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BACKGROUND: The genus Hydrocotyle Tourn. ex L. is a key group for further study on the evolution of Apiales, comprising around 170 species globally. Previous studies mainly focused on separate sections and provided much information about this genus, but its infrageneric relationships are still confusing. In addition, the genetic basis of its adaptive evolution remains poorly understood. To investigate the phylogeny and evolution of the genus, we selected ten representative species covering two of three diversity distribution centers and exhibiting rich morphology diversity. Comparative plastome analysis was conducted to clarify the structural character of Hydrocotyle plastomes. Positive selection analyses were implemented to assess the evolution of the genus. Phylogenetic inferences with protein-coding sequences (CDS) of Hydrocotyle and 17 related species were also performed. RESULTS: Plastomes within Hydrocotyle were generally conservative in structure, gene order, and size. A total of 14 regions (rps16-trnK, trnQ-rps16, atpI-atpH, trnC-petN-psbM, ycf3-trnS, accD-psaI-ycf4, petA-psbJ, rps12-rpl20, rpl16 intron, rps3-rpl16 intron, rps9-rpl22, ndhF-rpl32, ndhA intron, and ycf1a) were recognized as hotspot regions within the genus, which suggested to be promising DNA barcodes for global phylogenetic analysis of Hydrocotyle. The ycf15 gene was suggested to be a protein-coding gene for Hydrocotyle species, and it could be used as a DNA barcode to identify Hydrocotyle. In phylogenetic analysis, three monophyletic clades (Clade I, II, III) were identified with evidence of rapid radiation speciation within Clade I. The selective pressure analysis detected that six CDS genes (ycf1b, matK, atpF, accD, rps14, and psbB) of Hydrocotyle species were under positive selection. Within the genus, the last four genes were conservative, suggesting a relation to the unique evolution of the genus in Apiales. Seven genes (atpE, matK, psbH, ycf1a, ycf1b, rpoA, and ycf2) were detected to be under some degree of positive selection in different taxa within the genus Hydrocotyle, indicating their role in the adaptive evolution of species. CONCLUSIONS: Our study offers new insights into the phylogeny and adaptive evolution of Hydrocotyle. The plastome sequences could significantly enhance phylogenetic resolution and provide genomic resources and potential DNA markers useful for future studies of the genus.
Assuntos
Filogenia , Evolução Molecular , Genomas de Plastídeos , Apiaceae/genéticaRESUMO
BACKGROUND: Muscovy duck reovirus (MDRV) causes high morbidity and mortality in Muscovy ducklings at 10 days old and can persist in an infected flock until the ducklings of 6 weeks old. It shares common physicochemical properties with avian reovirus (ARV) and differs in coding assignment and pathogenicity. The ARV p17 protein has been shown to trigger autophagy via activation multiple signaling pathways, which benefits virus replication. Since MDRV lacks the p17 protein, whether and how MDRV induces autophagy remains unknown. The aim of this study was to explore whether MDRV induces autophagy and which viral proteins are involved in MDRV-induced autophagy. METHODS: The autophagosome-like structures in MDRV-infected cells was observed under transmission electron microscopy. MDRV-induced autophagy was examined by analyzing the LC3-II level and phosphorylated form of mammalian target of rapamycin (mTOR) by Western blot assays. The effects of 3-methyladenine, rapamycin, chloroquine on viral yields were measured with quantitative(q) real-time reverse transcription (RT)-polymerase chain reaction (PCR) and 50% tissue culture infective dose (TCID50) assays, respectively. Additionally, to determine which viral protein is responsible for MDRV-induced autophagy, both p10.8- and σNS-encoding genes of MDRV were cloned into the pCI-neo-flag vector and transfected into DF-1 cells for detection of LC3-II. RESULTS: The typical double-membrane vesicles containing cytoplasmic inclusions were visible in MDRV-infected immortalized chicken embryo fibroblast (DF-1) cells under transmission electron microscopy. Both primary Muscovy duck embryo fibroblasts (MDEF) and DF-1 cells infected with MDRV exhibited a significant increased levels of LC3-II accompanied with downregulation of phosphorylated form of mTOR, further confirming that MDRV is capable of inducing autophagy. Autophagy could be suppressed by 3-methylademine and induced by rapamycin and chloroquine. Furthermore, we found that σNS induces an increased levels of LC3-II, suggesting that the MDRV σNS protein is one of viral proteins involved in induction of autophagy. Both qRT-PCR and TCID50 assays showed that virus yield was increased in rapamycin treated DF-1 cells following MDRV infection. Conversely, when infected cells were pretreated with chloroquine, virus yield was decreased. CONCLUSIONS: The MDRV σNS nonstructural protein is responsible for MDRV-induced autophagy and benefits virus replication.
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Autofagia , Orthoreovirus Aviário/fisiologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Autofagossomos/ultraestrutura , Western Blotting , Linhagem Celular , Galinhas , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/análise , Reação em Cadeia da Polimerase em Tempo Real , Serina-Treonina Quinases TOR/análise , Carga ViralRESUMO
Muscovy duck reovirus (MDRV) causes high morbidity and mortality in ducklings. However, the molecular basis for pathogenesis of this virus remains poorly understood, and the complete genome sequence of Muscovy duck is lacking. Here we report the transcriptome profile of Muscovy ducks in response to MDRV infection. RNA sequencing technology was employed to obtain a representative complement of transcripts from the spleen of ducklings, and then differential gene expression was analyzed between MDRV-YB strain infected ducks and noninfected ducks. This analysis generated 65,536 unigenes. Of these, 6458 genes were found to be significantly differentially expressed between the infected and noninfected groups. The symptom and pathology of ducks infected with MDRV-YB was correlated with the greater proportion of decreased expression genes (4906) than increased expression (1552) level. Gene ontology analysis assigned differentially expressed genes to the categories: "biological processes," "cellular functions," and "molecular functions." Differentially expressed genes involved in the innate immune system were analyzed further, and 128 of these genes showed decreased expression and 86 showed increased expression between the infected and noninfected groups. These genes represented the Janus kinase-signal transducer and activator of transcription signaling pathway, and the retinoic acid-inducible gene I (RIG-I)-like and Toll-like receptor (TLR) signaling pathways and included interferon (IFN) α, IFNγ, interleukin 6, RIG-I, and TLR4. The data were verified by SYBR fluorescence quantitative polymerase chain reaction (SYBR-qPCR). Our findings offer new insight into the host immune response to MDRV infection.
Assuntos
Patos , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/veterinária , Reoviridae/classificação , Baço/metabolismo , Transcriptoma/imunologia , Animais , Doenças das Aves Domésticas/imunologia , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/metabolismoRESUMO
SUMMARY. An outbreak of egg-drop syndrome occurred on a Sheldrake duck farm in Longhai in Fujian Province, China, in 2012. The main clinical symptoms were sharply reduced egg production, crooked necks, and death. We isolated the virus from the sick ducks, identified it, and observed the histopathologic changes after viral infection. We detected viral RNA in the blood and feces of the infected ducks and developed a latex-agglutination diagnostic method to detect anti-Tembusu-virus antibodies. Our results show that the pathogenic virus is a Tembusu virus. The histopathologic changes included follicular cell degeneration and necrosis, follicular cavity filled with blood cells, massive necrosis in the brain, and degeneration and necrosis of the nerve and glial cells. When the transmission of the virus in the infected ducks was studied, the duck blood was positive for viral nucleic acid for up to 29 days, and the feces were positive for viral nucleic acid for up to 13 days. We successfully established a simple, rapid, and easy- to-use latex-agglutination diagnostic method for the detection of antibodies against duck Tembusu virus.
