Complete genome sequence of Frog virus 3, isolated from a strawberry poison frog (Oophaga pumilio) imported from Nicaragua into the Netherlands

Frog virus 3 was isolated from a strawberry poison frog (Oophaga pumilio) imported from Nicaragua via Germany to the Netherlands, and its complete genome sequence was determined. Frog virus 3 isolate Op/2015/Netherlands/UU3150324001 is 107,183 bp long and has a nucleotide similarity of 98.26% to the reference Frog virus 3 isolate

Development and validation of a two-step real-time RT-PCR for the detection of eel virus European X in European eel, Anguilla anguilla

AbstractEel virus European X (EVEX) is one of the most common pathogenic viruses in farmed and wild European eel (Anguilla anguilla) in the Netherlands. The virus causes a hemorrhagic disease resulting in increased mortality rates. Cell culture and antibody-based detection of EVEX are laborious and time consuming. Therefore, a two-step real-time reverse transcriptase (RT-)PCR assay was developed for rapid detection of EVEX. Primers and probe for the assay were designed based on a sequence of the RNA polymerase or L gene of EVEX. The real-time RT-PCR assay was validated both for use with SYBR Green chemistry and for use with a TaqMan probe. The assay is sensitive, specific, repeatable, efficient and has a high r2-value. The real-time RT-PCR assay was further evaluated by testing field samples of European eels from the Netherlands, which were positive or negative for EVEX by virus isolation followed by an indirect fluorescent antibody test. The real-time RT-PCR assay allows rapid, sensitive and specific laboratory detection of EVEX in RNA extracts from 10% eel organ suspensions and cell cultures with cytopathic effects, and is a valuable contribution to the diagnosis of viral diseases of eel

Identification and localization of the structural proteins of anguillid herpesvirus 1

Many of the known fish herpesviruses have important aquaculture species as their natural host, and may cause serious disease and mortality. Anguillid herpesvirus 1 (AngHV-1) causes a hemorrhagic disease in European eel, Anguilla anguilla. Despite their importance, fundamental molecular knowledge on fish herpesviruses is still limited. In this study we describe the identification and localization of the structural proteins of AngHV-1. Purified virions were fractionated into a capsid-tegument and an envelope fraction, and premature capsids were isolated from infected cells. Proteins were extracted by different methods and identified by mass spectrometry. A total of 40 structural proteins were identified, of which 7 could be assigned to the capsid, 11 to the envelope, and 22 to the tegument. The identification and localization of these proteins allowed functional predictions. Our findings include the identification of the putative capsid triplex protein 1, the predominant tegument protein, and the major antigenic envelope proteins. Eighteen of the 40 AngHV-1 structural proteins had sequence homologues in related Cyprinid herpesvirus 3 (CyHV-3). Conservation of fish herpesvirus structural genes seemed to be high for the capsid proteins, limited for the tegument proteins, and low for the envelope proteins. The identification and localization of the structural proteins of AngHV-1 in this study adds to the fundamental knowledge of members of the Alloherpesviridae family, especially of the Cyprinivirus genus

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10(-5) on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system

Proteomics and peptidomics in the search for (early) biomarkers of asthma

Occupational asthma (OA) is the most common work-related lung disease in the industrialized world and is characterized by reversible airway obstruction, non-specific airway hyperreactivity and airway inflammation. In 90% of the cases, a complaints-free latency period precedes the development of immunologically mediated OA resulting in late diagnosis of these workers. Proteomics and peptidomics techniques could help to find (new) proteins or peptides related to the pathophysiology and onset of OA, leading to novel biomarkers to identify sensitized workers. So far, theuse of these techniques in pulmonary research is limited. The research unit of Lung Toxicology has described a well-characterized and validated mouse model of chemical-induced asthma. Two dermal sensitizations and a single airway challenge with a chemical sensitizer, such as toluene diisocyanate resulted in several phenotypical characteristics of chemical-induced asthma, including lymphocyte proliferation and activation, non-specific airway hyperreactivity and neutrophilic lung inflammation. In this doctoral thesis we focused on:a) applying a proteomics approach to find biomarkers of sensitization caused by chemicals and biomarkers of chemical-induced asthma (Chapter 2 and 3)b) investigating the role of B lymphocytes during sensitization (Chapter 4)c) optimizing a label-free peptidomics approach to study peptide biomarkers (Chapter 5) We used two dimensional difference gel electrophoresis (2D-DIGE) to search for possible biomarkers of sensitization in auricular lymph nodes and serum of mice that were sensitized once andtwice. In the auricular lymph nodes, a large portion of the differentially expressed proteins are structural proteins (e.g. actin, vimentin), while another substantial group are immune related proteins (e.g. lymphocyte specific protein-1, coronin 1a). Most of the identified proteins in serum, after one or two sensitizations are involved in binding/transport (e.g. hemopexin, apolipoprotein E). Three potential biomarkers, lymphocyte specific protein-1, coronin 1a and hemopexin, were verified in an independent experiment by Western blot analysis or ELISA. In a next step, we aim to validate this set of proteins in TDI-exposed workerswith the potential of advancing early diagnosis of OA. To investigate possible biomarkers of disease (chemical-induced asthma), we compared the proteome changes in auricular lymph nodes, bronchoalveolar lavage (BAL) and serum of mice after the establishment of asthma. A majority of differentially expressed proteins (e.g. lymphocyte specific protein-1, vitamin D binding protein, apolipoprotein E, hemopexin and haptoglobin) could be directly linked to inflammation (both locally in the lung as systemically in serum), a hallmark of asthma. For the first time,a systematic proteomics approach was implemented using different samples obtained from the same mice in the setting of chemical-induced asthma.The role of B lymphocytes in the development of chemical-induced asthma is still unclear. Recently, it was shown that B lymphocytes from sensitized mice could carry a sensitized signature compared to B lymphocytes of naïve mice. We isolated B lymphocytes from sensitized and non-sensitized mice and studied this sensitized signature by using 2D-DIGE. The altered expression of cyclophilin A, cofilin 1 and zinc finger containing CCHC domain protein 11 in sensitized B lymphocytes could be linked directly to the function of B lymphocytes as initiators of T lymphocyte independent asthma-like responses. The low molecular weight proteome ( In conclusion, the introduction of proteomics and (in a lesser extent) peptidomics in a mouse model of chemical-induced asthma has led to the discovery of several potential biomarkers of sensitization and disease, encouraging further validation of these results in humans.status: publishe

Biomarker discovery in asthma and COPD: Application of proteomics techniques in human and mice – mini review

The use of advanced proteomics approaches in the search for biomarkers in chronic lung diseases, such as asthma and COPD, is rather limited. Asthma and COPD are complex disorders, which can be subdivided into several phenotypes. This results in a heterogeneity of differential expressed biological molecules. Furthermore, genetic differences between animals and humans make ‘translation’ of possible biomarkers challenging. Yet, the improved sensitivity and high throughput of proteomic techniques could be an important asset for(new) protein biomarker discovery in either human or animal models. We have reviewed the literature that reported the use of different proteomics approaches performed on samples obtained from humans and murine models in asthma and COPD research for the discovery of new biomarkers of diseases, biomarkers of sensitization or for the refinement of treatment. There is an increasing trend in the use of proteomics to explore new biomarkers of asthma or COPD. Although several murine models have been developed to study these lung diseases, and proteomics studies have been performed, ‘translation’ of identified candidate biomarkers into clinical studies is often lacking.status: publishe

Not All Mouse Strains Respond Equal in a Model of Chemical-Induced Asthma

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