The need to manage large amounts of data is a clear demand for laboratories nowadays. The use of Laboratory Information Management Systems (LIMS) to achieve this is growing each day. A LIMS is a complex computational system used to manage laboratory data with emphasis in quality assurance. Several LIMS are available currently. However, most of them have proprietary code and are commercialized with a high cost. Moreover, due to its complexity, LIMS are usually designed to comply with the needs of one kind of laboratory, making it very difficult to reuse a LIMS. In this work we describe the Sistema Integrado de Gerência de Laboratórios (SIGLa), an open source LIMS with a new approach designed to allow it to adapt its activities and processes to various types of laboratories.SIGLa incorporates a workflow management system, making it possible to create and manage customized workflows. For each new laboratory a workflow is defined with its activities, rules and procedures. During the execution, for each workflow created, the values of attributes defined in a XPDL file (which describe the workflow) are stored in SIGLa’s database, allowing then to be managed and retrieved upon request. These characteristics increase system’s flexibility and extend its usability to include the needs of multiple types of laboratories. To construct the main functionalities of SIGLa a workflow of a proteomic laboratory was first defined. To validate the SIGLa capability of adapting to multiples laboratories, on this paper we study theprocess and the needs of a microarray laboratory and define its workflow. This workflow has been defined in a period of about two weeks, showing the efficiency and flexibility of the tool.Using SIGLa it has been possible to construct a microarray LIMS in a few days illustrating the flexibility and power of the method proposed. With SIGLa’s development we hope to contribute positively to the area of management of complex data in laboratory by managing its large amounts of data, guaranteeing the consistence of the data and increasing the laboratory productivity. We also hope to make possible to laboratories with little resources to afford a high level system for complex data management. The advances in technologies present in biomedical research resulted in a large amount of data being generated by research, testing and commercial laboratories. With such large quantities of data, it becomes very difficult to control the quality of the processes and results generated. In order to address these issues the concept of a Laboratory Information Management Systems (LIMS) has been developed. LIMS are complex computational system used by a laboratory to manage its data. LIMS emphasizes quality assurance and aim to generate results in a consistent and trustworthy way . They alIn this work we describe the Sistema Integrado de Gerência de Laboratórios (SIGLa), an open source LIMS with a new approach designed to allow it to adapt its activities and processes to various types of laboratory. To make SIGLa adaptable we use a workflow management system incorporated to the system. In SIGLa, for each new laboratory a new workflow will be defined with its activities, rules and procedures. After that, a file with the workflow’s definitions will be loaded in the system, in order to allow SIGLa to manage the activities of the laboratory. The definition of a new workflow is simple enough to be executed by a user knowing all the procedures of the laboratory, but without any prior programming knowledge.http://luar.dcc.ufmg.br/sigla in the link SIGLa with the login guest and password guest. SIGLa has diverse functionalities that guarantee the quality of laboratory’s data and allow great flexibility in the construction of the workflow. With the development of SIGLa we hope to contribute positively to the area of management of complex laboratory data.The first workflow defined in SIGLA describes the activities of the proteomics’ laboratory of a UFMG Biochemistry laboratory . This woDue to the complexity of LIMS, usually they are developed by large companies. As examples of commercial LIMS we have SQL LIMS , LabSoftThere exist some LIMS that also incorporate diverse concepts of workflows, such as  and 12]12] that A workflow can be defined as the steps and tasks executed sequentially according to a set of rules and procedures in order to conclude a process. A workflow can be a sequential progression of activities,or a complex set of processes occurring concurrently and eventually impacting in others, according to a set of rules . A workfCurrently there are many workflow management systems, commercial and open-source. Some examples of these are the Enhydra Shark , ObjectWSIGLa is a LIMS focused on the workflow of laboratory activities. It guides the user through the execution of each activity, informing the next activity/activities that can be executed. For each activity SIGLa stores its attributes as an eletronic notebook. In just one view the user can visualize all the activities executed in a experiment. The details of each activity can be accessed by just clicking on the activity. SIGLa adapts its interface to each laboratory as it is capable of managing workflows defined in the XPDL standard. This is possible because SIGLa uses a workflow management system to control which activities have been executed and which ones are available for execution.It is an application with an easy to use interface that is easily adaptable to different types of laboratories, in contrast to most LIMS that support a single type of laboratory. To store and manage data on laboratory activities SIGLa uses workflows. In workflow based systems users define activities, transitions, actors and rules of transitions. In SIGLa it is possible (through the Together Workflow Editor) also to define further details for each entity. For example, during the laboratory workflow definition, the user can define the attributes of each activity, its types, the range of values that each attribute can assume, its formats or define auto-calculated attributes from other attributes. It’s also possible to define the inputs and outputs of each activity, to define the number of inputs and outputs, as well as the relation of these inputs and outputs with the experiments. In the workflow definition it is also possible to assign to each activity a documentation that contains standards, instruments calibration, procedures and registers associates to the activities.It’s important to notice that to successfully manage a laboratory it’s necessary to create a well defined workflow. It must contain all the experiments with its attributes, inputs and outputs clearly declared with its types, formats and specified sizes. The definition of workflow is a very important step in the process of quality assurance given by SIGLa. Once the activities, rules and procedures have been defined, the workflow editor generates a XPDL file with all the definitions. This file is loaded in SIGLa, then the LIMS will be ready to manage the laboratory activities. With this mechanism, practically any type of laboratory can define its activities and rules in an XPDL file and use SIGLa to manage all the laboratory information. For SIGLa’s initial development the workflow of a proteomics laboratory was defined . ProteomAfter defining the proteomic workflow and implementing the main functionalities of SIGLa we have defined a second workflow to validate the capability of SIGLa of adapting to multiples laboratories. With the support of UFMG Microarray Laboratory we have defined the microarray workflow. The technique of DNA microarrays is used to study gene expression on a large scale in several species. DNA microarrays are usually layers of glass, plastic or nylon which is deposited series of thousands of microscopic spots of oligonucleotides or cDNAs, each containing picomoles of a specific DNA sequence. The microarray slide is then used to detected expression level of mRNA related to the DNA printed on its surface by incubatiing of the microarray with a solution containing cDNA or RNA obtained from biological samples. .Microarray technique generates a large number of information, both laboratory data and image and data files. The microarray process has several steps, requiring the storage of information for each one of them. Usually this information is stored in lab books, which makes it difficult to access the information, since they are in chronological order only. After scanning microarray slides and image analysis, new and large image data and data files are produced and the number could reach hundreds of files. The organization of these data using laboratory notebooks or basic text files becomes very time-consuming. By using a single platform as SIGLa for this task is possible to keep all data organized making its manipulation more reliable. Moreover, the use of a web based platform such as SIGLas provides access to data for all members of a research group in a fast and efficient way.The current version of SIGLa is able to manage all the activities of a workflow and their attributes. An important feature that is not currently available, however, is the ability to call an external program to perform an automatic analysis such as gene sequence annotation. In this case, the analysis has to be performed externally. Its result, however, can be stored in SIGLa as an attribute of type file, and can be used later in the workflow. Automatic analysis execution will be available in the next version of SIGLa.not null, meaning that the value of the attribute must be always filled. It’s also possible to define the format of an attribute. The file type permits the association of files to an activity. For example, it’s possible o associate to the execution of the activity Data Analysis a file containing the annotation of the gene sequences in the microarray to be analyzed by another application. During the workflow definition we have also defined the inputs and outputs of the activities. Each activity generates outputs that will be inputs to the next activity. For example, in the case of RNA Extraction it generates the extracted RNA as output. In this case it is necessary to store the RNA total concentration, the RNA final volume, the Gel image and the dosage method. All these attributes are defined in the RNA Extraction activity, in the microarray workflow. After the RNA Extraction it’s possible to execute a Cleaning, an Amplification or a Labeling. In the workflow an input to these three activities was defined. In this way, the output of the RNA Extraction will be the input to the Cleaning, Amplification or Labeling activity. It’s also possible to set a property that can define the maximum and minimum number of each input and output of the activities.To implement the microarray process into SIGLa we have defined it’s workflow figure . On thisTo organize laboratory’s data, SIGLa has the following structure: A project has experiments where samples are analyzed. To begin a workflow execution the first step is to create a project and it’s associated experiments. Figure To execute an activity the user just needs to click in the correspondent gray box. Clicking on it, SIGLa will open an interface where the user can fill all the attributes that were defined in the workflow definition for this specific activity. When the user completes the activity, SIGLa checks the properties of each attribute defined in the workflow definition. If some attribute value is inconsistent, SIGLa presents an error message Figure . This avSIGLa controls the selection of specific inputs and outputs for each experiment registered in the system. When the activity is defined to have outputs,after the execution of the activity, SIGLa opens a window on the interface for inserting the outputs. As in the activity execution, in the output insertion SIGLa checks the properties of each attribute and makes the validation, guaranteeing data quality. When a activity is defined to have inputs resulting from previous activities, before the execution of the activity SIGLa opens an window on the interface for selecting the input(s). On this screen SIGLa validates the number of inputs that have been selected. Once the activity inputs and outputs were defined for an activity the information about them can be accessed by the link inputs and outputs shown in the figure SIGLa uses JAVA as programming language and has a basic architeture Figure . The WebOnce the microarray workflow has been loaded into SIGLa, a specialist on microarray executed several experiments to validate the system. The system was tested with real data from a microarray experiment with 12 initial samples of RNA and six microarray slides. All steps of the workflow have been completed for all 12 samples. The system was robust and easy to use during the test. The ability to see in one screen which parts have been executed and which ones have not made the process much simpler. After completing all the steps, the specialist could verify its data for any stage. This access to all information of an experiment is very useful when working with a technique complex and full of steps as microarray analysis. In addition, the system stores protocols and files generated during the execution of steps, such as pictures of gels or data files, making it a good tool for storing data in an experimental laboratory for the implementation and analysis of microarrays. SIGLa helped also with other important features such as allowing a choice between two or more possible options in a field, as the choice of fluorescent dyes Cy3 and Cy5, which facilitates the work of researchers when they are filling these fields. Moreover, it was possible to generate reports in PDF format with all the information of the experiment (Long and Short Report), which can be easily viewed and printed. The attribute values validation made by SIGLa was also very useful. In many moments it avoided filling the fields with wrong values. In addition, testing with real data allowed the specialist to make suggestions to improve the system, which will be analyzed and insert in the original design.It is important to notice that the period of time to fully create a complete microarray LIMS has been about two weeks. This illustrates that SIGLa is able to adapt itself to multiple laboratories in very little time. The process consists of defining the workflow in a graphical editor, and SIGLa automatically creates all the data structures needed to manage the laboratory. Any modification in the protocols, or the addition of new experiments takes only a few days making it possible to manage not only large quantities of data, but also different types of data efficiently.The need for fast and reliable data storage and management for biological laboratories is a reality nowadays. This need has been fulfillled only partially by available sistems given high costs or limitations of the available LIMS. In this work we present SIGLa, a system based on adaptable workflows, with an easy to use interface, that manages and guarantees the quality and integrity of laboratory data. Moreover SIGLa is not a solution designed for only one type of laboratory but several types, since the user can easily adapt it to the needs of his/her laboratory, simply by defining its workflow. On this paper we study the process and the needs of a microarray laboratory and define its workflow. This workflow has been used by researchers in real microarray experiments and they have reported that the system indeed has been very useful. With SIGLa’s development we hope to contribute positively to the area of management of complex data in laboratory by managing its large amounts of data, guaranteeing the consistence of the data and increasing the laboratory productivity. We also hope to make possible to laboratories with little resources to afford a high level system for complex data management.Project name: Sistema Integrado de Gerenciamento de Laboratórios (SIGLa)• http://www.luar.dcc.ufmg.br/siglaProject home page: • Operating system(s): Platform independent• Programming language: Java• Other requirements: Java 1.5.0 or higher• Any restrictions to use by non-academics: licence needed• The authors declare that they have no competing interests.On this work the decisions of implementation were made by AM, AFC and SC. AM, RK and VA wrote the java code. DDL and AFC gave the biological concepts for the implementation.


