Prof.MVDr. Vladimír Celer, DrSc.
THEMATIC RESEARCH FOCUS
- Porcine ssDNA viruses
- Porcine arteriviruses
- scFv antibodies
- Protein expression
To obtain novel information on the causes, mechanisms and spreading of infectious diseases in domestic animals.
CONTENT OF RESEARCH
Analysis of the causes, mechanisms and spread of infectious diseases in domestic animals
Characterisation of virulence factors of viruses
The main objective is to elucidate the pathogenesis of some single stranded DNA viruses and arteriviruses in sows. Model viruses for this study will be porcine circovirus 2, porcine anelloviruses and porcine reproductive and respiratory syndrome virus. Sequencing of virus strains from different clinical conditions will be used to define ORFs, which could have the impact on virus virulence. Virus genomes will then be cloned into plasmid vectors to obtain infectious molecular clones as the conserved genetic material of these model viruses. Transcription of potential virulence factors will be subsequently modified by site directed mutagenesis and by siRNA molecules in vitro and in vivo models. In vitro model will be performed on tissue culture transfected with modified molecular clones and then analysed by quantitation of mRNA expression. For in vivo models infection of specific host animals will be used to analyse virulence of molecular clones.
Specific inhibition of virus replication by single-chain variable fragment mediated intracellular immunisation
The main objective is to evaluate the protective effect of intracellular immunisation mediated by single-chain variable fragment derived intrabodies against different viruses. We expect that intrabodies would inhibit afferent parts of the viral life cycle as well as replication of the virus by blocking virus maturation.
Porcine circovirus 2 and porcine arterivirus (PRRRS) will be used as model viruses in this work package. Target virus proteins used for single-chain variable fragment selection will be expressed as recombinant proteins in Escherichia coli and Pichia pastoris expression systems. Recombinant single-chain variable fragment antibody fragments against these virus proteins (nonstructural proteins, nucleocapside) will be selected from appropriate phagemide library, e.g. Tomlinson, Griffi n1. Selected single-chain variable fragment clones will be characterised by sequencing and determination of affinity constant. Gene coding variable fragments responsible for antigen binding will be cloned into plasmid vectors enabling expression of antibody fragments in different cell compartments. Then, transgenic cell lines will be established and challenged with corresponding virus strains. Inhibition of virus replication will be monitored by virus titration, antigen capture assay and immunofluorescence. To monitor at the genomic level (virus genome and messenger ribonucleic acid) the different phases of the virus replication cycle real-time PCR and reverse transcriptase – PCR combined with Southern blotting will be applied.
KEY RESEARCH EQUIPMENT
PLANNED RESEARCH INFRASTRUCTURE
CURRENT RESEARCH INFRASTRUCTURE
The research infrastructure of Molecular virology group allows the routine use of cell cultures, sequencing and cloning, expression and purification of viral antigens. The laboratory is equipped with ultracentrifuges with suitable rotors, equipment for the automated subtraction and evaluation of ELISA tests, equipment for SDSPAGE, immunoblot and a thermal cyclic reactor for quantitative PCR. The laboratory is fully equipped for routine procedures associated with the cloning of virus genes into plasmid vectors and transformation or transfection of bacterial and eukaryotic cells, expression and the purification of recombinant proteins and other procedures.
- Porcine anelloviruses – prevalence, genotypization and pathogenesis (ME08108), Ministry of Education, Youth and Sports, 2008-2012, V. Celer, University of Veterinary and Pharmaceutical Sciences Brno.
- The role of small ORFs in the pathogenesis of porcine circovirus 2 diseases (GA524/09/0673), Czech Science Foundation, 2009-2012, V. Celer, University of Veterinary and Pharmaceutical Sciences Brno.
- Development of preparation with content of anti-sense oligonucleotides in nanoparticals for the local treatment of herpes virus infections caused by viruses HSV – 1 and HSV – 2 (FR-TI1/200), Ministry of Industry and Trade, 2009-2013, M. Krajícek, FAVEA, Ltd., R. Horváth, Genex CZ, Ltd., V. Celer, University of Veterinary and Pharmaceutical Sciences Brno.
- JAROSOVA, V., CELER, V., POGRANICHNIY, R. Prevalence and age distribution of porcine torque teno sus virus (TTSuV) in the Czech Republic. Folia microbiologica. 2011, 56(2). p. 90-94.
- ROSENBERGOVA, K., LANY, P., POSPISIL, Z., KUBICEK, O., CELER, V. Quantification of avian influenza virus in tissues of mute swans using TaqMan real time qRT-PCR. Veterinarni Medicina. 2009, 54(9), p. 435–443.
- LOBOVA, D., CIZEK, A., CELER, V. The selection of single-chain Fv antibody fragments specific to Bhlp 29.7 protein of Brachyspira hyodysenteriae. Folia Microbiologica. 2008, 53(6), p. 517-521.
- MOLINKOVA, D., SKLADAL, P., CELER, V. In vitro neutralization of Equid herpesvirus 1 mediated by recombinant antibodies. Journal of Immunological Methods. 2008, 333(1-2), p.186-191.
- TRUNDOVA, M., CELER, V. Expression of porcine circovirus 2 ORF2 gene requires codon optimized E. coli cells. Virus Genes. 2007, 34(2), p. 199-204.