Assuntos
Anticorpos Antivirais/sangue , Infecções por Flavivirus/veterinária , Flavivirus/isolamento & purificação , Testes de Fixação do Látex/veterinária , Doenças das Aves Domésticas/virologia , Animais , China/epidemiologia , Surtos de Doenças/veterinária , Patos , Flavivirus/classificação , Flavivirus/genética , Infecções por Flavivirus/diagnóstico , Infecções por Flavivirus/virologia , Testes de Fixação do Látex/métodos , Filogenia , Doenças das Aves Domésticas/sangue , Doenças das Aves Domésticas/diagnósticoRESUMO
OBJECTIVE: To investigate how the pretreatment of mice with Ganoderma spores affected the apoptosis of their splenic lymphocytes induced by dexamethasone after 19 days treatment. METHODS: Sixty Kunming mice were randomly divided into six groups: blank control groupdrenched with normal saline; a drug control group drenched with 150 mg/mL Ganoderma spores; a model group treated with saline; a low dose group with 50 mg/mL Ganoderma spores; a moderate dose group with 100 mg/mL Ganoderma spores; and a high dose group with 150 mg/mL Ganoderma spores. The effect of Ganoderma spores on apoptosis in spleen lymphocytes was analyzed. All groups were treated for 19 days. On day 20, the model group and the 3 treatment groups were intraperitoneally injected dexamethasone to induce apoptosis. Splenic index and apoptosis indes were employed to measure cell apoptosis. RESULTS: The results showed that Ganoderma spores reduced the splenic index to different degrees in each group and the best effect was seen in the high dose group (P < 0.05).Terminal dexynucleotidyl transferase (TdT)-mediated 2'-Deoxyuridine 5'-Triphosphate nick end labeling staining revealed that the apoptotic index in all groups administered Ganoderma spores differed significantly from the model group, and a dose-response was observed. Flow cytometric analysis indicated that spleen lymphocyte apoptosis in the model group was extensive. Each dose of Ganoderma spores inhibited dexamethasone-induced apoptosis in spleen lymphocytes, and a dose-response was observed as well. The highest dose of Ganoderma spores decreased Malondialdehyde content in serum induced by dexamethasone (P < 0.05). CONCLUSION: The findings imply that the pretreatment of the mice with Ganoderma spores could reduce the apoptosis rate induced by dexamethasone in their splenic lymphocytes.
Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Linfócitos/citologia , Substâncias Protetoras/farmacologia , Reishi/química , Baço/citologia , Esporos Fúngicos/química , Animais , Feminino , Linfócitos/efeitos dos fármacos , Masculino , Camundongos , Baço/efeitos dos fármacosRESUMO
Hydrocotyle pseudoconferta was an important medicinal plant. The complete plastid genome of this species was reported for the first time. The full length of the complete chloroplast genome is 153,302 bp, with a typical quadripartite organization: a large single-copy (LSC) region of 84,417 bp, a small single-copy (SSC) region of 18,767 bp, and a pair inverted repeat regions (IRa and IRb) with 25,059 bp for each. The complete chloroplast genome of H. pseudoconferta encoded 133 genes, comprising 86 protein-coding genes, 37 tRNA genes, 8 rRNA genes, and 2 pseudogenes. The phylogenetic analysis suggested the closest relationship between H. pseudoconferta and Hydrocotyle nepalensis.
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To investigate the effects of Hericium erinaceus polysaccharide (HEP) on immunity in Muscovy duck reovirus (MDRV)-infected ducklings and explore its mechanism of action, an MDRV contact-infection model was established. Then, we investigated the influence of HEP on morphology of main immune organs in MDRV-infected ducklings by HE staining, while antioxidant capacity (T-AOC, MDA), serum protein levels (TP, ALB, GLO), complement levels (C3, C4) and antibody levels (IgA, IgM, IgG) were detected. Apoptotic indexes (apoptosisi rate and FAS-L) were also quantified by TUNEL method and immunohistochemical staining. Meanwhile, FADD and CytC (apoptosis-related genes), were tested by quantitative RT-PCR. Results showed that HEP could reduce the injuries of immune organs caused by MDRV. Additionally, HEP markedly diminished MDA (p < 0.01), while significantly increased T-AOC, TP, ALB, GLO, C3, C4, IgA, IgM and IgG (p < 0.01 or p < 0.05). Then, HEP shifted apoptosis time to an early MDRV-infected stage and reduced apoptosis at later MDRV-infected stage. This was associated with changes of FADD and CytC. Collectively, our data suggested that HEP could reduce the immunesuppression by many ways, such as decreasing organs' injuries, improving antioxidant capacity, serum proteins levels, antibody levels and complement levels, while diminish the apoptosis by lowering the FADD and CytC.
