RESUMO
Ribonuclease (RNase) P is a ubiquitous ribozyme that cleaves the 5' leader from precursor tRNAs. Here, we report cryo-electron microscopy structures of the human nuclear RNase P alone and in complex with tRNAVal. Human RNase P is a large ribonucleoprotein complex that contains 10 protein components and one catalytic RNA. The protein components form an interlocked clamp that stabilizes the RNA in a conformation optimal for substrate binding. Human RNase P recognizes the tRNA using a double-anchor mechanism through both protein-RNA and RNA-RNA interactions. Structural comparison of the apo and tRNA-bound human RNase P reveals that binding of tRNA induces a local conformational change in the catalytic center, transforming the ribozyme into an active state. Our results also provide an evolutionary model depicting how auxiliary RNA elements in bacterial RNase P, essential for substrate binding, and catalysis, were replaced by the much more complex and multifunctional protein components in higher organisms.
Assuntos
Microscopia Crioeletrônica , RNA de Transferência/química , Ribonuclease P/química , Sítios de Ligação , Evolução Molecular , Células HEK293 , Holoenzimas/química , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Domínios Proteicos , Estrutura Terciária de Proteína , RNA de Transferência/metabolismo , Ribonuclease P/isolamento & purificação , Ribonuclease P/metabolismoRESUMO
Telomerase maintains chromosome ends from humans to yeasts. Recruitment of yeast telomerase to telomeres occurs through its Ku and Est1 subunits via independent interactions with telomerase RNA (TLC1) and telomeric proteins Sir4 and Cdc13, respectively. However, the structures of the molecules comprising these telomerase-recruiting pathways remain unknown. Here, we report crystal structures of the Ku heterodimer and Est1 complexed with their key binding partners. Two major findings are as follows: (1) Ku specifically binds to telomerase RNA in a distinct, yet related, manner to how it binds DNA; and (2) Est1 employs two separate pockets to bind distinct motifs of Cdc13. The N-terminal Cdc13-binding site of Est1 cooperates with the TLC1-Ku-Sir4 pathway for telomerase recruitment, whereas the C-terminal interface is dispensable for binding Est1 in vitro yet is nevertheless essential for telomere maintenance in vivo. Overall, our results integrate previous models and provide fundamentally valuable structural information regarding telomere biology.
Assuntos
Proteínas de Ligação a DNA/química , Simulação de Acoplamento Molecular , Proteínas de Saccharomyces cerevisiae/química , Telomerase/química , Homeostase do Telômero , Proteínas de Ligação a Telômeros/química , Sítios de Ligação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
High-throughput screening rapidly tests an extensive array of chemical compounds to identify hit compounds for specific biological targets in drug discovery. However, false-positive results disrupt hit compound screening, leading to wastage of time and resources. To address this, we propose ChemFH, an integrated online platform facilitating rapid virtual evaluation of potential false positives, including colloidal aggregators, spectroscopic interference compounds, firefly luciferase inhibitors, chemical reactive compounds, promiscuous compounds, and other assay interferences. By leveraging a dataset containing 823 391 compounds, we constructed high-quality prediction models using multi-task directed message-passing network (DMPNN) architectures combining uncertainty estimation, yielding an average AUC value of 0.91. Furthermore, ChemFH incorporated 1441 representative alert substructures derived from the collected data and ten commonly used frequent hitter screening rules. ChemFH was validated with an external set of 75 compounds. Subsequently, the virtual screening capability of ChemFH was successfully confirmed through its application to five virtual screening libraries. Furthermore, ChemFH underwent additional validation on two natural products and FDA-approved drugs, yielding reliable and accurate results. ChemFH is a comprehensive, reliable, and computationally efficient screening pipeline that facilitates the identification of true positive results in assays, contributing to enhanced efficiency and success rates in drug discovery. ChemFH is freely available via https://chemfh.scbdd.com/.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Software , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Reações Falso-Positivas , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , HumanosRESUMO
ADMETlab 3.0 is the second updated version of the web server that provides a comprehensive and efficient platform for evaluating ADMET-related parameters as well as physicochemical properties and medicinal chemistry characteristics involved in the drug discovery process. This new release addresses the limitations of the previous version and offers broader coverage, improved performance, API functionality, and decision support. For supporting data and endpoints, this version includes 119 features, an increase of 31 compared to the previous version. The updated number of entries is 1.5 times larger than the previous version with over 400 000 entries. ADMETlab 3.0 incorporates a multi-task DMPNN architecture coupled with molecular descriptors, a method that not only guaranteed calculation speed for each endpoint simultaneously, but also achieved a superior performance in terms of accuracy and robustness. In addition, an API has been introduced to meet the growing demand for programmatic access to large amounts of data in ADMETlab 3.0. Moreover, this version includes uncertainty estimates in the prediction results, aiding in the confident selection of candidate compounds for further studies and experiments. ADMETlab 3.0 is publicly for access without the need for registration at: https://admetlab3.scbdd.com.
