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1.
Fish Shellfish Immunol ; 98: 429-437, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31988017

RESUMO

Oxyeleotris marmoratus iridovirus (OMIV) and Oxyeleotris marmoratus rhabdovirus (OMRV) are the two major causative agents of disease leading to massive mortality and severe economic losses in marbled sleepy goby (Oxyeleotris marmoratus) industry. It's urgent to develop an effective vaccine against these fatal diseases. In this study, we developed bivalent inactivated vaccine against OMIV and OMRV and evaluated its protective effect in Oxyeleotris marmoratus. The intraperitoneally vaccinated fish were protected against challenge with OMIV and OMRV with both relative percent survival (RPS) of 100%. In addition, deep RNA sequencing was used to analyze the transcriptomic profiles of the spleen tissues at progressive time points post-vaccination with bivalent inactivated vaccine and challenge with OMIV and OMRV infection. Results showed that adaptive immune response was induced in Oxyeleotris marmoratus injected with bivalent inactivated vaccine. Furthermore, robust adaptive immune responses were also detected in vaccinated fish at 7 d and 2 d post-challenge with OMIV and OMRV. Taken together, these results indicated that bivalent inactivated vaccine activated adaptive immune responses in Oxyeleotris marmoratus, and provided protection against OMIV and OMRV lethal challenge.

2.
Microb Pathog ; 138: 103822, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669501

RESUMO

The virus inactivation test is a critical skill in inactivated vaccine production. Active viruses produced viral mRNA in susceptible cells or the host can be used to infer whether a DNA virus is replicating by RT-PCR. But it is generally difficult to avoid genomic DNA contamination in the samples. However, the use of primers spanning an intron is an effective alternative for virus inactivation test. Therein, a nested RT-PCR was developed to detect active ISKNV in the inactivated vaccine. At first, the transcriptome analysis of CPB cell infected with ISKNV revealed several gaps in some viral transcripts compared to ISKNV genome. One intron in ORF003L with 80 bp (designated IN-3) was confirmed by PCR and sequencing analysis. Then, two primer sets (primer A and primer B) spanning the IN-3 intron were designed to detect ISKNV transcription. The nested RT-PCR conditions were optimized with 0.4 µM primer A and 0.2 µM primer B, and 68 °C and 55 °C for annealing temperature, respectively. The sensitivity results indicated that the nested RT-PCR could detect one copy of live ISKNV propagating in CPB cells for seven days. The nested RT-PCR method was more sensitive and accurate than the method of blind passages in cells and fish challenge experiments. Together, above results indicate that this assay is a time-saving, labor-extensive and cost-effective for inactivation test of ISKNV in killed vaccine production.

3.
Fish Shellfish Immunol ; 97: 432-439, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31883470

RESUMO

As a high mortality disease, Infectious spleen and kidney necrosis virus (ISKNV) can cause massive economic damage on mandarin fish farming industry in China, which seriously hindered the development of mandarin fish farming industry. In this research, SWCNTs (single-walled carbon nanotubes) as a candidate for DNA vaccine carrier was vaccinated by immersion (1, 2, 5, 10, 20 mg/L) in juvenile mandarin fish. In muscle, spleen and kidney tissues, the results showed that transcription and expression of MCP gene can be detected in pcDNA-MCP and SWCNTs-pcDNA-MCP groups after bath immunization. The immune response (immune-related genes expression, serum antibody production, enzyme activities and C3 content) was significantly enhanced in fish which vaccinated with SWCNTs-pcDNA-MCP in comparison with those vaccinated with pcDNA-MCP alone. After 14 d challenge, the RPS (relative percentage survival) can be enhanced which using SWCNTs as a carrier in SWCNTs-pcDNA-MCP (82.4%) group at 20 mg/L (the highest vaccine dose) than the naked pcDNA-MCP (54.2%) group. This study reveals that functionalized SWCNTs could be a promising immersion DNA vaccine carrier in aquaculture.

