Tick-borne encephalitis virus

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Genomic and structural organization

The genome of flaviviruses is a single positive stranded RNA of10,8 kilobases (Kb) that serves three discrete roles within the life cycle, as 1/ the messenger RNA (mRNA) for translation of all viral proteins, 2/ a template during RNA replication, and 3/ genetic material packaged within new viral particles [18,19]. Furthermore, the genomic RNA is infectious by itself [20].
The genomic viral RNA (gRNA) is a single open reading frame (ORF) of3,400 codons, flanked by a 5’- and a 3’-noncoding regions (NCR) of respectively 100 nucleotides and 350 to 750 nucleotides [21]. The ORF encodes a polyproteinthat is processed by viral and host proteases into three structural and seven non-structural proteins (Figure 2). The 5’NCR carries two conserved stem-loop regions and a type I cap structure m7GpppAmpN2 [22–24]. The 3’NCR is made up of a stem-loop and two dumbbell sequences and, unlike cellular mRNAs, does not carry a poly-A tail [25]. However, for some variants and quasispecies of TBEV, an internal poly-A tract is included in the 3’-NCR variable region that enhances viral virulence [25–28]. Because they are located at the 5’ and 3’ ends of the flaviviral genome, the NCRs of flaviviruses play an important role in translation, RNA replication and packaging, as well as in immune modulation [29].
A unique ORF is expressed and the produced polyprotein is processed by cleavage. The viral protein prM, NS2A and NS3 are then subject to maturation. Specific marks highlight cleavage sites for host signalase (•), viral serine protease ( ), furin or related protease ( ), and unknown proteases (?) [Adapted from 30].
The incomplete degradation of the gRNA by the host 5′-3′ exoribonuclease 1 (XRN1) produces long subgenomic flaviviral RNA (sfRNA) of 300 to 500 nucleotides, originating from the three dimensional structures of 3’ NCR [31–33]. The produced sfRNAs interfere with RNAi complex mediators, inducing a decrease in siRNA response in tick cells [34] and/or in mammalian and mosquito cells [35,36]. Mosquito-borne flaviviruses sfRNAs also increase uncapped cellular mRNA stability by repressing XRN1 activity [37], interfere with type I interferon (IFN) signaling [38], and are essential for viral growth [39,40].
From a structural point of view, Flavivirus virions are small spherical particles of 35 nanometers (nm) to 55 nm, enclosing a 25 nm to 30 nm electron-dense core, that is presumed to contain one copy of the viral genome and hundreds of copies of Capsid (C) protein [41,42]. This nucleocapsid (NC) core is surrounded by a lipid envelope in which 180 copies of the Membrane (M, 8 kDa) and Envelope (E, 53 kDa) transmembrane proteins are anchored (Figure 3)[2,21,30,42]. The M protein produced during maturation of nascent virus particles within the secretory pathway is a small proteolytic fragment of the precursor prM protein [18]. The E glycoprotein, the major antigenic determinant on virus particles, is a class II viral fusion protein that forms an icosahedral network. It mediates binding and fusion during virus entry [43,44].
The viral nucleocapsid (in blue), formed by the positive-stranded RNA genome and protein C, is surrounded by a lipid envelope (in yellow) in which prM/M (in green) and E (in red) proteins are carboxy-terminally anchored. prM and E form heterodimers in immature particles, whereas infectious, mature virus particles carry the small protein M and homodimers of protein E [9].
TBEV is very stable under natural conditions and the three subtypes are genetically and antigenically very similar. TBEV-Fe and TBEV-Sib are phylogenetically more closely related to each other than to TBEV–Eu, but the degree of variation in the amino acid level between strains within TBEV-Eu and TBEV-FE subtypes remains low [17,45]. The amino acid variability between the three subtypes is in the range of variation within flaviviruses (3±6% to 5±6%), while the nucleotide level variability is higher (1% to 16±9%). Sequences of the E protein differ by no more than 2±2%, suggesting a selection pressure favoring the conservation of the E protein [46]. As a result of this close antigenic relationship, there is a high degree of cross-protection between the subtype strains in mice [47,48]. Moreover, neutralizing antibodies against TBEV can also provide a protection against the infection by some other flaviviruses, such as Omsk Hemorrhagic Fever Virus (OHFV) [49].

