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

Institut Pasteur, Unit of Epidemiology and Pathophysiology of Oncogenic Viruses, Paris, France
Clinical Microbiology and Infection (Impact Factor: 5.77). 10/2012; 19(2). DOI: 10.1111/1469-0691.12075
Source: PubMed


Several 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 10(5) -10(6) 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.

Download full-text


Available from: Tatjana Avsic Zupanc, Aug 21, 2015
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background: Resequencing DNA microarray (RMA) technology uses probes designed to identify a panel of viral sequences. It can be used for detecting emerging viruses by revealing the nucleotide polymorphisms within the target of interest. Objectives/study design: As a new tool for molecular diagnosis of arbovirus infection, high density PathogenID v2.0 RMA (PID2-RMA) was assessed for the detection and genetic analysis of dengue, West Nile, and Chikungunya viruses in spiked blood samples or sera from individuals infected with dengue virus. Viral RNAs extracted from biological samples were retrotranscribed into cDNA and amplified using the Phi 29 polymerase-based method. This amplified cDNA was used for hybridization on PID2-RMA. Results: A good specificity of RMA-based detection was demonstrated using a panel of arboviruses including Dengue, West Nile and Chikungunya viruses. This technology was also efficient for the detection and genetic analysis of the different serotypes of dengue virus in sera of infected patients. Furthermore, the mixing of dengue, West Nile and Chikungunya prototype viruses within a single sample of human blood did not interfere with the sensitivity of PID2-RMA. Conclusions: Our data show that high density PID2-RMA was suitable for the identification of medically important arboviruses. It appears to be particularly adapted to the genetic analysis of dengue, West Nile, and Chikungunya viruses in urgent clinical situations where the rapid identification and characterization of the pathogen is essential.
    No preview · Article · Dec 2012 · Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: For a few decades, the introduction and development of molecular methods in microbiology have shaped the detection and characterization of pathogens. Although serological and, more punctually, viral culture methods remain basic tools for viral diagnosis, molecular advances based on qPCR have brought a number of novel advantages, in terms of speed, specificity and costs. On the other hand, microarrays have demonstrated their own advantages by increasing drastically the capabilities of detection and characterization of a large range of viruses in a unique step. Nowadays, several microarray-based platforms exist that can be classified in different families according to the type of matrix (solid or liquid), the size and density of probes, the method used for visualizing hybridization results with the target and finally relative costs. The aims of this review will be to overview (i) basic concepts of the different technologies used and to enlighten differences, advantages and drawbacks of each type of platform and (ii) the applications in virology for the detection and characterization of viral agents
    Full-text · Article · Mar 2013 · Virologie
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.
    Full-text · Article · Jun 2014 · PLoS ONE
Show more