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
2016
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.
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