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Home » Archive » 2013

TDK conference 2013

Investigation of the virulence markers of West-Nile virus
Kuczmog Zita - year 5
SzIU, Faculty of Veterinary Science, Department of Microbiology and Infectious Diseases
Supervisors: Tamás Bakonyi DVM, Katalin Szentpáli-Gavallér DVM

Abstract:

Certain strains of the West Nile virus (WNV) are significantly different regarding their neuroinvasiveness. Construction of point-mutated infectious clones of a lineage 2 WNV strain that circulates in Eastern Europe allows us to identify possible genetic markers that can modify its virulence.

Construction of the full-length infectious clone was performed simultaneously in two different ways. First, the virus was propagated in African green monkey kidney epithelial (Vero) cells and a single-stranded complementer DNA (cDNA) was generated with genome-specific primers that were designed based on the the full-length genomic sequence of the virus.

The principle of the first way was the amplification of long overlapping PCR products by polymerase chain reactions (PCRs) which were joined with subsequent fusion PCRs covering the whole genome. The prepared full-length double-stranded cDNA was preceded by the human cytomegalovirus (CMV) promoter or the T7 bacteriophage promoter. The transcription was established in vivo by CMV promoter, after transfection into BHK-21 cells, while in the case of the T7 promoter transcription was performed in vitro using the mESSAGE mACHINE kit (Ambion) before transfection of baby hamster kidney BHK-21 cells.

The basis of the second way was the incorporation of the cDNA of the full-length virus into the low-copy number pBeloBAC plasmid that contained the CMV promoter. After digesting the overlapping PCR-products and the plasmid with the respective restriction enzymes, the cDNA containing the whole genome of the virus was cloned into the pBeloBAC plasmid in three consecutive steps to obtain CMV-WNV plasmid. Finally, CMV-WNV plasmid was propagated in E.coli DH10B competent cells. The transcription was established in vivo (by CMV promoter, after transfection into BHK-21 cells).

Lineage 1 viruses that have phenotypes of reduced virulence in mice and inefficient growth in cell culture have already been identified. Applying reverse genetic methods, these single amino acid alterations that were previously proven to lead to attenuation in vitro and in vivo, were generated in our lineage 2 infectious clone. Point mutations were inserted in the genome of WNV using PCR-based mutagenesis with specific oligonucleotides (forward and reverse complement oligos) containing the altered nucleotides. The newly synthesized, modified fragments were placed into the CMV-WNV plasmid by digestion with restriction endonucleases followed by ligation. The in vivo transcription was applied after transfection of BHK-21 cells.

Viruses with certain amino acid alterations showed different growth properties in Vero cells.

The research was supported by the VECTORIE EU FP7 project.



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