Assuntos
Patos/virologia , Hericium/química , Sistema Imunitário/efeitos dos fármacos , Polissacarídeos/uso terapêutico , Doenças das Aves Domésticas/tratamento farmacológico , Infecções por Reoviridae/veterinária , Imunidade Adaptativa/efeitos dos fármacos , Animais , Anticorpos Antivirais/sangue , Apoptose/efeitos dos fármacos , Proteínas Sanguíneas/análise , Citocromos c/análise , Avaliação Pré-Clínica de Medicamentos , Proteína de Domínio de Morte Associada a Fas/análise , Linfócitos/efeitos dos fármacos , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/metabolismo , Tecido Linfoide/patologia , Tecido Linfoide/virologia , Oxirredução , Polissacarídeos/isolamento & purificação , Polissacarídeos/farmacologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Distribuição Aleatória , Infecções por Reoviridae/tratamento farmacológico , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologiaRESUMO
Heteromorpha arborescens has long been recognized and cultivated as an important medicinal plant. We reported its complete plastid genome for the first time and reconstructed its phylogenetic position. The complete plastid genome was 157,172 bp in length with a typical quadripartite organization: a large single-copy (LSC) region of 86,436 bp, a small single-copy (SSC) region of 18,222 bp, and two inverted repeat regions (IRa and IRb), each of 26,257 bp. A total of 130 functional genes were recovered, consisting of 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The phylogenetic analysis suggested that H. arborescens is sister to other species except Cetella arborescens in Apiaceae with strong ultrafast support.
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Carya illinoinensis is rich in phenolic metabolites such as tannins and flavonols, but both the composition and the distribution of these nutritional constituents in most pecan organs were still unclear. In this experiment, a comprehensive qualification and quantification of phenolic metabolites in eight organs of pecan were conducted for the first time. Ninety-seven phenolic metabolites were identified, in which twelve were identified for the first time in pecan, including a series of ellagitannins with high molecular weight. Hydrolysable tannin was the dominant kind of phenolic metabolites in pecan. The metabolic profiles of tannins in pecan were extended. Thirty-three phenolic metabolites were quantified, among them the highest content was ellagic acid pentose in testa. From this experiment, we can see that the distribution of phenolic metabolites in pecan was organ-specific, tannins tend to accumulate in pecan testa with both diverse structures and high contents, while flavonols tend to accumulate in organs such as branch, bark, or leaf. Among all organs, testa contained the highest content of phenolics, which might play important roles in protecting pecan kernel from diseases and insects. A massive phenolic metabolites' matrix in different pecan organs was built in this experiment, which should be useful for related researches in the future and help provide a theoretical basis for using these organs as functional foods.
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OBJECTIVE: In order to find the new varieties with different horticultural characters, and investigate the mutation effects of seeds of Dioscorea zingerbrensis. METHOD: The seeds were carried by a satellite into space and recovered. The space mutation effect on the germination, seedling growth, chromosomes and rhizome diosgenin content of SP, populations of D. zingerbrensis were investigated. RESULT: Stimulated by space environment, the seed possessed the fast germinating characteristics. Germination rate showed no change. It was also found that a few plants were aneuploid or tetraploid. Fresh rhizome weight and rhizome diosgenin content in the second year plant were remarkably higher than those of the control. However, the increasing of third year plant was slow down, and rhizome diosgenin content in the third year plant declined simultaneously. CONCLUSION: The space environment showed stimulating effects on seed germination, fresh rhizome weight and rhizome diosgenin content.
