Viral infections of the central nervous system (CNS) represent a significant burden
to human health worldwide. Neurotropic viruses must travel from a body entry gate
up to the CNS, where they infect local cells and potentially cause neurological disorders.
The brain is protected from blood-borne pathogens by the so-called blood–brain barrier
(BBB), an endothelial cell wall exhibiting extremely low permeability. Bacteria, fungi,
parasites, and viruses have evolved various powerful strategies to reach the brain
[1–3]. However, it is unclear whether the different strategies coexist or a main pathway
prevails while the others are marginal. Here, we describe the main strategies for
viruses to cross the BBB, with a particular focus on flaviviruses, which represent
important emerging human neurotropic pathogens. The aim of this review is to put into
perspective the four main ways to cross the BBB and to re-visit these concepts in
the light of new technical developments.
Question 1. What are the different paths viruses can use to cross a tight endothelial
cell wall?
The four main ways described in the literature and represented in Fig 1 are:
Passive diffusion (the passive aggressive way): In this model, viruses passively diffuse
in-between endothelial cells. This is possible in loose or injured endothelia, or
upon induced permeabilization.
Endothelial cell infection (the energetic way): In this model, viral tropism is compatible
to endothelial cell infection. Virus replication in endothelial cells allows virus
release on the basolateral membrane of the endothelium, therefore releasing infectious
viral particles toward the adjacent tissue.
Virus transcytosis (the commuting way): In this model, endothelial cells are not infected
but still uptake circulating viral particles into nondegradative endosomal vesicles
that are then released on the other side of the endothelial cell wall.
Cell-associated virus transport (the Trojan horse way): In this model, viruses infect
or are carried by blood circulating cells, which undergo blood-to-tissue transmigration
throughout the endothelial cells (via paracellular or transcellular migration).
10.1371/journal.ppat.1008434.g001
Fig 1
Ways viruses can use to cross the BBB.
Illustration of a blood vessel and the four nonexclusive ways viruses may employ to
reach adjacent tissues: 1. Diffusion (passive-aggressive way): Viruses freely diffuse
when endothelium integrity is altered. 2. Infection (energetic way): The endothelium
is infected and viruses released on the other side. 3. Transcytosis (commuting way):
The circulating virions are endocytosed by endothelial cells and exocytosed on the
other side, in the absence of productive infection. 4. Cell transport (Trojan horse
way): Leukocytes are carrying the circulating viruses through the endothelial cell
wall. BBB, blood–-brain barrier.
Question 2. What is the interplay between the various pathways?
Numerous primary research studies have investigated viral dissemination to the CNS,
but none of them compared in parallel the different ways a virus can use to cross
the BBB. Since proving the existence of one way does not disproof the existence of
the others, one can speculate that they actually coexist and may be influenced and
triggered by each other.
For instance, passive diffusion (way 1) may be tightly connected to productive infection
of endothelial cells (way 2), as virus-induced cell death can result in BBB leakage.
Flaviviruses have been reported to infect endothelial cells, at least in vitro. However,
the picture is less clear in clinical samples and mouse models (for detailed reviews,
see [1, 4]). For instance, no endothelial cell infection was found in clinical samples
of a Zika virus (ZIKV)-infected brain from a fetus with severe microcephaly [5], and
thus, ZIKV crossing to the brain is likely independent of a productive BBB infection.
Way 1 involves passive diffusion of viral particles through the BBB, but addressing
it as “passive” is misleading. Indeed, for diffusion to happen, earlier virus-induced
perturbations should have occurred, which in turn caused the BBB to be leaky. This
apparently “passive” diffusion is rather a passive-aggressive strategy. A leaky endothelium
can be the result of either (A) the direct infection of endothelial cells (way 2)
that compromises endothelial impermeability, (B) the induction of a strong inflammatory
response—the so-called “cytokine storm”—or (C) the release of propermeable viral proteins
into the blood stream. These nonexclusive options have all been proposed for the Flaviviridae
family, and, in particular, the third scenario was recently mechanistically deciphered,
occurring through nonstructural protein 1 (NS1)-mediated vascular leakage [6, 7].
Virus transcytosis (way 3) is difficult to experimentally discriminate from productive
infection (way 2), as exemplified recently by Papa and colleagues. [8]. To date, no
evidence supports the existence of this pathway for Flaviviridae BBB crossing. Interestingly,
dengue virus (DENV) infection modulates transcytosis of soluble factors [9], but the
viral particle itself was not shown to be readily transported through this route.
The Trojan horse strategy (way 4) is an attractive and conceptually relevant option
that was proposed almost 40 years ago for the Visna virus [10]. It extends beyond
virology, as bacteria, parasites and synthetic nanodelivery carriers are also employing
this strategy [2, 11–13]. This pathway confers a significant advantage to the virus
because the carrier cells hide it from immune surveillance. The Trojan horse can be
a more efficient strategy to crossing the BBB than a cell-free virus. When comparing
cell-associated versus cell-free ZIKV dissemination using cerebral organoids beneath
an endothelial layer, it was shown that free virions were disseminating slower than
monocyte-associated ZIKV [5].
In these scenarios, it is very difficult to definitely ensure that one way precludes
all the others from happening. On the contrary, one way may instead trigger the other
ones. Then, the question is not anymore whether they coexist but rather which way
comes first and which way contributes the most to neuroinvasion.