Quercusalbus or Gorilla gorilla beringei, everyone recognizes we are speaking about white oak trees or mountain gorillas, respectively, and not northern red oak trees (Quercusrubra) or lowland gorillas (Gorilla gorillagorilla). However, taxonomy has not been, and still is not, without difficulties. Very early on, it was recognized that universal “codes” needed to be developed to guide the naming of biologic entities so that each name would be as unique and distinct as the object being named.Taxonomy, the science of identifying and naming entities, has long been an integral component of biologic sciences, both in botany and zoology. Not all biologists are actively engaged in taxonomy (only a few truly enjoy working in this field), yet everyone, even lay persons, recognizes the value of consistency and standardization in the naming of animate and inanimate objects. The naming of biologic entities is an exact tool that conveys a precise meaning and ensures maximal continuity and universality for present and future generations. It further confirms that when reference is given to a scientific name, such as  Virologists seem to be struggling with taxonomy more than scientists in other disciplines. Much of this struggle seems self-induced, and the article by van Regenmortel and Mahy, “Emerging Issues in Virus Taxonomy”The authors do continue to chip away at several fundamental issues, and they are to be commended for that. For instance, the authors acknowledge that viruses are biologic entities, they advocate applying species names in virus taxonomy, and they recognize that use of a binomial naming system is preferred. That virologists also recognize the value of using a combination of characters to define a species is not novel but reflects that elements other than morphologic features, i.e., host and geographic distribution, vector requirements, and molecular sequences, contribute to defining a species. Although seemingly at the very core of taxonomy, agreement on such basic principles is a major step, when one considers that not all virologists subscribe to standard biologic principles. As the authors note, virus taxonomy is an emerging discipline that allows working virologists to communicate without misunderstanding. However, the issue is larger than that, and virologists need to know that they are not only working to solve a problem in virology, but they are also accountable to the larger biologic community so that we can communicate clearly and effectively across disciplines. Only with consistency and uniformity will this communication occur. The overall goal should be to provide consistency not only within the field of virology, but also and more importantly, across the broader field of biology. In a recent article by Ashford, the need for consistency in defining terms was noted, and the author stated, “When we all agree on what we are talking about, we will understand each other better”  This is where this article (and seemingly most efforts to date on virus taxonomy) falls short. Inconsistencies in virus taxonomy—some perpetuated in this article, some introduced in it, indicate that, unless more attention is paid to what has gone before, virus taxonomy will never achieve the respect it deserves. For instance, insistence on italicizing names above genus level is out of character with most other zoologic disciplines. Similarly, the idea that viruses are unique and need their own set of rules appears presumptuous. All biologic entities are unique: humans are unique, a particular bacterium is unique, as are specific parasites, plants, algae, and the like. Viruses are different and distinct but occupy a spot along a continuum in the bigger biologic spectrum. Prions may be even more problematic than viruses to characterize and name.The most perplexing proposal offered in this article is the placement of the genus name after the species name. Taxonomy, and the more specific aspect of nomenclature, is based on a traditional system of naming organisms beginning with the highest order (kingdom) and descending to lowest order (species). Why virologists would wish to be discordant with the rest of biologic science is unclear, and no immediate value to such a system is evident. Virologists also need not worry about having a universally acceptable definition of a species before applying rigorous rules of taxonomy. Every discipline, botanical and zoologic, is wrestling with a working definition of what constitutes a species. Virologists should also not fret over whether a species concept only applies to sexually reproducing organisms. Other biologists are not so encumbered, as the International Code for Zoological Nomenclature  Other troubling areas of current taxonomy of viruses include the concept that viruses are abstract entities. How comforting will it be to patients with serious diarrhea caused by Norwalk virus to learn that their illness is caused by an abstract entity? Viruses cause disease just as parasites and bacteria cause disease, and we do not consider them abstract. Because parasites or bacteria are different from viruses in composition, life cycle, and the like does not make a virus an abstract entity. Developing scientific names for viruses has also provided some humorous fodder for other taxonomists. Using English rather than latinized words for names appears capricious and is dismissive of centuries of distinguished scientists and pioneers in the field, including virology. Lastly, virologists’ concern about having to demarcate and coin new names for an estimated 1,550 virus species is puzzling. One wonders what their response would be to naming and cataloguing in other disciplines, such as entomology, where there are >1 million recognized species, some 10,000 new species described each year, and an estimated 4–6 million species yet to be discovered and named ( From the perspective of a nonviral taxonomist, virologists might do well not to reinvent the wheel  Drs. van Regenmortel and Mahy are to be commended for trying to bring virus taxonomy to a higher order of consistency. However, given the controversies, virus taxonomy may not get it right


JADAS consists of four items; the joint count, the physician and the patient’s/parent’s global assessment and the erythrocyte sedimentation rate (ESR) as an inflammatory marker. C-reactive protein (CRP) is often a more rapid and available test than ESR and has been suggested as an alternative inflammatory marker. The aim of the study was to validate and compare the CRP et al (1). Cut-off for CRP was <10mg/ml and for ESR <20mm/H. Spearman’s rank order correlation rho was used to evaluate the correlations between CRP and ESR, and the JADAS27 based on CRP and ESR. Logistic regression was used to test the first JADAS score available as a predictor of DMARD treatment, and whether mean JADAS score was associated to treatment with methotrexate and biologic agents in children with at least three JADAS scores available.We included 514 consecutive cases of JIA from defined geographical areas of Denmark, Finland, Sweden and Norway with disease onset in 1997 to 2000. Clinical data and disease activity measures were registered according to a set protocol at regular follow-up visits from six to 147 months after onset. To calculate the JADAS, CRP was “normalized” to a value in the range 0-10 in a similar method as ESR, as described by Consolaro versus JADAS27-ESR (r=0.98). Bland-Altman plot of JADAS27-CRP versus JADAS27-ESR showed a high level of agreement (mean difference of 0.046 (CI -0.012 to 0.104)). Higher score of the first available JADAS27-CRP was significantly associated to treatment with methotrexate  and synthetic or biologic DMARDs  during the eight years follow-up after disease onset. Mean JADAS27-CRP also showed a significant association to treatment with methotrexate  and synthetic or biologic DMARDs  during the eight years follow-up after disease onset.Of the 514 children 66% were girls, 51% had oligoarticular disease six months after onset and 3% were rheumatoid factor positive. The first visit when both CRP and ESR were taken was chosen for analysis. Correlation between corresponding CRP and ESR values from each visit was moderate (r= 0.57). There was a high correlation between JADAS27-CRP JADAS based on CRP showed a high correlation with JADAS based on ESR in a multi-center Nordic cohort of juvenile idiopathic arthritis, indicating that CRP can be used as an alternative to ESR in the composite tool. High mean JADAS score during disease course was significantly associated to DMARD treatment in a prospective, longitudinal, multi-center Nordic JIA cohort. Further analyses on responsiveness to change of the JADAS-CRP are planned.Ellen Nordal: None; Marek Zak: None; Lillemor Berntson: None; Kristiina Aalto: None; Suvi Peltonieli: None; Susan Nielsen: None; Troels Herlin: None; Bjørn Straume: None; Anders Fasth: None; Marite Rygg: None.


Dengue has emerged as the most important life-threatening illness in the world, especially in Asian countries around Hong Kong, where the incidence of dengue hemorrhagic fever (DHF) is much greater than other continents. However, little is known about molecular and cellular processes sustaining egress of dengue virus in the host cell.To better understand the viral and cellular determinants of dengue virus egress, we have established a dengue VLP producing stable cell line (HeLa-prME) and demonstrated that dengue VLP was able to mimic the budding and egress process of dengue viruses so that it constitutes a safe and convenient tool for the study of egress of dengue virus. Under the support of RFCID grant, HeLa-prME cells were used to screen a siRNA library that included 122 cellular membrane trafficking genes.Our screen results revealed that knockdown of ADP-ribosylation factor (ARF) 1 and ARF6 had significant effects on VLP production by HeLa-prME cell. Experiments with other ARF proteins, which were not included in the siRNA library, showed that the ARF4/ARF5 double knockdown could inhibit VLP production but had no effect on secretion of other proteins such as soluble dengue E protein, suggesting the specificity of their involvement in VLP production. Further experiments using real virus showed that the depletion of ARF4/5 by siRNA could significantly reduced the replication of dengue 1 virus, dengue 4 virus and yellow fever virus, confirming the important role of ARF4 and ARF5 for not only dengue viruses but also other flaviviruses.Our study uncovered the importance of class II ARFs in the egress of dengue virus. Results from this project provided information on mechanism of dengue virus assembly and its dependence on cellular machineries.