Assuntos
Dioscorea/química , Dioscorea/genética , Meio Ambiente Extraterreno , Mutação , Extratos Vegetais/química , Dioscorea/fisiologia , Germinação , Plântula/química , Plântula/genética , Plântula/fisiologiaRESUMO
In the present study, the mechanisms underlying Muscovy duck reovirus (MDRV) p10.8 protein-induced ER stress and apoptosis in DF-1 cells and Muscovy duckling hepatic tissues were explored. On the fifth day post-infection, an increase in the mRNA levels of binding immunoglobulin protein (Bip) and X-box binding protein (XBP1), activation of XBP1/s, and an increase in percentage of apoptotic cells were observed in Muscovy duckling livers. The use of ER stress inducer Tunicamycin and ER stress inhibitor Tauroursodeoxycholic acid demonstrated that MDRV induces apoptosis via ER stress, leading to apoptosis. The use of Tunicamycin increased viral protein synthesis while Tauroursodeoxycholic acid reduced viral protein synthesis, suggesting that MDRV induces ER stress benefiting virus replication. The MDRV p10.8 is the major protein to induce ER stress and apoptosis. We found that p10.8 promotes the conversion of XBP1/u to XBP1/s and expands ER diameter, and increases the percentages of apoptotic cells in DF-1 and duckling liver tissues. To investigate the mechanism underlying the MDRV p10.8-induced ER stress and apoptosis, Western blot, siRNA, and co-immunoprecipitation (Co-IP) assays were performed. We found that the MDRV p10.8 protein up-regulates Bip, p-IRE1, XBP1s, and cleaved-caspase 3. Co-IP results reveal that the MDRV p10.8 protein disassociates the Bip/IRE1 complex. Inhibition of IRE1 by 4-methyl umbelliferone 8-carbaldehyde (4u8c) dramatically reversed the MDRV p10.8-modulated increase in levels of XBP1s and cleaved-caspase 3. Knockdown of XBP1 by siRNA reversed the increased level of p10.8-modulated cleaved-caspase 3. The present study provides mechanistic insights into the MDRV p10.8 protein induces ER stress, resulting in apoptosis via the Bip/IRE1/XBP1 pathway in DF-1 cells and duckling livers.
Assuntos
Apoptose , Doenças das Aves/virologia , Patos/virologia , Estresse do Retículo Endoplasmático , Orthoreovirus Aviário/fisiologia , Infecções por Reoviridae/veterinária , Animais , Linhagem Celular , Fibroblastos , Linfocinas/genética , Linfocinas/metabolismo , Infecções por Reoviridae/virologia , Replicação Viral , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Muscovy duck reovirus (MDRV) causes serious immunodeficiency in the intestinal mucosa, although the underlying histopathological mechanisms remain unclear. Thus, we investigated the impact of MDRV infection on intestinal morphology using hematoxylin and eosin staining. Immune-related cells were also quantified by staining with hematoxylin and eosin, toluidine blue, and periodic acid-Schiff stain, or by immunohistochemistry and cytochemistry for lectin. Similarly, CD4+ and CD8+ cells were quantified by flow cytometry, and the expression of several immune-related molecules was quantified by radioimmunoassay. We found that MDRV clearly damaged the intestinal mucosa, based on tissue morphology, villus length, villus width, intestinal thickness, villus height/crypt depth ratio, and villus surface area. MDRV also altered the density or distribution of lymphocytes, mastocytes, and goblet cells in the small intestinal mucosa, as well as microfold cells in Peyer's patches. In addition, MDRV markedly depleted CD4+ cells from the intestinal mucosa and lowered the CD4+:CD8+ ratio in peripheral blood. Moreover, MDRV diminished the levels of secretory IgA and mucosal addressin cell adhesion molecule-1 (p < 0.01), but elevated those of histamine and nitric oxide (p < 0.01 or p < 0.05). Finally, MDRV significantly suppressed IL-1ß, IL-4, IL-5, and IL-8 levels (p < 0.01 or p < 0.05) mid-infection. Collectively, our data suggest that MDRV severely damages the structure and function of the intestinal mucosa by modulating immune cells and immune-related factors, thus leading to local immunodeficiency. Our findings lay the foundation for further research on the pathogenesis of MDRV.
