Journal of Neuroimmunology 316 (2018) 65–73

Contents lists available at ScienceDirect

Journal of Neuroimmunology
journal homepage: www.elsevier.com/locate/jneuroim

Review Article

Functional failure of TLR3 and its signaling components contribute to herpes T

simplex encephalitis
⁎
Matylda Barbara Mielcarskaa, , Magdalena Bossowska-Nowickaa, Felix Ngosa Tokaa,b
a
Division of Immunology, Department of Preclinical Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Ciszewskiego 8 Str., 02-786 Warsaw,
Poland
b
Center for Integrative Mammalian Research, Ross University School of Veterinary Medicine, PO Box 334, Basseterre, Saint Kitts and Nevis

A R T I C L E I N F O A B S T R A C T

Keywords: Herpes simplex encephalitis (HSE) is a severe neurological disease in children and adults caused by herpes
TLR3 simplex virus. This review discusses recent findings on the role of Toll-like receptor 3 (TLR3) deficiencies in the
HSE HSE development. Critical checkpoints in the TLR3 signaling that contribute to innate response are discussed,
HSV-1 including the importance of TLR3 ligand recognition site and transportation in the cell. We also indicate un-
HSV-2
resolved issues in the TLR3 functioning that might lead to thorough understanding of immunity during HSE.
Innate immunity
Such a knowledge base will lead to discovery and design of a rationale therapeutic and preventive approach
against HSE.

1. Introduction
Herpes simplex virus type 1 (HSV-1) belongs to the Herpesviridae
family which contains at least eight species found to infect humans
(Sharma et al., 2016). HSV-1 is common, 80–85% of young adults
around the world are seropositive, while the wide majority have no
symptoms or infection signs are limited to mild herpes labialis (Wald and
Corey, 2007; Guo et al., 2011). However, in specific cases HSV-1, in a
major part, and herpes simplex virus type 2 (HSV-2), may produce a
life-threatening disease.
The immune system has an innate capacity to identify infectious
agents through pattern recognition receptors (PRRs), however, viruses
use cellular machinery to replicate and thus present a challenge for the
immune system to distinguish virus from self. To avoid response to self-
nucleic acids, molecular patterns presented by viruses are recognized
by PRRs, including Toll-like receptors (TLRs), in a precisely controlled
manner (Krishnan et al., 2007). Foreign nucleic acids are sensed by
endosomal TLRs, which belong to the most highly conserved immune
receptors in humans (Barreiro et al., 2009). TLRs were first described in
the fruit fly (Drosophila melanogaster) (Anderson et al., 1985) and soon
after in humans (Medzhitov et al., 1997), and are expressed in various
tissues of the body, including the central nervous system (CNS). TLR3
belongs to the group of TLRs present in prelysosomal organelles –
endosomes, and its localization as well as expression are highly regu-
lated (Barton and Kagan, 2009). Substantial expression of TLR3 occurs
in CNS-resident cells, e.g., microglia, neurons and oligodendrocytes
(Bsibsi et al., 2002, 2006; Peltier et al., 2010), which are permissive for
HSV-1 infection (Lafaille et al., 2012). By identifying viral double-
stranded RNA (dsRNA), TLR3 triggers a pathway leading to antiviral
response (Alexopoulou et al., 2001). Double-stranded RNA occurs as a
viral replication intermediate in the case of HSV-1, a double-stranded
DNA (dsDNA) virus (Jacobs and Langland, 1996). In this review we
provide an overview of HSV-1 infection and largely dwell on recent
developments in the failure of expression of a functional TLR3 thus
disrupting signaling mechanisms that lead to induction of an antiviral
response during infection with HSV-1. Further we discuss several per-
turbations in the TLR3 signaling pathway that impact infection with
herpes simplex virus (HSV).
2. HSE occurrence and clinical features
HSE is the most common sporadic viral encephalitis in the Western
World but with devastating neurological consequences. It occurs in 1–2
per 500,000 people per year, while about one third of all HSE cases
occurs in children and adolescents (Widener and Whitley, 2014; Maraş
Genç et al., 2016). HSE represents 10–20% of all viral CNS infections

Abbreviations: HSE, herpes simplex encephalitis; TLR3, Toll-like receptor 3; VSV, vesicular stomatitis virus; MCMV, murine cytomegalovirus; EE, early endosomes; LE, late endosomes;
EL, endolysosomal compartment; BFA, Brefeldin A; TGN, trans-Golgi network; KSHV, Kaposi's sarcoma-associated herpesvirus; DENV, dengue virus; MDCs, myeloid dendritic cells;
PBMCs, peripheral blood mononuclear cells; MEFs, mouse embryonic fibroblasts; NSCs, neural stem cells; iPSC-derived, induced pluripotent stem cell-derived; ESCRT-0, endosomal
sorting complex required for transport-0
⁎
Corresponding author.
E-mail address: matylda_mielcarska@sggw.pl (M.B. Mielcarska).

https://doi.org/10.1016/j.jneuroim.2017.12.011
Received 3 November 2017; Received in revised form 17 December 2017; Accepted 17 December 2017
0165-5728/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.B. Mielcarska et al.
(Rozenberg et al., 2011). This type of brain inflammation usually affects
healthy children between 6 and 36 months of age. In adult people over
the age of 50 years it probably reflects reactivation of latent virus in
infected neurons (De Tiège et al., 2008). During severe HSE more than
70% of patients without treatment die, 19–30% die despite the treat-
ment and only 9% of treated patients survive and recover to full health
(Aldea et al., 2003; Griffin, 2005; Gnann et al., 2015). Although it is
assumed that both HSV-1 and HSV-2 may be responsible for en-
cephalitis, HSE is generally reported to be caused by HSV-1 infection in
the vast majority of patients. The association between HSE and any
specific isolate of HSV-1 has not been shown so far. It is also possible
that HSE severity may be dictated not only by viral origin, but represent
more complex issues.
Symptoms allowing to qualify a patient as suffering from HSE in-
clude fever, encephalitis and focal neurologic signs, in both children
and adults. Symptoms and consequences of illness such as altered
mentation and changes of personality occur in almost all the sick. The
majority of patients who have undergone this type of brain infection
suffer from serious neurological sequelae: recurrent seizures, shakes
and mental retardation (Riancho et al., 2013; Gnann and Whitley,
2017; Modi et al., 2017). However, signs of other numerous nervous
system diseases may resemble HSE.
When a seronegative person is exposed to HSV through mucous
membranes or abraded skin, viral entry and replication at the infection
site is likely to follow. Subsequently, the virus is transported through
neurons to the dorsal root ganglia or trigeminal ganglia, where it may
replicate and become latent, which is very common (Zerboni et al.,
2013). Upon viral reactivation, lesions usually emerge in the form of
skin or mucocutaneous abrasions or ulcers. Both primary and recurrent
infections may contribute to the brain disease. One-third of HSE cases is
related to primary infection and the majority of patients is under
18 years of age. In some cases, particularly in immunosuppressed pa-
tients, infection may engage many organs and become a systemic dis-
ease. However, both in normal and immunosuppressed hosts the
probability of HSE occurrence is comparable (Whitley and Kimberlin,
2005).
Histopathological findings indicate that after reaching the CNS, HSV
replicates causing cell swelling, plasma membrane fragmentation and
nuclei degradation resulting in the formation of polynuclear giant cells
(Whitley and Kimberlin, 2005). Gliosis, satelitosis, cell necrosis and
neuronophagia are frequent (Whitley et al., 2007). Glial nodules, which
commonly appear within the second week of infection, are also a pa-
thological feature (Algahtani et al., 2016). Glial cells in particular,
appear to be very important in reactivity against HSV. They remove
cellular debris from the injury sites, since they belong to primary sca-
venger cells and have many common properties with tissue macro-
phages (Purves et al., 2001). This observation indicates an important
role for these cells during inflammatory processes in the CNS. Fur-
thermore, microglia are the main source of type I interferons (IFNs) in
the CNS cells during HSV infection (Reinert et al., 2016). The impact of
viral infection entails congestions, petechiae and hemorrhages, which
frequently localize in the temporal lobes (Chow et al., 2015).
People suffering from HSE and in particular patients with HSE and
TLR3 pathway deficiencies, are usually resistant to other infectious
diseases, including viral illnesses such as HSV-1-related diseases outside
the CNS (Abel et al., 2010; Sancho-Shimizu et al., 2011a). However, if
cells from organs other than the brain, e.g., dermal fibroblasts exhibit
direct defects in the TLR3 pathway, then they may also present im-
paired interferon-β (IFN)-β and IFN-λ production after stimulation with
synthetic dsRNA mimetic. Such deficiencies in cells may lead to high
susceptibility to HSV-1 or other viruses such as vesicular stomatitis
virus (VSV) infections (Herman et al., 2012). These findings indicate
that the association between TLR3 pathway and a range of other de-
fense strategies against HSV infection may be similar or vary in dif-
ferent types of cells and require further examination.
66
Journal of Neuroimmunology 316 (2018) 65–73
3. HSE association with the TLR3 pathway
Encephalitis caused by the HSV could be a serious complication
associated with primary infection or recurrence of the disease which
occurred in the past (Whitley and Kimberlin, 2005). In the infected cells
HSV constantly replicates for 18–24 h and virions at different stages of
assembly are present in many cellular compartments (Harley et al.,
2001). During this time the host cell mounts an effective first line of
defense; activated TLR3 elicits a signaling cascade leading to produc-
tion of IFNs, essential to initiate adaptive immune response (Akira
et al., 2001). Protective antiviral response during primary HSV-1 in-
fection in brain is dependent on TLR3-mediated induction of IFN-α, -β,
-λ, -γ production (Li et al., 2005; Zhang et al., 2008), which elicits the
synthesis of pro-inflammatory cytokines such as tumor necrosis factor
alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), inter-
leukin 8 (IL-8), chemokines (chemokine (CC motif) ligand 5 (CCL-5);
chemokine (CXC motif) ligand 10 (CXCL10)) and other molecules, such
as 2′-5′-oligoadenylate synthetase (2′,5′OAS) (Samuel, 2001). Those
cytokines and chemokines play a crucial role in limiting the spread of
the virus (Paludan et al., 2011). Deterioration of IFN production in the
infected cells results in increased virus replication and cell death (Upton
and Chan, 2014), what probably is responsible for the clinical signs of
HSE. There is also a hypothesis which designates an impaired antiviral
response to TLR3 defects, thus leading to aberrant control of HSV in the
brain and reactivation of latent virus, which can both contribute to HSE
and relapse of the disease. Immune reaction in the brains of patients,
where viral DNA, but no HSV antigens were found, persisted up to ten
years after acute onset of HSE (Lellouch-Tubiana et al., 2000). Conse-
quently, recurrence of HSE is particularly likely in the case of inborn
errors related directly to the TLR3 (Zhang et al., 2007; Guo et al., 2011)
or genes associated with the receptor signaling pathway (Herman et al.,
2012). Reactivation of the latent virus or post-infection inflammatory
response is considered fundamental causes of HSE relapse. Cranial
surgery, especially in the site of a previous herpetic infection, may
constitute a source of stimulus for recurrence, but no clear evidence has
been derived yet (Lo Presti et al., 2015). Indeed, there are multiple
stages in the cellular defense associated with TLR3 in particular, where
the imprecision or defect may facilitate virus replication and cause HSE.
3.1. TLR3 transportation to ligand recognition site – the role of UNC93B
Transportation of TLR3 in cells is a significant event, since the re-
ceptor recognizes its ligand in endosomes. In resting cells TLR3 can be
found in the endoplasmic reticulum (ER) where it co-localizes with
UNC93B, a transmembrane protein crucial for transportation not only
of TLR3, but also other endosomal TLRs (Meylan and Tschopp, 2006).
Association between TLR3 and UNC93B is not temporary, but persists
for a prolonged period of time, which indicates UNC93B can take part
in retaining TLR3 before transfer of the receptor to the endosomes oc-
curs (Brinkmann et al., 2007). After activation and with the participa-
tion of UNC93B, TLR3 is shipped to the endosomes (Kim et al., 2008).
Mutations in UNC93B which prevented signaling via Tlr3, Tlr7 and Tlr9
were found in mice that were susceptible to numerous infections
(Tabeta et al., 2006). Moreover, autosomal recessive deficiencies in
UNC93B, which resulted in lack of TLR responsiveness were found in
two children suffering from HSE. Subsequently, impaired IFN-α/β and
IFN-λ production in cells identifies these deficiencies as genetic
etiology of the disease in the cases referred to (Casrouge et al., 2006).
Furthermore, despite disabled TLR3, TLR7, TLR8 and TLR9 signaling,
the patients in the work cited here were not affected by viral infections
other than HSV-1 that caused HSE. On the other hand, the UNC93B-
deficient mice are highly susceptible to murine cytomegalovirus
(MCMV) and are less effective in eliminating bacterial infections
(Tabeta et al., 2006).
TLR3 may be transported from the ER to Golgi apparatus, then to
M.B. Mielcarska et al. Journal of Neuroimmunology 316 (2018) 65–73