Treatment modalities of chronic plaque psoriasis have dramatically changed over the past ten years with a still continuing shift from inpatient to outpatient treatment. This development is mainly caused by outpatient availability of highly efficient and relatively well-tolerated systemic treatments, in particular BioLogicals. In addition, inpatient treatment is time- and cost-intense, conflicting with the actual burst of health expenses and with patient preferences. Nevertheless, inpatient treatment with dithranol and UV light still is a major mainstay of psoriasis treatment in Germany. The current study aims at comparing the total costs of inpatient treatment and outpatient follow-up to mere outpatient therapy with different modalities  over a period of 12 months. To this end, a retrospective cost-of-illness study was conducted on 120 patients treated at the University Medical Centre Mannheim between 2005 and 2006. Inpatient therapy caused significantly higher direct medical, indirect and total annual costs than outpatient treatment . Its strong influence on cost levels was confirmed by regression analysis, with total costs rising by 104.3% in case of inpatient treatment. Patients receiving BioLogicals produced the overall highest costs, whereas outpatient treatment with classic systemic antipsoriatic medications was less cost-intense than other alternatives.  Psoriasis is a chronic inflammatory disease affecting about 2% of the population in Europe and Northern America. In Germany, an estimated 1.6 million individuals suffer from this disease that implicates considerable physical, psychological and social burden for patients but also a major financial burden for health economy –6. In thGiven the rising expenses for health, economic analyses comparing the different treatment modalities are imperative. Such analyses performed in the Angloamerican context mostly concentrated on cost efficiency of different outpatient alternatives ,17–19, aThe present cost-of-illness analysis was carried out as a 12-month retrospective bottom-up study with a separate view of inpatient and outpatient therapy. Three hundred ninety-one patients treated for psoriasis between January 2005 and July 2006 either as inpatients or outpatients at the Department of Dermatology of the University Medical Centre Mannheim, University of Heidelberg, were contacted by mail in December 2006. Of 202 patients answering back (return rate: 51.7%), 149 gave written informed consent to participate. One hundred twenty patients were available for personal interviews and could be included in the study. The personal interview was conducted by one interviewer from February until July 2007 using a 17-page standardized questionnaire see , focussiThe study was conducted according to the ethics principles expressed in the Declaration of Helsinki and approved by the Medical Ethics Committee II of the University Medical Centre Mannheim, University of Heidelberg, Mannheim, Germany. All patients gave written informed consent for participation in the study.For statistical analysis the cohort (n=120 patients) was stratified into inpatients and outpatients as well as according to the most intense outpatient treatment modality, i.e. topical therapy, phototherapy , classic systemic therapy  or biological therapy . The majority of patients (68%) received a combination of two or more treatment modalities  and multivariate regression analysis. In the latter, logarithmic values for direct medical costs, costs for outpatient medication, costs for outpatient visits and diagnostics, indirect costs through loss of working time and total costs were taken as dependent variables. After logarithmic transformation Kolmogoroff-Smirnoff tests presented normal distribution of all cost variables , for phototherapy and systemic therapy  as well as for full time employment, part time employment and minor employment (reference group: not working) were incorporated. All independent variables were concomitantly integrated into the analysis.>37.5 hours per week) and 13.3% part time (19.5-37.5 hours per week) whereas 40% had no employment at all because of retirement (25.8%), unemployment  or homemaking . The vast majority had compulsory health insurance.Among the patients included into the study, 55% were male and 45% female . The meaNinety percent of the patients suffered from plaque psoriasis, and the mean disease duration was 22 years. Mean PASI scores were 13.5 for inpatients and 8.9 for mere outpatients. According to the classification criteria of the Committee for Medicinal Products for Human Use [t-test) and a higher impact on life quality as assessed by DLQI .When inpatients were compared to outpatients, the former had a longer disease duration with an earlier onset, more severe psoriasis . After demission from hospital former inpatients sought medical consultation for psoriasis somewhat more frequently than mere outpatients . Inpatients were absent from work for more than one month during the assessment period whereas mere outpatients only lost two working days due to psoriasis  required more than one hospital stay during the 12-month observation period leading to a mean total hospitalization time of 27.9 days. Eighty-four percent of inpatients were treated with dithranol and phototherapy. Mean patient-reported remission time after inpatient treatment was 104.3 days. Remarkably, despite intense topical and phototherapy during the hospital stay a high percentage of inpatients required phototherapy (55%), classic systemic therapy (35%) or biologicals (10%) after demission.Regarding the different treatment modalities prescribed in an outpatient setting, either for mere outpatients or as follow-up therapy after inpatient treatment, 93% of all patients were treated with topical therapy, including medical skin care products, 45% with phototherapy, 25% with classic systemic therapy and 6.7% with biologicals. Details of the different regimens are indicated in When costs were analyzed for the whole patient collective, mean total annual costs per patient added up to 7,092 €, including average direct medical costs of 4,978 €, direct non-medical costs of five hundred and ninety-eight € and indirect costs through loss of working time of 1,515 € . In the t-test). This was due to (a) higher total direct medical costs including costs for hospitalization  and (b) higher indirect costs through loss of working time . Unexpectedly, however, costs for outpatient medication in the time following hospitalization were also significantly higher for former inpatients than for mere outpatients , leading to more than twofold higher direct medical costs for ambulatory treatment .For further cost analysis and comparison the cohort was stratified into inpatients and outpatients. Total annual costs for inpatients were more than as fourfold high as those for mere outpatients . Both in the inpatient and in the outpatient group, classic systemic therapy produced significantly higher direct medical costs in the outpatient sector than topical or phototherapy . Furthermore, outpatients receiving phototherapy had higher indirect costs due to loss of working time than those receiving topical or systemic therapy . Patients received these therapies for an average of only four months within the assessment period. Today, there is a trend to long-term therapy with biologicals with presumably higher direct medical costs. Moreover, medication costs for most biologicals have been rising considerably more than the general inflation rate over the last years .Also according to other recent studies, total costs increased after introduction of biologicals, as the rise in medication costs could not fully outweigh possible decreases in costs for hospitalizations or outpatient admissions ,26,31,34In a German study comparing inpatient treatment to outpatient biological therapy with efalizumab, total annual costs for the patient group receiving efalizumab were more than threefold higher than for the inpatient group . HoweverFonia et al.  showed aIn a study by Ghatnekar et al.  biologicOnly in the American study of Bhosle et al. , which wOverall, introduction of biologicals into the treatment of psoriasis has led to a considerable raise in costs. However, patients are significantly more satisfied and compliant with biologicals than with other treatments . MoreoveOur study demonstrates impressively that costs for inpatient therapy exceed costs for all conventional outpatient alternatives by far. Remarkably, former inpatients also required high expenses for outpatient follow-up treatments, with 55% needing additional phototherapy, 35% classic systemic therapy and 10% biologicals. According to our data inpatient therapy may still be less expensive than biological therapy. However, inpatient treatment with dithranol and UV light cannot substitute for biological treatment, as the effect of inpatient treatment only had a considerably short duration of less than four months. As a consequence, several inpatients of our cohort were prescribed biologicals after demission. Our primary hypothesis that time- and cost-intense inpatient treatment might lead to long term remission of psoriasis and therefore to decreased follow-up costs had to be rejected.The enormous costs produced by inpatient therapy may in part be justified by the fact that inpatients were more severely affected than mere outpatients, with higher PASI scores and higher prevalence of psoriatic arthritis and comorbidities . It was Willingness of patients to be hospitalized is decreasing as effective and well-tolerated outpatient treatment options are becoming accessible. In a recent study analysing patient preferences for psoriasis treatments it was shown that patients attach great value to outpatient treatment . The treOur study has several limitations. As data were collected shortly after the introduction of biologicals into the German market, the subgroup of patients receiving biologicals was too small to explicitly compare costs for outpatient biological therapy to inpatient therapy with appropriate statistical methods. Our cost data stem from 2005/2006. Since then the price of etanercept slightly decreased by 2-3%, whereas the price for infliximab increased by 8-19%. In addition, some of the most efficient biologicals, i.e. adalimumab and ustekinumab, were not available at the time of data collection. Our study was conducted as a monocentric cost analysis, impairing generalization of our results. Moreover, direct comparison of inpatient and outpatient costs is hampered by the fact that inpatients were more severely affected than outpatients and presented higher PASI scores, higher prevalence of psoriatic arthritis and higher rates of comorbidities. The well-known human capital approach was used for measuring indirect costs, as it allowed the inclusion of the societal value of honorary activities. With regard to the high proportion of unemployment within our study collective (40%) that approach seemed to estimate indirect costs in the most appropriate way. Nevertheless this approach tends to overestimate opportunity costs in the long run, why a supplement by e.g. a friction cost approach could have been reasonable.A major strength of our study is that it provides the first all-encompassing cost analysis including all available treatment modalities. Our analysis reflects the real-world cost situation, in which prescription of combination regimens is frequent. Costs have been collected not only by chart review, allowing detailed calculation of direct medical costs, but also in personal interviews, which provided precise information on individual costs and indirect costs. Our study shows that the costs of inpatient therapy are significantly higher than those of outpatient treatment. Inpatient treatment did not result in decreased follow-up costs, as the duration of remission was relatively short and several patients required systemic treatment after demission. Patients receiving biologicals produced the overall highest costs. Topical treatment, phototherapy or classic systemic medications were significantly less costly when prescribed in an outpatient setting, as expected. Mere outpatient treatment with classic systemic anti-psoriatic medications turned out to be less cost-intense than phototherapy. Despite high requirements for laboratory testing and monitoring for adverse effects, this option is attractive from the economic perspective.Questionnaire S1Cost-of-illness in Psoriasis .(PDF)Click here for additional data file.Table S1Costs are normally distributed when using logarithmic values .(DOC)Click here for additional data file.Table S2Comparison to other German cost-of-illness studies.(DOC)Click here for additional data file.