4.
Biomolecules ; 9(9)2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480692

RESUMO

Glucose is a main carbon and energy source for virus proliferation and is usually involved in the glycolysis, pentose phosphate pathway (PPP), and tricarboxylic acid cycle (TCA cycle) pathways. In this study, we investigated the roles of glucose-related metabolic pathways during the replication of infectious spleen and kidney necrosis virus (ISKNV), which has caused serious economic losses in the cultured Chinese perch (Siniperca chuatsi) industry. We found that ISKNV infection enhanced the metabolic pathways of the PPP and the TCA cycle at the early stage of the ISKNV infection cycle and enhanced the glycolysis pathway at the late stage of the ISKNV infection cycle though the comprehensive analysis of transcriptomics, proteomics, and metabolomics. The advanced results proved that ISKNV replication induced upregulation of aerobic glycolysis at the late stage of ISKNV infection cycle and aerobic glycolysis were required for ISKNV multiplication. In addition, the PPP, providing nucleotide biosynthesis, was also required for ISKNV multiplication. However, the TCA cycle involving glucose was not important and necessary for ISKNV multiplication. The results reported here provide new insights into viral pathogenesis mechanism of metabolic shift, as well as antiviral treatment strategies.

5.
Metabolites ; 9(9)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487859

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV) has caused serious economic losses in the cultured mandarin fish (Siniperca chuatsi) industry in China. Host metabolism alteration induced by disease infection may be the core problem of pathogenesis. However, to date, little is known about the disease-induced fish metabolism changes. In this study, we first reported ISKNV, the fish virus, induced metabolism alteration. The metabolomics profiles of Chinese perch brain cells (CPB) post-ISKNV infection at progressive time points were analyzed using the UHPLC-Q-TOF/MS technique. A total of 98 differential metabolites were identified. In the samples harvested at 24 hours post-infection (hpi; the early stage of ISKNV infection), 49 differential metabolites were identified comparing with control cells, including 31 up-regulated and 18 down-regulated metabolites. And in the samples harvested at 72 hpi (the late stage of ISKNV infection), 49 differential metabolites were identified comparing with control cells, including 27 up-regulated and 22 down-regulated metabolites. These differential metabolites were involved in many pathways related with viral pathogenesis. Further analysis on the major differential metabolites related to glucose metabolism and amino acid metabolism revealed that both glucose metabolism and glutamine metabolism were altered and a metabolic shift was determined from glucose to glutamine during ISKNV infection cycle. In ISKNV-infected cells, CPB cells prefer to utilize glucose for ISKNV replication at the early stage of infection, while they prefer to utilize glutamine to synthetize lipid for ISKNV maturation at the late stage of infection. These findings may improve the understanding of the interaction between ISKNV and host, as well as provide a new insight for elucidating the ISKNV pathogenic mechanism.

6.
Microb Pathog ; 135: 103617, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31283962

RESUMO

The bluegill sunfish, Lepomis macrochirus, is an important aquacultural and recreational species in southern China because of its excellent taste, rapid growth rate, and good looks. At present, few pathogens are known to affect the bluegill sunfish. However, an iridovirus-like disease recently caused heavy losses to the bluegill sunfish aquaculture industry in Guangdong, China. We report that a virus, designated BSMIV-SD-20171020, was isolated from diseased bluegill sunfish in China. The isolate was efficiently propagated in a Chinese perch brain (CPB) cell line. The cytopathic effect was observed, the MCP gene PCR amplified, and the virus observed with electron microscopy. Its viral titer in CPB cells reached 104.13 TCID50 mL-1. The mortality rate was 100% when bluegill sunfish were challenged with BSMIV-SD-20171020 at a dose of 103.13 TCID50/fish. A histopathological examination revealed basophilic hypertrophied cells in the intestine, liver, and spleen. A nucleotide sequence alignment and phylogenetic analysis of the major capsid protein revealed that isolate BSMIV-SD-20171020 is the species Infectious spleen and kidney necrosis virus (ISKNV), in the genus Megalocytivirus.


