@phdthesis{VinaRodriguez2018, author = {Ariel Vi{\~n}a Rodr{\´i}guez}, title = {RNA virus detection and identification using techniques based on DNA hybridization}, journal = {Nachweis und Identifizierung von RNA-Viren mittels DNA-Hybridisierungs-Techniken}, url = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-21758}, pages = {112}, year = {2018}, abstract = {Humanity is constantly confronted with the emergence and reemergence of infectious diseases. Many of them produce large or devastating epidemics, like AIDS (HIV) and Ebola. Others have been long neglected, yet pose immediate threats to global public health as evidences the abrupt emergence of Zika virus in South America and its association with microcephaly in babies. The examples illustrate, that many of these diseases are provoked by RNA viruses. One of the first steps in understanding and eliminating those threats is the development of sensitive and rapid diagnostic methods. A general and relatively rapid method is the direct detection and examination of the agent’s genome. However, the nature of (re)emerging RNA viruses poses a series of very specific problems for the design of such methods. Therefore, a systematic approach was proposed for the design of DNA-hybridization-base methods to detect and characterize RNA viruses that will have both a high sensitivity and a specificity sufficiently broad to detect, per reaction, down to a single copy of any of the possible variants of the viral genome. Following this approach a series of assays were designed, developed or adapted and put into use for detection and characterization of important RNA viruses. One of those viruses is West Nile virus (WNV), which after its explosive introduction into USA become the most widespread flavivirus throughout the world and, consequently, many countries began an intensive monitoring. While existing assay detected predominantly the Lineage 1, in Europa Lineage 2 was expected. Two new RT-qPCR for the detection of both lineages were developed, and reportedly used by independent laboratories. Due to more than 50000 associated deaths per year, the Hepatitis E virus also received an increasing attention to elucidate novel routes of transmission. This virus (especially genotype 3) has the zoonotic potential of transmission from pigs and wild boar to humans. RT-qPCR and nested qPCR for detection and characterization of this virus as well as a methodology for subtyping were developed and the first detected case of subtype 3b in a German wild animal was documented. In addition a novel assay for flaviviruses conformed by a RT-qPCR coupled with a low density DNA microarray was developed, which enabled the identification of WNV in mosquitoes from Greece. A RT-qPCR suitable for surveillance and diagnostic of all known variants of Venezuelan equine encephalitis virus was developed too. A causative agent of hemorrhagic infections, the Ngari virus, was detected and characterized in animal samples from Mauritania. These achievements were supported by the development of software applications for selection and visualization of primers and probes from aligned DNA sequences and for modeling of DNA hybridizations using unaligned sequences. In conclusion a general methodology for rapid development of sensitive diagnostic methods based in DNA-hybridization technics (PCR, sequencing and microarray) was stablished and successful applications are reported.}, language = {en} }