Binding and entry

The first step of Flavivirus entry involves the interaction of the E glycoprotein with cellular attachment factors that concentrate and recruit the virus, and primary receptors that bind the viral particles and induce endocytosis [50,51].
Negatively charged sulfated Glycoaminoglycans (GAG), which are abundantly expressed on numerous cell types, are utilized as attachment factors by several flaviviruses [52–56]. For TBEV, the GAG protein Heparan Sulfate (HS) mediates viral attachment but CHO epithelial cell lines lacking HS are still highly susceptible to TBEV, suggesting that other surface molecules, which might be laminin binding protein (LBP) [57] and human αVβ3 integrin [58], are involved in viral attachment and entry [59,60].
After attachment, TBEV entry into the cell can occur through clathrin-mediated endocytosis [61] or through micropinocytosis, a clathrin-independent endocytosis [62]. This might be dependent on cell type and serotype [63]. So far, the entry receptors of TBEV are not deciphered. The virus utilizes either a ubiquitous receptor molecule or multiple receptors for cell entry, as TBEV infection has been observed in a variety of host cells and as it circulates in nature between arthropod vectors and their vertebrate hosts [59].
The internalized virus is then transported to the early/intermediate endosome that matures into a late endosome. The low pH in the endosome, at an optimum of pH=5.4±1, triggers conformational rearrangements of the class II fusion E protein [43,64], inducing the fusion of the viral particle with endosomal membrane and gRNA delivery into the cytoplasm (Figure 4) [65,66].
(1) Endocytosis. (2) Fusion with the membrane of the endosome and release of viral genome into the cytoplasm. (3) Translation of the polyprotein from viral genomic RNA and synthesis of negative strand RNA. (4) Genome replication in the ER. (5) Viral particle assembly and packaging of newly synthesized positive strands RNA. (6) Maturation of virions in the trans-Golgi Network and transport via the secretory pathway. (7) Release of mature virions from the cell. ER=endoplasmic reticulum. PM=plasma membrane [67]

Replication, assembly, maturation, and release.

Flaviviruses replicate in the cytoplasm of infected cells. The released gRNA acts as a template for mRNA synthesis [19]. The capped 5’-end of the viral genome, aided by 3’UTR elements, triggers the recruitment of eukaryotic initiation factors to form the ribosome complex [41]. The RNA is directly translated into a single polyprotein of 3411 amino acids and is then co- and post-translationally cleaved by NS2B/NS3 viral serine protease and host-encoded proteases such as signalase and furin [68,69]. The cleavage results into three structural proteins: C, prM, and E, and seven non-structural (NS) proteins: NS1 (glycoprotein), NS2A, NS2B (protease component), NS3 (protease, helicase and NTPase activity), NS4A, NS4B, NS5 (RNA-dependent polymerase) (Figure 2) [10,70,71].
The newly produced NS viral proteins remodel the rough endoplasmic reticulum (RER) membrane, forming membrane curvatures and invaginations where the replication complex forms, becoming viral replication hubs (Figure 4) [72–75]. NS3 and NS5 proteins will bind to the 5’ cap structure of the gRNA and induce negative sense RNA synthesis that acts as a template for multiple rounds of capped positive-stranded viral RNA synthesis [41]. Intermediates of dsRNA are formed between positive and negative strands [76,77] and the replication occurs in a semi-conservative and asymmetric way, where positive-strands accumulate in a large excess over negative-strands [30,78]. The NS5 acts as a regulatory element of viral gRNA synthesis and replication (Reviewed by [79]) and TBEV NS5 mutant viral genome fails to replicate [80].
The assembly process is probably coupled with the replication and is initiated by association of C-dimers with newly synthesized viral gRNA, on the cytoplasmic side of the ER membrane, to form a nucleocapsid precursor [81]. The NC buds into the ER, thus acquiring an envelope. The assembly of NC and E-prM heterodimers forms immature non-infectious viral particles [71,82].
The immature viral particles accumulate in the ER lumen and are transported via the host secretory pathway to the Golgi. Maturation of TBEV occurs in the acidic environment of the late trans-Golgi network, when prM is cleaved by a cellular furin protease [69,83], associated with E protein conformational rearrangements [30,43]. The mature particles are released in the extracellular matrix by fusion of the transport vesicles with the plasma membrane (Figure 4) [9]. While flaviviral RNA synthesis is detected after three to six hours of infection, the release of infectious viral particles begins after 12 hours [21].

Transmission and epidemiology of tick-borne encephalitis virus

As an arbovirus, TBEV is maintained in nature by hard ticks, which act as vectors of the virus [84,85]. In addition to tick bites, it can be transmitted to humans by consumption of unpasteurized dairy products from infected livestock [86]. Several confirmed or suspected cases of TBEV transmission by exposure to raw milk or dairy products were reported. While viremia in TBEV-infected livestock remains low [86-88], LGTV, a closely related virus, is stable for several days in milk at room temperature, and cheese making processes are likely to reduce viral loads [90], which suggests a similar stability for TBEV. Other transmission routes were also reported in rare or single occurrences, such as laboratory infection [91] and person-to-person transmission after an organ transplantation [92] or blood transfusion [93]. Transmission to infants through breastfeeding was suggested to be possible for several flaviviruses such as DENV [96], WNV [97], or YFV [98], but unlikely for ZIKV [94,95]. Even though reviews evoke this transmission route for TBEV [84,99], no evidence of TBEV presence in maternal milk or breastfeeding transmission was reported. Transmission through sexual intercourse was also reported for ZIKV and suggested for WNV and YFV, as they have been detected in semen [100], but there is no data supporting sexual transmission of TBEV in humans [101].