Question 3. Which way comes first? A chicken-and-egg situation
For the Japanese encephalitis virus (JEV) and West Nile virus (WNV), reports suggest
that first, virions enter the CNS with an integer BBB (by an unknown way) and, in
a second step, disrupt the BBB through inflammatory signals [14, 15]. In the case
of DENV, the Harris lab reported that vascular permeability was not cytokine-dependent
but rather mediated by the flavivirus NS1 protein [6, 16]. In this latter case, mouse
injection of the NS1 protein from WNV, DENV, and ZIKV (but not JEV) showed increased
brain vasculature leakage at three days postinjection. One scenario could be that
DENV infects cells (different from endothelial cells), leading to NS1 release to the
blood stream, therefore inducing vascular leakage and increased passive diffusion
of the virus to the BBB. Yet, it remains unknown whether the kinetics of NS1-mediated
vascular leakage precedes or follows inflammation-induced BBB permeability.
In the Trojan horse strategy, the question is rather to determine whether the immune
infected cells found in the brain correspond to the first wave of invasion or to secondary
infiltrations responding to an already established brain infection. On one hand, immune
cells readily migrate to the brain upon inflammation cues (for review, see [17]),
but on the other hand, the virus may need to first reach the brain to induce this
neuro-inflammation. These two options are both relevant but particularly challenging
to discriminate in vivo. Although immune cells transmigrate vastly less in the absence
of stimuli, it was previously observed by microscopic analyses of mouse brain tissue
sections that a low (but nonnull) number of monocytes can patrol across the BBB [18].
Moreover, monocytes are efficient cellular Trojan horses for drug delivery to the
brain in the absence of immunological signals [13]. Thus, it is reasonable to think
that inflammation plays a significant role in viral BBB crossing and in the worsening
of virus-induced neuropathology, but it may not be critical for initial cerebral colonization.
These observations highlight the importance of studying the temporality of events
in greater details to map the sequential appearance of viral neuroinvasion, BBB leakage,
and inflammation under infection conditions in relevant in vivo or ex vivo models.
Question 4. What are the upcoming methodological approaches to study viral BBB crossing?
From an experimental point of view, studying all four pathways simultaneously is very
challenging. In vivo, most of the flavivirus neuroinvasion studies have been performed
in mice (see for instance [6, 8, 14, 15, 19]), focusing on a single way. In fixed
samples, one could monitor several neuroinvasion pathways in parallel (for instance
endothelial infection (way 2) and leukocyte infiltration (way 4)), but it would not
discriminate whether it is the cause or the consequence of neuroinvasion. Spatiotemporal
dynamics is an attractive approach to address such limitation, requiring 3D imaging
of live infected mice. Although a few labs have achieved such challenging in vivo
imaging in principle [20, 21], the low number of events, the thickness of the region
to be imaged, and the relatively large time window of imaging are technical limitations
for the use of the mouse model. In contrast, the transparency and the numerous tools
available for imaging and genetic manipulation represent interesting advantages for
the use of the zebrafish embryo in neuroinvasion studies. Recently, a zebrafish embryo
model was developed to observe the transmigration through endothelia of ZIKV-infected
human monocytes [5], a xenotypic transfer previously characterized [22]. The zebrafish
model was also used to study neuroinvasion of two arboviruses, showing that chikungunya
virus (CHIKV) was mainly using endothelial infection (way 2), while Sindbis virus
(SINV) was mostly following peripheric axonal transport to reach the brain [23]. Although
very attractive for spatiotemporal cell biology analyses, zebrafish infection does
not recapitulate neuropathology observed in mammals, and thus, other complementary
approaches are still needed.
Besides in vivo models, emerging in vitro systems of human endothelial barriers represent
appealing strategies to gain further molecular insights onto viral neuroinvasion.
To discriminate between the four ways presented in Fig 1, an ideal system should combine
the following requirements: (A) measure endothelial permeability in real time, (B)
have a reporter system to monitor productive infection, (C) track single viral particles,
and (D) recapitulate bloodstream-like sheer stress. Moreover, the system should closely
mimic an actual vasculature, including the possibility to coculture various cell types
involved in BBB formation and maintenance and preserving the tube-shaped 3D architecture
of the endothelial cells.
BBB spheroids made of human brain microvascular endothelial cells (HBMEC) were used
to study ZIKV-induced THP-1 cells transmigration [24]. Recently, more physiological
techniques taking advantage of human pluripotent stem cell (hPSC)-derived BBB organoids
have been developed to obtain 3D human blood vessels with morphological, functional,
and molecular features of human microvasculature [25]. The most evolved in vitro technique
to date is probably the functional vasculature-like system grown within human cortical
organoids derived from embryonic stem cells [26].
A common limitation of the organoid approach however is that they do not allow the
application of a luminal flow. In that regard, original lithography-based microfluidic
chips are being employed to combine vessel architecture and flow dynamics [27–29].
These innovative systems could provide quantitative temporal and spatial information
to evaluate in parallel the contribution of each of the ways described in Fig 1. Although
very attractive, these new methods may not be straightforward to implement in neophyte
labs, but their rapid democratization should lead to exciting new discoveries in a
near future.
Conclusions
Common and divergent mechanisms have been evolved by the Flavivirus genus members
to reach the CNS. We propose that these ways of crossing the BBB are not mutually
exclusive but that they likely coexist and contribute to each other. However, a major
difficulty faced by researchers includes the fact that the envisioned pathways (Fig
1), as well as unforeseen ones, are difficult to experimentally segregate and individually
interrogate. The powerful in vitro BBB models recently developed could pave the way
toward important breakthroughs in the coming years. We believe that engineering better
in vitro cell walls will be key to spatiotemporally disentangle the mechanisms leading
to flavivirus neuroinvasion.