Investigation of hantavirus in western regions of Turkey: Investigation of the presence of hanta virus in rodents in Thrace region by using polymerase chain reaction

Hantavirus is the RNA virus which has three-segmented and negative-strand genomes. It causes two major syndrome in humans. The first is hemorrhagic fever with renalyndrome (HFRS) in Asia and Europe, the other is hantaviruspulmoner syndrome (HPS) in USA. Hantavirus is carried by rodent species and each rodent species is unique to only one Hantavirus species. Serological and molecular methods are used for the diagnosis of hantavirus. Immun Fluorescence Antibody Test (IFAT), Enzyme-Linked Immuno Sorbent Assay (ELISA), Focus Reduction Neutralization Test and Stripİmmunoblot method are generally used as serological methods, also IFAT and ELISA are used to detect high avidity IgG antibodies in epidemiological studies. Molecular methods are highly important for rapid diagnosis because hantavirus infections can cause death within 12 to 24 hours. Since viral RNA presences in low-levels in tissue samples of human and rodents, the nested reverse transcription polymerase chain reaction (nested-RT-PCR) which is performed by specific primers to regions with high homology is necessary. In this study, the rodents collected from Thrace Region have been screened for the presence of Dobrava, Saarema and Puumala viruses by using optimized nested-RT-PCR. Specific primers for each three species were designed and synthesized prior to the optimization study. RNA extraction from rodent tissues have been performed according to extraction kit manufacturer's instructions. It has been decided in the light of the literature that PCR method is performed as nested PCR and concentration of MgCl2, cDNA, DMSO, primers, product and Tm were optimized respectively for Dobrova virus. These conditions also have been tested for each Saaremaa and Puumala viruses to the end that method optimization was finalized for three viruses, too. Then, cDNAs obtained from the RNA extracted from tissues have been screened for the presence of these three viruses. The positive results were not found for three serotypes in result of molecular screening. PCR method which is optimized in this study may also be used for further diagnostic and immunological studies. Keywords: Hantavirus, nested-RT-PCR, optimization