Institute of Diagnostic Virology

Greifswald, Germany

Institute of Diagnostic Virology

Greifswald, Germany
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Hechinger S.,Institute of Diagnostic Virology | Wernike K.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
Veterinary Research | Year: 2013

Schmallenberg virus (SBV), an arthropod borne pathogen, spread rapidly throughout the majority of Europe since 2011. It can cause a febrile disease, milk drop, diarrhea, and fetal malformation in ruminants. SBV, a member of the Simbu serogroup within the genus Orthobunyavirus, is closely related to Akabane virus (AKAV) and Aino virus (AINOV) among others. In the present study, 4 Holstein-Friesian calves were immunized twice four weeks apart with a multivalent, inactivated vaccine against AKAV and AINOV. Another 4 calves were kept as unvaccinated controls. All animals were clinically, serologically and virologically examined before and after challenge infection with SBV. AKAV- and AINOV-specific neutralizing antibodies were detected one week before challenge infection, while SBV-specific antibodies were detectable only thereafter. SBV genome was detected in all vaccinated animals and 3 out of 4 controls in serum samples taken after challenge infection. In conclusion, the investigated vaccine was not able to prevent an SBV-infection. Thus, vaccines for other related Simbu serogroup viruses can not substitute SBV-specific vaccines as an instrument for disease control. © 2013 Hechinger et al.; licensee BioMed Central Ltd.

Beer M.,Institute of Diagnostic Virology | Conraths F.J.,Institute of Epidemiology | Van Der Poel W.H.M.,Wageningen University
Epidemiology and Infection | Year: 2013

SUMMARY In 2011, a novel orthobunyavirus of the Simbu serogroup, the Schmallenberg virus (SBV), was discovered using a metagenomic approach. SBV caused a large epidemic in Europe in ruminants. As with related viruses such as Akabane virus, it appears to be transmitted by biting midges. Transplacental infection often results in the birth of malformed calves, lambs and goat kids. In more than 5000 farms in Germany, The Netherlands, Belgium, France, UK, Italy, Spain, Luxembourg, Denmark and Switzerland acute infections of adult ruminants or malformed SBV-positive offspring were detected, and high seroprevalences were seen in adult ruminants in the core regions in The Netherlands, Germany and Belgium. The discovery of SBV, the spread of the epidemic, the role of vectors, the impact on livestock, public health issues, SBV diagnosis and measures taken are described in this review. Lessons to be learned from the Schmallenberg virus epidemic and the consequences for future outbreaks are discussed. Copyright © Cambridge University Press 2012.

Aebischer A.,Institute of Diagnostic Virology | Wernike K.,Institute of Diagnostic Virology | Hoffmann B.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
Journal of Clinical Microbiology | Year: 2014

Over the past few years, there has been an increasing demand for rapid and simple diagnostic tools that can be applied outside centralized laboratories by using transportable devices. In veterinary medicine, such mobile test systems would circumvent barriers associated with the transportation of samples and significantly reduce the time to diagnose important infectious animal diseases. Among a wide range of available technologies, high-speed real-time reverse transcriptase quantitative PCR (RT-qPCR) and the two isothermal amplification techniques loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) represent three promising candidates for integration into mobile pen-side tests. The aim of this study was to investigate the performance of these amplification strategies and to evaluate their suitability for field application. In order to enable a valid comparison, novel pathogen-specific assays have been developed for the detection of Schmallenberg virus and bovine viral diarrhea virus. The newly developed assays were evaluated in comparison with established standard RT-qPCR using samples from experimentally or field-infected animals. Even though all assays allowed detection of the target virus in less than 30 min, major differences were revealed concerning sensitivity, specificity, robustness, testing time, and complexity of assay design. These findings indicated that the success of an assay will depend on the integrated amplification technology. Therefore, the application- specific pros and cons of each method that were identified during this study provide very valuable insights for future development and optimization of pen-side tests. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Wernike K.,Institute of Diagnostic Virology | Hoffmann B.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
Journal of Clinical Microbiology | Year: 2013

Detection of several pathogens with multiplexed real-time quantitative PCR (qPCR) assays in a one-step setup allows the simultaneous detection of two endemic porcine and four different selected transboundary viruses. Reverse transcription (RT)-qPCR systems for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2), two of the most economically important pathogens of swine worldwide, were combined with a screening system for diseases notifiable to the World Organization of Animal Health, namely, classical and African swine fever, foot-and-mouth disease, and Aujeszky's disease. Background screening was implemented using the identical fluorophore for all four different RT-qPCR assays. The novel multiplex RT-qPCR system was validated with a large panel of different body fluids and tissues from pigs and other animal species. Both reference samples and clinical specimens were used for a complete evaluation. It could be demonstrated that a highly sensitive and specific parallel detection of the different viruses was possible. The assays for the notifiable diseases were even not affected by the simultaneous amplification of very high loads of PRRSV- and PCV2-specific sequences. The novel broad-spectrum multiplex assay allows in a unique form the routine investigation for endemic porcine pathogens with exclusion diagnostics of the most important transboundary diseases in samples from pigs with unspecific clinical signs, such as fever or hemorrhages. The new system could significantly improve early detection of the most important notifiable diseases of swine and could lead to a new approach in syndromic surveillance.

Drager C.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology | Blome S.,Institute of Diagnostic Virology
Archives of Virology | Year: 2015

Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates. © 2015, Springer-Verlag Wien.