Table 1
Known deficiencies in TLR3 and genes of proteins involved in TLR3 signaling pathway identified in children and adults suffering from HSE. AD – autosomal dominant, AR – autosomal
recessive, n k – not known.

Gene Mutation Inheritance model Age at which HSE occurred – child/adult References

TLR3 p. P554S AD child Zhang et al., 2007

TLR3 p. P554S AD child Zhang et al., 2007
TLR3 p. P554S/E746X AR child Guo et al., 2011
TLR3 p. G743D + R811D AD child, adult Lim et al., 2014
TLR3 p. L360P AD child, adult Lim et al., 2014
TLR3 p. R867Q AR adult Lim et al., 2014
TLR3 p. L297 V nk adult Mørk et al., 2015
TLR3 p. L297 V nk adult Sironi et al., 2017
TLR3 c.-8 + 6 T > C nk adult Sironi et al., 2017
UNC93B c. 1034del4 AR child Casrouge et al., 2006
UNC93B c. 781 G > A AR child Casrouge et al., 2006
TRIF p. R141X AR child Sancho-Shimizu et al., 2011b
TRIF p. S186 L AD child Sancho-Shimizu et al., 2011b
TRIF p. A568T nk adult Mørk et al., 2015
TRIF p. S160F nk adult Mørk et al., 2015
TRAF3 p. R118W AD child Pérez de Diego et al., 2010
TBK1 p. G159A AD child Herman et al., 2012
TBK1 p. D50A AD child Herman et al., 2012
TBK1 p. I207V nk adult Mørk et al., 2015
IRF3 p. R285Q AD child Andersen et al., 2015; Mørk et al., 2015
IRF3 p. A277T nk adult Mørk et al., 2015

early endosomes (EE), which mature into late endosomes (LE). These in
turn may fuse with lysosomes to form the endolysosomal compartment
(EL), required for dsRNA recognition. However, mechanisms regulating
detailed steps of TLR3 sorting pathway still remain unclear and stages
described above may occur with the exception of EE/LE or LE (Tsai
et al., 2015). Concurrently, it should be highlighted that during infec-
tion, HSV conquers and remodels membrane composition and circula-
tion simultaneously, which may impair host response (Hollinshead
et al., 2012). It has not yet been precisely defined which cytoplasmic
compartments are exploited by HSV in its production pathway, after
exiting the nucleus, where genome replication and capsid assembly
occurs (Baines, 2011). HSV particles may accumulate in organelles
which co-fractionate with Golgi apparatus network and endosomes
(Harley et al., 2001). Partial evidence is demonstrated by addition of
Brefeldin A (BFA) to cells infected with HSV-1, which causes dis-
assembly of the Golgi complex, arrests maturation and secretion of the
HSV-1 virions (Cheung et al., 1991). Furthermore, endocytic compart-
ments are involved in the envelopment of HSV-1 capsids and viral
glycoproteins were recently implicated to be internalised from the
membranes of organelles located on the trans side of the Golgi appa-
ratus (trans-Golgi network, TGN) (Turcotte et al., 2005; Hollinshead
et al., 2012). Early and recycling endosomes co-localize with Kaposi's
sarcoma-associated herpesvirus (KSHV) particles upon viral fusion
(Greene and Gao, 2009). Moreover, adding lysosome maturation in-
hibitors restrains TLR3-mediated response in immune cells (De
Bouteiller et al., 2005), although it may also hinder the viral cycle,
which requires endosomes to overtake the host cells. For instance,
noninfectious enveloped HSV particles accumulate in cells after the
addition of chloroquine or bafilomycin A (Akula et al., 2003). In sum-
mary, the endosomal compartment is a fundamental area where HSV
nucleic acid can occur at different phases of replication cycle and
dsRNA may interact with TLR3. However, the knowledge regarding
TLR3 transportation in CNS cells is limited, while its aberrations may
possibly influence the repercussions of HSV infection, including HSE.
The exact time and space in which the HSV replication intermediate –
viral dsRNA is available and capable of activating TLR3 pathway needs
further investigation.
3.2. TLR3 cleavage in endolysosomes
Prior to launching TLR3 pathway another significant process has to
take place. TLR3 may be present in cells in a number of forms, full
length and two other forms, produced after cleavage by lysosomal ca-
thepsins B and H, which is essential for the receptor signaling (Garcia-
Cattaneo et al., 2012). Moreover, cleaved forms of the receptor have
more than two times longer half-life in human embryonic kidney (HEK)
293T cells and modify TLR3 transportation to subpopulations of en-
dolysosomes (Qi et al., 2012). Newly discovered L360P TLR3 mutation
localized in the immediate vicinity of the enzyme cleavage site
(323–356 amino acid residues), results in a mutant protein resistant to
cleavage. Significantly, cells carrying such a mutation could not confer
poly(I:C) (polyinosinic:polycytidylic acid, dsRNA mimetic) responsive-
ness, and secretion of IFN-β, IFN-λ, and IL-6 was severely impaired
(Lim et al., 2014), confirming that cleaved TLR3 may represent the
primary form of the signaling receptor (Toscano et al., 2013). However,
TLR3 cleavage not always leads to the disintegration of the receptor, on
the contrary, C- and N-terminal fragments may not dissociate and
generate stable “cleaved/associated” TLR3 in endolysosomes. In fact,
this form of the receptor also recognizes dsRNA and transduces a signal
(Toscano et al., 2013). Importantly, the expression of a TLR in a single
cell type may indicate a distinctive role for this molecule in a restricted
setting (Muzio et al., 2000), therefore investigation regarding functions
of TLR3 forms in relation to HSV infection and HSE development in the
CNS cells would yield valuable information. In relation to HSV, there
are no studies indicating that deficiency in cathepsin B and H in humans
exacerbates infection with HSV or leads to severe HSE. Therefore, this is
another area awaiting scientific evaluation.
3.3. TLR3 pathway “checkpoints”
Defects due to mutations in TLR3 and various proteins in the TLR3
signaling pathway often contribute to defective antiviral response,
which may have serious outcomes for the host. Such a consequence
although rare may increase susceptibility to viral encephalitis following
HSV infection. Dysfunctional TLR3 pathway elements may appear as
autosomal dominant or autosomal recessive partial defects due to allelic
variability. Deficiencies indicated as contributing causative factors of
HSE in children as well as in adults are presented in Table 1. However, a
closer look at the detailed trail leading to anti-HSV-1 defense is needful
to provide hints for investigating checkpoints in TLR3 pathway. Fig. 1
shows those significant checkpoints that have been marked to date.