Assuntos
Descoberta de Drogas , Internet , Software , Descoberta de Drogas/métodos , Humanos , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismoRESUMO
Machine learning-based scoring functions (MLSFs) have become a very favorable alternative to classical scoring functions because of their potential superior screening performance. However, the information of negative data used to construct MLSFs was rarely reported in the literature, and meanwhile the putative inactive molecules recorded in existing databases usually have obvious bias from active molecules. Here we proposed an easy-to-use method named AMLSF that combines active learning using negative molecular selection strategies with MLSF, which can iteratively improve the quality of inactive sets and thus reduce the false positive rate of virtual screening. We chose energy auxiliary terms learning as the MLSF and validated our method on eight targets in the diverse subset of DUD-E. For each target, we screened the IterBioScreen database by AMLSF and compared the screening results with those of the four control models. The results illustrate that the number of active molecules in the top 1000 molecules identified by AMLSF was significantly higher than those identified by the control models. In addition, the free energy calculation results for the top 10 molecules screened out by the AMLSF, null model and control models based on DUD-E also proved that more active molecules can be identified, and the false positive rate can be reduced by AMLSF.
Assuntos
Proteínas , Proteínas/metabolismo , Bases de Dados Factuais , Ligantes , Simulação de Acoplamento Molecular , Ligação ProteicaRESUMO
The conserved shelterin complex caps chromosome ends to protect telomeres and regulate telomere replication. In fission yeast Schizosaccharomyces pombe, shelterin consists of telomeric single- and double-stranded DNA-binding modules Pot1-Tpz1 and Taz1-Rap1 connected by Poz1, and a specific component Ccq1. While individual structures of the two DNA-binding OB folds of Pot1 (Pot1OB1-GGTTAC and Pot1OB2-GGTTACGGT) are available, structural insight into recognition of telomeric repeats with spacers by the complete DNA-binding domain (Pot1DBD) remains an open question. Moreover, structural information about the Tpz1-Ccq1 interaction requires to be revealed for understanding how the specific component Ccq1 of S. pombe shelterin is recruited to telomeres to function as an interacting hub. Here, we report the crystal structures of Pot1DBD-single-stranded-DNA, Pot1372-555-Tpz1185-212 and Tpz1425-470-Ccq1123-439 complexes and propose an integrated model depicting the assembly mechanism of the shelterin complex at telomeres. The structure of Pot1DBD-DNA unveils how Pot1 recognizes S. pombe degenerate telomeric sequences. Our analyses of Tpz1-Ccq1 reveal structural basis for the essential role of the Tpz1-Ccq1 interaction in telomere recruitment of Ccq1 that is required for telomere maintenance and telomeric heterochromatin formation. Overall, our findings provide valuable structural information regarding interactions within fission yeast shelterin complex at 3' ss telomeric overhang.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Telomerase , Proteínas de Transporte/genética , DNA de Cadeia Simples , Ligação Proteica , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Complexo Shelterina , Telomerase/genética , Telômero/genética , Telômero/metabolismo , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismoRESUMO
Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbß, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at â¼11 Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association of GPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.