Mononegavirales. In the combined analysis of 63 representative nucleoprotein (N) sequences from four viral families  we predict the regions of protein disorder, intra-residue contact and co-evolving residues. Correlations between location and conservation of predicted regions illustrate a strong division between families while high- lighting conservation within individual families. These results suggest the conserved regions among the nucleoproteins, specifically within Rhabdoviridae and Paramyxoviradae, but also generally among all members of the order, reflect an evolutionary advantage in maintaining these sites for the viral nucleoprotein as part of the transcription/replication machinery. Results indicate conservation for disorder in the C-terminus region of the representative proteins that is important for interacting with the phosphoprotein and the large subunit polymerase during transcription and replication. Additionally, the C-terminus region of the protein preceding the disordered region, is predicted to be important for interacting with the encapsidated genome. Portions of the N-terminus are responsible for N∶N stability and interactions identified by the presence or lack of co-evolving intra-protein contact predictions. The validation of these prediction results by current structural information illustrates the benefits of the Disorder, Intra-residue contact and Compensatory mutation Correlator (DisICC) pipeline as a method for quickly characterizing proteins and providing the most likely residues and regions necessary to target for disruption in viruses that have little structural information available.The goal of this Bioinformatic study is to investigate sequence conservation in relation to evolutionary function/structure of the nucleoprotein of the order  Mononegavirales, in their list of Bioterrorism Agents/Diseases, however, structural knowledge of these agents is limited. Mononegavirales is composed of four viral families: Bornaviridae contains the Borna Disease Virus (BDV), which affects the nervous system and the brain in many animals, including cows and rats, and endogenous borna-like nucleoprotein elements sequences exist within the human genome Paramyxoviridae includes Sendai Virus (SENV), which typically affects rats and mice, and two viruses that cause childhood epidemics, Measles Virus (MeV) and Mumps Virus (MuV). Filoviridae has only two members, Ebolavirus and Marburgvirus that cause hemorrhagic fevers with mortality rates up to 90% in humans Rhabdoviridae contains Rabies Virus (RABV) and Vesicular Stomatitis viruses, which are both able to pass from their animal hosts to cause disease in humans, as do many Mononegavirales. Vesicular Stomatitis virus (VSV) is the model for the Rhabdoviridae family, and the prototype for most of the investigation of transcription and replication for the entire order of MononegaviralesHuman Immunodeficiency Virus and influenza The Centers for Disease Control and Prevention have included the Ebola and Marburg viruses, both negative-strand RNA viruses belonging to the order Mononegavirales, the RdRp complex is composed of the negative-sense RNA genome and three proteins: nucleoprotein (N), phosphoprotein (P) and the large subunit polymerase protein (L). The RNA genome of this complex is always found associated with the nucleoprotein as the RNP. This structure is resistant to nucleases, even during synthesis Negative-strand RNA viruses are unique in that their RNA genomes are always encapsidated by a viral coded nucleoprotein to form a ribonucleoprotein (RNP) complex. This complex serves as the template for viral RNA synthesis and forms the structural core of the viruses when packaged into virions Respiratory Syncytial Virus (RSV). The VSV crystal was isolated with a 90-nucleotide strand of RNA associated with 10 copies of the nucleoprotein forming a truncated RNP in the shape of a cylinder/ring The nucleoprotein plays a critical role by polymerizing to cover the entire length of the genome, thereby protecting it from ribonuclease digestion The current proposed mechanism for VSV RNA synthesis suggests that a portion of the nucleoprotein temporarily dissociates from the RNA allowing the polymerase access to the genome. This is supported by the crystal structure of the nucleoprotein from VSV that shows the neighboring lobe interactions provide more stability than the positively charged residues of the RNA binding cavity The use of Bioinformatic methods has been implemented to produce models of the individual intra-protein contacts and disorder for the nucleoprotein in the study presented here. The results of protein disorder prediction, correlated mutations, sequence conservation, and intra-residue prediction methods have been correlated to characterize the nucleoproteins based on the data these approaches generate from the protein sequence information. The purpose of evaluating the regions of disorder within a protein is that such areas are observed to be binding sites for protein-ligand interactions. Upon association with the partner ligand the protein assumes a secondary structure as observed using x-ray crystallography Filoviruses, which was different from the most recent tree created using portions of the polymerase Filoviridae, Clade II contains Paramyxoviridae and Clade III is Rhabdoviridae; all clades show posterior probabilities (PP) of 1.To explore the relationship of the evolution of the nucleoprotein within the viral families and among the entire order a phylogenetic reconstruction was implemented. The multiple alignment of all 63 N sequences was generated by manual curation of a MAFFT alignment Filoviruses group with each other and Lake Victoria Marburgvirus (MARV) branches from the Ebolaviruses at a PP of 1.Examination of Clade I reveals that BDV clades with Filoviridae at a PP of 0.98. The Paramyxoviridae branching into the subfamilies Paramyxovirinae and Pneumovirinae  sits outside the human viruses with a PP of 1. The Paramyxovirinae subfamily branches into two subclades. The first contains the Rubulavirus, Avulaviruses with the unclassified Tioman Virus (TIOV). The Rubulaviruses and Avulaviruses relationships are highly supported by PP of 1 throughout their topology. TIOV groups within the Rubulaviruses. The second is made up of Respirovirus, Henipaviruses, Morbilliviruses and the five unclassified viruses: Fer-de-lance Virus (FDLV), Tupaia Virus (TUPV), Mossman Virus (MOSV), Beilong Virus (BEIV), and JV with a PP of 1. FDLV is an outgroup to the Henipaviruses and Morbilliviruses at a PP of 0.81. Both MOSV and TUPV group with Henipaviruses with PPs of 0.86 respectively. With a low PP of 0.53, BEIV and J Virus (JV) form their own group outside the Morbillivirues. The Morbilliviruses and Respiroviruses resolve relationships with PPs from 0.8–1.0.Clade II shows ovirinae . Within Rhabdoviridae in Clade III reveals high PPs across all genera. Within Clade III there are two subclades. The first subclade is composed of the Ephemroviruses, Vesiculoviruses and Lyssaviruses. The currently unassigned Flanders Virus (FLAV) branches with Bovine Ephemeral Fever Virus (BEFV) with a PP of 1 suggesting it belongs to the Ephemeroviruses. Siniperca Chuatsi Rhabdovirus (SCRV) groups between the Ephemeroviruses and the other Vesiculosviruses with a PP of 0.99. Lyssaviruses are an outgroup to the Ephemeroviruses and Vesiculoviruses with a PP of 1.0. The second subclade contains the Cytorhabdoviruses, Nucleorhabdoviruses and the Novirhabdovirues. The Novirhabdoviruses are an outgroup to the plant viruses Cytorhabdoviruses and Nucleorhabdoviruses at a PP of 0.96.Examination of the Bornaviridae sequence displays four regions of disorder with the largest being in the N and C-terminals  Vesicular Stomatitis Indiana Virus (VSIV) N∶RNA & P complex (pdb id – 3HHZ) For a more specific look at the nucleoprotein interaction with the phosphoprotein a recent crystal structure of the Filoviridae are closer to each other than they are to Rhabdoviridae or Paramyxoviridae , which indicate they are closer to one another than to any other sequence in the study. This result is consistent between BEASTv1.5.4 and MrBayes3.1 analyses.The expected . The relParamyxovirinae subfamily reveals some clear relationships of the previously unclassified viruses. Menangle Virus (MENV) and the unclassified TIOV branch together within the Rubulavirus. The association of MENV with the Rubulaviruses is supported by earlier molecular characterization and phylogenetic analysis Henipaviruses and Morbilliviruses. Previous results agree with this observation as the nucleoprotein gene FDLV was shown to branch between the Henipaviruses, Rubulaviruses and MorbillivirusesHenipaviruses and Morbilliviruses. The relationship of MOSV and TUPV grouping is supported by previous phylogenetic work and the results from this study agree with the previous N results Henipaviruses and Morbilliviruses is supported by previous phylogenetic analysis The phylogenetic reconstruction of the Mononegavirales viral nucleoproteins contain the largest portion of disordered residues (Paramyxoviruses but does correspond with being a disordered region (Paramyxoviruses (Newcastle Disease Virus (NCDV) the C-terminal region at residues, 376–489, appear to be unnecessary when it comes to forming an eleven-subunit ring of the nucleocapsid, suggesting this region functions separately from the formation of the N-RNA structure Paramyxoviridae in this region.Disordered or intrinsically unstructured proteins (IUPs) are able to exist without a defined secondary structure. It has been shown that these IUPs can assume a secondary structure after interacting with their binding ligand. Such regions of disorder within proteins are observed to be binding sites for proteins assuming a secondary structure that is observed under x-ray crystallography when in association with the partner ligand residues . This ild region . NCDV waoviruses . AdditioRhabdoviridae the trend is less neatly organized, as the divergence of these sequence is more than that observed in the other families, but still highlights the flexibility in the C-terminus. In addition to the C-terminal disorder observed in the other families, a region within the first 20 amino acids of the Rhabdoviridae sequences in the N-terminus is observed to contain disorder. In Lettuce Necrotic Yellow Virus (LNYV) this disordered region is larger than the corresponding disorder predictions of the other Rhabdoviruses, even the other Cytorhabdoviruses SCRV and Sonchus Yellow Net Virus (SYNV) (Orchid Fleck Virus (OFV) showed the closest match in size to this N-terminal disorder regions. OFV had been classified as a tentative Rhabdovirus, but has since been removed due to possessing a bipartite genome. OFV appears to go against the main trend of the other Rhabdoviruses and the viral order by displaying a large disordered region in the N-terminus (results not shown). As OFV is not in the family any longer these results are likely due to the existence of the OFV genome as bipartite negative-sense RNA that could require some further flexibility in function/structure compared to the non-segmented genomes. As LNYV is a single-stranded virus the similarity is either a coincidence or an undetermined link.In s (SYNV) . The regFiloviridae displays a longer region of disorder in the C-terminus compared to the other families    also disMononegavirales with the same disorder and CICP predictions highlights their regions of interaction.Based on the distribution of the large disordered regions of the C-terminus of the nucleoproteins being at the fringes of the nucleocapsid-ring complex  it can bRhabdoviridae and Paramyxoviridae, and more generally the other viruses in Mononegavirales, have similar functional/structural regions corresponding specifically to those regions showing conservation in disorder and co-evolution even though they may have weak amino acid sequence conservation. Specifically the C-terminal end of the nucleoprotein is predicated to be involved with binding to the phosphoprotein in a manner