Assuntos
Infecções por Vírus de DNA/veterinária , Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Iridoviridae/classificação , Iridoviridae/isolamento & purificação , Perciformes/virologia , Animais , Aquicultura , Encéfalo , Proteínas do Capsídeo/classificação , Proteínas do Capsídeo/genética , Linhagem Celular , China , Infecções por Vírus de DNA/patologia , Doenças dos Peixes/patologia , Peixes , Iridoviridae/genética , Iridoviridae/patogenicidade , Rim/patologia , Rim/virologia , Fígado/patologia , Fígado/virologia , Percas , Filogenia , Análise de Sequência de DNA/veterinária , Baço/patologia , Baço/virologia
7.
Fish Shellfish Immunol ; 92: 133-140, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31173860

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV) cause a high mortality disease which lead to significant economic loss on mandarin fish in China. There is no effective drug or vaccine against this fatal disease at present. Meanwhile, many drugs and vaccines had no effect in many cases account of several impenetrable barriers (cell, skin and gastrointestinal tract). Here we reported an immersion subunit vaccine system (SWCNTs-MCP) encoding MCP gene of ISKNV based on single-walled carbon nanotubes (SWCNTs). To evaluate its efficacy against ISKNV, we found a stronger and longer duration immune response (serum antibody production, enzyme activities and immune-related genes expression) can be induced in fish vaccinated with SWCNTs-MCP in comparison with those vaccinated with MCP alone. Importantly, SWCNTs can increase the immune protective effect of naked subunit vaccine by ca. 23.8%. Thereby, this study demonstrates that SWCNTs as a promising carrier for subunit vaccine might be used to vaccinate large-scale juvenile mandarin fish by bath administration approach.


Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata , Iridoviridae/imunologia , Nanotubos de Carbono , Perciformes/imunologia , Vacinação/veterinária , Vacinas Virais/imunologia , Animais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/veterinária , Vacinas de Subunidades/imunologia
8.
Microb Pathog ; 129: 146-151, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30731189

RESUMO

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


Assuntos
Doenças dos Peixes/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções por Rhabdoviridae/veterinária , Rhabdoviridae/fisiologia , Transcrição Genética , Replicação Viral , Animais , Encéfalo/virologia , Percas , RNA Viral/genética , Reprodutibilidade dos Testes , Rhabdoviridae/genética , Infecções por Rhabdoviridae/virologia , Sensibilidade e Especificidade
9.
Fish Shellfish Immunol ; 84: 299-303, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30308292

RESUMO

MicroRNAs (miRNAs) are small noncoding RNAs that have been reported to play important roles in virus replication. Snakehead vesiculovirus (SHVV) is a new rhabdovirus isolated from diseased hybrid snakehead and has caused heavy economical losses in cultured snakehead fish in China. Our previous study has revealed that miR-214 inhibited SHVV replication, but the underline mechanism was not completely understood. In this study, glycogen synthase (GS) gene was identified as a target gene of miR-214. Overexpression of miR-214 reduced cellular GS gene expression. Knockdown of GS by siRNA, similar to the overexpression of miR-214, inhibited SHVV replication. Moreover, we found that siGS-mediated inhibition of SHVV replication could be restored by reducing cellular miR-214 level via using miR-214 inhibitor, indicating that miR-214 inhibited SHVV replication at least partially via targeting GS. This study provided information for understanding the molecular mechanism of SHVV pathogenicity and a potential antiviral strategy against SHVV infection.


Assuntos
Doenças dos Peixes/fisiopatologia , Proteínas de Peixes/genética , Glicogênio Sintase/genética , MicroRNAs/genética , Perciformes , RNA Viral/genética , Infecções por Rhabdoviridae/veterinária , Animais , Doenças dos Peixes/virologia , Proteínas de Peixes/metabolismo , Glicogênio Sintase/metabolismo , MicroRNAs/metabolismo , RNA Viral/metabolismo , Infecções por Rhabdoviridae/fisiopatologia , Infecções por Rhabdoviridae/virologia , Vesiculovirus/genética , Vesiculovirus/fisiologia
10.
Fish Shellfish Immunol ; 84: 1075-1082, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30423456

RESUMO

MicroRNAs are non-coding RNAs, which widely participate in biological processes. In recent years, Siniperca chuatsi rhabdovirus (SCRV) has caused mass mortality in Chinese perch (Siniperca chuatsi). To identify specific miRNAs involved in SCRV infection, deep sequencing of microRNA on Chinese perch brain cell line (CPB) with or without SCRV infection were performed at 6 and 12 h post of infection (hpi). Totally 382 miRNAs were identified, including 217 known miRNA aligned with zebrafish miRNAs and 165 novel miRNAs by MiRDeep2 program. Of which 15 and 35 differentially-expressed miRNAs were determined respectively to 6 and 12 hpi. Nine miRNAs were selected randomly from the differentially-expressed miRNAs and validated by quantitative real-time PCR (qRT-PCR). These results were consistent with the microRNA sequencing results. Besides, target genes of 98 differentially-expressed miRNAs were predicted. Three of miRNAs (miR-122, miR-214, miR-135a) were selected, and its effects were analyzed in CPC cells transfected with appropriate miRNA mimics/inhibitors to evaluate its regulation effects by qRT-PCR and western blot. The results demonstrated that miR-214 inhibited the replication of SCRV, while miR-122 promoted the replication of SCRV and there was no correlation between the miR-135a and SCRV replication. These results will pave a new way for the development of effective strategies against the SCRV infection.


Assuntos
Encéfalo/virologia , Doenças dos Peixes/imunologia , Imunidade Inata/genética , MicroRNAs/genética , Percas , Infecções por Rhabdoviridae/veterinária , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Doenças dos Peixes/virologia , MicroRNAs/metabolismo , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Infecções por Rhabdoviridae/virologia , Análise de Sequência de RNA/veterinária
11.
BMC Microbiol ; 18(1): 113, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30200876

RESUMO

BACKGROUND: Fish culture in rice paddies can contribute to increasing yields of rice and surplus fish products. Environmental impacts and food-safety issues have become important topics in aquaculture, and organic foods currently were paid attention by researchers and industry practitioners. But the mechanism of differences in quality of Loach (Paramisgurnus dabryanus) reared in rice fields and ponds remains largely uncharacterized. In this study,digestive enzyme activity, intestinal mucosa cells and the gut microbial community of loach were determined under the two separate cultivation modes. RESULTS: The levels of intestinal digestive enzyme activity of fish reared in the paddy-cultivated mode (PACM) were higher (P < 0.05) than those in the pond-cultivated mode (POCM). It was extremely significant (P < 0.01) for the activity of lipase in the liver, foregut and midgut, and for the activities of amylase and trypsin in the hindgut. Acid mucous cells in the loach foregut in PACM were fewer than in POCM (P < 0.01). In summer, the abundance of the Firmicutes, Lactobacillus spp., Aeromonas hydrophila, Enterobacteriaceae and Streptococcus spp. in loach intestinal mucosa in PACM was higher than in POCM. In fall, the abundance of total bacteria, the Bacteroidetes, Bifidobacterium and Enterobacteriaceae in the intestinal mucosa in PACM was likewise higher than in POCM. These differences were significant (P < 0.05 or P < 0.01) between loach in the two separate culture modes for all microorganisms except for A. hydrophila and Streptococcus spp. In addition, quantitative PCR assays showed that some microorganisms presented consistently similar abundances in the gut as in the culture water. CONCLUSIONS: These results showed some enzymatic activities involved in digestion in liver and intestine of loach in PACM were higher than those in POCM, as using digestive enzyme analysis and histological observation of intestinal sections. These findings suggest most of the microorganisms examined in the gut mucosa of loach in the two culture modes significantly differed in abundance between summer and fall. However, some pathogenic bacteria in the gut, particularly A. hydrophila, presented lower abundance in PACM in fall, yet did not differ in abundance between loach in the two cultivation modes.