Assuntos
Complexo Glicoproteico GPIb-IX de Plaquetas , Trombose , Humanos , Complexo Glicoproteico GPIb-IX de Plaquetas/química , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombina/metabolismo , Microscopia Crioeletrônica , Plaquetas/metabolismo , Trombose/metabolismoRESUMO
Half-life is a significant pharmacokinetic parameter included in the excretion phase of absorption, distribution, metabolism, and excretion. It is one of the key factors for the successful marketing of drug candidates. Therefore, predicting half-life is of great significance in drug design. In this study, we employed eXtreme Gradient Boosting (XGboost), randomForest (RF), gradient boosting machine (GBM), and supporting vector machine (SVM) to build quantitative structure-activity relationship (QSAR) models on 3512 compounds and evaluated model performance by using root-mean-square error (RMSE), R2, and mean absolute error (MAE) metrics and interpreted features by SHapley Additive exPlanation (SHAP). Furthermore, we developed consensus models through integrating four individual models and validated their performance using a Y-randomization test and applicability domain analysis. Finally, matched molecular pair analysis was used to extract the transformation rules. Our results revealed that XGboost outperformed other individual models (RMSE = 0.176, R2 = 0.845, MAE = 0.141). The consensus model integrating all four models continued to enhance prediction performance (RMSE = 0.172, R2 = 0.856, MAE = 0.138). We evaluated the reliability, robustness, and generalization ability via Y-randomization test and applicability domain analysis. Meanwhile, we utilized SHAP to interpret features and employed matched molecular pair analysis to extract chemical transformation rules that provide suggestions for optimizing drug structure. In conclusion, we believe that the consensus model developed in this study serve as a reliable tool to evaluate half-life in drug discovery, and the chemical transformation rules concluded in this study could provide valuable suggestions in drug discovery.
Assuntos
Aprendizado de Máquina , Relação Quantitativa Estrutura-Atividade , Meia-Vida , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Farmacocinética , Máquina de Vetores de SuporteRESUMO
Liver microsomal stability, a crucial aspect of metabolic stability, significantly impacts practical drug discovery. However, current models for predicting liver microsomal stability are based on limited molecular information from a single species. To address this limitation, we constructed the largest public database of compounds from three common species: human, rat, and mouse. Subsequently, we developed a series of classification models using both traditional descriptor-based and classic graph-based machine learning (ML) algorithms. Remarkably, the best-performing models for the three species achieved Matthews correlation coefficients (MCCs) of 0.616, 0.603, and 0.574, respectively, on the test set. Furthermore, through the construction of consensus models based on these individual models, we have demonstrated their superior predictive performance in comparison with the existing models of the same type. To explore the similarities and differences in the properties of liver microsomal stability among multispecies molecules, we conducted preliminary interpretative explorations using the Shapley additive explanations (SHAP) and atom heatmap approaches for the models and misclassified molecules. Additionally, we further investigated representative structural modifications and substructures that decrease the liver microsomal stability in different species using the matched molecule pair analysis (MMPA) method and substructure extraction techniques. The established prediction models, along with insightful interpretation information regarding liver microsomal stability, will significantly contribute to enhancing the efficiency of exploring practical drugs for development.
Assuntos
Inteligência Artificial , Microssomos Hepáticos , Microssomos Hepáticos/metabolismo , Animais , Camundongos , Ratos , Humanos , Aprendizado de Máquina , Descoberta de Drogas/métodos , Preparações Farmacêuticas/metabolismo , Preparações Farmacêuticas/químicaRESUMO
Mirizzi syndrome is a serious complication of gallstone disease. It is caused by the impacted stones in the gallbladder neck or cystic duct. One of the features of Mirizzi syndrome is severe inflammation or dense fibrosis at the Calot's triangle. In our clinical practice, bile duct, branches of right hepatic artery and right portal vein clinging to gallbladder infundibulum are often observed due to gallbladder infundibulum adhered to right hepatic hilum. The intraoperative damage of branches of right hepatic artery occurs more easily than that of bile duct, all of which are hidden pitfalls for surgeons. Magnetic resonance cholangiopancreatography (MRCP) and endoscopic retrograde cholangiopancreatography (ERCP) are the preferable tools for the diagnosis of Mirizzi syndrome. Anterograde cholecystectomy in Mirizzi syndrome is easy to damage branches of right hepatic artery and bile duct due to gallbladder infundibulum adhered to right hepatic hilum. Subtotal cholecystectomy is an easy, safe and definitive approach to Mirizzi syndrome. When combined with the application of ERCP, a laparoscopic management of Mirizzi syndrome by well-trained surgeons is feasible and safe. The objective of this review was to highlight its existing problems: (1) low preoperative diagnostic rate, (2) easy to damage bile duct and branches of right hepatic artery, and (3) high concomitant gallbladder carcinoma. Meanwhile, the review aimed to discuss the possible therapeutic strategies: (1) to enhance its preoperative recognition by imaging findings, and (2) to avoid potential pitfalls during surgery.