important to transcription/replication and not necessarily important to the formation of the nucleocapsid for every virus evaluated in this study. Also, it appears that evolution has constrained the function of some binding proteins not simply through sequence conservation but through conserving regions to remain disordered. These disorder and CICP residue presence and absence findings are validated by the existing experimental and crystal structure information for RABV  using the E-INS-i strategy. Each family alignment was manually curated to ensure optimal alignments. For the alignment of the entire order, each independent family alignment were organized into one FASTA file and submitted to the MAFFT ver. 6 alignment server using the E-INS-i strategy Filoviridae, Rhabdoviridae and Paramyxoviridae were also used in the model. The following viral proteins were included in the study: SEBOV, Sudan Ebola Virus (YP_138520.1); ZEBOV, Zaire Ebola Virus (NP_066243.1); REBOV, Reston Ebola Virus (NP_690580.1); MARV, Lake Victoria Marburgvirus (NP_042025.1); BDV, Borna Virus (NP_042020.1); HMPNV, Human Metapneumovirus (YP_012605.1); AVPNV, Avian Pneumovirus (AAT58236.1); HRSVB1, Human Respiratory Syncytial Virus B1 (NP_056858.1); HRSVA2, Human Respiratory Syncytial Virus A2 (P03418); HRSVS2, Human Respiratory Syncytial Virus S2 (AAC57022.1); RSV, Respiratory Syncytial Virus (NP_044591.1); BRSV, Bovine Respiratory Syncytial Virus (NP_048050.1); PNVM15, Pneumonia Virus of Mice 15 (AAW02834.1); PNVMJ3666, Pneumonia Virus of Mice J3666 (YP_173326.1); MuV, Mumps Virus (NP_054707.1); TIOV, Tioman Virus (NP_665864.1); MENV, Menangle Virus (YP_415508.1); SPIV41, Simian Parainfluenza Virus 41 (YP_138504.1); HPIV2, Human Parainfluenza Virus 2 (NP_598401.1); SPIV5, Simian Parainfluenza Virus 5 (YP_138511.1); AVPMV6, Avian Paramyxovirus 6 (NP_150057.1); GPV, Goose Paramyxovirus SF02 (NP_872273.1); NCDV, Newcastle Disease Virus (NP_071466.1); TUPV, Tupaia Paramyxovirus (NP_054690.1); FDLV, Fer-de-lance Virus (NP_899654.1); NIPH, Nipah Virus (NP_112021.1); HV, Hendra Virus (NP_047106.1); MOSV, Mossman Virus (NP_958048.1); BEIV, Beilong Virus (YP_512244.1); JV, J Virus (YP_338075.1); CDV, Canine Distemper Virus (NP_047201.1); PDV, Phocine Distemper Virus (CAA53376.1); DMV, Dolphin Morbillivirus (NP_945024.1); PDPRV, Peste-des-petits-ruminants Virus (YP_133821.1); MeV, Measles Virus (NP_056918.1); RPV, Rinderpest Virus (YP_087120.2); HPV1, Human Parainfluenza Virus 1 (NP_604433.1); SENV, Sendai Virus (NP_056871.1); BPV3, Bovine Parainfluenza Virus 3 (NP_037641.1); HPV3, Human Parainfluenza Virus 3 (NP_067148.1); FLAV, Flanders Virus (AAN73283.1); BEFV, Bovine Ephemeral Fever Virus (NP_065398.1); SCRV, Siniperca Chuatsi Rhabdovirus (YP_802937.1); ISFV, Isfahan Virus (Q5K2K7); CHPV, Chandipura Virus (P11211); SVCV, Spring Viremia of Carp Virus (NP_116744.1); VSNJV, Vesicular Stomatitis New Jersey Virus (P04881); VSIV, Vesicular Stomatitis Indiana Virus (NP_041712.1); VSSJV, Vesicular Stomatitis San Juan Virus (P03521); ABLV, Australian Bat Lyssavirus (NP_478339.1); RABV, Rabies Virus (NP_056793.1); MOKV, Mokola Lyssavirus (YP_142350.1); NCMV, Northern Cereal Mosaic Virus (NP_057954.1); LNYV, Lettuce Necrotic Yellows Virus (YP_425087.1); SYNV, Sonchus Yellow Net Virus (NP_042281.1); MFSV, Maize Fine Streak Virus (YP_052843.1); RYSV, Rice Yellow Stunt Virus (NP_620496.1); MMV, Maize Mosiac Virus (YP_052850.1); TVCV, Taro Vein Chlorosis Virus (YP_224078.1); SNAKV, Snakehead Virus (NP_050580.1); VHSV, Viral Hemorrhagic Septicemia Virus (NP_049545.1); HIRV, Hirame Virus (NP_919030.1); IHNV, Infectious Hematopoietic Necrosis Virus (NP_042676.1).The multiple sequence alignments for each family were created by submitting the sequences to the MAFFT ver.6 server  was selected for comparison. The amino acid sequence information from the protein database file was extracted for individual nucleoprotein subunits and aligned with the corresponding amino acid sequence used in the predictions. The aligned positions were then used to map the appropriate prediction to the crystal structure with a color to highlight the corresponding residue. Chimera To explore predicted features that may point to protein-protein interaction the crystal structure of the VSIV N∶RNA & P complex (pdb id – 3HHZ) was used. The nucleoproteins in the complex were mapped using the same method as above. MolProbity all-atom-contact analysis Table S1List of predicted Disordered and Coevolving/Intra-residue contact residues for each virus. Results are organized by family and in the same order as the phylogenetic tree (Bornaviridae B.) Filoviridae C.) Paramyxoviridae D.) Rhabdoviridae.tic tree . The num(DOCX)Click here for additional data file.


Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as a preferential site for primary replication. Bovine herpesvirus type 1 (BoHV-1) may replicate at both sites and cause two major clinical entities designated as infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB) in cattle. It has been hypothesized that subtype 1.1 invades preferentially the upper respiratory mucosa whereas subtype 1.2 favors replication at the peripheral genital tract. However, some studies are in contrast with this hypothesis. A thorough study of primary replication at both mucosae could elucidate whether or not different BoHV-1 subtypes show differences in mucosa tropism. We established bovine respiratory and genital organ cultures with emphasis on maintenance of tissue morphology and viability during in vitro culture. In a next step, bovine respiratory and genital mucosa explants of the same animals were inoculated with several BoHV-1 subtypes. A quantitative analysis of viral invasion in the mucosa was performed at 0 h, 24 h, 48 h and 72 h post inoculation (pi) by measuring plaque latitude and penetration depth underneath the basement membrane. All BoHV-1 subtypes exhibited a more profound invasion capacity in respiratory tissue compared to that in genital tissue at 24 h pi. However, at 24 h pi plaque latitude was found to be larger in genital tissue compared to respiratory tissue and this for all subtypes. These similar findings among the different subtypes take the edge off the belief of the existence of specific mucosa tropisms of different BoHV-1 subtypes.In general, members of the  Alphaherpesviruses in general have a broad epithelial tropism. BoHV-1, the known etiological agent of infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis/balanoposthitis (IPV/IPB) in cattle, may replicate in both respiratory and genital mucosa. However, respiratory and genital infections have been assigned to different BoHV-1 strains in the past [Although there is proof of the existence of the virus as early as 1941, BoHV-1 was first isolated in 1955 ,3. In thAn attempt was made to see if BoHV-1 could be subdivided into distinct types with different tropisms i.e. whether a correlation could be found between IBR and IPV on the one hand and distinct virus subtypes on the other hand. A classification was made of different BoHV-1 subtypes using restriction endonuclease digestion and reactivity tests on a panel of monoclonal antibodies. BoHV-1.1 was associated with respiratory disease and abortion whereas BoHV-1.2 was regarded as a genital type -11. At tA good way to elucidate the relationship between the distinct viral BoHV-1 subtypes and the clinical entities would be to test isolates representing the different subtypes on similar groups of animals under identical conditions. Practical, ethical and financial reasons make this in vivo approach difficult to perform. For that purpose, suitable in vitro systems resembling the in vivo situation and implementing the three R's principle of Russell and Burch (1959), are needed to study primary host-virus interactions.The aim of the present study was to evaluate quantitatively the replication characteristics of different BoHV-1 subtypes in in vitro systems of bovine respiratory and genital mucosa explants. Previously, we optimized an in vitro bovine respiratory organ culture . Here, wAt the slaughterhouse, seven different female slaughter animals between 3 and 5 years old were selected. Criteria to include the animals in this experiment were based on both female reproductive hormone level and BoHV-1 specific serological status. At slaughter, a thorough palpation and visual inspection of the ovaries was performed to select cows with a clear marked corpus luteum. Moreover, blood was collected at slaughter. On all sera, a progesterone determination and a complement-dependent seroneutralization (SN)-test were performed to determine peripheral blood progesterone levels and BoHV-1 specific antibody titers respectively.2). Genital tissue from four animals was used for an extensive morphometry and viability analysis. Both respiratory and genital tissue of three other cows was used to evaluate BoHV-1 subtype replication characteristics.From seven different cows, proximal trachea and vestibulum vaginae were collected at the local abattoir immediately after slaughter. A similar set up was used for bovine respiratory and genital mucosal explants. Briefly, tissues were immediately placed in phosphate buffered saline (PBS), supplemented with 1 μg/mL gentamycin , 1 mg/mL streptomycin , 1 mg/mL kanamycin , 1000 U/mL penicillin  and 5 μg/mL fungizone  for transportation to the laboratory. Respiratory and genital mucosa was stripped from the underlying layers. Small square tissue pieces were made and placed on fine-meshed gauze for culture. The explants were cultivated in serum-free medium (50% DMEM (Invitrogen)/50% Ham's F-12 GlutaMAX (Invitrogen)) supplemented with 0.1 mg/mL streptomycin (Certa), 100 U/mL penicillin  and 1 μg/mL gentamycin (Invitrogen) for up to 96 h , based on Terminal deoxynucleotidyl transferase mediated dUTP Nick End labeling (TUNEL). The test was performed according to the manufacturer's guidelines. The amount of TUNEL-positive cells was counted from five randomly chosen fields of 100 cells in both epithelium as well as lamina propria. An analysis was made at 0 h, 24 h, 48 h, 72 h and 96 h of cultivation.At 0 h, 24 h, 48 h, 72 h and 96 h of cultivation, explants of genital tissue were gathered for further analysis. A thorough morphometrical assessment was performed by means of light microscopy, transmission electron microscopy and scanning electron microscopy in an analogous way as for the previously optimized bovine respiratory organ culture.Fixation of explants was performed at the different time points by submerging them in a phosphate-buffered 3.5% formaldehyde solution for 24 h. Embedding in paraffin and further processing happened according to standard methods. As a parameter for the effect of in vitro culture on the epithelial morphometry, epithelial thickness was measured by means of a haematoxylin-eosin staining. At 40×magnification, five randomly selected places in five randomly chosen fields were measured in each explant. Next, a reticulin staining to evaluate continuity and thickness of the basement membrane was carried out. Five randomly chosen places in five randomly chosen zones were measured in each sample. Finally, the structure of the connective tissue was evaluated by means of a Van Gieson staining. In five randomly chosen fields, the relative amounts of collagen and nuclei were calculated in a defined region of interest (ROI) by setting a threshold. All measurements and calculations were performed using the Cell F Software linked to a BX61 light microscope  (magnification 40×).2 , mounted on metal stubs, platinum-coated  and examined by a Jeol JSM 5600 LV scanning electron microscope (Jeol). The integrity of the epithelium was examined using 1 500× and 5 000× magnification.After gathering the explants at the defined time points, fixation of the explants in a HEPES-buffer containing 2% paraformaldehyde and 2.5% glutaraldehyde solution during 24 h was followed by a post-fixation step in an un-buffered 1% osmium tetroxide solution for 2 h. Then, the fixed explants were dehydrated through ascending grades of