Etablierung allgemein anwendbarer Influenza A-Virusnachweise auf Basis immunkompetenter Peptid-Epitope im Vergleich zu RT-PCR aus Saliva

The influenza virus continues to be a great danger to the health mankind. Within defined intervals it comes to pandemics effected by the virus. These can lead to thousands of deaths. The treatment, diagnosis and prophylaxis by vaccination can be difficult due to the high variability of the virus. An antigenic shift may lead to the development of a virus strain resistant against the vaccination. The antigenic shift was e.g. the reason for the emergence of the Spanish flu with millions of casualties. Therefore, further research on the virus and the improvement of diagnostics and prevention is necessary. In the first section of this paper, two potential proteins were investigated that could be deduced as an alternative reading frame by means of the primary sequence of the influenza virus genome. The expression products of these open reading frames (ORF) were not described until now. To identify the putative expression products it was neccessary to isolate the coding sequences by PCR first and to clone them into a bacterial expression vector eventually. The expressed proteins from this vector should be used to determine the prevalence of antibodies against these proteins in human sera. Furthermore, these proteins should be used for the production of polyclonal antibodies in rabbits. The expressed proteins were isolated for this purpose and presented in their pure form. Despite the use of different purification protocols and expression vectors, the proteins were expressed indeed but could not be reproduced sufficiently in quantity and purity to conduct further investigations. The second part dealt with saliva as a possible material for influenza diagnosis. The advantage of Saliva is the easier extraction compared to a throat swab. First, the stability of influenza virus in saliva was investigated. We could show that even after 120 hours, influenza virus RNA can be detected without reducing the detection limit in Saliva. As next step, the sensitivity and specificity were determined. For this purpose, 57 saliva samples from infected patients, which were detected in throat swabs by an influenza quick test, were examined by RT-PCR. In addition, 20 samples from healthy persons were analyzed. This resulted in a sensitivity of 56% and a specificity of 100%. Saliva can certainly not replace the throat swab as a test material, but could be an alternative, e.g. as part of epidemiological investigations or in the absence of compliance for a throat swab.