Rohrs S.,Institute of Diagnostic Virology | Kalthoff D.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
Vaccine | Year: 2014

Highly pathogenic avian influenza viruses of subtype H5N1 sporadically cause severe disease in humans and involve the risk of inducing a pandemic by gaining the ability for human-to-human transmission. In naïve poultry, primarily gallinaceous birds, the virus induces fatal disease and the used inactivated vaccines occasionally are unable to provide efficient and early onset of protection. Therefore, optimized vaccines must be developed and evaluated in model systems. In our study, we tested a novel H5 neuraminidase-deleted influenza A virus variant to analyze the induction of a very early onset of immunity. Ferrets, mice and chickens were each immunized with a single vaccine dose seven, three and one day before lethal challenge infection, respectively. Sound protection was conferred in 100% of animals immunized seven days prior to challenge infection. In these animals, no clinical signs were observed, and no challenge virus RNA was detected by real-time RT-PCR analyses of swabs, nasal washings, and organ samples. Moreover, the attenuated modified-live virus variant protected all chickens, mice, and ferrets as early as three days after vaccination against severe clinical signs. Chickens and ferrets developed hemagglutinin-specific antibodies after seven days, but no neuraminidase-specific antibodies, making this kind of neuraminidase-negative strain suitable for the DIVA ("differentiating vaccinated from infected animals") strategy. © 2014 Elsevier Ltd.

Blome S.,Institute of Diagnostic Virology | Gabriel C.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
Virus Research | Year: 2013

African swine fever (ASF) is among the most important viral diseases that can affect domestic and feral pigs. Both clinical signs and pathomorphological changes vary considerably depending on strain virulence and host factors. Acute infections with highly virulent virus strains lead to a clinical course that resembles a viral haemorrhagic fever that is characterized by pronounced depletion of lymphoid tissues, apoptosis of lymphocyte subsets, and impairment of haemostasis and immune functions. It is generally accepted that most lesions can be attributed to cytokine-mediated interactions triggered by infected and activated monocytes and macrophages, rather than by virus-induced direct cell damage. Nevertheless, most pathogenetic mechanisms are far from being understood. This review summarizes the current knowledge and discusses implications and research gaps. © 2012 Elsevier B.V.

Hoper D.,Institute of Diagnostic Virology | Hoffmann B.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
PLoS ONE | Year: 2011

Driven by the impact of influenza A viruses on human and animal health, much research is conducted on this pathogen. To support this research, we designed an all influenza A-embracing reverse transcription-PCR (RT-PCR) for the generation of DNA from influenza A virus negative strand RNA genome segments for full-length genome deep sequencing on a Genome Sequencer FLX instrument. For high reliability, the RT-PCRs are designed such that every genome segment is divided into two amplicons and for the most variable segments redundancy is included. Moreover, to minimize the risk of contamination of diagnostic real-time PCRs by sequencing amplicons, RT-PCR does not generate amplicons that are amenable to RT-qPCR detection. With the presented protocol we were able to generate virtually all amplicons (99.3% success rate) from isolates representing all so far known 16 hemagglutinin and 9 neuraminidase subtypes and from an additional 2009 pandemic influenza A H1N1 virus. Three isolates were sequenced to analyze the suitability of the DNA for sequencing. Moreover, we provide a short R script that disambiguates the sequences of the primers used. We show that using unambiguous primer sequences for read trimming prior to assembly with the genome sequencer assembler software results in higher quality of the final genome sequences. Using the disambiguated primer sequences, high quality full-length sequences for the three isolates used for sequencing trials could be established from the raw data in de novo assemblies. © 2011 Höper et al.

Beer M.,Institute of Diagnostic Virology
Veterinary research | Year: 2014

The arthropod-borne Schmallenberg virus (SBV), family Orthobunyaviridae, emerged in Europe in 2011. SBV is associated with a mild disease in adult ruminants but fetal malformation after an infection during a critical phase of pregnancy. A number of inactivated vaccines have been developed; their efficacy after two injections was demonstrated. To make the vaccination of sheep more efficient and economic the effect of a single immunization with one of these vaccines was investigated in the present study. Five vaccinated sheep and five additional control sheep were inoculated with SBV three weeks after vaccination and the results of a competitive ELISA, a standard microneutralization test and an SBV-specific real-time RT-PCR confirmed vaccine efficacy by demonstrating complete inhibition of viral replication in immunized animals.

Scheuch M.,Institute of Diagnostic Virology | Hoper D.,Institute of Diagnostic Virology | Beer M.,Institute of Diagnostic Virology
BMC bioinformatics | Year: 2015

BACKGROUND: Fuelled by the advent and subsequent development of next generation sequencing technologies, metagenomics became a powerful tool for the analysis of microbial communities both scientifically and diagnostically. The biggest challenge is the extraction of relevant information from the huge sequence datasets generated for metagenomics studies. Although a plethora of tools are available, data analysis is still a bottleneck.RESULTS: To overcome the bottleneck of data analysis, we developed an automated computational workflow called RIEMS - Reliable Information Extraction from Metagenomic Sequence datasets. RIEMS assigns every individual read sequence within a dataset taxonomically by cascading different sequence analyses with decreasing stringency of the assignments using various software applications. After completion of the analyses, the results are summarised in a clearly structured result protocol organised taxonomically. The high accuracy and performance of RIEMS analyses were proven in comparison with other tools for metagenomics data analysis using simulated sequencing read datasets.CONCLUSIONS: RIEMS has the potential to fill the gap that still exists with regard to data analysis for metagenomics studies. The usefulness and power of RIEMS for the analysis of genuine sequencing datasets was demonstrated with an early version of RIEMS in 2011 when it was used to detect the orthobunyavirus sequences leading to the discovery of Schmallenberg virus.

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