alcohol and critical point dried with COAfter an overnight fixation step at 4 °C in Karnovsky's fixative , the explants were rinsed in 0.1 M sodium cacodylate buffer pH 7.4 for 8 h. Next, an overnight post-fixation step in 2% osmium tetroxide at 4 °C was performed. Further, the tissues were dehydrated stepwise in ascending grades of alcohol before they were embedded in a low viscosity embedding (LVR) medium . Finally, ultrathin sections of embedded material were cut, using a diamond knife on an Ultramicrotome Ultracut EM UC6 . Afterwards, a Leica Microsystems EM staining was performed and samples were analyzed on a JEM-1010 transmission electron microscope (Jeol) operating at 60 kV.Different strains were used, representing the known different genotypes of BoHV-1. The Cooper strain, which is considered as the prototype 1.1 subtype, was isolated in Colorado . Another7 TCID50/mL) was added. The submerged explants were incubated for 1 h . Before the tissues were placed back again on their gauze, they were thoroughly washed. In that way, different explants, both proximal trachea and vestibulum vaginae from the same animal were infected with the different strains. Samples were collected at 0 h, 24 h, 48 h and 72 h pi. At every time point, 2 respiratory and 2 genital explants from each animal for each strain were collected. Finally, all gathered explants were embedded in a cryoprotection medium ) and frozen at -70 °C.After 24 h of cultivation, explants were inoculated with the different strains. The explants were taken from their gauze and placed in a 24-well plate after rinsing with warm medium. In each well, 1 mL of virus-containing medium (10® (Molecular Probes (Invitrogen)) were used. Next, an FITC®-labeled goat anti-IBR polyclonal antiserum  directed against viral proteins was applied. Mounted samples were analyzed by means of a confocal microscope . Using the software program ImageJ, dissemination characteristics were monitored.From all collected samples, cryosections were produced, fixed in methanol  and kept at -20 °C until staining. An immunofluorescence staining was performed to evaluate plaque latitude and plaque penetration depth underneath the BM. Firstly, to stain the BM, mouse anti-collagen VII antibodies (Sigma) and goat anti-mouse Texas RedP values of ≤ 0.05 were considered significant.The data obtained were assessed using SPSS software (ANOVA) to evaluate the variance. The results shown represent means + standard deviation of quadruple and triple independent experiments of respectively viability/morphometry analysis and analysis of viral subtype replication characteristics. The results with All animals included in the experiment had a peripheral blood progesterone (P4) level of > 1 ng/mL, suggesting they were in the luteal phase of the reproductive cycle. Three animals showing an Sn-titer of < 2 for BoHV-1 specific antibodies were selected for the study of BoHV-1 subtype dissemination characteristics. The four other animals were used for a thorough morphometry and viability analysis. The latter animals had an Sn-titer ranging from 2 to 96.We saw no major differences in the occurrence of apoptosis in the epithelium with increasing times of in vitro culture. However, a small significant increase in apoptotic epithelial cells was observed at 96 h of cultivation. For the connective tissue, we noticed a small increase in the occurrence of apoptosis during culture time (to 7.7 ± 2.7 at 96 h). Values of the effect of in vitro culture on the viability of bovine genital mucosa explants are given in Table No significant changes were noticed in the epithelial thickness of the vestibulum vaginae measured during cultivation. A clear non-keratinized stratified squamous epithelium was visible for all samples Figure . HoweverThe evaluation of epithelial integrity by scanning the surface of the epithelium showed no significant changes in epithelial morphology during in vitro culture for up to 96 h. Surface cells had an irregular pavement-like appearance Figure . MoreoveOverall integrity was evaluated by means of transmission electron microscopy. The epithelial structure was maintained during the entire cultivation period (96 h) Figure . StartinAfter analysis of the reticulin stained sections, significant changes were not observed regarding the thickness of the lamina reticularis during in vitro culture Figure .The continuity and integrity of the lamina densa of the basement membrane were evaluated by means of transmission electron microscopy. During the 96 h of in vitro cultivation, no significant changes in lamina densa continuity and integrity were found Figure .Using a Van Gieson staining, no significant changes were noticed in relative percentage of collagen and nuclei in the connective tissue with increasing time after sampling Figure .Individual plaques were visible starting from 24 h pi for all strains in all tissues. All different BoHV-1 subtypes were found to spread in a plaquewise manner in both the respiratory and genital mucosa derived from three animals. The analysis of plaque latitude and penetration depth underneath the BM was performed at different time points pi for all BoHV-1 subtypes on proximal trachea and vestibulum vaginae Figure .Individual plaque measurement was performed at 0 h, 24 h, 48 h and 72 h pi Figure .At 0 h pi, no viral plaques were visible. A significant increase in plaque latitude was observed between 0 h, 24 h, 48 h and 72 h for all strains on the different tissues.Interestingly, we observed a significant difference in plaque latitude between proximal trachea and vestibulum vaginae at 24 h pi. This was noticed for all different BoHV-1 subtypes. Lateral spread was more clear in vestibulum vaginae compared to the proximal trachea. At 48 h pi, the Lam and Schönböken strain still exhibited a higher dissemination capacity in the epithelium of the vagina compared to the trachea whereas Cooper and K22 did not. All strains no longer showed a significant difference in plaque latitude when comparing proximal trachea and vestibulum vaginae at 72 h pi Figure .When comparing the strains, no significant differences between the subtypes could be observed at 0 h, 24 h, 48 h and 72 h pi. All did exhibit a higher average plaque latitude at 24 h pi in vestibulum vaginae compared to proximal trachea Figures -6a.The average invasion depth of different BoHV-1 subtypes was measured at 0 h, 24 h, 48 h and 72 h pi Figure .At 0 h pi, no vertical spread was observed for all different BoHV-1 subtypes in both proximal trachea and vestibulum vaginae. All strains included showed an increase in plaque penetration depth between 0 h, 24 h, 48 h and 72 h pi in proximal trachea and vestibulum vaginae. This increase was significant for all strains on all tissues between 48 h and 72 h pi.Both respiratory strains Cooper and Lam showed a clear advantage on proximal trachea compared to the vestibulum vaginae. The average invasion depth was significantly higher in respiratory tissue than in genital tissue and this at 24 h (Lam en Cooper) and 48 h (Cooper) pi. Interestingly, for both designated genital 1.2-strains Schönböken and K22, we did not see a significant difference when comparing both target tissues. However, we must mention that for both genital strains at 24 h pi, not one plaque crossed the BM in the genital tract whereas some plaques did in the respiratory tract. At 48 h (except Cooper) and 72 h pi, no significant differences were found when comparing all strains on proximal trachea and vestibulum vaginae Figures -6b.We observed no significant differences amongst strains at 0 h, 24 h, 48 h and 72 h pi. The penetration depth of the Cooper strain in the proximal trachea was significantly higher than the penetration depth of the Schönböken strain in the proximal trachea only at 24 h pi.In this study, we developed and utilized bovine respiratory and genital organ culture systems to reconstruct key elements in BoHV-1 mucosal invasion and moreover, to shed some light on the current classification of different BoHV-1 subtypes with particular tropisms. Because of their in vivo relevance, these in vitro models are highly appreciated since laboratory animal use is diminished and confounding factors such as individual animal variation and environmental factors are excluded. Therein lies their strength, when comparing an array of strains/subtypes on viral behavior at mucosal entry ports. Until now, organ cultures of bovine vestibulum vaginae have not been described.Monodelphis domestica, describing the remarkable fact as cells damaged by the previous desquamation process or not yet differentiated cells [The first part of the current study consisted of the development of an organ culture of bovine vestibulum vaginae. Some interesting facts were seen when evaluating tissue morphology. One cow showed highly active columnar to cuboidal cells in the stratified columnar to cuboidal epithelial zones containing a PAS positive substance. It is noteworthy that at the time of sampling, this cow was in a transition phase of the reproductive cycle namely from a progesterone to an estrogen dominance or vice versa (P4 1.03 ng/mL), this mucin production might be an outcome of estrogen influence on the epithelium . These hed cells . Howevered cells . OverallIn the second part of the study, bovine respiratory and genital mucosa explants from the same animals were inoculated with several BoHV-1 subtypes. We clearly saw that both respiratory and genital mucosa are susceptible to infection with all different BoHV-1 subtypes. This dual tropism is also seen in closely related animal alphaherpesviruses with either emphasis on the genital tract such as Caprine herpesvirus type 1 and Cervid herpesvirus type 2; or on the respiratory tract such as Cervid herpesvirus type 1 and Suid herpesvirus type 1 (PrV) -31. HoweNext, a thorough analysis on the dissemination characteristics of different BoHV-1 subtypes on both respiratory and genital mucosa divulged important information on the current phylogenetic classification. Looking at average plaque penetration depth, key findings were twofold. Firstly, the so called respiratory subtypes Cooper and Lam invaded significantly deeper in respiratory tissue compared to genital tissue at 24 h and 48 h pi. However, secondly, in general all BoHV-1 subtypes exhibited a more profound invasion capacity in respiratory tissue compared to that in genital tissue at 24 h pi. It is known that massive accumulation of neutrophils and activation of macrophages reach peaks between 24-48 h post BoHV-1 infection . Thus, tOur findings are in line with the vision of McKercher. He believes that virulent respiratory strains arose out of the less virulent genital strains and that in general the virus may be limited in invasion capacity due to biochemical and physiological characteristics of the vagina with emphasis on barrier function ,7.We can conclude that the respiratory and genital organ culture is suitable to study BoHV-1 invasion at primary entry ports and that they can be extrapolated to other species and viruses. Taken together, our findings and the existing knowledge on BoHV-1 classification, clearly take the edge off the belief of the existence of specific mucosa tropisms of different BoHV-1 subtypes and make the phylogenetic BoHV-1 subdivision rather farfetched. The question arises whether this could be a difference in virulence between strains, as seen in all other viruses, rather than a difference in mucosa tropism?The authors declare that they have no competing interests.LS set up the study design, carried out the optimization of the organ culture as well as the infection experiments and the processing of all samples, performed the statistical analysis and drafted the manuscript. APV assisted in the set up of both experiments and sampling. WVdB participated in the analysis and the interpretation of all the morphological results. SG took part in the design of the study and critically analyzed all the results. HJN coordinated the study and participated in its design. All authors read and approved the final manuscript.


