Browsing by discipline "Elintarvike- ja ympäristövirologia"
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(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.
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(University of HelsinkiHelsingin yliopistoHelsingfors universitet, 2007)Rotaviruses are double-stranded RNA-viruses. They are members of the family Reoviridae and are the cause of severe gastroenteritis in young humans and animals. Primary infection is the most severe and reinfections usually produce only mild symptoms. Worldwide about 600 000 children die every year from rotavirus infection, and this figure represents about 5 % of all deaths in children younger than five years. Virtually all children will be infected by the time they reach one year of age. Because primary infection is so severe, rotavirus vaccines have been developed. Surveillance studies are needed to monitor the vaccine impact on circulating viral strains. It is important to know whether some strains disappear and new strains, possibly from animals, emerge. This rotavirus genotyping study is part of a long-term European surveillance study, which tries to define and monitor rotavirus types circulating in Europe. Proteins VP4, VP6 and VP7 are the primary antigens of rotaviruses. Rotavirus is classified into serogroups A-G based on inner layer protein VP6. Group A-C rotaviruses are human pathogenes. Most human rotavirus diseases are caused by group A rotavirus. The group A rotaviruses are further classified into G and P types based on the outer layer proteins VP4 and VP7. 15 G and 14 P types have been identified in humans. In this study we established G and P types for 200 rotavirus samples by using nested-PCR. Materials consisted of stool samples originated from HUSLAB virology department and Kuopio University Hospital. Rotavirus positive stool samples taken from children during years 2005-2007. RNA was extracted from stool suspensions and it was translated to DNA and amplified by nested-PCR. As first step RNA was transcripted to cDNA by reverse transcription. cDNA was amplified from gene segment areas that code for VP4 and VP7 proteins. As second step these PCR-products were amplified using G and P type-specific primers. Final PCR products were G and P typed by using agarose electrophoresis. We find that most common G types in 2005-2007 were G1 and G9; other G types was also found. Most common P type was P[8], which constituted 90 % of all viruses. The most common G and P combinations were G1P[8] and G9P[8]. In epidemic season 2006-2007 the most common G types in Helsinki were G1 and G9. In Kuopio predominant stains were G2 and G9. The most common G and P combinations in epidemic season 2006-2007 were G1P[8] and G9P[8] in Helsinki and G9P[8] and G2P[4] in Kuopio. In recent years the most common G types in Europe have been G1-G4 and G9. Finnish genotype distribution is mainly congruent with G and P types in other European countries.
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