Molecular studies on the virus diversity and virus distribution in bats in Germany and development of a novel Enzyme-linked Immunosorbent Assay for the detection of henipavirus antibodies

Bats are receiving considerable scientific attention recently since they were found to carry a broad diversity of agents that are potential pathogens for humans and livestock. Some highly pathogenic viral agents like members of the Henipavirus genus were shown to have their natural reservoir in bats of the Pteropus genus. In the case of the zoonotic Hendra and Nipah viruses (HeV; NiV), spillover events to livestock and humans have occurred repeatedly. For Germany, some studies had already been conducted revealing certain viral families to be present in indigenous bats, including coronaviruses, astroviruses and paramyxoviruses. To better understand the virus ecology, diversity and potential transmission dynamics within and between bat colonies or from bats to other mammals, the first objective of this thesis was to investigate samples of German bats for the presence of these virus families. The second objective of the thesis was to develop tools for serological diagnostics of Hendra virus, an Australian bat-associated zoonotic paramyxovirus. In the first set of studies saliva, fecal and urine samples from indigenous vespertilionid bats from four different species were collected across Germany and RNA was extracted. More than 950 samples from 653 bats were analyzed by hemi-nested RT-PCRs targeting highly conserved genome regions of the three different viral families, namely astroviruses, coronaviruses and paramyxoviruses. Highest detection rates were observed for astroviruses with an overall rate of 25.8% and a maximum of up to 65% in some local bat colonies. Coronaviruses and paramyxoviruses generally displayed lower detection rates ranging between 1.4% and 3.1%. Obtained sequences were phylogenetically analyzed and compared to bat-associated and other sequences available in the GenBank database. Our findings for most of the astrovirus and coronavirus sequences from our study strongly suggest a bat species-specificity, independent of the spatial distance. With respect to astroviruses, we did not find evidence for interspecies transmission between the four analyzed bat species. Interestingly, some of the generated astrovirus sequences were clearly assigned to other mammalian and avian astroviruses from the database, among others to human astroviruses (HAstV) raising the question whether bats might act as intermediate hosts for these viruses. Since sequence data of henipa-like viruses as well as serological evidence for infections with these viruses have been found in African fruit bats and domestic pigs, the previously assumed limitation of henipavirus occurence to the Australasian region needed to be extended. For the development of diagnostic tools for future serological surveillance studies in Australia and Africa where fruit bats are abundant, it was of special interest to establish the stable and reliable expression of a recombinant HeV G protein. Therefore, a novel system based on the stable transfection of eukaryotic protozoa, Leishmania tarentolae, was chosen. This expression system soon delivered promising rapid cell growth, high cell densities and protein yield and mammalian-type glycosylation. A truncated, Strep-tag labeled and soluble version of the HeV attachment protein was expressed, purified and its authenticity was confirmed by immunoblotting and mass spectrometry. Although cloning strategy suggested the protein to be secreted in the cell culture medium, the protein was only detected in cell lysates. However, the protein showed to be functional in several assays interacting with the HeV cell entry receptor ephrin B2. Antigenicity was preserved in the recombinant protein as it induced cross-neutralizing antibodies in immunized rabbits. Serum samples from NiV and HeV infected pigs (cross-) reacted with the Leishmania-derived HeV G protein, indicating a potential use in serological assays for surveillance studies. This thesis contributes to a better understanding of the diversity and distribution of viruses harbored by indigenous bats as well as to getting insights into virus ecology, virus-host interplay and potential virus transmission. Furthermore, it has provided the basis for future serological surveillance studies in henipavirus-affected countries by expressing a functional recombinant form of the HeV glycoprotein G. This protein serves as an excellent basis for the establishment of valuable diagnostic tools to identify possible intermediate hosts and to further investigate the potential distribution of henipa-like and henipaviruses in these affected countries. With respect to Public Health, the identification of potential intermediate hosts as well as of virus transmission routes plays an important role in predicting zoonotic spillover events and possible health threats.