Assuntos
Amilases/metabolismo , Bactérias/isolamento & purificação , Cipriniformes/microbiologia , Microbioma Gastrointestinal , Mucosa Intestinal/enzimologia , Tripsina/metabolismo , Animais , Aquicultura , Bactérias/classificação , Bactérias/genética , Cipriniformes/crescimento & desenvolvimento , Mucosa Intestinal/microbiologia , Estações do Ano
12.
Environ Microbiol ; 20(9): 3442-3456, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30136361

RESUMO

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.


Assuntos
Aeromonas veronii/metabolismo , Aeromonas veronii/patogenicidade , Toxinas Bacterianas/metabolismo , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Sepse/veterinária , Aeromonas/isolamento & purificação , Aeromonas veronii/genética , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , China , Infecções por Bactérias Gram-Negativas/microbiologia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/toxicidade , Sepse/microbiologia , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Fatores de Virulência/toxicidade , Peixe-Zebra/microbiologia
13.
Fish Shellfish Immunol ; 79: 102-111, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29733959

RESUMO

Infectious spleen and kidney necrosis virus (ISKNV) has caused significant losses in the cultured mandarin fish (Siniperca chuatsi) industry. The molecular mechanisms that underlie interaction between ISKNV and hosts are not fully understood. In this study, the proteomic profile of CPB cells at progressive time points after ISKNV infection was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ). A total of 2731 proteins corresponding to 6363 novel peptides (false discovery rate <0.01) were identified. In the samples harvested 24 h (early-stage) and 72 h (late-stage) post-infection, 232 and 199 differentially expressed proteins were identified comparing with mock-infected cells, respectively. Western-blotting analysis of several proteins as G6PDH, ß-tubulin and RPL11 were done to validate iTRAQ data. Among those differentially expressed proteins, several glucose metabolism-related enzymes, including glucose-6-phosphate dehydrogenase (G6PDH), pyruvate dehydrogenase phosphatase (PDP) and fumarate hydratase (FH), were up-regulated, while pyruvate dehydrogenase kinase (PDK) and enolase (ENO) were down-regulated at 24 h poi, suggesting that ISKNV enhanced glucose metabolism in CPB cells in early-stage infection. Simultaneously, expression of apoptosis-related proteins including Caspase 8, phosphoinositide 3-kinases (PI3Ks), and regulatory-associated protein of mTOR-like isoform X3 changed upon ISKNV infection, indicating that ISKNV induced apoptosis of CPB cells. Autophagy-related proteins including LC3 and PI3Ks were up-regulated at 24 h poi, indicating that ISKNV induced autophagy of CPB cells in early-stage infection. These findings may improve the understanding of ISKNV and host interaction and help clarify its pathogenesis mechanisms.


Assuntos
Apoptose , Autofagia , Doenças dos Peixes/imunologia , Glucose/metabolismo , Perciformes/imunologia , Transdução de Sinais , Animais , Linhagem Celular , Infecções por Vírus de DNA/imunologia , Iridoviridae/fisiologia , Proteômica
14.
Fish Shellfish Immunol ; 78: 26-34, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29621635