67
M.B. Mielcarska et al. Journal of Neuroimmunology 316 (2018) 65–73

Fig. 1. Schematic representation of TLR3 fate and signaling pathway in cells. TLR3 ligand – dsRNA may be obtained from HSV-infected cells or appear as replication intermediate during
HSV infection of the cell. Subsequently, dsRNA is delivered to the endosome, where it associates with the receptor. Upon dimerization and phosphorylation, TLR3 triggers signaling
cascades leading to the activation of the transcription factors: interferon regulatory factor 3 (IRF3), nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), initiating the production of
type I IFNs and inflammatory cytokines. Proteins involved in the above signaling pathways, in which reported deficiencies may lead to the onset of HSE, are highlighted in red. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Deficiencies in TLR3 and associated signaling molecules
The TLR3 molecule contains an ectodomain (ECD) facing the in-
terior of an endosome and a cytoplasmic Toll/interleukin-1 receptor
(TIR) domain that associates with TIR domains of adapter proteins in-
volved in the signaling pathway. Upon engagement of ECDs with li-
gand, which should contain at least 40–50 base pairs in length, TLR3
undergoes dimerization, leading to conformational changes in TLR3
cytoplasmic domains, enabling their phosphorylation, thus facilitating
downstream signaling (De Bouteiller et al., 2005; Liu et al., 2008). Li-
gand-dependent phosphorylation of specific tyrosine residues of the
TLR3 cytoplasmic domain is indispensable for proper implementation
of the signaling pathway. Tyr759 and Tyr858 are most crucial among five
tyrosine residues (Sarkar et al., 2003, 2004, 2007). Src, Epidermal
Growth Factor Receptor (EGFR) and Bruton's Tyrosine Kinase (BTK) are
protein tyrosine kinases (PTKs) responsible for TLR3 phosphorylation.
Mutations of kinases or critical TLR3 tyrosine residues result in im-
paired nuclear factor-κB (NF-κB) and interferon regulatory factor 3
(IRF3) signaling branches, e.g., BTK-deficient macrophages are unable
to conquer the dengue virus (DENV) infection, where dsRNA is pro-
duced as replication intermediate (Chattopadhyay and Sen, 2014).
Chemical inhibitors of Src, EGFR and BTK block TLR3-mediated
downstream activation of transcription factors. Although EGFR-di-
rected antibodies are currently used in solid tumors therapy, inhibition
of EGFR may permit increased viral replication and therefore increase
the risk of infection (Altan and Burtness, 2015). Consequently, directing
attention towards deficiencies associated with incomplete receptor
phosphorylation should not be omitted among HSE patients.
Several mutations affecting TLR3 cleavage region, critical for
dsRNA binding and receptor multimerization, were recently found
among patients suffering from HSE and proved to be dominantly
negative, as they resulted in loss of TLR3 function (Zhang et al., 2007;
Lim et al., 2014). HSV-1 seropositive relatives of these patients, car-
rying the same heterozygous mutations did not suffer from HSE, de-
monstrating incomplete clinical penetrance. In HSE patients carrying
heterozygous TLR3 mutations, the response to poly(I:C) was impaired
in fibroblasts, but normal or only partially weakened in myeloid den-
dritic cells (MDCs) or keratinocytes, suggesting that not all cells may
require intact TLR3 to respond to HSV-1 infection. It is likely that an-
tiviral response in the mentioned cells may have been mediated by
TLR3-independent mechanisms. Conversely, natural killer (NK) cells or
CD8+ α/β T cells of those patients presented an impaired dsRNA re-
sponse in the form of IFN-β and -λ production, which may underline
their role in HSE pathogenesis. However, people lacking NK and T cell
subsets are not necessarily highly susceptible to HSV-1 infection and do
not suffer from HSE (Buckley, 2004). By inference, poly(I:C) response
rates in various cell types with different levels of TLR3 deficits may
explain the reduction of HSE lesions in the brain, and the lack of dis-
seminated disease. Another HSE patient (Guo et al., 2011) carrying two
heterozygous TLR3 loss-of-function alleles had a mother who was het-
erozygous for one mutation but presented normal response of fibro-
blasts to poly(I:C) in the form of IFN-β and -λ production. Two defi-
ciencies resulting from the lack of TIR domain and a mutation in the
TLR3 ligand binding site, caused total loss of TLR3 function. Never-
theless, peripheral blood mononuclear cells (PBMCs) exhibited normal
responses to poly(I:C) stimulation as well as normal levels of HSV-1
replication. A similar observation was made in PBMCs of an adult HSE
patient who presented another rare mutation in TLR3 (Mørk et al.,
2015). An identical mutation in TLR3 was found in an Italian patient
suffering from HSE (Sironi et al., 2017). This underscores the pre-
sumption that functionally impaired IFN responses may be critical only
in certain cell types, such as CNS resident cells. Interestingly, Tlr3-