Assuntos
Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/virologia , Orthoreovirus Aviário/imunologia , Infecções por Reoviridae/imunologia , Fatores Etários , Animais , Contagem de Linfócito CD4 , Citocinas/imunologia , Patos/virologia , Duodeno , Fibroblastos/virologia , Histamina/análise , Imunoglobulina A Secretora/análise , Intestino Delgado/imunologia , Óxido Nítrico/análise , Orthoreovirus Aviário/patogenicidade , Doenças das Aves Domésticas/virologia , Infecções por Reoviridae/patologia , Carga ViralRESUMO
Avian leukosis virus (ALV) induces multiple avian tumors, growth decrease and immune suppression. Previously, a novel natural recombinant ALV isolate FJ15HT0 was proven to be associated with significant body weight decrease, immune suppression and lymphocytoma in infected SPF chickens. In order to uncover the interaction between virus and host, we compared differences in the transcriptomes of the thymuses from the mock chickens and simulated congenitally infected chickens at 5days (d), 13d and 21d of age by RNA-seq analysis of the thymuses. Signaling pathways including cytokine-cytokine receptor interactions, peroxisome proliferator-activated receptor (PPAR) signaling pathway, Janus tyrosine kinase/signal transducers and activators of transcription (Jak-STAT) signaling pathway and fatty acid degradation were involved in the interaction between FJ15HT0 and SPF chickens. Interestingly, fold change of ciliary neurotrophic factor receptor α (CNTFRα) in infected donor collected from 2d to 21d showed a significant positive correlation with the corresponding expression of the viral gp85 gene in thymuses (r=0.656, P<0.01) and in livers (r=0.525, P<0.05). It will provide new insights for the molecular pathogenesis of ALV infection.
Assuntos
Vírus da Leucose Aviária/genética , Leucose Aviária/genética , Proteínas Aviárias/genética , Doenças das Aves Domésticas/genética , Timo/virologia , Transcrição Gênica , Animais , Leucose Aviária/imunologia , Leucose Aviária/patologia , Leucose Aviária/virologia , Vírus da Leucose Aviária/crescimento & desenvolvimento , Vírus da Leucose Aviária/metabolismo , Proteínas Aviárias/imunologia , Peso Corporal , Galinhas , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/genética , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/imunologia , Citocinas/genética , Citocinas/imunologia , Ácidos Graxos/metabolismo , Interações Hospedeiro-Patógeno , Janus Quinases/genética , Janus Quinases/imunologia , Metabolismo dos Lipídeos , Fígado/imunologia , Fígado/virologia , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/patologia , Doenças das Aves Domésticas/virologia , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/imunologia , Transdução de Sinais , Organismos Livres de Patógenos Específicos , Timo/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
To elucidate the effect of Hericium erinaceus polysaccharide (HEP) on the intestinal mucosal immunity in normal and Muscovy duck reovirus (MDRV)-infected Muscovy ducklings, 1-day-old healthy Muscovy ducklings were pretreated with 0.2g/L HEP and/or following by MDRV infection in this study, duodenal samples were respectively collected at 1, 3, 6, 10, 15 and 21day post-infection, tissue sections were prepared for observation of morphological structure and determination of intestinal parameters (villus height/crypt depth ratio, villus surface area) as well as counts of intraepithelial lymphocytes (IELs), goblet cells, mast cells. Additionally, dynamics of secretory immunoglobin A (sIgA), interferon-γ (IFN-γ) and interleukin-4 (IL-4) productions in intestinal mucosa were measured with radioimmunoassay. Results showed that HEP significantly improved intestinal morphological structure and related indexes, and significantly inhibited the reduction of intestinal mucosal IELs, goblet cells and mast cells caused by MDRV infection. Furthermore, HEP significantly increased the secretion of sIgA, IFN-γ and IL-4 to enhance intestinal mucosal immune functions. Our findings indicate that HEP treatment can effectively repair MDRV-caused injures of small intestinal mucosal immune barrier, and improve mucosal immune function in sick Muscovy ducklings, which will provide valuable help for further application of HEP in prevention and treatment of MDRV infection.