Assuntos
Colelitíase , Síndrome de Mirizzi , Humanos , Síndrome de Mirizzi/diagnóstico por imagem , Síndrome de Mirizzi/cirurgia , Colangiopancreatografia Retrógrada Endoscópica , Colelitíase/cirurgia , Colecistectomia , Ductos BiliaresRESUMO
Hematotoxicity has been becoming a serious but overlooked toxicity in drug discovery. However, only a few in silico models have been reported for the prediction of hematotoxicity. In this study, we constructed a high-quality dataset comprising 759 hematotoxic compounds and 1623 nonhematotoxic compounds and then established a series of classification models based on a combination of seven machine learning (ML) algorithms and nine molecular representations. The results based on two data partitioning strategies and applicability domain (AD) analysis illustrate that the best prediction model based on Attentive FP yielded a balanced accuracy (BA) of 72.6%, an area under the receiver operating characteristic curve (AUC) value of 76.8% for the validation set, and a BA of 69.2%, an AUC of 75.9% for the test set. In addition, compared with existing filtering rules and models, our model achieved the highest BA value of 67.5% for the external validation set. Additionally, the shapley additive explanation (SHAP) and atom heatmap approaches were utilized to discover the important features and structural fragments related to hematotoxicity, which could offer helpful tips to detect undesired positive substances. Furthermore, matched molecular pair analysis (MMPA) and representative substructure derivation technique were employed to further characterize and investigate the transformation principles and distinctive structural features of hematotoxic chemicals. We believe that the novel graph-based deep learning algorithms and insightful interpretation presented in this study can be used as a trustworthy and effective tool to assess hematotoxicity in the development of new drugs.
Assuntos
Aprendizado Profundo , Simulação por Computador , Aprendizado de Máquina , Algoritmos , Descoberta de DrogasRESUMO
Several outbreaks of duck hepatitis A virus type 1 (DHAV-1), which were characterized by yellow coloration and hemorrhage in pancreatic tissues, have occurred in China. The causative agent is called pancreatitis-associated DHAV-1. The mechanisms involved in pancreatitis-associated DHAV-1 infection are still unclear. Transcriptome analysis of duck pancreas infected with classical-type DHAV-1 and pancreatitis-associated DHAV-1 was carried out. Deep sequencing with Illumina-Solexa resulted in a total of 53.9 Gb of clean data from the cDNA library of the pancreas, and a total of 29,597 unigenes with an average length of 993.43 bp were generated by de novo sequence assembly. The expression levels of D-3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which are involved in glycine, serine, and threonine metabolism pathways, were significantly downregulated in ducks infected with pancreatitis-associated DHAV-1 compared with those infected with classical-type DHAV-1. These findings provide information regarding differences in expression levels of metabolism-associated genes between ducks infected with pancreatitis-associated DHAV-1 and those infected with classical-type DHAV-1, indicating that intensive metabolism disorders may contribute to the different phenotypes of DHAV-1-infection.
