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Usutu virus and West Nile virus use a transcellular route of neuroinvasion across an in vitro model of the human blood-brain barrier

This publication is part of the project ‘Preparing for vector-borne virus outbreaks in a changing world: a One Health Approach’ (NWA.1160.1S.210) which is (partly) financed by the Dutch Research Council (NWO).

Barry Rockx – PI One Health PACT:

New pre-print! Great work from @_El_Marshall_ starting to write up her PhD projects. Here she aimed to identify the capacity of USUV to employ haematogenous routes of neuroinvasion and compare with WNV.

She showed that both USUV & WNV can use transcellular invasion in the absence of barrier disruption. USUV’s reduced replicative capacity within the BBB may explain its lower impact on human health compared to WNV. Yet, given access, USUV may still cause neurological disease. Very cool work as part of the @OneHealthPact @MarionKoopmans

Authors: Eleanor M. Marshall, Marion KoopmansBarry Rockx


West Nile virus (WNV) leads to thousands of cases of severe neurological disease in humans each year. Usutu virus (USUV) is closely related to WNV, but rarely induces disease in humans. We hypothesised that USUV is less able to cross the blood-brain barrier, and is therefore less likely to infect the brain. Therefore, we developed an in vitro BBB model consisting of primary human brain microvascular endothelial cells (BMECs), pericytes and astrocytes. Both USUV and WNV invaded across the in vitro BBB via a transcellular mechanism in absence of barrier disruption. USUV replicated to lower titres than WNV but induced a comparable cytokine and chemokine response, with modulation of key factors associated with barrier function and immune-cell migration. In conclusion, USUV appears attenuated in its ability to replicate at this interface compared with WNV, but further work must to done to identify key determinants underlying the differing clinical presentations.


Many arthropod-borne viruses (arboviruses) can cause severe neurological disease in humans, but how these viruses bypass the numerous physical and immunological barriers in place to protect the central nervous system (CNS), is not well understood. West Nile virus (WNV) is a mosquito-borne flavivirus that causes thousands of cases of neuroinvasive disease in many regions of the world every year, including North America1, and increasingly in Europe2, with a continued emergence in new regions each transmission season. Usutu virus (USUV) is phylogenetically closely related to and co-circulates with WNV in Europe, but since the first case of human infection identified in 1981, there have only been around 100 documented cases of USUV induced disease and no reports of fatal infection3. WNV and USUV therefore appear to differ in their ability to cause severe disease in humans, which could stem from many factors, including the ability of the virus to gain access to the CNS via one or more routes of neuroinvasion. WNV is thought to use multiple routes of neuroinvasion, including both transcellular and paracellular invasion across the blood-brain barrier (BBB)4. The potential for use of these haematogenous routes of neuroinvasion by USUV is not well understood.

The BBB is a semipermeable selective border composed of tightly joined brain microvascular endothelial cells (BMECs), ensheathed by pericytes and astrocytes. This barrier is considered an important interface for invasion of viruses into the CNS from the blood. Such invasion can occur via a transcellular route in which the virus must infect or be transported across the cells of the barrier, or via a paracellular route in which virus is able to enter between the cells of a disrupted barrier, either as free virions or within infected immune cells in a so-called Trojan horse mechanism of invasion4.

Here, we aimed to determine whether USUV and WNV differ in their capacity to invade the CNS across the BBB. We employed a triple co-cultured in vitro transwell system, using primary human BMECs, astrocytes and pericytes, to recapitulate the human BBB and investigate the mechanism of viral invasion across this barrier. Identifying key differences and similarities between these two viruses will shed light on the essential virus or host factors that underlie viral neuroinvasion, and thereby aid in assessment of the future risk posed by USUV. We also provide a robust, biologically relevant platform that can be employed to evaluate the neuroinvasive capacity of emerging viruses and live-attenuated vaccine candidates, and to develop therapeutic strategies working to prevent viral neuroinvasion across the BBB.

Read the whole publication here.