Molecular diagnostic and genetic characterization of highly pathogenic viruses: application during Crimean–Congo haemorrhagic fever virus outbreaks in Eastern Europe and the Middle East

AbstractSeveral haemorrhagic fevers are caused by highly pathogenic viruses that must be handled in Biosafety level 4 (BSL–4) containment. These zoonotic infections have an important impact on public health and the development of a rapid and differential diagnosis in case of outbreak in risk areas represents a critical priority. We have demonstrated the potential of a DNA resequencing microarray (PathogenID v2.0) for this purpose. The microarray was first validated in vitro using supernatants of cells infected with prototype strains from five different families of BSL-4 viruses (e.g. families Arenaviridae, Bunyaviridae, Filoviridae, Flaviviridae and Paramyxoviridae). RNA was amplified based on isothermal amplification by Phi29 polymerase before hybridization. We were able to detect and characterize Nipah virus and Crimean–Congo haemorrhagic fever virus (CCHFV) in the brains of experimentally infected animals. CCHFV was finally used as a paradigm for epidemics because of recent outbreaks in Turkey, Kosovo and Iran. Viral variants present in human sera were characterized by BLASTN analysis. Sensitivity was estimated to be 105–106 PFU/mL of hybridized cDNA. Detection specificity was limited to viral sequences having ˜13–14% of global divergence with the tiled sequence, or stretches of ˜20 identical nucleotides. These results highlight the benefits of using the PathogenID v2.0 resequencing microarray to characterize geographical variants in the follow-up of haemorrhagic fever epidemics; to manage patients and protect communities; and in cases of bioterrorism

Complex evolution and epidemiology of Dobrava-Belgrade hantavirus: definition of genotypes and their characteristics.

Dobrava-Belgrade virus (DOBV) is a human pathogen that has evolved in, and is hosted by, mice of several species of the genus Apodemus. We propose a subdivision of the species Dobrava-Belgrade virus into four related genotypes – Dobrava, Kurkino, Saaremaa, and Sochi – that show characteristic differences in their phylogeny, specific host reservoirs, geographical distribution, and pathogenicity for humans

Biosafety standards for working with Crimean-Congo haemorrhagic fever virus

In countries from which Crimean-Congo haemorrhagic fever (CCHF) is absent, the causative virus CCHF virus (CCHFV) is classified as a hazard group 4 agent and handled in containment level 4. In contrast, most endemic countries out of necessity have had to perform diagnostic tests under biosafety level (BSL) -2 or -3 conditions. In particular, Turkey and several of the Balkan countries have safely processed more than 100000 samples over many years in BSL-2 laboratories. It is therefore advocated that biosafety requirements for CCHF diagnostic procedures should be revised, to allow the required tests to be performed under enhanced BSL-2 conditions with appropriate biosafety laboratory equipment and personal protective equipment used according to standardized protocols in the affected countries. Downgrading of CCHFV research work from Cl-4, BSL-4 to Cl-3, BSL-3 should also be considered.Additional co-authors: Gülay Korukluoglu, Pieter Lyssen, Ali Mirazimi, Johan Neyts, Matthias Niedrig, Aykut Ozkul, Anna Papa, Janusz Paweska, Amadou A Sall, Connie S Schmaljohn, Robert Swanepoel, Yavuz Uyar, Friedemann Weber, Herve Zelle

An evaluation of serological methods to diagnose tick-borne encephalitis from serum and cerebrospinal fluid

Background: Tick-borne encephalitis (TBE) is an infectious disease endemic to large parts of Europe and Asia. Diagnosing TBE often relies on the detection of TBEV-specific antibodies in serum and cerebrospinal fluid (CSF) as viral genome is mostly not detectable once neurological symptoms occur. Objectives: We evaluated the performance of TBEV IgM and IgG ELISAs in both serum and CSF of confirmed TBEV patients and discuss the role of (CSF) serology in TBEV diagnostics. Study design: For the assay evaluation we collected specimen from confirmed TBEV patients. Assay specificity was assessed using sera from patients with a related flavivirus infection or other acute infection. A selected ELISA assay was used to analyze TBEV-specific antibodies in CSF and to evaluate the use in confirming TBE diagnosis. Results: In this study the overall sensitivity of the IgM TBEV ELISAs was acceptable (94 -100 %). Four out of five IgM ELISA's demonstrated an excellent overall specificity from 94 -100% whereas a low overall specificity was observed for the IgG TBEV ELISAs (30-71%). Intrathecal antibody production against TBEV was demonstrated in a subset of TBE patients. Conclusions: In four out of five ELISAs, IgM testing in serum and CSF of TBE patients is specific and confirmative. The lack of IgG specificity in all ELISAs emphasizes the need of confirmatory testing by virus neutralisation, depending on the patient's background and the geographic location of exposure to TBEV. A CSF-serum IgG antibody index can support the diagnosis specifically in chronic disease or once IgM has disappeared

The first clinical case due to AP92 like strain of Crimean-Congo Hemorrhagic Fever virus and a field survey

<p>Abstract</p> <p>Background</p> <p>Crimean-Congo Hemorrhagic Fever (CCHF) is a fatal infection, but no clinical case due to AP92 strain was reported. We described the first clinical case due to AP92 like CCHFV.</p> <p>Methods</p> <p>A case infected by a AP92 like CCHFV was detected in Balkanian part of Turkey. Diagnosis was confirmed by RT-PCR and sequencing. A human serologic and tick survey studies were performed in the region, where the case detected.</p> <p>Results</p> <p>Thirty eight individuals out of 741 were found to be anti CCHFV IgM positive. The attack rate for overall CCHFV was calculated as 5.2%. In univariate analyses, CCHFV IgM positivity was found to be associated with the age (p < 0.001), male gender (p = 0.001), agricultural activity (p = 0.036), and history of tick bite (p = 0.014). In multivariate analysis, older age (OR: 1.03, CI:1.01–1.05, p < 0.001), male gender were found to be the risk factors (OR: 2.5, CI:1.15–5.63, p = 0.020) for CCHFV infection.</p> <p>Conclusion</p> <p>This is the first human case with AP92 like CCHFV infection. Furthermore, this is the first report of AP92 like strain in Turkey. In the region, elderly males carry the highest risk for CCHFV infection.</p

Construction and Nonclinical Testing of a Puumala Virus Synthetic M Gene-Based DNA Vaccine

Puumala virus (PUUV) is a causative agent of hemorrhagic fever with renal syndrome (HFRS). Although PUUV-associated HFRS does not result in high case-fatality rates, the social and economic impact is considerable. There is no licensed vaccine or specific therapeutic to prevent or treat HFRS. Here we report the synthesis of a codon-optimized, full-lengthMsegment open reading frame and its cloning into a DNA vaccine vector to produce the plasmid pWRG/PUU-M(s2). pWRG/PUU-M(s2) delivered by gene gun produced high-titer neutralizing antibodies in hamsters and nonhuman primates. Vaccination with pWRG/ PUU-M(s2) protected hamsters against infection with PUUV but not against infection by related HFRS-associated hantaviruses. Unexpectedly, vaccination protected hamsters in a lethal disease model of Andes virus (ANDV) in the absence of ANDV crossneutralizing antibodies. This is the first evidence that an experimental DNA vaccine for HFRS can provide protection in a hantavirus lethal disease model

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs