Abstract
Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.
Highlights
Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia
Productive TBEV replication was detected in the form of released virions on day 3 p.i
Viral antigen accumulated in association with rough endoplasmic reticulum (RER) alterations, such as large whorl formations (Fig. 2B, white arrows), or in places that exhibited a local loss of network structure (Fig 2C)
Summary
Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. We directly examined the pathology that occurs after TBEV infection in human primary neurons. In the present study, we exploited the advantages of high-pressure freezing and freeze-substitution techniques to improve the preservation of virally modified structures in TBEV-infected neurons. To the best of our knowledge, this study was the first to visualize the architecture of cellular components involved in TBEV replication and transport in neurons and the major ultrastructural changes that occur in response to TBEV infections. These novel data revealed the neuronal injury caused by TBEV infections, independent of the immune system response. Our results may facilitate the development of novel strategies for treating this serious human neuroinfection
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