New Insights in the Tropism and Dissemination of Encephalitic Paramyxoviridae – NITRODEP

The use of a combination organotypic brain culture and reverse genetic allows us following, in real time by fluorescent microscopy and flow cytometry, the tropism and the dissemination of wild type, receptor blind and viruses with their viral glycoproteins expression placed under the control of cellular factors in a preserved tri-dimensional architecture.

The central nervous system cells activation state seems to be more responsible for the susceptibility to the infection than the expression of known receptors. The tropism and dissemination of Measles virus is independent of the known receptor. Astrocytes and microglia depends on their activation state. Additionally, microglia susceptibility rely on the type 1 interferon pathway. The encephalitic measles virus (F L454W) can infect cells even at low temperature and in absence of known receptor. To date, Nipah virus can infect all test cells in the CNS. Measles virus dissemination relies on the H protein and its ability to bind a receptor through the position 533. Both F and H seem to be necessary to the neural invasion while it doesn’t exclude a potential very low alternative cell-to-cell dissemination.

We have characterized/determined the best conditions to infect our ex vivo model.The central nervous system cells activation state seems to be more responsible for the susceptibility to the infection than the expression of known receptors. The tropsim and dissemination of Measles virus is independent of the known receptor. The encephalitic measles virus (F L454W) can infect cells even at low temperature and in absence of known receptor. To date, Nipah virus can infect all test cells in the CNS. We aim at analyzing the role of the viral glycoproteins in the dissemination to pave the way for the design a new drugs able to target viral glycoproteins and activating the cerebral immune response in order to limit the invasion of the central nervous system by these letal viral infections.

Through this project we have characterized the organotypic sections to standardize their use in neurovirology. This allowed us to demonstrate that the susceptibility of Astrocytes and microglia depends on the interferon response and on their activation state (Welsch et al, Paper 1 submitted). We have also shown that instability of the measles virus fusion protein is sufficient to override the expression of any input receptor (Jurgens, Mathieu et al., Mbio 2015). Nevertheless, the dissemination of the measles virus is based on its attachment protein and especially on a binding zone to a known receptor which is not expressed in the brain (Welsch et al. paper2 in process). Finally, the organotypic system evaluated the effect of new antiviral compounds against Nipah virus and measles virus in the brain tissue (Mathieu et al, Expert Rev Anti Infect Ther 2015, Bloyet et al, j virol 2016)

A large number of RNA viruses are neurotropic and can cause a variety of neurological disorders. The outcome of neurotropic viral infection depends on the virus and the infected neurons. In neurons, many viruses undergo a switch to a persistent infection, which is often accompanied by a drastic reduction in production of cell-free viruses. However, despite reduction of extracellular viruses, many neurotropic viruses can efficiently, disseminate within the Central Nervous System (CNS) suggesting a cell-to-cell transmission pathway that differs from the canonical spread in the periphery. The overall goal of this application is to elucidate how neurotropic RNA viruses can spread within the CNS. We will focus on two human pathogens, measles (MV) and Nipah (NiV) virus that belong to closely related single stranded RNA virus Paramyxoviridae family, with a common transcription and replication strategy. MV is restricted to humans leading in most of cases to mild, although sometimes severe and deadly disease. NiV has very high pathogenicity in humans (lethality up to 90%), can infect different animal species and requires BSL4 facility for the analysis. While Nipah virus is a new recently emerged virus, measles is re-emerging with regular outbreaks around the world, including industrialized countries. Both viruses could infect neuronal cells and cause lethal encephalitis as well as persistent brain infection, giving a relapse several months or years after primary infection. However, while CNS infection in measles is a rare event, in Nipah it is very frequent. Viral neuro-pathogenesis remains in both cases poorly understood.
In the proposed studies, a combination of brain organotypic and neuronal cell culture system from transgenic mouse models will be used to analyze the spreading of these two neurotropic RNA viruses in the CNS with the expectation that comparison of these two viral infections will bring further insights in each of them. I aim at answering the five related questions (i) Do the viruses use known or alternative cellular receptor(s) to enter and spread throughout the CNS? (ii) Which glycoprotein(s) is(are) necessary/involved in entry/cell-cell spreading? (iii) How the viral core is transported from virus entry to the virus exit site assuming the cell-cell spreading? (iv) Are viral protein constituents properly assembled to give rise to virus particles responsible for the cell-cell spreading? (v) What governs the possible selectivity of virus propagation through limited neuron network(s)? In vitro and ex vivo MV and NiV infection studies will benefit from the following tools: (i) primary cultures and brain explant cultures from transgenic murine models expressing or not known viral entry receptors, (ii) molecular tools blocking the viral entry/fusion machinery and spread (iii) recombinant viruses either expressing glycoproteins blind for their receptors and/or lacking protein constituents the envelop. In addition, to identify the neuron subsets targeted by these viruses, we will build recombinant viruses expressing two fluorescent proteins with one made sensitive to the silencing by brain specific miRNA(s). The results from the proposed studies will ultimately be used to determine how acute and persistent RNA viral infections evolve to induce CNS disease. Elucidating the state of the virus during infection and mechanism of its interneuronal spread will hopefully pave the way for the design of therapies able to interfere with these processes and thus prevent fatal neurologic disorders induced by these viruses.