We diagnosed 400 possible IgM-positive cases of chikungunya virus in Bo, Sierra Leone, during July 2012–January 2013 by using lateral flow immunoassays. Cases detected likely represent only a small fraction of total cases. Further laboratory testing is required to confirm this outbreak and characterize the virus. Aedes spp. mosquitoes, were frequent in sub-Saharan Africa and southern and Southeast Asia during the 1950s–1970s, but the infection largely disappeared in the 1980s; only sporadic cases were observed (Outbreaks of infection with chikungunya virus (CHIKV), an alphavirus that is transmitted by bites of infected The primary symptoms of CHIKV infection are high fever (>38.5°C [102°F]) and severe pain in the distal joints of the extremities or the lumbar spine. A maculopapular rash, sensorineural impairment, severe headache, and other nonspecific symptoms may also occur. Symptoms usually resolve within 1–2 weeks after onset of fever, but for a sizeable proportion of patients, arthralgia and arthritis become chronic and pain persists for years  in Bo, Sierra Leone, initiated a 1-year infectious disease surveillance program to identify the diversity of pathogens causing febrile illness in the city. A tiered analysis approach was used. First, all specimens from febrile study participants were tested for ≈12 infections with various pathogens, including CHIKV, by commercially available test kits. Specimens that showed negative results in this first round of testing were further tested by using cultures, multiplex PCR, and resequencing pathogen microarrays. The research protocol was approved by Njala University, George Mason University, the Liverpool School of Tropical Medicine, the US Naval Research Laboratory, and the Sierra Leone Ethics and Scientific Review Committee.Leptospira spp., Salmonella enterica Serovar typhi, and syphilis.During July 7, 2012–January 10, 2013, MHRL conducted first-tier lateral flow immunoassay (LFI) tests of blood samples from all 932 outpatients ≥5 years of age who had been clinically examined at the hospital, were found to have febrile illness, and consented to having blood drawn for laboratory testing. LFI test kits  were used for diagnosis of IgM against CHIKV; IgG and IgM against dengue virus and hepatitis A virus; hepatitis B virus surface antigen, hepatitis C virus, HIV-1/2, and antibodies against these viruses; and IgG and IgM against Most patients reported that they had sought medical care within several days after the onset of their febrile illnesses. Levels of IgM against CHIKV are usually detectable by immunochromatographic methods within a few days after infection and persist for ≈3–4 months  of 932 febrile patients were positive by LFI for CHIKV . Ages ofwww.ushahidi.com) to compile case reports. Details about the patients who were positive for CHIKV were uploaded to the MHRL website (www.mhrlsl.com/GIA/ushahidi) and, if possible, were geolocated on an open street map (www.openstreetmap.org) that linked to a map of Bo created by MHRL for health research purposes (On July 28, MHRL launched an Ushahidi-based website (purposes . Of the ,Results of attempts by the US Naval Research Laboratory to confirm the LFI results by using semi-nested reverse transcription PCR on fast technique for analysis of nucleic acid–preserved samples were inconclusive, possibly because of genetic sequence variation from well-characterized strains or because of the timing of specimen collections. Viral loads for humans with CHIKV infection decrease after the second day of symptoms, and viral titers may be low after the fifth day (Aedes spp. mosquito–borne infections (,Our results suggest that an outbreak of chikungunya virus occurred in Sierra Leone. The exact time of the reemergence of this virus cannot be pinpointed, but retrospective analyses of outpatient charts suggested that, on the basis of syndromic criteria, the first cases occurred in January 2012 and the outbreak peaked during the rainy season in 2012. Other outbreaks reported in central and west Africa have also occurred during the rainy season, which is typical for


Alexander the Great died in Babylon in 323 BC. His death at age 32 followed a 2-week febrile illness. Speculated causes of death have included poisoning, assassination, and a number of infectious diseases. One incident, mentioned by Plutarch but not considered by previous investigators, may shed light on the cause of Alexander’s death. The incident, which occurred as he entered Babylon, involved a flock of ravens exhibiting unusual behavior and subsequently dying at his feet. The inexplicable behavior of ravens is reminiscent of avian illness and death weeks before the first human cases of West Nile virus infection were identified in the United States. We posit that Alexander may have died of West Nile encephalitis. Alexander the Great died in the ancient Mesopotamian city of Babylon, on June 10, of 323 BC . His dea. Plutarch mentions that Aristotle (Alexander’s tutor) procured arsenic to poison Alexander . Diseases endemic to the area (present-day Iraq)  were not mentioned by chroniclers of Alexander’s death. Also not reported was illness among his troops, mainly Macedonians and local recruits. Descriptions of Alexander’s illness do not include common disease signs .Alexander’s death occurred in late spring, upon his return to Babylon from the Indian subcontinent. Environmental conditions were unremarkable , causes rapid death, accompanied by skin and muscle lesions and bleeding.Typhoid fever and its complications have also been thoroughly considered (Schistosoma haematobium infection (Acanthamoeba spp. (pathogenic free-living amoebae) and Naegleria spp. cause meningoencephalitis, which is acquired during bathing, an activity in which Alexander reportedly participated with compulsion. Acanthamoebae are cosmopolitan but prefer compromised hosts. Moreover, death from naegleriasis usually occurs within a week of onset, and encephalitis caused by acanthamoebae causes death only after a prolonged period of symptoms.Other suggested diagnoses include When Alexander’s clinical symptoms were listed on GIDEON , first isolated from a febrile patient in Uganda in 1937 , >98% had fever, 46% cognitive changes, and 17% abdominal pain or myalgias. Nearly 18% became comatose , onset of disease in humans and birds, including inexplicable avian die-offs, could have occurred earlier in the summer. We reread Plutarch and saw the following passage about Alexander’s entrance into Babylon: “… when he arrived before the walls of the city he saw a large number of ravens flying about and pecking one another, and some of them fell dead in front of him.” (dāgil işşūri) were common in Asia Minor at the time. These diviners considered birds as oracles. Greek Kulturkreis and Babylonian Alalakh tablets mention auguries based on the behavior of birds, particularly fighting birds, to predict the future (Bird observers (Corvus corax) and not crows (Corvus corone sardonius or other crow species). No ravens were at the Bronx Zoo in 1999 . However, in the United States today, New World crows  are among the birds most susceptible to fatal West Nile virus infections. One wonders if an influx of migratory birds might have served as reservoirs of West Nile virus and infected ravens in Babylon, causing a massive die-off.Current geographic distribution of corvids indicates that these likely were ravens (Corvus corone sardonius) for the first time, demonstrating experimental infection of birds with viremia as high as 109, and death rates of 100% (3.5 and could subsequently serve as West Nile virus vectors to humans of any age. This early epidemiologic work provided an early clue in New York City in 1999, when both exotic and domestic birds signaled the introduction of West Nile virus disease to the New World (Pathogenicity of West Nile virus for corvids was established 50 years ago. Work et al., assigned by the Rockefeller Foundation to study arboviruses in Egypt, isolated 23 West Nile virus strains from blood samples of febrile children in the Sindbis area and found that the virus caused illness in more children and young adults than in older adults. In addition, and particularly germane to our hypothesis, they isolated West Nile virus from a hooded crow (lexander the Great died in late spring in the semi-tropical, urban area of present-day Baghdad. Explanations for his death have included poisoning, enteric and parasitic diseases, influenza, and poliomyelitis. Our diagnosis, as well as previous alternative diagnoses, may be subject to author bias, errors in translation, and a paucity of clinical information. We assumed that he died in late spring in Babylon after a 2-week illness that included fever and signs suggestive of encephalitis. We presumed that diseases now endemic to Iraq were also present in ancient Mesopotamia. Recent scholarly thought has been ingenious and rigorous, given the sparseness of available information. Nonetheless, earlier diagnoses did not include West Nile virus encephalitis. Previous considerations omitted an event that was carefully recorded by Plutarch and which, before 1999, would have been considered irrelevant: the erratic behavior and observable deaths of numerous ravens outside the walls of Babylon. This observation might now be construed as an important clue. If this observation is included as part of Alexander’s illness, West Nile virus encephalitis complicated by flaccid paralysis becomes an alternative diagnosis. It is possible that, in the 3rd century BC, disease caused by West Nile virus arrived in Mesopotamia for the first time in recorded history, killing indigenous birds and an occasional human and causing only incidental febrile illness in many others. Over subsequent centuries the virus may have devolved, becoming less pathogenic for indigenous birds, while retaining its potential as a dangerous human pathogen. This is speculative, but in 1999, a “natural experiment” did occur when this Old World epizootic strain was introduced into the United States. What has been observed in the ongoing North American epizootic and epidemic might be similar to what happened in Babylon many years ago. We now know that unexplained bird die-offs can presage human cases of disease caused by West Nile virus. In 323 BC, a similar event might have been considered an omen of Alexander the Great’s death. In this instance, the oracles would have been correct.