Assuntos
Vírus da Hepatite do Pato/patogenicidade , Hepatite Viral Animal/virologia , Interações Hospedeiro-Patógeno/genética , Infecções por Picornaviridae/veterinária , Doenças das Aves Domésticas/virologia , Aminoácidos/genética , Aminoácidos/metabolismo , Animais , Patos/virologia , Expressão Gênica , Hepatite Viral Animal/genética , Hepatite Viral Animal/metabolismo , Hepatite Viral Animal/patologia , Pâncreas/citologia , Pâncreas/patologia , Pâncreas/virologia , Pancreatite/patologia , Pancreatite/virologia , Infecções por Picornaviridae/metabolismo , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/metabolismo , Doenças das Aves Domésticas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNARESUMO
Recently, a novel goose astrovirus (N-GoAstV) was discovered in China, with the transmission route of N-GoAstV unclear. In this study, we developed a TaqMan-based real-time RT-PCR (qRT-PCR) assay for the detection of N-GoAstV infection. After the optimization of the qRT-PCR assay conditions, the results demonstrated that the lower limit of detection for N-GoAstV was 33.4 copies/µL. No cross-reactivity was observed with other goose-origin viruses. Intra-assay and inter-assay variability were ≤1.36% and 2.34%, respectively. N-GoAstV was detected in both field samples, embryos and newly hatched goslings by qRT-PCR assay, provided the view that N-GoAstV may be both horizontally and vertically transmitted. The established qRT-PCR method showed high specificity, sensitivity, and reproducibility, which can be used in future investigations on the pathogenesis and epidemiology of N-GoAstV.
Assuntos
Infecções por Astroviridae/veterinária , Avastrovirus/isolamento & purificação , Doenças das Aves/virologia , Gansos/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Infecções por Astroviridae/virologia , Avastrovirus/classificação , Avastrovirus/genética , China , Sensibilidade e EspecificidadeRESUMO
Plant domestication was a pivotal accomplishment in human history, which led to a reduction in genetic diversity of crop species; however, there was less research focus on how this reduced genetic diversity of crops in affecting rhizosphere microbial communities during crop domestication process. Here, we used high-throughput sequencing to explore the different effects of crops domestication on rhizosphere microbial community structure of rice (Oryza sativa L. and Oryza rufipogon Griff.) and soybean (Glycine max L. and Glycine soja Sieb. et Zucc.). Results indicated that rhizosphere fungal communities are more strongly influenced by crop domestication than bacterial communities. There was a stronger relationship for fungi and bacteria in the cultivated crops than in the wild relatives. Results also showed that the wild varieties had a higher abundance of beneficial symbionts and a lower abundance of pathogens comparing with the cultivated varieties. There was a similar tendency for both rice and soybean in rhizosphere microbial communities by comparing wild crops and their cultivated varieties. In conclusion, crop domestication had a stronger effect on the fungal communities than on the bacterial communities and had improved the microbial relationship in rhizosphere of cultivated crops.
Assuntos
Produtos Agrícolas , Glycine max/microbiologia , Microbiota , Oryza/microbiologia , Rizosfera , Fenômenos Fisiológicos Bacterianos , Fungos/fisiologia , Variação GenéticaRESUMO
The fungicides used to control diseases in cereal production can have adverse effects on non-target microbial communities, with possible consequences for plant health and productivity. Although we know that fungicides affect microbial community structure and soil activities, it is unclear how crop cultivars have altered the impact of fungicides on rhizomicrobiomes. In this study, the rhizosphere bacterial and fungal communities and structures of cultivated crops and their wild relatives were studied by Illumina MiSeq sequencing analysis. The results indicated that the rhizomicrobiome communities of wild crops reacted more strongly to fungicides than that of their cultivated relatives. Furthermore, fungal community composition was more affected by fungicides than bacterial community composition. Remarkably, the same trend was observed in both soybean and rice with regard to the influence of crop cultivar on the response of the rhizomicrobiome to fungicide application, although the level of the response was not similar. We report for the first time that the rhizomicrobiomes of wild crops reacted more strongly to fungicides than the rhizomicrobiomes of cultivated crops.
Assuntos
Produtos Agrícolas/microbiologia , Fungicidas Industriais/farmacologia , Microbiota/efeitos dos fármacos , Microbiologia do Solo , Bactérias/genética , Bactérias/isolamento & purificação , Fungos/genética , Fungos/isolamento & purificação , Oryza/microbiologia , Rizosfera , Glycine max/microbiologiaRESUMO
Duck adenovirus 3 (DAdV-3) is a newly identified duck adenovirus that has recently emerged in China. The incidence of duck infection caused by this virus is very high, with very large economic losses to the poultry industry. Thus, there is an urgent need for a serological assay for the specific detection of DAdV-3. To this end, prokaryotic expression of the fiber2 protein of DAdV-3 was used as a coating antigen to establish an indirect enzyme linked immunosorbent assay (ELISA) method for the specific detection of antibodies against DAdV-3. The method was found to be specific, repeatable and more sensitive than the agarose gel precipitation test (AGP). This indirect ELISA method based on the recombinant fiber2 protein may be used for the clinical detection of DAdV-3 infection and for monitoring antibody levels after vaccine immunization and is of great significance for the effective prevention and control of the disease.
