The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the... Show moreThe sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. lmmunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays. Show less
Hybrid zones occur where two species meet and produce offspring (hybrids). Typically, hybrids show a considerable reduction in fitness. In this thesis two hybrid zones are treated.Two species of... Show moreHybrid zones occur where two species meet and produce offspring (hybrids). Typically, hybrids show a considerable reduction in fitness. In this thesis two hybrid zones are treated.Two species of banded newts (Ommatotriton nesterovi and O. ophryticus) are thought to meet in a hybrid zone in the north of Turkey. In this thesis I confirm the species status of the two banded newt species based on mitochondrial DNA and two nuclear DNA sequences. The location of the hybrid zone is narrowed down to a 60 km wide region. If a hybrid zone is present between O. nesterovi and O. ophryticus, it is narrower than 60 km and it may be geographically stable. An introduced population of hybrid banded newts in Spain provides evidence that the two species can produce fertile offspring. This increases the likelihood that a hybrid zone indeed exists between the two species.Common and spined toads (Bufo bufo and B. spinosus) meet in an 800 km long hybrid zone that runs diagonally across France. A genetic footprint north of the hybrid zone was previously recorded and linked to southward hybrid zone movement, with B. bufo overtaking B. spinosus. To test hypotheses of hybrid zone movement, a transect in northwest France was studied with 31 nuclear markers. The contrasting results suggest that stronger reproductive isolation on the B. spinosus side of the hybrid zone than on the B. bufo side, may be more likely than hybrid zone movement.To continue the research on the Bufo hybrid zone, two distant transects, one in the northwest and one in the southeast of France, were studied using 1200 nuclear markers. Asymmetries which were previously found in the literature and in this thesis, were confirmed for the hybrid zone in northwest France, but not in the southeast, where the gene flow appears to be symmetric, indicating the hybrid zone is stable here. Barrier markers, genetic regions which may be associated with barrier genes, were identified by their relatively restricted gene flow. The barrier markers present in both transects suggest that the two species have evolved a universal genetic barrier to gene flow. The differences in patterns of gene flow between the transects may be caused by genetic divergence within B. bufo, documented in previous phylogeographical work. We can clearly no longer think of hybrid zones as static upon formation; hybrid zones evolve! With the increasing availability of genome resources, detection of more detailed patterns of differential introgression along the genome in hybrid zone studies will reveal the genetic architecture of speciation and the evolution of hybrid zones. Show less
The mechanisms of the evolution and development of the heart in metazoans are highlighted, starting with the evolutionary origin of the contractile cell, supposedly the precursor of cardiomyocytes.... Show moreThe mechanisms of the evolution and development of the heart in metazoans are highlighted, starting with the evolutionary origin of the contractile cell, supposedly the precursor of cardiomyocytes. The last eukaryotic common ancestor is likely a combination of several cellular organisms containing their specific metabolic pathways and genetic signaling networks. During evolution, these tool kits diversified. Shared parts of these conserved tool kits act in the development and functioning of pumping hearts and open or closed circulations in such diverse species as arthropods, mollusks, and chordates. The genetic tool kits became more complex by gene duplications, addition of epigenetic modifications, influence of environmental factors, incorporation of viral genomes, cardiac changes necessitated by air-breathing, and many others. We evaluate mechanisms involved in mollusks in the formation of three separate hearts and in arthropods in the formation of a tubular heart. A tubular heart is also present in embryonic stages of chordates, providing the septated four-chambered heart, in birds and mammals passing through stages with first and second heart fields. The four-chambered heart permits the formation of high-pressure systemic and low-pressure pulmonary circulation in birds and mammals, allowing for high metabolic rates and maintenance of body temperature. Crocodiles also have a (nearly) separated circulation, but their resting temperature conforms with the environment. We argue that endothermic ancestors lost the capacity to elevate their body temperature during evolution, resulting in ectothermic modern crocodilians. Finally, a clinically relevant paragraph reviews the occurrence of congenital cardiac malformations in humans as derailments of signaling pathways during embryonic development. Show less