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Imunidade/efeitos dos fármacos , Polissacarídeos/farmacologia , Infecções por Reoviridae/tratamento farmacológico , Animais , Autofagia/efeitos dos fármacos , Basidiomycota/química , Patos/imunologia , Patos/virologia , Imunidade/imunologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Polissacarídeos/química , Reoviridae/efeitos dos fármacos , Reoviridae/patogenicidade , Infecções por Reoviridae/veterinária , Infecções por Reoviridae/virologiaRESUMO
Avian Leukosis Viruses (ALVs) are associated with neoplasias, immune suppression and reduced performance in chicken flocks. In the present study, a naturally occurring recombinant strain of ALV (FJ15HT0) was isolated from an infected flock of Chinese "Hetian" chickens, and was subsequently identified as an exogenous ALV by immuno-fluorescence assay (IFA), PCR and following entire proviral DNA nucleotide sequencing. This isolate is revealed as a novel recombinant virus, lacking viral oncogenes, with the gp85 (93.4%) of subgroup B, the U3 (92.1%) and R (95.2%) region of subgroup J, the U5 (93.8%) region and 5'UTR (95.7%) of subgroup C, as well as the gp37 (90.6%) and 3' (92.2%) of ALV-E. The simulative congenital infection with this isolate in SPF chickens resulted in significant weight loss (P<0.05) and a significant reduction in the humoral immune response to the live NDV vaccine (P<0.05), but not to the inactive AIV-H5 vaccine (P>0.05). Foci of lymphocytomas were observed in tissues of congenitally infected chickens at 11 weeks post-hatch, demonstrating the acute oncogenicity of the isolate.
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Vírus da Leucose Aviária/imunologia , Leucose Aviária/virologia , Galinhas/virologia , Doenças das Aves Domésticas/virologia , Provírus , Pseudolinfoma/veterinária , Animais , Vírus da Leucose Aviária/genética , Vírus da Leucose Aviária/isolamento & purificação , Sequência de Bases , Galinhas/crescimento & desenvolvimento , Galinhas/imunologia , Terapia de Imunossupressão/veterinária , Pseudolinfoma/virologia , Recombinação Genética , Alinhamento de Sequência/veterinária , Organismos Livres de Patógenos Específicos , Vacinação/veterinária , Aumento de PesoRESUMO
In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.
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Infecções por Coronavirus/veterinária , Patos/virologia , Vírus da Bronquite Infecciosa/isolamento & purificação , Doenças das Aves Domésticas/virologia , Sequência de Aminoácidos , Animais , Embrião de Galinha , Infecções por Coronavirus/virologia , Feminino , Vírus da Bronquite Infecciosa/classificação , Vírus da Bronquite Infecciosa/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
Muscovy duck reovirus (MDRV) is a highly pathogenic virus in waterfowl and causes significant economic loss in the poultry industry worldwide. Because the host innate immunity plays a key role in defending against virus invasion, more and more attentions have been paid to the immune response triggered by viral infection. Here we found that the genomic RNA of MDRV was able to rapidly induce the production of interferons (IFNs) in host. Mechanistically, MDRV infection induced robust expression of IFNs in host mainly through RIG-I, MDA5 and TLR3-dependent signaling pathways. In addition, we observed that silencing VISA expression in 293T cells could significantly inhibit the secretion of IFNs. Remarkably, the production of IFNs was reduced by inhibiting the activation of NF-κB or knocking down the expression of IRF-7. Furthermore, our study showed that treatment of 293T cells and Muscovy duck embryo fibroblasts with IFNs markedly impaired MDRV replication, suggesting that these IFNs play an important role in antiviral response during the MDRV infection. Importantly, we also detected the induced expression of RIG-I, MDA5, TLR3 and type I IFN in Muscovy ducks infected with MDRV at different time points post infection. The results from in vivo studies were consistent with those in 293T cells infected with MDRV. Taken together, our findings reveal that the host can resist MDRV invasion by activating innate immune response involving RIG-I, MDA5 and TLR3-dependent signaling pathways that govern IFN production.
Assuntos
Patos , Imunidade Inata , Orthoreovirus , Infecções por Reoviridae/veterinária , Animais , Antivirais , Patos/virologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Interferons/farmacologia , NF-kappa B/metabolismo , Infecções por Reoviridae/virologia , Transdução de Sinais , Viroses , Replicação Viral/efeitos dos fármacosRESUMO
Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.