Periodic Reporting for period 1 - Viroglimmage (Whole brain visualisation of the neuroinflammatory response during acute neurotropic flavivirus infection and its implications for virus-induced neurodegeneration)
Reporting period: 2023-01-01 to 2024-12-31
Flaviviruses are globally distributed pathogens and cause severe illness in millions of people worldwide. Several members of the flavivirus family, as well as many more viruses from other families, are neurotropic, which means that they can infect the central nerve system, including the brain. In Europe and Sweden, tick-borne encephalitis virus (TBEV) is the medically most important flavivirus, which is transmitted by ticks. In most cases infections are asymptomatic but, TBEV can also cause severe encephalitis potentially leading to death. Furthermore, 50% of patients who survive tick-borne encephalitis suffer from chronic neurological complications. Unfortunately, there are currently no antiviral drugs available for TBEV or the other neurotropic flaviviruses and treatment remains limited to supportive and symptomatic care.
A big issue is that the viral actions in the brain, which lead to this devastating disease are poorly understood, which largely relies on the lack of suitable imaging technology. Although confocal and electron microscopy have provided invaluable knowledge in infection biology, these microscopic imaging techniques do not qualify to visualize viral actions and consequent pathology at the level of the whole brain. Therefore, we are in desperate need of whole brain imaging techniques, capable of visualizing the course of viral infection and the pathological brain changes that go hand in hand. Such techniques can then improve our understanding od viral pathogenesis and aid the development of effective therapies and preventative measures, thereby lowering mortality and incidence of chronic neurological complications.
To be able to study viral actions in the brain and its consequences more accurately, the project aimed to optimize and develop whole brain 3D imaging protocols and validated quantification pipelines to study the link between viral infection, virus-induced neuroinflammation and virus-induced neurodegenerative morbidities.
A big issue is that the viral actions in the brain, which lead to this devastating disease are poorly understood, which largely relies on the lack of suitable imaging technology. Although confocal and electron microscopy have provided invaluable knowledge in infection biology, these microscopic imaging techniques do not qualify to visualize viral actions and consequent pathology at the level of the whole brain. Therefore, we are in desperate need of whole brain imaging techniques, capable of visualizing the course of viral infection and the pathological brain changes that go hand in hand. Such techniques can then improve our understanding od viral pathogenesis and aid the development of effective therapies and preventative measures, thereby lowering mortality and incidence of chronic neurological complications.
To be able to study viral actions in the brain and its consequences more accurately, the project aimed to optimize and develop whole brain 3D imaging protocols and validated quantification pipelines to study the link between viral infection, virus-induced neuroinflammation and virus-induced neurodegenerative morbidities.
Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) are 3D optical imaging techniques that visualize fluorescently labeled molecules in optically transparent tissues. Studying virus infection at the level of the whole brain in 3D, significantly increases the complexity of the pre-processing protocols, data acquisition and analysis in comparison with the usual 2D confocal microcopy images. Whole mount immunohistochemistry for 3D optical imaging is a time-consuming and tedious procedure requiring meticulous optimization for every antibody and every antibody combination. First, preprocessing and whole mount immunohistochemistry for virus-infection alone, was further optimized to obtain the best possible whole brain 3D images. To perform detailed anatomical brain mapping of viral infection in the whole brain, an MR-based brain template and its corresponding atlas were developed along with a pipeline to coregister and normalize OPT images to the brain template. Based on these resources, an atlas-based quantification protocol was established, allowing the calculation of viral infection metrics in al 336 regions of interest. Thereby, we established the first of its kind 3D optical brain imaging and quantification pipeline for viral brain mapping along with quantification. The established resources and pipelines were also used to compare the influence of structural viral proteins on the whole brain distribution of three related viruses (TBEV, LGTV and ChLGTV). Afterwards, we aimed to optimized dual-label whole mount immunolabeling for simultaneous imaging of virus infection and virus-induced neuroinflammation. Several neuroinflammatory markers were tested but many were unsuitable for use in an imaging system with such high resolution. Eventually we identified Galectin3 as a very promising target for the visualization of virus-induced neuroinflammation and developed dual label immunohistochemistry protocols and imaging regimens for simultaneous imaging of virus infection and neuroinflammation. These protocols are currently being used to determine the course of neuroinflammation in acute flavirus infection and experiments in specific knock out models are planned 2025 to investigate the role of the microglia/macrophage population.
Applying the developed technologies and image analysis pipelines, our results reveal that the interferon response of the innate immune system influences both the whole brain distribution and the cellular tropism of tick-borne flavivirus and that the whole brain distribution cannot be attributed to the structural viral proteins alone. For neuroinflammation imaging, we found that the most commonly used targets for clinical imaging of neuroinflammation do not work for high-resolution 3D optical imaging since they are also expressed on other cell then microglia, which also provides important information regarding clinical images. We identified Galectin-3 as a very promising marker for visualization of virus-induced neuroinflammation and found that the neuroinflammatory response in fatal flaviviral encephalitis is dominated by macrophage infiltration rather than microglial activation.
In conclusion, the viroglimmage project has significantly advanced tissue-preprocessing protocols, image analysis and quantification pipelines of whole brain 3D optical imaging of neurotropic flaviviral infection and has applied these for the visualization and quantification of viral distribution of distinct neurotropic flaviviruses in the whole mouse brain, which allowed us to provide novel insights in flavivirus induced brain pathology. It has also pioneered new tools for simultaneous visualization of viral infection and virus-induced neuroinflammation. This new technology opens a new road to investigate the detrimental effects of lingering neuroinflammation after viral clearance, which in the future will help us understand why so many patients surviving tick-borne encephalitis experience life-long neurological complications.
In conclusion, the viroglimmage project has significantly advanced tissue-preprocessing protocols, image analysis and quantification pipelines of whole brain 3D optical imaging of neurotropic flaviviral infection and has applied these for the visualization and quantification of viral distribution of distinct neurotropic flaviviruses in the whole mouse brain, which allowed us to provide novel insights in flavivirus induced brain pathology. It has also pioneered new tools for simultaneous visualization of viral infection and virus-induced neuroinflammation. This new technology opens a new road to investigate the detrimental effects of lingering neuroinflammation after viral clearance, which in the future will help us understand why so many patients surviving tick-borne encephalitis experience life-long neurological complications.