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Browsing by Author "Rönnqvist, Maria"

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  • Rönnqvist, Maria (University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2009)
    In humans influenza viruses cause infectious respiratory disease, which can in worst case be lethal. Particularly virulent are those influenza A virus strains against which the patient has no previous immunity. It is known that birds serve as reservoir for all influenza viruses. Approximately ten years ago it was found that bird influenza A virus (H5N1) can be transmitted from birds to man causing serious infection. Very little is known about the significance of water in spread of influenza viruses. However it is known that influenza A viruses in birds multiply in the gut. Particularly duck-type of birds excrete large amounts of viruses to lakes and oceans in their feces. It has been shown that influenza viruses could stay infectious as long as 30 days if the water is salt free, moderately alkaline and cool. It is presumed that influenza viruses can survive over the winter buried in ice and be still infectious when the ice melts in spring. The goal of this study was to develop a filtrating method that could be used to detect influenza A viruses from water samples. Three commercial filters, Zetapor, Millipore (SMWP) and Sartorius (D5F), were compared. The concentration of viruses is based on electric interaction between the filters and the viruses. An additional goal was to figure out if the results of the filtration could be improved by dipping the filter to chickens, pigs or cows serum before the filtration. These sera differ from each other in carbohydrate chain sialic acid bonding. In this study human influenza A virus strain (H3N2) and bird influenza A virus strain (H5N2) were used. The desired amount of the virus was added to distilled water and the specimen was poured into the filtrating system that worked on negative pressure. To test the receptor specificity Millipore filters were incubated in inactivated chicken, pig or cow serum before filtration. The viruses were eluted from the filter membrane using a lysis buffer from RNA purification kit (QIAamp viral mini RNA kit). In the buffer the genome of the virus was released from its structural components. RNA was purified using the same kit and detected by real-time RT-PCR. In this study Millipore filter was the most efficient in filtrating influenza viruses. Virus recovery using this filter varied between 62,1% and 65,9%. It was 2,5 to 21,2 times more efficient than the other two filters. The second best filter was Zetapor (9,5-24,7%), although the difference between Zetapor and Sartorius (3,1-18,6%) was minimal. Dipping the filter into the sera showed no difference to the results compared to filters without serum. Millipore nitrocellulose filter would be a good choice for filtrating lake and sea waters because of its large pore size. Because the amounts of the viruses in natural waters are most likely very small, each step in the method should be optimized to further increase the sensitivity of the filtration method.