WORKINGGROUP 5
  • Participants:

    Participant
    Organization
    E-mail
    Phone number
    Fax
    Anders Sj?stedt  Ume? University  anders.sjostedt@climi.umu.se  +46907851125  +46907852225 
    Forsman Mats  FOI, Swedish Defence Research Agency  mats.forsman@foi.se  +4690106669  +4690106800 
    Frank Vandenbussche  CODA-CERVA-VAR  frvan@var.fgov.be  +32(0)2 379 05 12  - 
    Gernot Schmoock  Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses  gernot.schmoock@fli.bund.de  +049-3641-804467  +049-3641-804228 
    Isabel Jado  Instituto de Salud Carlos III  ijado@isciii.es  -  - 
    Janusz Tadeus Paweska  Nation Inst for Communicable Diseases of the NHS, Sandringham, South Afrika  januszp@nicd.ac.za  +27 11 3866336  +27 11 38823741 
    Laura Brum  -  laura.brum@insa.min-saude.pt  -  - 
    M. David ALBERT  AFSSA  d.albert@alfort.AFSSA.FR  -  - 
    Mandy Elschner, Chair  Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses  mandy.elschner@fli.bund.de  +049-3641-804428  +049-3641-804228 
    Manfred Weidmann  Institute of Virology, G?ttingen  mweidma@gwdg.de  +49-551-3899-406  +49-551-3899-439 
    Nadya Brankova  Nat Center of Infections and Pararsitic Diseases  nbrankova@abv.bg  +359 2 944 6999  - 
    Nigel J. Silman  HPA  nigel.silman@hpa.org.uk  -  - 
    Nora Madani  AFSSA  n.madani@alfort.afssa.fr  -  - 
    Patrick Butaye  CODA-CERVA-VAR  pabut@var.fgov.be  + 32 (0)2 379 04 15  + 32 (0)2 379 06 70 
    Paula Pilo  University of Berne, Inst of Vet Bacteriology  paula.pilo@vbi.unibe.ch  +41 31 631 2491  +41 31 631 2634 
    Pierre-Alain Fontayne  WIV-ISP  PAFonteyne@iph.fgov.be  +32(0)2 642 55 33  - 
    Plamen Padeshki  National Center of Infectious and Parasitic Diseases, Dep of Microbiology  ppadeshki@yahoo.com  -  - 
    Raquel Escudero  Instituto de Salud Carlos III  rescude@isciii.es  -  - 
    Todor Kantardjev  National center of infectious and parasitic diseases  kantardj@ncipd.netbg.com  +359 2 846 55 20  +359 888 567 660 


  • Description:
    WG5 Microbiology (bacteriology, mycology and virology).
    • Objective:
      Selection of a range of pathogens, including multiple strains of each species. Inactivated material such as DNA/RNA (genomic research), cell-free extracts (antigenicity and proteomics) will be prepared. To definition the taxonomical relations between the closely related microorganisms. To develop primers to allow random amplification of viral RNA from a mammalian RNA background.
    • Description:
      This work package will bring together experts in a range of pathogenic microorganisms. The bacterial pathogens that will be studied are Bacillus anthracis, Brucella spp., Burkholderia spp. (B.mallei & B.pseudomallei), Coxiella burnetti (and closely related Rickettsiae), Yersinia pestis (plus Y. pseudotuberculosis), Francisella tularensis, and Clostridium botulinum. The yeasts and filamentous fungi under investigation will be Aspergillus fumigatus and A. flavus, Blastomyces dermatitidis, Candida albicans, Cladophialophora bantiana and C. devriesii, Coccidioides immitis and C. posadasii, Cryptococcus gatti and C. neoformans, Exophiala dermatitidis, Fusarium verticillioides, Histoplasma capsulatum, Paracoccidioides brasiliensis, Penicillium marneffei, Pseudallescheria boydii, and Ramichloridium mackenziei. Viruses from the RNA virus families Bunyaviridae, Filoviridae, Arenaviridae, Togaviridae, Paramyxoviridae, Orthomyxoviridae and from the DNA virus family of the Poxviridae are prime candidates. Virus strains of emerging viruses are not easily available. Since the anthrax attacks in the US, virus collections independent of the US and former Soviet research establishment, have become very important to guarantee free research in Europe. The coordination of culture tasks and growth protocols as well as quality control and storage of the extracts could be the focal point of a new European collection of emerging microorganisms. The acquisition of new strains from around the world therefore is also a necessary task. These viruses could be deposited and curated within the pre-exisiting National Collection of Pathogenic Viruses (NCPV) housed within the Health Protection Agency (Porton Down site) in the UK.

      Cell-free extracts (DNA extracts, fractions:cell wall, cytoplasmic extract, cell filtrates) from each of the strains examined will be prepared for use by the participants. Such extracts need to be validated as free of infectious agents and suitable for use in BSL (ACDP) level 2 laboratories and below.
      For use in antigenicity and immunogenicity studies, live, killed or fixed whole cells of each strain may also be prepared. These cells are potentially of use for raising polyclonal antisera or MAb, and also screening antisera produced from either cell-free extracts. Specific attention will be made on the large genomes of these organisms and the attribution of these to the post-genomic activities.
      Optimisation of protocols for preparation of these deliverables will be done in close collaboration with the concerning WG?s in order to obtain high quality products.
      The strain collection also will be further characterised with emphasis upon defining unique regions allowing identification at the genus or species level and below. New developments in the taxonomy of these species will be followed in order to be prepared when new closely related species are described which might be of interest in the differentiation of the species under investigation. Multiple geographic isolates of microorganisms of interest will be needed. All of these strains will be examined using conventional microbiological techniques (morphology, staining, biochemistry, molecular techniques e.g.16S rRNA gene sequence, RFLP, AFLP, PCR-Restriction, etc.) and all strains under investigation will be fully characterised, both microbiologically and genetically, and a genotype assigned (where an appropriate genotyping scheme exists).
      Another approach with the aim of defining unique DNA regions within the genomes of interest would be to apply such techniques as fluorescent AFLP (Amplified Fragment Length Polymorphism) and other similar technologies (e.g. Random amplified polymorhic DNA (RAPD), tandem repeats, IS elements, SNP?s, REA, etc.). It has been observed that within the profiles of different organisms unique genus, species or strain specific restriction fragments have been identified. The species-specific unique DNA fragments will be of major interest. The specificity of the fragments will be evaluated by reverse line blot hybridisation.
      DNA preparations will also be amplified using PCR with consensus primer pairs. Examples of such primer pairs are those widely reported for amplification of 16S and 23S rRNA genes. Primers to amplify other genes, conserved within a particular species, will also be employed (eg. gyrA, sod,?). Also intergenic spacers between conserved regions will be investigated. Focus will be set on the intergenic spacers between the tRNA genes (possible sites for insertion of virulence associated genes) and 16S-23S intergenic spacers and the internally transcribed spacer region 2 between the 5.8S and 28S rRNA genes for fungi.
      The identification of viruses always has to occur against a mammalian background. It is very difficult to enrich viruses from patient material. An approach to identify viruses by hybridisation arrays from clinical or environmental sources can only work if viral material can be selectively amplified. Since RNA viruses are notoriously diverse, it seems impossible to define specific targets for each pathogenic RNA virus. Therefore the aim is to design and test random primers for RAPD that specifically match viral RNA. In order to be able to design primers for random amplification that specifically match to RNA viruses bioinformatic approaches that allow tetranucleotide analysis up to decanucleotide analysis need to be investigated. The approach published by Yap et.al (2003) seems to be appropriate to define such RNA virus specific tenmers for RAPD. To use this approach computer facilities for the calculation need to be identified as at least a Sun Fire server with 24 CPUs each running at 900 Mhz is needed for the calculation.
      The resulting primers will be distributed to test them on the viral RNA extracts of the viruses from their collections. PCR trials will follow to come up with universal protocols to amplify viral RNA from cell cultures. PCR bands generated by this approach will be ligated into plasmids and sequenced. This will happen in close collaboration with WG 4. The viral sequences obtained will then be spotted onto the arrays of the WG1.
      The family of the Poxviridae is composed of dsDNA viruses with genomes ranging from 130-375Kb. In the genus Orthhopoxvirus (OPV) camelpox virus (CMPV) is most closely related to variola virus VAR (Gubser and Smith, 2002). Both viruses are host specific. The high frequency of genomic recombination in OPVs or simple mutations in the CMPV genes coding for the virus virulence factors or for virus coat proteins interacting with the cell receptor-binding sites could cause a breakthrough in the host species barrier. These mutations could occur naturally or artificially. The CMPV antigens and genome will be analysed, looking for unique genes and proteins responsible for the genes mentioned above. Other OPVs are to be included for differential diagnosis such as monkeypox, cowpox and vaccinia virus as these are all infectious to humans. These segments could be used in the early diagnosis of potential dangerous mutants or hybrid viruses.
      Genomic libraries of the viruses under investigation will be constructed. The libraries should not contain large fragments in order not to loose their differential capacities. Differences between these viruses under investigation could be missed when larger fragments would be investigated, since these would allow more non specific hybridisation.
      Furthermore, for testvalidation this group has all means for the validation of an eventual diagnostic test coming out of this research. Initial estimates of repeatability and of analytical sensitivity and specificity may be investigated. The application itself should delineate the minimum acceptable requirements for diagnostic sensitivity and specificity.
  • Actions:

    Action
    Description
    download
    EU green paper on Bio preparedness Report on the meeting dealing with the EU green paper on bio-preparedness
    Reimbursment rules for training course COST rules for training courses
    Strain Collection database Strain Collection Database is now available
    Training School Date and programm for 2nd training school in G?ttingen, 2008


  • Publications:

    Name
    Year
    Title
    Periodical
    Volume
    Page
    Pubmed
    Isabel Jado, Raquel Escudero, Horacio Gil, Mar?a Isabel Jim?nez-Alonso, Rita Sousa, Ana L. Garc?a-P?rez, Manuela Rodr?guez-Vargas, Bruno Lobo, Pedro Anda 2006 A molecular method for the identification of Rickettsia species in clinical and environmental samples J Clin Microbiol 44 4572-4576 link to pubmed
    Rosa de los R?os Mart?n, Juan Carlos Sanz Moreno, Fernando Mart?n Mart?nez, M? ?ngeles T?bar Beteg?n, Marta Cort?s Garc?a y Raquel Escudero Nieto. 2006 Q fever outbreak in an urban area following a school-farm visit. Med Clin (Barc) 162 573-575. link to pubmed
    Sanz JC, de los Rios R, Martin F, Tebar MA, Jado I, Anda P. Application of four ELISA techniques (two for IgM and two for IgG) for serological diagnosis of an outbreak of Q fever. Enferm Infecc Microbiol Clin. 2006 24: 2006 Application of four ELISA techniques (two for IgM and two for IgG) for serological diagnosis of an outbreak of Q fever. Enferm Infecc Microbiol Clin. 24 178-81 link to pubmed
    J.E. Burton, O.J. Oshota and N.J. Silman 2006 Differential identification of Bacillus anthracis from environmental Bacillus species using microarray analysis. Journal of Applied Microbiology 101 754?763 link to pubmed
    Spiegel M, Schneider K, Weber F, Weidmann M, Hufert FT 2006 Interaction of SARS Coronavirus with Dendritic Cells General Virology 87 1953-60 link to pubmed
    Weidmann M, Schmidt P, Hufert FT, Krivanec K, and Meyer H 2006 Tick borne encephalitis virus in Clethrionomys glareolus in the Czech Republic Vector-Borne and Zoonotic Diseases (accepted) - link to pubmed
    Weidmann M, Hufert FT, Sall AA 2006 Viral load among patients infected with Marburgvirus in Angola J Clin Virol (accepted) - link to pubmed
    Henrik Andersson, Blanka Hartmanov?, Rhonda KuoLee, Patrik Ryd?n, Wayne Conlan, Wangxue Chen, Anders Sj?stedt. 2006 Transcriptional profiling of host responses in mouse lungs following aerosol infection with type A Francisella tularensis. J. Med. Microbiol. 55 263-271 link to pubmed
    Henrik Andersson, Blanka Hartmanov?, Erik B?ck, Henrik Eliasson, Mattias Landfors, Linda N?slund, Patrik Ryd?n, Anders Sj?stedt. 2006 Transcriptional profiling of the peripheral blood response during tularemia. Genes Immun. 7 503-513 link to pubmed
    Henrik Andersson, Blanka Hartmanov?, Patrik Ryd?n, Laila Noppa, Linda N?slund, Anders Sj?stedt. 2006 A microarray analysis of the murine macrophage response to infection with Francisella tularensis LVS. J. Med. Microbiol. 55: 1023-1033. link to pubmed
    Patrik Ryd?n, Henrik Andersson, Mattias Landfors, Linda N?slund, Blanka Hartmanov?, Laila Noppa and Anders Sj?stedt. 2006 Evaluation of microarray data analysis methods using spike-in experiments. BMC Bioinformatics 7 300-317 link to pubmed
    Jane E. Burton, O. James Oshota, Emma North, Michael J. Hudson, Natasha Polyanskaya, John Brehm, Graham Lloyd, Nigel J. Silman* 2005 Development of a multipathogen oligonucleotide microarray for detection of Bacillus anthracis. Molecular and Cellular Probes 19 349?357 link to pubmed
    Weidmann M, Schmidt P, Vackova M, Krivanec K, Munclinger P, Hufert FT 2005 Identification of genetic evidence for dobrava virus spillover in rodents by nested reverse transcription (RT)-PCR and TaqMan RT-PCR. J Clin Microbiol 43 808-812 link to pubmed
    Isabel Jado, Jos? A. Oteo, Mikel Ald?miz, Horacio Gil, Raquel Escudero, Valvanera Ibarra, Joseba Portu, Aranzazu Portillo, Mar?a J. Lezaun, Cristina Garc?a-Amil, Isabel Rodr?guez-Moreno, Pedro Anda. 0 Rickettsia monacensis, a new rickettsia species causing human disease. Emerg Infect Dis (in press). - - link to pubmed