia , Infecções por Rhabdoviridae/genética , Infecções por Rhabdoviridae/patologia , Poluentes Químicos da Água/toxicidade
15.
Virol J ; 16(1): 110, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481132

RESUMO

BACKGROUND: Iridoviruses are large DNA viruses that cause diseases in fish, amphibians and insects. Singapore grouper iridovirus (SGIV) is isolated from cultured grouper and characterized as a ranavirus. ICP46 is defined to be a core gene of the family Iridoviridae and SGIV ICP46 was demonstrated to be an immediate-early (IE) gene associated with cell growth control and could contribute to virus replication in previous research. METHODS: The transcription start site (TSS) and 5'-untranslated region (5'-UTR) of SGIV ICP46 were determined using 5' RACE. The core promoter elements of ICP46s were analyzed by bioinformatics analysis. The core promoter region and the regulation model of SGIV ICP46 promoter were revealed by the construction of serially deleted promoter plasmids, transfections, drug treat and luciferase reporter assays. The identification of virion-associated transcriptional transactivator (VATT) that interact with SGIV ICP46 promoter and their binding site on promoter were performed by electrophoretic mobility shift assays (EMSA), DNA pull-down assays and mass spectrometry (MS). RESULTS: SGIV ICP46 was found to have short 5'-UTR and a presumptive downstream promoter element (DPE), AGACA, which locates at + 36 to + 39 nt downstream of the TSS. The core promoter region of SGIV ICP46 located from - 22 to + 42 nt relative to the TSS. VATTs were involved in the promoter activation of SGIV ICP46 and further identified to be VP12, VP39, VP57 and MCP. A 10-base DNA sequence "ATGGCTTTCG" between the TSS and presumptive DPE was determined to be the binding site of the VATTs. CONCLUSION: Our study showed that four VAATs (VP12, VP39, VP57 and MCP) might bind with the SGIV ICP46 promoter and be involved in the promoter activation. Further, the binding site of the VATTs on promoter was a 10-base DNA sequence between the TSS and presumptive DPE.


Assuntos
Regiões Promotoras Genéticas , Ranavirus/genética , Transativadores/genética , Transativadores/metabolismo , Vírion/genética , Animais , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Doenças dos Peixes/virologia , Peixes/virologia , Espectrometria de Massas , Fases de Leitura Aberta , Transcrição Genética , Replicação Viral
16.
J Fish Dis ; 42(11): 1543-1552, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31515819

RESUMO

Tilapia lake virus (TiLV)-suspected samples of tilapia were collected from grow-out ponds located with clinical signs and mortality ranged from 5% to 50%. The reverse transcription-polymerase chain reaction (RT-PCR) assay revealed the presence of TiLV in the disease outbreak ponds. Cell lines were developed from heart, gill and eye of Mozambique tilapia and characterized. Morphologically, these cell lines are composed of epithelioid cells. The optimum growth of these cells was observed at 28°C and 20% concentration of FBS. After cryopreservation, 70%-90% of cells were found to be viable. The cells of all three cell lines were found to be positive to fibronectin and pancytokeratin. PCR amplification of 16S rRNA and COI of O. mossambicus confirmed the origin of these cell lines from O. mossambicus. Heart and gill cell lines were found to be highly susceptible to TiLV and found to be useful for its isolation from infected fish samples. The experimental infection was carried out in O. niloticus and O. mossambicus using the TiLV propagated in susceptible cell lines. The RT-PCR results revealed the presence of TiLV in brain, gill, liver, kidney, spleen, eye, muscle, intestine and heart of experimentally infected O. niloticus and O. mossambicus.


Assuntos
Suscetibilidade a Doenças/veterinária , Doenças dos Peixes/virologia , Infecções por Vírus de RNA/veterinária , Vírus de RNA/fisiologia , Tilápia , Animais , Linhagem Celular , Infecções por Vírus de RNA/virologia
17.
J Fish Dis ; 42(10): 1391-1399, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31381181

RESUMO

A cell line (PaF) derived from the fin tissue of silver pomfret (Pampus argenteus) was established and characterized in this study. The cell line has been subcultured for more than 50 times in Dulbecco's modified Eagle's medium (DMEM) containing 15% foetal bovine serum (FBS) since the initial primary culture. PaF cells grew well at temperatures from 24°C to 28°C in DMEM supplemented with 15% FBS. Partial amplification and sequence analysis of the cytochrome B gene indicated that PaF originated from silver pomfret. Cytogenetic analysis demonstrated that the modal chromosome number was 48. A significant cytopathic effect was observed in PaF cells during viral haemorrhagic septicaemia virus (VHSV) infection, and the VHSV replication was confirmed by qRT-PCR and viral titre assays. In contrast, PaF cells were resistant to red-spotted grouper nervous necrosis virus infection. Moreover, PaF cells could respond to VHSV and lipopolysaccharide treatments, as indicated by the expression of immune-related genes, TLR5 and TLR9. In conclusion, the establishment of PaF cell line will provide an appropriate in vitro tool for the study of mechanisms of pathogen-silver pomfret interaction.


Assuntos
Linhagem Celular/fisiologia , Peixes , Nodaviridae/fisiologia , Novirhabdovirus/fisiologia , Replicação Viral , Nadadeiras de Animais , Animais , Doenças dos Peixes/virologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Septicemia Hemorrágica Viral/virologia , Lipopolissacarídeos/fisiologia , Infecções por Vírus de RNA/veterinária , Infecções por Vírus de RNA/virologia
18.
J Fish Dis ; 42(11): 1563-1572, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31441949

RESUMO

Fish rhabdoviruses are a family of viruses responsible for large-scale fish die-offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT-PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT-PCR. Electron microscopy showed the presence of bullet-shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole-genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen-free largemouth bass juveniles. VHSVLB2018-infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%-93.3% by 7 days post-infection. VHSVLB2018 was re-isolated from dead fish and confirmed by RT-PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.


Assuntos
Doenças dos Peixes/diagnóstico , Novirhabdovirus/isolamento & purificação , Infecções por Rhabdoviridae/veterinária , Sequência de Aminoácidos , Animais , Bass , Doenças dos Peixes/virologia , Novirhabdovirus/classificação , Filogenia , Infecções por Rhabdoviridae/diagnóstico , Infecções por Rhabdoviridae/virologia , Alinhamento de Sequência , Proteínas Virais/análise
19.
Microb Cell Fact ; 18(1): 142, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434565

RESUMO

BACKGROUND: Hirame novirhabdovirus (HIRRV) can infect a wide range of marine and freshwater fish, causing huge economic losses to aquaculture industry. Vaccine development, especially oral vaccine, has become an effective and convenient way to control aquatic infectious diseases. HIRRV glycoprotein (G), an immunogenic viral protein is a potential vaccine candidate for prevention of the disease. Here, we aimed to construct a recombinant Lactococcus lactis strain expressing HIRRV-G on the cell surface as an oral vaccine to prevent HIRRV. RESULTS: Glycoprotein gene of HIRRV was successfully cloned and expressed in L. lactis NZ9000 in a surface-displayed form, yielding Ll:pSLC-G. An approximately 81 kDa recombinant G protein (containing LysM anchoring motif) was confirmed by SDS-PAGE, western blotting and mass spectrometry analysis. The surface-displayed G protein was also verified by immunofluorescence and flow cytometry assays. Furthermore, to evaluate the potential of Ll:pSLC-G as oral vaccine candidate, flounders were continuously fed with commercial diet pellets coated with 1.0 × 109 cfu/g of induced Ll:pSLC-G for 1 week. Four weeks later, booster vaccination was performed with the same procedure. Compared with the controls, Ll:pSLC-G elicited significantly higher levels of specific IgM against HIRRV in flounder gut mucus at the second week and in serum at the fourth week (p < 0.05). Meanwhile, oral immunization with Ll:pSLC-G could provide 60.7% protection against HIRRV infection and a significantly lower virus load was detected than the controls on the third day post-challenge (p < 0.01). Moreover, on the first day post 1-week feeding, approximately 104-105 recombinant L. lactis cells were detected in every gram of foregut, midgut and hindgut of flounder, which were mainly localized at the bottom of gut mucus layer; and on day 21, 102-103 L. lactis cells could still be recovered. CONCLUSIONS: HIRRV-G protein was successfully expressed on the surface of L. lactis cells, which could trigger mucosal and humoral immune response of flounder and provide considerable immune protection against HIRRV. It suggests that genetically engineered L. lactis expressing G protein can be employed as a promising oral vaccine against HIRRV infection.


Assuntos
Doenças dos Peixes/prevenção & controle , Doenças dos Peixes/virologia , Linguado/imunologia , Glicoproteínas/imunologia , Novirhabdovirus/imunologia , Proteínas Virais/imunologia , Viroses/prevenção & controle , Animais , Clonagem Molecular , Doenças dos Peixes/imunologia , Engenharia Genética , Glicoproteínas/genética , Lactococcus lactis/genética , Proteínas Recombinantes/imunologia , Vacinação/veterinária , Vacinas Sintéticas/imunologia , Proteínas Virais/genética , Viroses/veterinária
20.
Genet Sel Evol ; 51(1): 47, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31455244

RESUMO

BACKGROUND: Infectious hematopoietic necrosis (IHN) is a disease of salmonid fish that is caused by the IHN virus (IHNV). Under intensive aquaculture conditions, IHNV can cause significant mortality and economic losses. Currently, there is no proven and cost-effective method for IHNV control. Clear Springs Foods, Inc. has been applying selective breeding to improve genetic resistance to IHNV in their rainbow trout breeding program. The goals of this study were to elucidate the genetic architecture of IHNV resistance in this commercial population by performing genome-wide association studies (GWAS) with multiple regression single-step methods and to assess if genomic selection can improve the accuracy of genetic merit predictions over conventional pedigree-based best linear unbiased prediction (PBLUP) using cross-validation analysis. RESULTS: Ten moderate-effect quantitative trait loci (QTL) associated with resistance to IHNV that jointly explained up to 42% of the additive genetic variance were detected in our GWAS. Only three of the 10 QTL were detected by both single-step Bayesian multiple regression (ssBMR) and weighted single-step GBLUP (wssGBLUP) methods. The accuracy of breeding value predictions with wssGBLUP (0.33-0.39) was substantially better than with PBLUP (0.13-0.24). CONCLUSIONS: Our comprehensive genome-wide scan for QTL revealed that genetic resistance to IHNV is controlled by the oligogenic inheritance of up to 10 moderate-effect QTL and many small-effect loci in this commercial rainbow trout breeding population. Taken together, our results suggest that whole genome-enabled selection models will be more effective than the conventional pedigree-based method for breeding value estimation or the marker-assisted selection approach for improving the genetic resistance of rainbow trout to IHNV in this population.


Assuntos
Doenças dos Peixes/genética , Vírus da Necrose Hematopoética Infecciosa , Oncorhynchus mykiss/genética , Infecções por Rhabdoviridae/veterinária , Animais , Teorema de Bayes , Cruzamento , Cruzamentos Genéticos , Resistência à Doença/genética , Doenças dos Peixes/virologia , Pesqueiros , Estudo de Associação Genômica Ampla/veterinária , Herança Multifatorial , Oncorhynchus mykiss/virologia , Locos de Características Quantitativas , Infecções por Rhabdoviridae/genética
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