Microscopy of hepatitis viruses
A microscopy unit was established at the Heidelberg Partner Site for research groups from the research field “Hepatitis” and other research fields including “Emerging Infections”, “Malaria and Neglected Tropical Diseases” and “HIV” in particular. The working group microscopy of hepatitis viruses enables research groups to conduct their analyses and investigate live cells and tissues infected with hepatitis viruses and other pathogens at the required biosafety levels 2 and 3. Using the imaging methods provided for at this working group, researchers have been able to decode the mechanisms of action of different antiviral substances and have also been able to characterise the various interactions between viruses and host cells.
In addition, electron and correlative microscopy methods are available for use in joint projects. Furthermore, training in light and electron microscopy is provided.
Hepatitis viruses that are pathogenic for humans, such as the hepatitis B virus (HBV) and the hepatitis C virus (HCV), may only be investigated in biosafety level 3 laboratories. Biosafety level 3 laboratories are only available on a limited basis due to the elaborate technical requirements, and advanced microscopy techniques are rarely available within these laboratories as they are very costly. This presents a major obstacle to research as the viruses and many other pathogens can only be studied in live cells.
See more about the Hepatitis C virus life cycle
For this reason, Professor Bartenschlager established the working group microscopy of hepatitis viruses within his department to enable the investigation of cells infected with hepatitis and other viruses at high biosafety levels. This working group provides the opportunity to conduct microscopic analysis of live cells, for example, to study the dynamics of an infection or the efficacy of antiviral drugs. It has enabled scientists to demonstrate that daclatasvir and other NS5A inhibitors block hepatitis C virus replication by preventing the virus from building replication factories. Our working group also played a crucial role in describing a new pathway of HCV spread within a cell cluster through extracellular vesicles. These vesicles utilise the cell's own lipoprotein system and escape the antiviral antibodies of the immune system.
Correlative light and electron microscopy (CLEM) of liver cells infected with genetically modified hepatitis C viruses (HCV) and treated with the daclatasvir drug. Top left: Fluorescence microscopy of the live cells. The cell marked with a white-dotted square is enlarged in the centre image. Top right: Superposition of electron microscope image (EM) and a fluorescence image of the same cell. EM images were produced of the areas marked in yellow, which are visible in the centre row. The centre row shows EM images of cells that were infected with daclatasvir-resistant HCV. The treatment had no effect on the development of replication factories (blue arrows). The bottom row shows EM images of cells that were infected with daclatasvir-sensitive HCV. It shows that treatment with daclatasvir completely inhibited the development of replication factories (identifiable by the absence of vesicles with a double membrane). Vesicles can always be found near lipid droplets (LD).
We also enable researchers from joint projects to carry out analysis on cells or tissues that are infected with other pathogens. For example, the structure of Zika virus’ replication factories was deciphered in close collaboration with the research field “Emerging Infections”, and it was demonstrated that inhibitors of the cytoskeleton prevent the replication of the virus in human neuronal cells.
In numerous collaborative projects, our working group was also able to decipher the mechanism of action of a new inhibitor against the Dengue virus and identify a target against the human Norovirus. These results were achieved in particular with the help of correlative microscopy, a combination of light and electron microscopy. During the SARS-CoV-2 pandemic, our working group identified the viral and cellular components required for the formation of SARS-CoV-2 replication factories. Both the SARS-CoV-2 proteins and the required cellular components can serve as candidates for targeted therapy of SARS-CoV-2 infection. The working group was also instrumental in the development of image-based analytical methods for the characterisation of SARS-CoV-2 virions.
Electron tomography of Zika virus (ZIKV) induced replication factories in human neuron precursor cells. The cells were fixed 24 hours after infection, bedded in epoxy resin and analysed by means of electron tomography. Left: Single tomography images showing ZIKV-induced vesicles (Ve) in the rough endoplasmic reticulum (ER) and virions (Vi). Scale: 100 nm. Right: 3D surface model of the area marked in yellow. ER membranes are light blue, virus induced vesicles are dark blue, virions golden and intermediate filaments are red. Scale length: 200 nm.
An important feature of our working group is that it provides training in the use of light and electron microscopy. The training is offered in close collaboration with the HIV Microscopy Unit and includes training on the use of both established and new microscopy methods as well as analysis procedures and quantification of light microscope images.