Assuntos
Infecções por Adenoviridae/virologia , Adenoviridae/metabolismo , Patos/virologia , Ensaio de Imunoadsorção Enzimática/métodos , Doenças das Aves Domésticas/virologia , Adenoviridae/imunologia , Infecções por Adenoviridae/imunologia , Animais , Anticorpos Antivirais/imunologia , China , Patos/imunologia , Doenças das Aves Domésticas/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Vacinas Virais/imunologiaRESUMO
Both Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) can cause high mortality and morbidity in Muscovy ducklings. MDPVs and GPVs share high nucleotide identity, which can cause errors during differential diagnosis. In this study, the NS genes of both MDPVs and GPVs were chosen for the design of specific primers after genetic comparison. Only three primers (GF1, MF1 and MGR1) were designed for the duplex PCR assay: GF1 is specific for GPV only; MF1 is specific for MDPV only; and MGR1 is highly conserved for both MDPV and GPV. After a series of optimization experiments, the duplex PCR assay amplified a 161-bp fragment specifically for GPV, a 1197-bp fragment specifically for MDPV, and two fragments (161-bp and 1197-bp) for both GPV and MDPV. The lowest detection limit was 103 copies/µl. No amplification was obtained using nucleic acids from other pathogens (including DAdV-A, DuCV, DEV, GHPV, R.A., E. coli., P.M. and S.S.) occurring in Muscovy ducks. Application of the duplex PCR assay in field samples showed that even one-day-old Muscovy ducklings were both MDPV-positive and GPV-positive. In conclusion, a duplex PCR assay for the simultaneous detection and differentiation of MDPV and GPV was established using only three highly specific primers. Our finding suggested that country-wide vaccination with MDPV and GPV vaccines in waterfowls are necessary.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Infecções por Parvoviridae/veterinária , Parvovirus/classificação , Doenças das Aves Domésticas/virologia , Proteínas não Estruturais Virais/genética , Animais , Diagnóstico Diferencial , Patos , Gansos , Limite de Detecção , Parvovirinae , Parvovirus/genética , Parvovirus/isolamento & purificação , Filogenia , Especificidade da EspécieRESUMO
Recently, infectious disease outbreaks characterized by swelling and hemorrhagic liver and kidneys occurred in Muscovy ducklings in China. Four viruses were isolated and identified as adenoviruses by transmission electron microscopy (TEM) and polymerase chain reaction (PCR). Sequence analysis identified the new isolates as duck adenovirus 3 (DAdV-3), species Duck aviadenovirus B. The pathogenicity of the new isolate DAdV-3 FJGT01 was investigated using challenge experiments. The gross lesions in the animal experiment were similar to the clinical lesions observed in the diseased ducks. TEM examination of liver sample showed that virions accumulated and arranged in crystal lattice formations in the nuclei of hepatocytes. The present study provides new information about the epidemiology and characteristics of duck adenovirus associated with Muscovy ducklings.