68
M.B. Mielcarska et al.
deficient mice infected with different viruses, manifest varied infection
phenotypes similar to humans, however, they are highly susceptible to
infection of the CNS caused by HSV-2, although it does not translate
into lower survival rates (Reinert et al., 2012).
3.4.1. TRIF
TLR3 associates with TIR-domain-containing adapter-inducing in-
terferon-β (TRIF) (Yamamoto et al., 2003) through TIR domains. Two
TRIF deficiencies, autosomal dominant and autosomal recessive were
reported among children suffering from HSE, which resulted in im-
paired or abolished TLR3 pathway, respectively (Sancho-Shimizu et al.,
2011b). Although the patients in question had no history of other in-
fections, their dermal fibroblasts presented impaired responses to
viruses and exhibited decreased production of IFN-β and -λ after poly
(I:C) stimulation. Similar assays performed with cells of a third HSE
patient carrying TRIF mutation did not reveal any deleterious effects
(Sancho-Shimizu et al., 2011b). Consequently, clinical penetrance of
these deficiencies seems to be incomplete because some TRIF-deficient
and HSV-1 infected people do not suffer from HSE. Another two mis-
sense heterozygous TRIF mutations were identified in two HSE patients
aged over 60 years (Mørk et al., 2015). PBMCs from both patients
showed significantly reduced production of IFN-β after HSV-1 infection.
One of the patients had greatly reduced CXCL10 and TNF-α expression
both after poly(I:C) stimulation and HSV-1 infection. Studies in Trif-
deficient mice demonstrate increased susceptibility to MCMV, and
during infection with other viruses resembled the Tlr3-deficient mice
infection phenotype (Pérez de Diego and Rodriguez-Gallego, 2014).
Such mice are defective in Tlr3- and Tlr4-mediated activation of IRF3
and IFN-β production (Yamamoto et al., 2003), which underlines a vital
role of this adaptor molecule in facilitating antiviral host defense.
TRIF subsequently interacts with signalosome containing Tumor
necrosis factor-receptor-associated factor 3 (TRAF3), NF-κB-activating
kinase-associated protein 1 (NAP1), Tumor necrosis factor-receptor-
associated factor 6 (TRAF6) and Receptor interacting serine/threonine
kinase protein 1 (RIP1) (Oganesyan et al., 2006; Sasai et al., 2005;
Jiang et al., 2004; Meylan et al., 2004). Upon TLR3 stimulation TRAF6
and RIP1 activate NF-κB, the transcription factor for IFN-β and proin-
flammatory cytokine genes (Blasius and Beutler, 2010).
3.4.2. TRAF3
TRAF3 plays an important role downstream of TLR3, TLR4, TLR7
and TLR8 activation leading to antiviral response in the form of IFN-α,
IFN-β and IFN-λ production (Hoebe and Beutler, 2006). Importantly,
TRAF3 also participates in the dsRNA recognition by cytosolic receptors
such as RIG-I and MDA5 (Oganesyan et al., 2006). TRAF3 autosomal
dominant deficiency has been reported in an adult who suffered from
HSE in childhood (Pérez de Diego et al., 2010). According to Pérez de
Diego, mutation in TRAF3 allele disturbed one of the TRAF3 zinc-finger
domains and despite normal mRNA expression it prevented production
of a stable protein. Upon stimulation with poly(I:C), dimerization of
IRF3 was affected and accumulation of the NF-κB in the nucleus was
impaired in patient's cells. Moreover, those cells did not respond by
IFN-β and IFN-λ production, therefore predisposition to HSE occurred
through impairment of TLR3-mediated IFNs production. Weak but de-
tectable residual TRAF3 responses were observed in the patient. Ge-
netically deficient TRAF3-knockout mice only live up to 10 days due to
the progressive depletion of peripheral white blood cells. However, it is
still possible to use mice with TRAF3-deficient specific cell types or
tissues (Xie et al., 2007). Mice with TRAF3-deficient myeloid cells
(neutrophils, eosinophils, basophils, monocytes, macrophages and
monocyte-derived dendritic cells (DCs); M-TRAF−/− mice) exhibited
altered systemic responses after poly(I:C) injection. Serum levels of IFN-
β decreased 6 h after poly(I:C) inoculation, but production of IL-6 in-
creased at 2 and 6 h after inoculation, whereas IL-12 levels were
markedly increased 2 h after injection (Lalani et al., 2015). Mice with
TRAF3-deficient B lymphocytes (B-TRAF−/− mice) displayed
69
Journal of Neuroimmunology 316 (2018) 65–73
development of spontaneous and highly penetrant B lymphomas
(Moore et al., 2012), however, mice with TRAF3 overexpression in B
cells exhibited autoimmune diseases, systemic inflammation and were
also predisposed to cancers (Zapata et al., 2009).
3.4.3. TBK1
TRIF and TRAF3 association leads to recruitment of two kinases,
TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), which play
important roles by coordinating the activation of IRF3 and NF-κB
(Fitzgerald et al., 2003). Murine Tbk1, besides participation in the Tlr3
pathway has been shown to take part in cytosolic dsRNA or dsDNA
sensing pathways, also resulting in antiviral defense (McWhirter et al.,
2004; Takaoka et al., 2007). Two heterozygous, autosomal dominant
mutations of TBK1 have been found in children suffering from HSE
(Herman et al., 2012). The loss of kinase functionality is due to loss of
activity or stability, possibly because mutations occurred in areas
strictly conserved across species. High titers of HSV-1 replication were
observed in fibroblasts from both patients, and the defect of TLR3-
mediated response initiated by dsRNA was observed in cells with de-
ficiency in TBK1 activity. On the contrary, response of the same cells to
infection with other dsDNA or single-stranded RNA (ssRNA) viruses was
comparable to that of healthy individuals. PBMCs of both patients
produced normal amounts of IFN-α in response to HSV-1 infection.
These results suggest that residual IFN is sufficient against common
viruses when mediated by other receptors and/or TLR3 pathway with
partial TBK1 deficiency. Interestingly, one of the parents of the HSE
patient, who had identical heterozygous mutation resulting in loss of
kinase activity, never suffered from HSE, thus, the clinical penetration
of this TBK1 deficiency was incomplete. A 50-year-old HSE patient
infected with HSV-2 and a mutation in TBK1 presented a significant
reduction in expression of CXCL10 and TNF-α in PBMCs after HSV-1
infection (Mørk et al., 2015). However, animals with Tbk1 knockout die
in perinatal period (Bonnard et al., 2000), but Tbk1−/− mouse em-
bryonic fibroblasts (MEFs) or macrophages show impaired production
of α/β IFNs in response to dsRNA or HSV-1 infection respectively
(Hemmi et al., 2004; Perry et al., 2004).
3.4.4. IRF3
IRF3, a decisive molecule in innate immune responses, upon phos-
phorylation and dimerization acts as a nuclear transcription factor in
several type I and III IFN-inducing pathways (Honda and Taniguchi,
2006). The importance of a functional IRF3 in CNS cells stresses the fact
that IRF3-deficient mice have increased risk of HSV-1 infection in the
brain (Menachery et al., 2010). Similarly, murine neural stem cells
(NSCs) lacking functional Tlr3, exhibited decreased Irf3 phosphoryla-
tion level as well as IFN-β expression after HSV-1 infection, highlighting
the dependency on Tlr3 and Irf3 in antiviral response (Sun et al., 2015).
Recently, an adolescent patient suffering from HSE was been found to
carry an autosomal dominant IRF3 deficiency by haploinsufficiency,
due to inability of phosphorylation and subsequent dimerization of the
protein, resulting in an impaired TLR3-TRIF pathway (Andersen et al.,
2015). Because of the mutation IFN-β expression was severely impaired
in fibroblasts of the patient after poly(I:C) stimulation or HSV-1 infec-
tion. Surprisingly, IRF3 protein expression in PBMCs from the patient
was comparable to healthy controls, however, significant reduction of
CXCL10 was observed in these cells after poly(I:C) stimulation, and IFN-
α2, -β, -λ1 and CXCL10 expression was highly abrogated after HSV-1
infection. Nevertheless, the father of the patient was found to be a
healthy carrier of the mutation. Furthermore, another IRF3 mutation
has been found in an adult who experienced HSE (Mørk et al., 2015).
Patient's PBMCs after poly(I:C) stimulation exhibited significantly re-
duced CXCL10 expression and highly diminished IFN-β as well as
CXCL10 response after HSV-1 infection.
Overall, the data suggest that while TLR3 may be largely diversified