RESUMO

The emergence of multi antibiotic resistance by the pathogens and toxic impacts on host metabolism has opened new perspectives to rational novel vaccine techniques. Outbreaks of Aeromonas hydrophila in aquaculture caused high mortality throughout the world and resulted in the extensive economic loss in the aquaculture industry. In this study, we report the efficacy of anti-A. hydrophila IgY antibodies by passive vaccination and its prophylactic or therapeutic effects against A. hydrophila in blunt snout bream. Inactivated A. hydrophila immunized hens produced effective IgY antibodies that were stable at temperatures less than 60 °C or the pH value was >4. The specific IgY can be bound directly to A. hydrophila that efficiently agglutinated and inhibited the bacterial growth in a dose-dependent manner. The specific IgY had significantly enhanced the phagocytosis activity of macrophages and resulted in rapid bacterial clearance. Anti-A. hydrophila IgY antibodies significantly increased macrophage mediated respiratory burst, including nitric oxide and superoxide anion production and subsequently killed the pathogen. Histopathological studies of intestine and spleen from vaccinated blunt-snout bream challenged with A. hydrophila showed the structural integrity of the organs was maintained intact from the bacterial injury. In addition, the prophylactic and therapeutic immunization, protected the blunt snout bream and the survival is approximately about 60% and 50%, respectively. These data suggest that specific IgY has the potential for protecting blunt snout bream against A. hydrophila infection and show promise for the future development of harmless vaccines.


Assuntos
Proteínas Aviárias/imunologia , Cyprinidae/imunologia , Doenças dos Peixes/imunologia , Imunidade Inata/efeitos dos fármacos , Imunoglobulinas/imunologia , Aeromonas hydrophila/fisiologia , Animais , Galinhas , Gema de Ovo/imunologia , Infecções por Bactérias Gram-Negativas/imunologia , Distribuição Aleatória
15.
Microb Pathog ; 112: 269-273, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28987623

RESUMO

Ranavirus has become a noticeable threat to both farmed and natural populations of fish and amphibians. Herein, we reported that 3 strains of novel viruses, designated as ScRIV-GM-20150902, CmRIV-XT-20150917 and ScRIV-ZS-20151201, were isolated from diseased Chinese perch and snakehead fish in China. Efficient propagation of these isolates were determined in Chinese perch brain (CPB) cell line by the means of cytopathic effect observation, PCR amplification and electron microscopy observation. And their viral titers in CPB cells reached 108.13 TCID50 ml-1, 107.71 TCID50 ml-1 and 107.94 TCID50 ml-1, respectively. While the challenge experiment results showed that 3 isolates resulted in 100% mortality of Chinese perch after virus infection. Electron microscopy analysis showed that two kinds of viral inclusion bodies (intracytoplasmic and intranuclear inclusion body) were observed in infected CPB cells. Sequence alignment and phylogenetic analysis of major capsid protein gene sequences of isolates revealed that these isolates belonged to the species Santee-Cooper Ranavirus.


Assuntos
Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Peixes/virologia , Percas/virologia , Ranavirus/classificação , Ranavirus/isolamento & purificação , Ranavirus/patogenicidade , Animais , Ascite/patologia , Ascite/virologia , Sequência de Bases , Encéfalo/patologia , Encéfalo/virologia , Proteínas do Capsídeo/genética , Linhagem Celular , China , Infecções por Vírus de DNA/patologia , Infecções por Vírus de DNA/virologia , DNA Viral , Corpos de Inclusão Viral , Mesentério/patologia , Mesentério/virologia , Microscopia Eletrônica de Transmissão , Filogenia , Ranavirus/genética , Alinhamento de Sequência , Virulência
16.
Microb Pathog ; 111: 422-430, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28890148

RESUMO

In spite of the quite common co-infections of viruses in the cultured fish, most of the previous studies have just simply focused on the infection of a single pathogen. In this report, we observed that about 13% of cultured Chinese perch have been co-infected by infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdovirus (SCRV). Furthermore, Chinese perch could co-infected by ISKNV and SCRV by intraperitoneally injection with the two viruses. Interestingly, we revealed that the two viruses could even co-infect a single cell of Chinese perch in vivo and a single Chinese perch brain cells (CPB) cell in vitro. The dynamic co-infected viruses loads in the different tissues of Chinese perch showed dependent. When CPB cells were infected with the same 10 MOI of SCRV and ISKNV, the replication of SCRV overwhelmed the replication of ISKNV. When the MOI of ISKNV (10 MOI) was 10,000 times of MOI of SCRV (0.001 MOI), the dynamic virus loads of the two viruses in CPB cells indicated that co-infections could synergistically stimulate both viruses replication at the late time points but not at early time points. The co-infections of ISKNV and SCRV in the cultured Chinese perch will shed a new light on the prevention of the viral diseases of Chinese perch. The development of multivalent vaccine which could be effective for preventing against the co-infections of the viruses is highly needed.