Viral encephalitis (VE) and viral meningitis (VM) have been notifiable infectious diseases under surveillance in the Republic of Ireland since 1981. Laboratories have reported confirmed cases by detection of viral nucleic acid in cerebrospinal fluid since 2004. To determine the prevalence of these diseases in Ireland during 2005–2008, we analyzed 3 data sources: Hospital In-patient Enquiry data  accessed through Health Intelligence Ireland, laboratory confirmations from the National Virus Reference Laboratory, and events from the Computerised Infectious Disease Reporting surveillance system. We found that the national surveillance system underestimates the incidence of these diseases in Ireland with a 10-fold higher VE hospitalization rate and 3-fold higher VM hospitalization rate than the reporting rate. Herpesviruses were responsible for most specified VE and enteroviruses for most specified VM from all 3 sources. Recommendations from this study have been implemented to improve the surveillance of these diseases in Ireland.  Encephalitis and meningitis are serious inflammatory diseases of the brain that require hospitalization for many patients and are a substantial cause of illness. Although the etiologic agent is not identified for most cases  passive surveillance system, during 2005–2008, reflected the incidence of VE and VM in Ireland. CIDR is the real-time Internet-based surveillance system for 93.9% of all notifiable infectious diseases reportable by statute in Ireland. We compared cases reported to CIDR with laboratory detection of cases from the National Virus Reference Laboratory (NVRL) and cases identified from hospitalized patient discharge information in the Hospital In-Patient Enquiry (HIPE) scheme. In 2005, eight (24%) of the 34 public hospital laboratories, in addition to the national reference laboratories, were connected directly to CIDR. This connection increased to 16 (47%) in 2008. Hospital/community physicians who manage VE or VM patients using laboratories which were not directly connected to CIDR, were obligated to report notifiable diseases to CIDR through the local department of public health.www.healthatlasireland.ie/f1live).The data sources and the process of reporting notifiable diseases in Ireland are shown in A previous study suggested an underestimation of the incidence of acute encephalitis and VM in the northeastern part of Ireland  more than once with the same or different diagnoses. A HIPE discharge record is generated after a patient is discharged from, or dies, in the hospital , fourth edition diagnosis codes A83, A84, A85, A86, B00.4, B01.1, B02.0, B05.0, and B26.2 were defined as VE. Discharges associated with the codes A87, B00.3, B01.0, B02.1, B05.1, and B26.1 were defined as VM. We determined the proportion of these codes recorded in any of the diagnosis fields and in the primary diagnosis field only.Hospitalizations in which a lumbar puncture (ICD-10-AM code 39000–00) was listed were extracted from HIPE for comparison with the number of CSF samples received at NVRL for virologic testing and with those in which a virus was detected from patients with symptoms of acute encephalitis or meningitis. Deaths were determined by searching for patients on HIPE who “died with post mortem” or “died no post mortem.”www.cso.ie/en/statistics/population). To prevent the identification of individual hospital in-patients, when 5 or fewer discharges or patients for a particular diagnosis were found, we depicted the number as ≤5.Denominator data for population-based incidence rates  correspond to published Irish Census 2006 Principle Demographic Results ; 95% CIs were also calculated. Male-to-female ratios (MFRs) are presented as the number of male patients to 1 female patient.The statistical significance of the difference in proportions was tested by using the χInvestigation of CNS infection is primarily based on detection of HSV-1 DNA, HSV-2 DNA, or VZV DNA. When appropriate, on the basis of clinical manifestations/underlying clinical issues and in collaboration with the clinical teams managing a patient’s condition, testing can be performed for human herpesvirus 6 DNA, Epstein-Barr virus DNA, cytomegalovirus DNA, JC virus DNA, enterovirus RNA, enterovirus 71 RNA, measles virus RNA, mumps virus RNA, and lymphocytic choriomeningitis virus RNA. When encephalitis or meningitis is suspected because of arboviral infection, the infection is diagnosed by detecting IgM in serum, CSF, or both. On the basis of the patients’ clinical and travel history, tests were performed for serologic evidence of the following: West Nile virus, Venezuelan equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, dengue virus, eastern equine encephalitis virus, western equine encephalitis virus, St. Louis encephalitis virus, Powassan virus, La Crosse virus, and tick-borne encephalitis virus.In addition to the investigation of CSF, molecular analysis and culture of fecal samples were also performed. The detection of enterovirus provides additional circumstantial evidence for the viral etiology of the CNS manifestations.NVRL does not routinely receive convalescent-phase serum samples; therefore, investigations of acute- or convalescent-phase serum are uncommon. Intrathecal antibody testing is not performed at NVRL. On the rare occasion when such specimens are sent to an international reference laboratory, the results of such testing would be known to NVRL if the specimens were originally sent through NVRL to that laboratory.Herpesvirus DNA is often not detectable in CSF from encephalitis/meningitis patients. It can nonetheless be confirmed through evidence of elevated specific intrathecal IgG. The absence of such testing in our analysis may have led to underdetection of meningitis attributable to herpes group viruses, particularly VZV, which other studies have found to be the most common viral cause for VM, including a 2001 study in Finland  WinPath  and configured for upload to CIDR.Between 2005 and 2008, a total of 40 VE events and 341 VM events were reported to CIDR . Of thesVE (62.5%) and VM (58.7%) occurred more frequently in male patients . The greatest age-standardized incidence rates (ASIR) of VE (27.5%) and VM (29.9%) were for children <4 years of age (The highest number of VE events were caused by HSV (40.0%), followed by VZV (27.5%) and mumps virus (12.5%) as shown in No deaths from either VE or VM were reported to CIDR. VM reporting was greatest in the month of August; overall rates were highest in August 2006  and a mean annual discharge rate of 3.51/100,000 population (95% CI 2.15–4.87); A total of 83.0% of discharges of patients with VE and 92.2% of those with VM were considered primary diagnosis discharges. The mean length of stay for a patient discharged with VE was 15.3 days , and mean length of stay in an intensive care unit was 1.2 days . The average cost per discharge for VE was €9,783.31. For those with VM, the mean length of stay was 6.4 days , the mean length of stay in an intensive care unit was 0.2 days , and the average cost per discharge was €4,612.77.Male patients accounted for 53.4% of all VE patients and 53.0% of VM patients . The ASIR of patients hospitalized with VE was highest among elderly (80–84 years of age) and very young patients  of persons hospitalized with VE, viral cause was unknown . When anThirty-three hospitalized patients with a diagnosis of VE died, giving a case-fatality ratio (CFR) of 7.9% , panel AA peak of both VE and VM hospitalizations occurred in the summer months, most notably in July and August .The NVRL received 6,502 CSF specimens collected during 2005–2008. Of those, 1.3% tested positive for a virus causing encephalitis, and 4.2% tested positive for a virus causing meningitis . A listi>55 years of age , as were those indicating VM . The highest ASIR of laboratory confirmations for viruses causing encephalitis was from patients s of age , panel As of age , panel Bs of age , panel As of age , panel BWe analyzed the differences between hospital activity, laboratory confirmations, and events created by using several parameters. When these factors were compared by age of patient, we found a significant difference in the distribution of VE laboratory confirmations and events .Of VE cases with a specified cause, which should have been reported to public health authorities according to the case definitions in use at the time, a significant difference was found in the distribution of VE cases by causative agent between laboratory confirmations and events . Lessthan half of laboratory confirmations and 18.5% of hospitalizations were reported. 2 = 139.83, p<0.001) and laboratory confirmations and events . Only 9.2% of patients with illness of unspecified cause were reported. On the other hand, the number of enteroviral events or laboratory confirmations was 4-fold higher than the number of hospitalizations. No deaths had been reported to CIDR, in contrast to the 33 VE deaths and ≤5 VM deaths recorded in HIPE.Among VM cases for which an organism was specified, a significant difference was found in the distribution of cases by causative agent between hospitalizations and events . The laboratories conducting the testing probably contributed to the underreporting. Underreporting or misclassification of diagnoses by hospital physicians without confirmatory tests may also have occurred.Surveillance data are routinely used to quantify the incidence of disease and to identify outbreaks or emerging viral diseases, and this underidentification of VE and VM in Ireland is of concern. With the emergence of West Nile fever in Italy , with peaks in the 0–4 and 15–19 year age groups, which had also been found in Denmark . It is also possible that alternative noninvasive diagnostic tools were used in patients for whom lumbar punctures were contraindicated, such as magnetic resonance imaging or electroencephalographic testing , improved laboratory reporting, and clinical guidelines have been put in place, to evaluate whether there has been an effect by these changes. Analysis of the referral source of clinical diagnoses and laboratory results to CIDR, specimen type, and laboratory testing performed would facilitate better understanding of the proportion of difference between HIPE and CIDR because of underreporting. Such analysis would also provide feedback and education to the partners involved in health protection as an aid in highlighting the value of the surveillance of these diseases in Ireland and the detection of possible threats to public health.