Assuntos
Infecções por Adenoviridae/veterinária , Aviadenovirus/isolamento & purificação , Patos/virologia , Doenças das Aves Domésticas/virologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/virologia , Animais , Aviadenovirus/classificação , Aviadenovirus/genética , Aviadenovirus/patogenicidade , Fígado/patologia , Fígado/virologia , Filogenia , Doenças das Aves Domésticas/patologia , VirulênciaRESUMO
Duck hepatitis A virus type 1 (DHAV-1) causes acute hepatitis with high morbidity and mortality in ducklings of the genera Cairina and Anas and is characterized by ecchymotic haemorrhage and necrosis of the liver surface. Since September 2011, a new subtype of DHAV-1 (named pancreatitis-type DHAV-1) has been isolated. This new subtype is characterized by yellowish or haemorrhagic pancreatitis, but with no significant pathological changes in the liver. To further investigate the difference in pathogenicity between hepatitis-type DHAV-1 and pancreatitis-type DHAV-1, we infected Muscovy ducklings with a hepatitis-type DHAV-1 strain, FZ86, or a pancreatitis-type DHAV-1 strain, MPZJ1206, and then compared the resulting gross lesions, histopathological changes, viral distribution and cellular apoptosis in the liver and pancreas of Muscovy ducklings. The results suggested that FZ86 induced a more efficient viral propagation in the liver than MPZJ1206, and the gross and histopathological lesions were also limited to the liver. However, MPZJ1206 induced more effective viral replication in the pancreas than FZ86. The MPZJ1206-infected Muscovy ducklings showed an obviously yellowed and haemorrhagic pancreas, but with no significant pathological changes in the liver. Furthermore, FZ86 induced notable hepatocyte apoptosis and increased the expression of caspase-3 in the liver, whereas MPZJ1206 caused apoptosis in a large number of acinar epithelial cells and elevated the expression of caspase-3 in the pancreas. Taken together, these results demonstrated that pancreatitis-type DHAV-1 has many new pathogenic features which distinguish it from the hepatitis-type DHAV-1. RESEARCH HIGHLIGHTS Pancreatitis-type DHAV-1 (MPZJ1206) was characterized by pancreatic haemorrhage and yellow discolouration, but with no obvious haemorrhage and necrosis in the liver. Pancreatitis-type DHAV-1 (MPZJ1206) exhibits many new pathogenic features which distinguish it from the hepatitis-type DHAV-1 (FZ86).
Assuntos
Patos , Vírus da Hepatite do Pato/patogenicidade , Hepatite Viral Animal/virologia , Pancreatite Necrosante Aguda/veterinária , Infecções por Picornaviridae/veterinária , Doenças das Aves Domésticas/virologia , Animais , Vírus da Hepatite do Pato/classificação , Hepatite Viral Animal/patologia , Fígado/patologia , Pâncreas/patologia , Pancreatite Necrosante Aguda/patologia , Pancreatite Necrosante Aguda/virologia , Infecções por Picornaviridae/patologia , Infecções por Picornaviridae/virologia , Doenças das Aves Domésticas/patologiaRESUMO
BACKGROUND: Classic goose parvovirus (cGPV) causes high mortality and morbidity in goslings and Muscovy ducklings. Novel GPV (N-GPV) causes short beak and dwarfism syndrome (SBDS) in Cherry Valley ducks, Pekin ducks and Mule ducks. Both cGPV and N-GPV have relatively strict host specificity, with obvious differences in pathogenicity. Specific detection of cGPV and N-GPV may result in false positives due to high nucleotide similarity with Muscovy duck parvovirus (MDPV). The aim of this study was to develop a highly specific, sensitive, and reliable TaqMan real-time PCR (TaqMan qPCR) assay for facilitating the molecular detection of cGPV and N-GPV. RESULTS: After genetic comparison, the specific conserved region (located on the NS gene) of cGPV and N-GPV was selected for primer and probe design. The selected regions were significantly different from MDPV. Through a series of optimization experiments, the limit of detection was 50.2 copies/µl. The assay was highly specific for the detection of cGPV and N-GPV and no cross-reactivity was observed with E. coli., P.M., R.A., S.S., MDPV, N-MDPV, DAdV-A, DEV, GHPV, DHAV-1, DHAV-3, ATmV, AIV, MDRV and N-DRV. The assay was reproducible with an intra-assay and inter-assay variability of less than 2.37%. Combined with host specificity, the developed TaqMan qPCR can be used for cGPV and N-GPV in differential diagnoses. The frequency of cGPV in Muscovy duckling and goslings was determined to be 12 to 44%, while N-GPV frequency in Mule ducks and Cherry Valley ducks was 36 to 56%. Additionally, fluorescence-positive signals can be found in Mule duck embryos and newly hatched Mule ducklings. These findings provide evidence of possible vertical transmission of N-GPV from breeding Mule ducks to ducklings. CONCLUSIONS: We established a quantitative platform for epidemiological investigations and pathogenesis studies of cGPV and N-GPV DNA that was highly sensitive, specific, and reproducible. N-GPV and cGPV infections can be distinguished based on host specificity.