for dsRNA responses in other cells, the CNS relies on TLR3 pathway to
respond to viral dsRNA and TLR3-dependent IFN induction as a
M.B. Mielcarska et al. Journal of Neuroimmunology 316 (2018) 65–73

cardinal consequence. Nevertheless, it has been observed that, in the broader immune responses than during natural infection (Johnston
fibroblasts of patients who experienced HSE and were diagnosed with et al., 2011). For example, viral envelope glycoproteins present in
mutations in TLR3, UNC93B1, TRIF, TRAF3 and TBK1, the production certain vaccines may enhance the production of neutralizing antibodies
of IFN-β and IFN-λ was also considerably impaired following HSV-1 (Önnheim et al., 2016; Awasthi et al., 2014; Zhang et al., 2014). In
infection (Lim et al., 2014). TLR3 and TLR3 pathway-related defi- order to achieve fuller antiviral protection, innovative adjuvants are
ciencies examined in induced pluripotent stem cell-derived (iPSC-de- added to vaccines to stimulate tissue resident memory CD4+ and CD8+
rived) neurons and oligodendrocytes resembled the phenotype of fi- T cells, which play an indispensable role in potent antiviral control
broblasts from patients with these deficiencies (Lafaille et al., 2012). (Long et al., 2014; Shin and Iwasaki, 2013). Further, chemokines are
Therefore, cells other than CNS-resident may also provide a molecular added to vaccines to improve memory CD8+ T cell-transportation to
substance of HSE pathogenesis and HSE-related phenotypes may pos- areas of viral infection (Shin and Iwasaki, 2012). In addition, novel
sibly be observed in a variety of host cells. vaccine platforms as well as delivery methods are still being in-
Deficiencies in single genes associated with TLR3 signaling pathway vestigated. A comprehensive list of vaccines against HSV currently in
may lead to impaired production of type I and III IFNs and various clinical trials or in research and development is reviewed by Johnston
cytokines that provide antiviral defense against HSV infection. The et al. (2016).
cellular phenotype associated with the HSV infection often corresponds
with decreases in production or function of antiviral proteins, and are
5. Conclusions
manifested among some people in the form of clinical consequences and
contribute to HSE predisposition. Mutations are often observed in single
Following discovery that the serious consequences of HSV infection
alleles, as the total deficiency of many TLR3-related proteins would be
may have a genetic background, there has been an ongoing process of
lethal, due to the participation in signaling pathways from multiple
identifying cellular components in which defects may favor the onset of
receptors. The first assumption of HSE association with genetic muta-
HSE. Well reported cases are those of children with HSE having mu-
tions emerged after the discovery of autosomal recessive signal trans-
tations in the TLR3 gene which suggests that TLR3 is a principal com-
ducer and activator of transcription 1 (STAT-1) deficiency in patients
ponent in defense against this disease. However, careful consideration
who suffered from HSE and exhibited incapacitated IFN-α/β, -γ and -λ
should also be given to the abundant mutations in single, im-
responses of cells to viral infection (Dupuis et al., 2003). Importantly,
munologically relevant genes associated with the TLR3 signaling
other signaling pathways which induce IFN production may also be
pathway upon receptor activation. Close examination of the fate of the
related to HSE, e.g., stimulator of interferon genes (STING)-defective
receptor at ligand recognition sites, as well as upstream events of TLR3
mice are highly susceptible to acute form of HSE (Reinert et al., 2016).
egress from the ER may identify factors facilitating signaling required
Pertinent to this, NF-κB essential modulator (NEMO) deficiency in
for induction of immunity in the CNS during HSV infection. For ex-
combination with impaired TLR3-mediated IFN production was found
ample, human deficiencies in the cathepsins and their role in TLR3
among patients suffering from HSE (Audry et al., 2011). Furthermore,
cleavage has not been investigated regarding development and severity
TRIF, which plays vital role in TLR3 signaling, determines the proper
of HSE. Cathepsins may constitute important components that facilitate
functioning of STING signaling pathway. Triggered by HSV, this
activation of TLR3. Presumably, cytoplasmic factors other than
pathway may also lead to the emergence of adequate antiviral defense
UNC93B may have significant influence on the TLR3 transportation
(Wang et al., 2016).
cells. In our laboratory, we have investigated the contribution of the
Cases discussed in this review may explain why HSV becomes CNS-
ESCRT-0 protein complex (endosomal sorting complex required for
hostile only in a small number of people, hence, predisposition to HSE
transport-0) to TLR3 transportation, and it appears that it may parti-
may be due to different single-gene defects, which may be functionally
cipate in TLR3 delivery to endosomes (unpublished), as is the case for
or immunologically related. Defective innate immunity to HSV may
other endosomal TLRs – TLR7 and TLR9 (Lee and Barton, 2014).
increase the risk of HSE both in children and adults and particular at-
Patients with a confirmed TLR3 deficiency or other HSE-predis-
tention in HSE should be directed towards careful monitoring of people
posing mutations should be carefully observed as HSE can be a result of
with diagnosed deficits in TLR3-related genes. However, no solid ex-
defects in other innate receptor pathways associated with induction of
planations can be offered for other infections or non-infectious diseases
IFN production. Genome sequencing in patients with HSE may con-
which may occur among HSE patients. Patients with inborn TLR3/TLR3
tribute to testing for mutations in TLR3/TLR3 signaling pathway and if
pathway errors may develop encephalitis or remain healthy, therefore
found, should be fully investigated. Although HSE recurrence is rare
clinical penetrance of the above mutations for HSE may be incomplete.
(Abel et al., 2010), there is a hypothesis that the disease appears more
Factors such as the environment, other infections, immunodeficiency
frequently among people with inborn errors of TLR3 immunity (Lim
status or other factors associated directly with the patient may be re-
et al., 2014). It is not known whether there are TLR3/TLR3 signaling-
garded as comparably important for HSE incidence (Zhang et al., 2013).
associated mutations common for recurrent HSE, yet a significant
On the other hand, HSE recurrence may occur more frequently among
number of TLR3-deficient patients have suffered from HSE more than
patients with congenital defects in the TLR3 pathway. For example,
once (Lim et al., 2014). The role of the host immune response in the
recurrent HSE was observed in 46,15% of such patients, however,
development of HSE has still not been precisely defined and HSV should
further studies are necessary to confirm this inclination (Lim et al.,
be considered as a challenging opponent. Intensive research is going on
2014).
towards creating a vaccine able to prevent primary HSV infection and
thus prevent subsequent development of HSE and associated con-
4. Progress on HSV vaccine development
sequences (Stanfield and Kousoulas, 2015; Johnston et al., 2016).
Based on the data presented in this review, HSE should be con-
Just as viral resistance and timing of treatment initiation are lim-
sidered as not only a rare disease, but also as a more complex disease
iting factors in the efficiency of drug-based therapies, vaccine devel-
with genetic defects, particularly related to the function of TLR3 and its
opment is considerably more difficult due geographical diversity of
signaling pathways in CNS resident cells.
HSV-1 and HSV-2 strains, complexity of the replication cycle and la-
tency. Current efforts are directed towards the design of preventive
vaccines that can protect against infection, or therapeutic vaccines that Funding
can reduce or eliminate clinical signs of infection (Johnston et al.,
2016). Emphasis is put on ensuring that future generations of vaccines, This work was supported by the National Science Centre, Poland [Nr
which may encounter both types of HSV, stimulate stronger and UMO-2016/23/N/NZ6/02499].