Assuntos
Coinfecção/veterinária , Doenças dos Peixes/virologia , Iridoviridae/fisiologia , Percas/virologia , Rhabdoviridae/fisiologia , Animais , Células Cultivadas , Coinfecção/virologia , Iridoviridae/genética , Iridoviridae/isolamento & purificação , Rhabdoviridae/genética , Rhabdoviridae/isolamento & purificação , Replicação Viral
17.
Fish Shellfish Immunol ; 70: 536-544, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28923524

RESUMO

A number of size variants of the p53 protein have been described in mammal, but little is known about alternative splicing of p53 expression and function in the fish. In our previous study, the immune defense and antiviral responses of p53 had been determined in mandarin fish (Siniperca chuatsi). However, the role of its splicing variants remains unknown. In the present study, the organization of mandarin fish p53 (Sc-p53) genome sequence was determined and a novel splice variant was characterized. The Sc-p53 genomic sequence was composed of 5543 bp, containing 11 exons and 10 introns, which was similar to other species. Then, a 1106 bp full-length cDNA of a novel splice variant p53 from mandarin fish (designed as Sc-p53I6) was cloned and characterized. Quantitative real-time PCR assays revealed that Sc-p53I6 was expressed in all tissues examined, and it was most abundant in the gill, hemocyte and hind kidney. Western blotting analysis revealed that Sc-p53I6 protein was abundant in liver, trunk kidney, hind kidney, stomach and heart. In addition, the regulation of Sc-p53I6 gene expression after virus infection was determined and characterized. The results showed twice rise expression pattern of Sc-p53I6 in CPB cells and spleen of mandarin fish in response to infectious kidney and spleen necrosis virus (ISKNV). However, a different expression pattern, once rise, of Sc-p53I6 in response to Siniperca chuatsi rhabdovirus (SCRV) infection was found. The mRNA expression of Sc-p53I6 was significantly up-regulated in CPB at 4 h and spleen of mandarin fish at 12 h post-infection. These results will shed a new light on antiviral response mechanisms of p53 in mandarin fish.


Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Perciformes/genética , Perciformes/imunologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia , Processamento Alternativo , Animais , Linhagem Celular , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Iridoviridae/fisiologia , RNA Mensageiro/genética , Rhabdoviridae/fisiologia , Infecções por Rhabdoviridae/imunologia , Distribuição Tecidual
18.
Arch Virol ; 162(9): 2829-2834, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28550433

RESUMO

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses which infects mammals, birds, reptiles, fish, insects and plants. Herein, we reported the isolation and characterization of 6 novel viruses from diseased fish collected from China including SCRV-QY, SCRV-SS, SCRV-GM, CmRV-FS, MsRV-SS, OmbRV-JM. The typical clinical symptom of diseased fish was hemorrhaging. Efficient propagation of these isolates in a Chinese perch brain cell line was determined by means of observation of cytopathic effect, RT-PCR and electron microscopy. Sequence alignment and phylogenetic analysis of the complete G protein sequences revealed that these isolates were clustered into one monophyletic lineage belonging to the species Siniperca chuatsi rhabdovirus.


Assuntos
Doenças dos Peixes/virologia , Variação Genética , Rhabdoviridae/genética , Rhabdoviridae/patogenicidade , Animais , China/epidemiologia , Doenças dos Peixes/epidemiologia , Doenças dos Peixes/patologia , Peixes
19.
J Aquat Anim Health ; 29(2): 89-94, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28379065

RESUMO

Grass Carp reovirus (GCRV) is one of the most pathogenic agents among aquareovirus isolates and has the ability to cause a severe epidemic outbreak of hemorrhagic disease, thus resulting in both a high mortality rate during the culture of Grass Carp Ctenopharyngodon idella and an enormous economic loss. Aptamers have been demonstrated to have strong promising applications in antiviral drug development. In the present study, a complementary DNA fragment encoding the S10 gene of GCRV was cloned. The S10 protein was expressed and purified. Aptamers for S10 protein were selected by the method of selective evolution of ligands by exponential enrichment (SELEX), and their characteristics and antiviral actions were examined. All targeting-selected aptamers formed a similar structure, forming a 5-7 base loop at the terminus. The results show that the aptamers could inhibit the GCRV infection. The most significant inhibitory effect was obsereved when the aptamers were added to the cell culture for 1 h before the cells were infected by GCRV. Our data showed that these novel molecular agents could be considered suitable candidates for anti-GCRV therapy. Received August 23, 2016; accepted February 5, 2017.


Assuntos
Aptâmeros de Nucleotídeos/antagonistas & inibidores , Carpas , Doenças dos Peixes/prevenção & controle , Infecções por Reoviridae/veterinária , Reoviridae/genética , Animais , Infecções por Reoviridae/prevenção & controle
20.
Microb Pathog ; 107: 380-389, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28416382

RESUMO

In recent years, mandarin fish had a high mortality rate associated with abnormal swimming, exophthalmia, corneal opacity and eye hemorrhage on a fish farm located at Foshan city, Guangdong province, China. Three isolates of Gram-positive, chain-forming cocci were recovered from moribund fish, and designated as SS131025-1, SS131025-2, and SS131025-3. These isolates were identified as Streptococcus uberis according to their morphologic and physio-biochemical characteristics as well as phylogenetic analysis based on their 16S rRNA and GapC gene sequences. The pathogenicity of S. uberis to mandarin fish was determined by challenge experiments. Results of artificial challenge showed S. uberis infected healthy mandarin fish and lead to death by eyeball injection or immersion route, and the LD50 of SS131025-1 with eyeball injection was 2.0 × 106.42 CFU per fish. Moreover extracellular product (ECP) of the isolated S.uberis induced CPB cell apoptosis and cause death of mandarin fish. In addition, these S. uberis strains could also infect tilapia, but not grass carp and crucian carp, and grew in brain-heart infusion broth with an optimal temperature of 37 °C, pH of 7.0, and salinity of 0%. Antibiotic sensitivity testing indicated that these isolates were susceptible to rifampicin and furazolidone but resistant to 20 kinds of antibiotics. Histopathologically, infection with S. uberis could cause serious pathological changes in brain tissues such as vacuoles in matrix, swollen mitochondria with lysis of cristae and disintegration, and lots of coccus was observed both under electron and light microscope. These results shed some light on the pathogenicity of the isolates and how to prevent and control S. uberis infection in mandarin fish.


Assuntos
Doenças dos Peixes/microbiologia , Peixes/microbiologia , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/veterinária , Streptococcus/isolamento & purificação , Streptococcus/patogenicidade , Animais , Antibacterianos/farmacologia , Antígenos de Bactérias/genética , Apoptose , Proteínas de Bactérias/genética , Encéfalo/microbiologia , Encéfalo/patologia , Carpas/microbiologia , China , DNA Bacteriano , Modelos Animais de Doenças , Genoma Microbiano , Coração/microbiologia , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Filogenia , RNA Ribossômico 16S/genética , Infecções Estreptocócicas/mortalidade , Streptococcus/genética , Streptococcus/fisiologia , Temperatura Ambiente , Tilápia/microbiologia , Virulência
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