70
M.B. Mielcarska et al.
Acknowledgments
We thank Dr. Ryszard A. Mielcarski for helpful discussions on TLR3
signaling mechanisms as well as the art work.
References
Abel, L., Plancoulaine, S., Jouanguy, E., Zhang, S.Y., Mahfoufi, N., Nicolas, N., Sancho-
Shimizu, V., Alcaïs, A., Guo, Y., Cardon, A., Boucherit, S., Obach, D., Clozel, T.,
Lorenzo, L., Amsallem, D., Berquin, P., Blanc, T., Bost-Bru, C., Chabrier, S., Chabrol,
B., Cheuret, E., Dulac, O., Evrard, P., Héron, B., Lazaro, L., Mancini, J., Pedespan,
J.M., Rivier, F., Vallée, L., Lebon, P., Rozenberg, F., Casanova, J.L., Tardieu, M.,
2010. Age-dependent Mendelian predisposition to herpes simplex virus type 1 en-
cephalitis in childhood. J. Pediatr. 157, 623–629.
Akira, S., Takeda, K., Kaisho, T., 2001. Toll-like receptors: critical proteins linking innate
and acquired immunity. Nat. Immunol. 2, 675–680.
Akula, S.M., Naranatt, P.P., Walia, N.S., Wang, F.Z., Fegley, B., Chandran, B., 2003.
Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) infection of human
fibroblast cells occurs through endocytosis. J. Virol. 77, 7978–7990.
Aldea, S., Joly, L.M., Roujeau, T., Oswald, A.M., Devaux, B., 2003. Postoperative herpes
simplex virus encephalitis after neurosurgery: case report and review of the litera-
ture. Clin. Infect. Dis. 36, e96–9.
Alexopoulou, L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of double-
stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413,
732–738.
Algahtani, H., Shirah, B., Hmoud, M., Subahi, A., 2016. Nosocomial herpes simplex en-
cephalitis: a challenging diagnosis. J. Infect. Public Health 10, 343–347.
Altan, M., Burtness, B., 2015. EGFR-directed antibodies increase the risk of severe in-
fection in cancer patients. BMC Med. 13, 37.
Andersen, L.L., Mørk, N., Reinert, L.S., Kofod-Olsen, E., Narita, R., Jørgensen, S.E.,
Skipper, K.A., Höning, K., Gad, H.H., Østergaard, L., Ørntoft, T.F., Hornung, V.,
Paludan, S.R., Mikkelsen, J.G., Fujita, T., Christiansen, M., Hartmann, R., Mogensen,
T.H., 2015. Functional IRF3 deficiency in a patient with herpes simplex encephalitis.
J. Exp. Med. 212, 1371–1379.
Anderson, K.V., Jürgens, G., Nüsslein-Volhard, C., 1985. Establishment of dorsal-ventral
polarity in the Drosophila embryo; genetic studies on the role of the Toll gene pro-
duct. Cell 42, 779–789.
Audry, M., Ciancanelli, M., Yang, K., Cobat, A., Chang, H.H., Sancho-Shimizu, V.,
Lorenzo, L., Niehues, T., Reichenbach, J., Li, X.X., Israel, A., Abel, L., Casanova, J.L.,
Zhang, S.Y., Jouanguy, E., Puel, A., 2011. NEMO is a key component of NF-κB- and
IRF-3-dependent TLR3-mediated immunity to herpes simplex virus. J. Allergy Clin.
Immunol. 128, 610–617.
Awasthi, S., Huang, J., Shaw, C., Friedman, H.M., 2014. Blocking herpes simplex virus 2
glycoprotein E immune evasion as an approach to enhance efficacy of a trivalent
subunit antigen vaccine for genital herpes. J. Virol. 88, 8421–8432.
Baines, J.D., 2011. Herpes simplex virus capsid assembly and DNA packaging: a present
and future antiviral drug target. Trends Microbiol. 19, 606–613.
Barreiro, L.B., Ben-Ali, M., Quach, H., Laval, G., Patin, E., Pickrell, J.K., Bouchier, C.,
Tichit, M., Neyrolles, O., Gicquel, B., Kidd, J.R., Kidd, K.K., Alcaïs, A., Ragimbeau, J.,
Pellegrini, S., Abel, L., Casanova, J.L., Quintana-Murci, L., 2009. Evolutionary dy-
namics of human Toll-like receptors and their different contributions to host defense.
PLoS Genet. 5, e1000562.
Barton, G.M., Kagan, J.C., 2009. A cell biological view of Toll-like receptor function:
regulation through compartmentalization. Nat. Rev. Immunol. 9, 535–542.
Blasius, A.L., Beutler, B., 2010. Intracellular toll-like receptors. Immunity 32, 305–315.
Bonnard, M., Mirtsos, C., Suzuki, S., Graham, K., Huang, J., Ng, M., Itié, A., Wakeham, A.,
Shahinian, A., Henzel, W.J., Elia, A.J., Shillinglaw, W., Mak, T.W., Cao, Z., Yeh, W.C.,
2000. Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-
kappaB-dependent gene transcription. EMBO J. 19, 4976–4985.
Brinkmann, M.M., Spooner, E., Hoebe, K., Beutler, B., Ploegh, H.L., Kim, Y.M., 2007. The
interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial
for TLR signaling. J. Cell Biol. 177, 265–275.
Bsibsi, M., Ravid, R., Gveric, D., Van Noort, J.M., 2002. Broad expression of Toll-like
receptors in the human central nervous system. J. Neuropathol. Exp. Neurol. 61,
1013–1021.
Bsibsi, M., Persoon-Deen, C., Verwer, R.W., Meeuwsen, S., Ravid, R., Van Noort, J.M.,
2006. Toll-like receptor 3 on adult human astrocytes triggers production of neuro-
protective mediators. Glia 53, 688–695.
Buckley, R.H., 2004. Molecular defects in human severe combined immunodeficiency and
approaches to immune reconstitution. Annu. Rev. Immunol. 22, 625–655.
Casrouge, A., Zhang, S.Y., Eidenschenk, C., Jouanguy, E., Puel, A., Yang, K., Alcais, A.,
Picard, C., Mahfoufi, N., Nicolas, N., Lorenzo, L., Plancoulaine, S., Sénéchal, B.,
Geissmann, F., Tabeta, K., Hoebe, K., Du, X., Miller, R.L., Héron, B., Mignot, C., de
Villemeur, T.B., Lebon, P., Dulac, O., Rozenberg, F., Beutler, B., Tardieu, M., Abel, L.,
Casanova, J.L., 2006. Herpes simplex virus encephalitis in human UNC-93B defi-
ciency. Science 314, 308–312.
Chattopadhyay, S., Sen, G.C., 2014. Tyrosine phosphorylation in Toll-like receptor sig-
naling. Cytokine Growth Factor Rev. 25, 533–541.
Cheung, P., Banfield, B.W., Tufaro, F., 1991. Brefeldin A arrests the maturation and egress
of herpes simplex virus particles during infection. J. Virol. 65, 1893–1904.
Chow, F.C., Glaser, C.A., Sheriff, H., Xia, D., Messenger, S., Whitley, R., Venkatesan, A.,
2015. Use of clinical and neuroimaging characteristics to distinguish temporal lobe
herpes simplex encephalitis from its mimics. Clin. Infect. Dis. 60, 1377–1383.
De Bouteiller, O., Merck, E., Hasan, U.A., Hubac, S., Benguigui, B., Trinchieri, G., Bates,
71
Journal of Neuroimmunology 316 (2018) 65–73
E.E., Caux, C., 2005. Recognition of double-stranded RNA by human Toll-like re-
ceptor 3 and downstream receptor signaling requires multimerization and acidic pH.
J. Biol. Chem. 280, 38133–38145.
De Tiège, X., Rozenberg, F., Héron, B., 2008. The spectrum of herpes simplex encephalitis
in children. Eur. J. Paediatr. Neurol. 12, 72–81.
Dupuis, S., Jouanguy, E., Al-Hajjar, S., Fieschi, C., Al-Mohsen, I.Z., Al-Jumaah, S., Yang,
K., Chapgier, A., Eidenschenk, C., Eid, P., Al, Ghonaium A., Tufenkeji, H., Frayha, H.,
Al-Gazlan, S., Al-Rayes, H., Schreiber, R.D., Gresser, I., Casanova, J.L., 2003.
Impaired response to interferon-alpha/beta and lethal viral disease in human STAT1
deficiency. Nat. Genet. 33, 388–391.
Fitzgerald, K.A., McWhirter, S.M., Faia, K.L., Rowe, D.C., Latz, E., Golenbock, D.T., Coyle,
A.J., Liao, S.M., Maniatis, T., 2003. IKKepsilon and TBK1 are essential components of
the IRF3 signaling pathway. Nat. Immunol. 4, 491–496.
Garcia-Cattaneo, A., Gobert, F.X., Müller, M., Toscano, F., Flores, M., Lescure, A., Del
Nery, E., Benaroch, P., 2012. Cleavage of Toll-like receptor 3 by cathepsins B and H is
essential for signaling. Proc. Natl. Acad. Sci. U. S. A. 109, 9053–9058.
Gnann, J.W., Whitley, R.J., 2017. Herpes simplex encephalitis: an update. Curr. Infect.
Dis. Rep. 19, 13.
Gnann, J.W., Sköldenberg, B., Hart, J., Aurelius, E., Schliamser, S., Studahl, M., Eriksson,
B.M., Hanley, D., Aoki, F., Jackson, A.C., Griffiths, P., Miedzinski, L., Hanfelt-Goade,
D., Hinthorn, D., Ahlm, C., Aksamit, A., Cruz-Flores, S., Dale, I., Cloud, G., Jester, P.,
Whitley, R.J., 2015. Herpes simplex encephalitis: lack of clinical benefit of long-term
valacyclovir therapy. Clin. Infect. Dis. 61, 683–691.
Greene, W., Gao, S.J., 2009. Actin dynamics regulate multiple endosomal steps during
Kaposi's sarcoma-associated herpesvirus entry and trafficking in endothelial cells.
PLoS Pathog. 5, e1000512.
Griffin, D.E., 2005. Encephalitis, myelitis, and neuritis. In: Mandell, G.L., Bennett, J.E.,
Dolin, R. (Eds.), Mandell, Bennett, & Dolin: Principles and Practice of Infectious
Diseases, 6th Edition. Churchill Livingstone, New York, pp. 1144–1150.
Guo, Y., Audry, M., Ciancanelli, M., Alsina, L., Azevedo, J., Herman, M., Anguiano, E.,
Sancho-Shimizu, V., Lorenzo, L., Pauwels, E., Philippe, P.B., Pérez de Diego, R.,
Cardon, A., Vogt, G., Picard, C., Andrianirina, Z.Z., Rozenberg, F., Lebon, P.,
Plancoulaine, S., Tardieu, M., Doireau, V., Jouanguy, E., Chaussabel, D., Geissmann,
F., Abel, L., Casanova, J.L., Zhang, S.Y., 2011. Herpes simplex virus encephalitis in a
patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective
immunity. J. Exp. Med. 208, 2083–2098.
Harley, C.A., Dasgupta, A., Wilson, D.W., 2001. Characterization of herpes simplex virus-
containing organelles by subcellular fractionation: role for organelle acidification in
assembly of infectious particles. J. Virol. 75, 1236–1251.
Hemmi, H., Takeuchi, O., Sato, S., Yamamoto, M., Kaisho, T., Sanjo, H., Kawai, T.,
Hoshino, K., Takeda, K., Akira, S., 2004. The roles of two IkappaB kinase-related
kinases in lipopolysaccharide and double stranded RNA signaling and viral infection.
J. Exp. Med. 199, 1641–1650.
Herman, M., Ciancanelli, M., Ou, Y.H., Lorenzo, L., Klaudel-Dreszler, M., Pauwels, E.,
Sancho-Shimizu, V., Pérez de Diego, R., Abhyankar, A., Israelsson, E., Guo, Y.,
Cardon, A., Rozenberg, F., Lebon, P., Tardieu, M., Heropolitanska-Pliszka, E.,
Chaussabel, D., White, M.A., Abel, L., Zhang, S.Y., Casanova, J.L., 2012.
Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex
encephalitis of childhood. J. Exp. Med. 209, 1567–1582.
Hoebe, K., Beutler, B., 2006. TRAF3: a new component of the TLR-signaling apparatus.
Trends Mol. Med. 12, 187–189.
Hollinshead, M., Johns, H.L., Sayers, C.L., Gonzalez-Lopez, C., Smith, G.L., Elliott, G.,
2012. Endocytic tubules regulated by Rab GTPases 5 and 11 are used for envelop-
ment of herpes simplex virus. EMBO J. 31, 4204–4220.
Honda, K., Taniguchi, T., 2006. IRFs: master regulators of signalling by Toll-like receptors
and cytosolic pattern-recognition receptors. Nat. Rev. Immunol. 6, 644–658.
Jacobs, B.L., Langland, J.O., 1996. When two strands are better than one: the mediators
and modulators of the cellular responses to double-stranded RNA. Virology 219,
339–349.
Jiang, Z., Mark, T.W., Sen, G., Li, X., 2004. Toll-like receptor 3-mediated activation of NF-
kappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing
IFN-beta. Proc. Natl. Acad. Sci. U. S. A. 101, 3533–3538.
Johnston, C., Koelle, D.M., Wald, A., 2011. HSV-2: in pursuit of a vaccine. J. Clin. Invest.
121, 4600–4609.
Johnston, C., Gottlieb, S.L., Wald, A., 2016. Status of vaccine research and development
of vaccines for herpes simplex virus. Vaccine 34, 2948–2952.
Kim, Y.M., Brinkmann, M.M., Paquet, M.E., Ploegh, H.L., 2008. UNC93B1 delivers nu-
cleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234–238.
Krishnan, J., Selvarajoo, K., Tsuchiya, M., Lee, G., Choi, S., 2007. Toll-like receptor signal
transduction. Exp. Mol. Med. 39, 421–438.
Lafaille, F.G., Pessach, I.M., Zhang, S.Y., Ciancanelli, M.J., Herman, M., Abhyankar, A.,
Ying, S.W., Keros, S., Goldstein, P.A., Mostoslavsky, G., Ordovas-Montanes, J.,
Jouanguy, E., Plancoulaine, S., Tu, E., Elkabetz, Y., Al-Muhsen, S., Tardieu, M.,
Schlaeger, T.M., Daley, G.Q., Abel, L., Casanova, J.L., Studer, L., Notarangelo, L.D.,
2012. Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient
CNS cells. Nature 491, 769–773.
Lalani, A.I., Moore, C.R., Luo, C., Kreider, B.Z., Liu, Y., Morse 3rd, H.C., Xie, P., 2015.
Myeloid cell TRAF3 regulates immune responses and inhibits inflammation and
tumor development in mice. J. Immunol. 194, 334–348.
Lee, B.L., Barton, G.M., 2014. Trafficking of endosomal Toll-like receptors. Trends Cell
Biol. 24, 360–369.
Lellouch-Tubiana, A., Fohlen, M., Robain, O., Rozenberg, F., 2000. Immunocytochemical
characterization of long-term persistent immune activation in human brain after
herpes simplex encephalitis. Neuropathol. Appl. Neurobiol. 26, 285–294.
Li, K., Chen, Z., Kato, N., Gale Jr., M., Lemon, S.M., 2005. Distinct poly(IeC) and virus-
activated signaling pathways leading to interferon-beta production in hepatocytes. J.
M.B. Mielcarska et al.
Biol. Chem. 280, 16739–16747.
Lim, H.K., Seppänen, M., Hautala, T., Ciancanelli, M.J., Itan, Y., Lafaille, F.G., Dell, W.,
Lorenzo, L., Byun, M., Pauwels, E., Rönnelid, Y., Cai, X., Boucherit, S., Jouanguy, E.,
Paetau, A., Lebon, P., Rozenberg, F., Tardieu, M., Abel, L., Yildiran, A., Vergison, A.,
Roivainen, R., Etzioni, A., Tienari, P.J., Casanova, J.L., Zhang, S.Y., 2014. TLR3 de-
ficiency in herpes simplex encephalitis: high allelic heterogeneity and recurrence
risk. Neurology 83, 1888–1897.
Liu, L., Botos, I., Wang, Y., Leonard, J.N., Shiloach, J., Segal, D.M., Davies, D.R., 2008.
Structural basis of toll-like receptor 3 signaling with double-stranded RNA. Science
320, 379–381.
Lo Presti, A., Weil, A.G., Niazi, T.N., Bhatia, S., 2015. Herpes simplex reactivation or
postinfectious inflammatory response after epilepsy surgery: case report and review
of the literature. Surg. Neurol. Int. 6, 47.
Long, D., Skoberne, M., Gierahn, T.M., Larson, S., Price, J.A., Clemens, V., Baccari, A.E.,
Cohane, K.P., Garvie, D., Siber, G.R., Flechtner, J.B., 2014. Identification of novel
virus-specific antigens by CD4 + and CD8 + T cells from asymptomatic HSV-2 ser-
opositive and seronegative donors. Virology 464-465, 296–311.
Maraş Genç, H., Uyur Yalçın, E., Sayan, M., Bayhan, A., Öncel, S., Arısoy, E.S., Kara, B.,
2016. Clinical outcomes in children with herpes simplex encephalitis receiving
steroid therapy. J. Clin. Virol. 80, 87–92.
McWhirter, S.M., Fitzgerald, K.A., Rosains, J., Rowe, D.C., Golenbock, D.T., Maniatis, T.,
2004. IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-defi-
cient mouse embryonic fibroblasts. Proc. Natl. Acad. Sci. U. S. A. 101, 233–238.
Medzhitov, R., Preston-Hurlburt, P., Janeway Jr., C.A., 1997. A human homologue of the
Drosophila Toll protein signals activation of adaptive immunity. Nature 388,
394–397.
Menachery, V.D., Pasieka, T.J., Leib, D.A., 2010. Interferon regulatory factor 3-dependent
pathways are critical for control of herpes simplex virus type 1 central nervous
system infection. J. Virol. 84, 9685–9694.
Meylan, E., Tschopp, J., 2006. Toll-like receptors and RNA helicases: two parallel ways to
trigger antiviral responses. Mol. Cell 22, 561–569.
Meylan, E., Burns, K., Hofmann, K., Blancheteau, V., Martinon, F., Kelliher, M., Tschopp,
J., 2004. RIP1 is an essential mediator of Toll-like receptor 3-induced NF-kappa B
activation. Nat. Immunol. 5, 503–507.
Modi, S., Mahajan, A., Dharaiya, D., Varelas, P., Mitsias, P., 2017. Burden of herpes
simplex virus encephalitis in the United States. J. Neurol. 264, 1204–1208.
Moore, C.R., Liu, Y., Shao, C., Covey, L.R., Morse 3rd, H.C., Xie, P., 2012. Specific deletion
of TRAF3 in B lymphocytes leads to B-lymphoma development in mice. Leukemia 26,
1122–1127.
Mørk, N., Kofod-Olsen, E., Sørensen, K.B., Bach, E., Ørntoft, T.F., Østergaard, L., Paludan,
S.R., Christiansen, M., Mogensen, T.H., 2015. Mutations in the TLR3 signaling
pathway and beyond in adult patients with herpes simplex encephalitis. Genes
Immun. 16, 552–566.
Muzio, M., Polentarutti, N., Bosisio, D., Manoj Kumar, P.P., Mantovani, A., 2000. Toll-like
receptor family and signalling pathway. Biochem. Soc. Trans. 28, 563–566.
Oganesyan, G., Saha, S.K., Guo, B., He, J.Q., Shahangian, A., Zarnegar, B., Perry, A.,
Cheng, G., 2006. Critical role of TRAF3 in the Toll-like receptor-dependent and -in-
dependent antiviral response. Nature 439, 208–211.
Önnheim K., Ekblad M., Görander S., Bergström T., Liljeqvist J., 2016, Vaccination with
the secreted glycoprotein G of herpes simplex virus 2 induces protective immunity
after genital infection. Viruses 8, 110.
Paludan, S.R., Bowie, A.G., Horan, K.A., Fitzgerald, K.A., 2011. Recognition of herpes-
viruses by the innate immune system. Nat. Rev. Immunol. 11, 143–154.
Peltier, D.C., Simms, A., Farmer, J.R., Miller, D.J., 2010. Human neuronal cells possess
functional cytoplasmic and TLR-mediated innate immune pathways influenced by
phosphatidylinositol-3 kinase signaling. J. Immunol. 184, 7010–7021.
Pérez de Diego, R., Rodriguez-Gallego, C., 2014. Chapter 34 – other TLR pathway defects.
In: Sullivan, K.E., Stiehm, E.R. (Eds.), Stiehm's Immune Deficiencies. Elsevier, pp.
692.
Pérez de Diego, R., Sancho-Shimizu, V., Lorenzo, L., Puel, A., Plancoulaine, S., Picard, C.,
Herman, M., Cardon, A., Durandy, A., Bustamante, J., Vallabhapurapu, S., Bravo, J.,
Warnatz, K., Chaix, Y., Cascarrigny, F., Lebon, P., Rozenberg, F., Karin, M., Tardieu,
M., Al-Muhsen, S., Jouanguy, E., Zhang, S.Y., Abel, L., Casanova, J.L., 2010. Human
TRAF3 adaptor molecule deficiency leads to impaired Toll-like receptor 3 response
and susceptibility to herpes simplex encephalitis. Immunity 33, 400–411.
Perry, A.K., Chow, E.K., Goodnough, J.B., Yeh, W.C., Cheng, G., 2004. Differential re-
quirement for TANK-binding kinase-1 in type I interferon responses to toll-like re-
ceptor activation and viral infection. J. Exp. Med. 199, 1651–1658.
Purves, D., Augustine, G.J., Fitzpatrick, D., Katz, L.C., LaMantia, A.S., McNamara, J.O.,
Williams, S.M., 2001. Chapter 1 – neuroglial cells. In: Neuroscience, 2nd Edition.
Sinauer Associates, Sunderland (MA).
Qi, R., Singh, D., Kao, C.C., 2012. Proteolytic processing regulates Toll-like receptor 3
stability and endosomal localization. J. Biol. Chem. 287, 32617–32629.
Reinert, L.S., Harder, L., Holm, C.K., Iversen, M.B., Horan, K.A., Dagnæs-Hansen, F.,
Ulhøi, B.P., Holm, T.H., Mogensen, T.H., Owens, T., Nyengaard, J.R., Thomsen, A.R.,
Paludan, S.R., 2012. TLR3 deficiency renders astrocytes permissive to herpes simplex
virus infection and facilitates establishment of CNS infection in mice. J. Clin. Invest.
122, 1368–1376.
Reinert, L.S., Lopušná, K., Winther, H., Sun, C., Thomsen, M.K., Nandakumar, R.,
Mogensen, T.H., Meyer, M., Vægter, C., Nyengaard, J.R., Fitzgerald, K.A., Paludan,
S.R., 2016. Sensing of HSV-1 by the cGAS–STING pathway in microglia orchestrates
antiviral defence in the CNS. Nat. Commun. 7, 13348.
Riancho, J., Delgado-Alvarado, M., Sedano, M.J., Polo, J.M., Berciano, J., 2013. Herpes
simplex encephalitis: clinical presentation, neurological sequelae and new prognostic
factors. Ten years of experience. Neurol. Sci. 34, 1879–1881.
Rozenberg, F., Deback, C., Agut, C., 2011. Herpes simplex encephalitis: from virus to
72
Journal of Neuroimmunology 316 (2018) 65–73
therapy. Infect. Disord. Drug Targets 11, 235–250.
Samuel, C.E., 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778–809.
Sancho-Shimizu, V., Pérez de Diego, R., Jouanguy, E., Zhang, S.Y., Casanova, J.L., 2011a.
Inborn errors of anti-viral interferon immunity in humans. Curr. Opin. Virol. 1,
487–496.
Sancho-Shimizu, V., Pérez de Diego, R., Lorenzo, L., Halwani, R., Alangari, A., Israelsson,
E., Fabrega, S., Cardon, A., Maluenda, J., Tatematsu, M., Mahvelati, F., Herman, M.,
Ciancanelli, M., Guo, Y., AlSum, Z., Alkhamis, N., Al-Makadma, A.S., Ghadiri, A.,
Boucherit, S., Plancoulaine, S., Picard, C., Rozenberg, F., Tardieu, M., Lebon, P.,
Jouanguy, E., Rezaei, N., Seya, T., Matsumoto, M., Chaussabel, D., Puel, A., Zhang,
S.Y., Abel, L., Al-Muhsen, S., Casanova, J.L., 2011b. Herpes simplex encephalitis in
children with autosomal recessive and dominant TRIF deficiency. J. Clin. Invest. 121,
4889–4902.
Sarkar, S.N., Smith, H.L., Rowe, T.M., Sen, G.S., 2003. Double-stranded RNA signaling by
Toll-like receptor 3 requires specific tyrosine residues in its cytoplasmic domain. J.
Biol. Chem. 278, 4393–4396.
Sarkar, S.N., Peters, K.L., Elco, C.P., Sakamoto, S., Pal, S., Sen, G.C., 2004. Novel roles of
TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.
Nat. Struct. Mol. Biol. 11, 1060–1067.
Sarkar, S.N., Elco, C.P., Peters, K.L., Chattopadhyay, S., Sen, G.C., 2007. Two tyrosine
residues of Toll-like receptor 3 trigger different steps of NF-kappa B activation. J.
Biol. Chem. 282, 3423–3427.
Sasai, M., Oshiumi, H., Matsumoto, M., Inoue, N., Fujita, F., Nakanishi, M., Seya, T., 2005.
Cutting edge: NF-kappaB-activating kinase-associated protein 1 participates in TLR3/
Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory
factor 3 activation. J. Immunol. 174, 27–30.
Sharma, V., Mobeen, F., Prakash, T., 2016. Comparative genomics of herpesviridae family
to look for potential signatures of human infecting strains. Int. J. Genomics 2016,
9543274.
Shin, H., Iwasaki, A., 2012. A vaccine strategy protects against genital herpes by estab-
lishing local memory T cells. Nature 491, 463–467.
Shin, H., Iwasaki, A., 2013. Generating protective immunity against genital herpes.
Trends Immunol. 34, 487–494.
Sironi, M., Peri, A.M., Cagliani, R., Forni, D., Riva, S., Biasin, M., Clerici, M., Gori, A.,
2017. TLR3 mutations in adult patients with herpes simplex virus and varicella-zoster
virus encephalitis. J. Infect. Dis. 215, 1430–1434.
Stanfield, B., Kousoulas, K.G., 2015. Herpes simplex vaccines: prospects of live-attenuated
HSV vaccines to combat genital and ocular infections. Curr. Clin. Microbiol. Rep. 2,
125–136.
Sun, X., Shi, L., Zhang, H., Li, R., Liang, R., Liu, Z., 2015. Effects of Toll-like receptor 3 on
herpes simplex virus type-1-infected mouse neural stem cells. Can. J. Microbiol. 61,
201–208.
Tabeta, K., Hoebe, K., Janssen, E.M., Du, X., Georgel, P., Crozat, K., Mudd, S., Mann, N.,
Sovath, S., Goode, J., Shamel, L., Herskovits, A.A., Portnoy, D.A., Cooke, M.,
Tarantino, L.M., Wiltshire, T., Steinberg, B.E., Grinstein, S., Beutler, B., 2006. The
Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-
like receptors 3, 7 and 9. Nat. Immunol. 7, 156–164.
Takaoka, A., Wang, Z., Choi, M.K., Yanai, H., Negishi, H., Ban, T., Lu, Y., Miyagishi, M.,
Kodama, T., Honda, K., Ohba, Y., Taniguchi, T., 2007. DAI (DLM-1/ZBP1) is a cy-
tosolic DNA sensor and an activator of innate immune response. Nature 448,
501–505.
Toscano, F., Estornes, Y., Virard, F., Garcia-Cattaneo, A., Pierrot, A., Vanbervliet, B.,
Bonnin, M., Ciancanelli, M.J., Zhang, S.Y., Funami, K., Seya, T., Matsumoto, M., Pin,
J.J., Casanova, J.L., Renno, T., Lebecque, S., 2013. Cleaved/associated TLR3 re-
presents the primary form of the signaling receptor. J. Immunol. 190, 764–773.
Tsai, S.Y., Segovia, J.A., Chang, T.H., Shil, N.K., Pokharel, S.M., Kannan, T.R., Baseman,
J.B., Defrêne, J., Pagé, N., Cesaro, A., Tessier, P.A., Bose, S., 2015. Regulation of TLR3
activation by S100A9. J. Immunol. 195, 4426–4437.
Turcotte, S., Letellier, J., Lippé, R., 2005. Herpes simplex virus type 1 capsids transit by
the trans-Golgi network, where viral glycoproteins accumulate independently of
capsid egress. J. Virol. 79, 8847–8860.
Upton, J.W., Chan, F.K., 2014. Staying alive: cell death in anti-viral immunity. Mol. Cell
54, 273–280.
Wald, A., Corey, L., 2007. Chapter 36 – persistence in the population: epidemiology,
transmission. In: Arvin, A., Campadelli-Fiume, G., Mocarski, E., Moore, P.S.,
Roizman, B., Whitley, R.J., Yamanishi, K. (Eds.), Human Herpesviruses: Biology,
Therapy, and Immunoprophylaxis. Cambridge University Press, Cambridge.
Wang, X., Majumdar, T., Kessler, P., Ozhegov, E., Zhang, Y., Chattopadhyay, S., Barik, S.,
Sen, G.C., 2016. STING requires the adaptor TRIF to trigger innate immune responses
to microbial infection. Cell Host Microbe 20, 329–341.
Whitley, R.J., Kimberlin, D.W., 2005. Herpes simplex encephalitis: children and adoles-
cents. Semin. Pediatr. Infect. Dis. 16, 17–23.
Whitley, R.J., Kimberlin, D.W., Prober, C.G., 2007. Chapter 32 – Pathogenesis and dis-
ease. In: Arvin, A., Campadelli-Fiume, G., Mocarski, E., Moore, P.S., Roizman, B.,
Whitley, R.J., Yamanishi, K. (Eds.), Human Herpesviruses. Biology, Therapy, and
Immunoprophylaxis. Cambridge University Press, Cambridge.
Widener, R.W., Whitley, R.J., 2014. Chapter 11 – Herpes simplex virus. In: Tselis, A.C.,
Booss, J. (Eds.), Handbook of Clinical Neurology. Neurovirology Vol 123. pp.
251–263.
Xie, P., Stunz, L.L., Larison, K.D., Yang, B., Bishop, G.A., 2007. TRAF3 is a critical reg-
ulator of B cell homeostasis in secondary lymphoid organs. Immunity 27, 253–267.
Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O.,
Sugiyama, M., Okabe, M., Takeda, K., Akira, S., 2003. Role of adaptor TRIF in the
MyD88-independent toll-like receptor signaling pathway. Science 301, 640–643.
Zapata, J.M., Llobet, D., Krajewska, M., Lefebvre, S., Kress, C.L., Reed, J.C., 2009.
Lymphocyte-specific TRAF3 transgenic mice have enhanced humoral responses and
M.B. Mielcarska et al.
develop plasmacytosis, autoimmunity, inflammation, and cancer. Blood 113,
4595–4603.
Zerboni, L., Che, X., Reichelt, M., Qiao, Y., Gu, H., Arvin, A., 2013. Herpes simplex virus 1
tropism for human sensory ganglion neurons in the severe combined im-
munodeficiency mouse model of neuropathogenesis. J. Virol. 87, 2791–2802.
Zhang, S.Y., Jouanguy, E., Ugolini, S., Smahi, A., Elain, G., Romero, P., Segal, D., Sancho-
Shimizu, V., Lorenzo, L., Puel, A., Picard, C., Chapgier, A., Plancoulaine, S., Titeux,
M., Cognet, C., von Bernuth, H., Ku, C.L., Casrouge, A., Zhang, X.X., Barreiro, L.,
Leonard, J., Hamilton, C., Lebon, P., Héron, B., Vallée, L., Quintana-Murci, L.,
Hovnanian, A., Rozenberg, F., Vivier, E., Geissmann, F., Tardieu, M., Abel, L.,
Casanova, J.L., 2007. TLR3 deficiency in patients with herpes simplex encephalitis.
Science 317, 1522–1527.
73
Journal of Neuroimmunology 316 (2018) 65–73
Zhang, S.Y., Boisson-Dupuis, S., Chapgier, A., Yang, K., Bustamante, J., Puel, A., Picard,
C., Abel, L., Jouanguy, E., Casanova, J.L., 2008. Inborn errors of interferon (IFN)-
mediated immunity in humans: insights into the respective roles of IFN-alpha/beta,
IFN-gamma, and IFN-lambda in host defense. Immunol. Rev. 226, 29–40.
Zhang, S.Y., Herman, M., Ciancanelli, M.J., Pérez de Diego, R., Sancho-Shimizu, V., Abel,
L., Casanova, J.L., 2013. TLR3 immunity to infection in mice and humans. Curr. Opin.
Immunol. 25, 19–33.
Zhang, P., Xie, L., Balliet, J.W., Casimiro, D.R., Yao, F., 2014. A herpes simplex virus 2
(HSV-2) glycoprotein D-expressing nonreplicating dominant-negative HSV-2 virus
vaccine is superior to a gD2 subunit vaccine against HSV-2 genital infection in guinea
pigs. PLoS One 9, e101373.