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Risk areas and endemic zone

Tick-borne encephalitis Virus endemic zone spreads From Central, Northern and Eastern Europe to the Russian Far East, including Mongolia, northern China, and Japan (Figure 5) [84,102,103]. Endemic zone of TBEV-Eu (red), TBEV-FE (yellow) and overlapping areas (orange) are represented [104].
Since the 1990s, TBEV is expanding to previously unaffected areas such as Czech Republic, Germany, Norway, Slovenia, Sweden, Switzerland [105–107] and more recently, the Netherlands [108]. Endemic zones are also expanding in altitude, up to 1500 meters above sea level, as reported in Austria and Slovakia [109,110], However, it is possible that the attention drawn to the disease may have led to a higher number of registered cases [111]. A review from Charrel et al. [112] mapped at least 25 European and 7 Asian countries where TBEV is present. In France, fewer than 10 cases are reported yearly, mainly in the Alsace region, with a significant increase in 2016 [113]. As a consequence of the higher risk of TBE in Western Europe, it is listed as a disease under surveillance in the European Union (EU), and it joined the list of notifiable diseases in September 2012 [114–116]. Prior to the EU decision, the type of TBE cases routinely reported, the source and type of case-based data surveillance, the case definition, and the laboratory test used for diagnosis were country-dependent [107]. This might lead to a high number of underdiagnosed or unreported TBE cases, and in highly endemic countries, the number of reported cases does not adequately reflect the real risk of infection [111]. However, thanks to efficient vaccination campaigns, the disease incidence in some endemic countries, such as Austria, decreased importantly [117].
Between 1976 and 1989, TBEV was widely undiagnosed and fewer than 40,000 cases were reported in Europe, with an average of about 2,700 per year. TBEV reported incidence has increased since the 1990s, and between 1990 and 2007, nearly 160,000 TBE cases were documented worldwide, with an average of about 8,700 cases per year. This corresponds to an increase of TBE morbidity by more than 300% [111]. The most recent data about the morbidity worldwide [84] and in 20 EU member states [107] are from 2010. By crossing data from different reviews and databases [84,111,118–120], we were able to estimate the number of cases worldwide from 1985 to 2010 (Figure 6). Yearly, 10,000 to 12,000 clinical cases of TBE is often given as a reference, but although this estimate is above those recent data (about 6000 cases in 2010), this figure is believed to significantly underestimate the actual total, due to the high rate of asymptomatic cases [103].
Interestingly, the increase of the TBEV risk in Europe might be partly due to climate change, affecting vector biology, pathogen transmission [121] and density of hosts on which ticks feed [122]. Other factors might as well be involved, such as political or sociological changes and human behavior which influenced the tick bite exposure of humans and vaccination acceptance [121,123–127]. Those factors may have created favorable living conditions for ticks and thus led to a further spread of tick-borne diseases.
Symptomatic infection occurs in all age groups and genders but the case distribution may vary by region [103]. However, it is often more severe in adults and elderly people and the seroconversion rate following vaccination decreases after the age of 50 [128,129].

Amplifying and spreading hosts

Ticks feed on a large span of hosts. In general, immature stages of ticks (larvae and nymphs) feed on small mammals and birds, while adults feed on large animals such as ungulates and livestock [130]. TBEV transmission to a vertebrate host occurs mainly after a tick bite, during a blood meal [131], and is indirectly facilitated by tick saliva through its contained analgesic, anti-inflammatory and anti-coagulant substances that allow the blood meal to be unnoticed [132].
Small rodents and insectivores, mainly yellow-necked field mice (Apodemus flavicollis) and bank voles (Myodes glareolus), act as a the main reservoir of TBEV, transmitting the virus to feeding ticks and amplifying tick populations [133,134]. In bank voles, TBEV RNA was found in the brain, where it can cause marginal clinical symptoms and mild meningoencephalitis [135,136]. Moreover, infected laboratory male mice can transmit TBEV to females through the sexual route, inducing increased embryonal mortality [137]. The virus is also transmitted vertically in Myodes red voles, through placenta to embryo [133]. This suggests a possible mechanism for tick-free long-term maintenance of TBEV in rodent hosts [138].
Birds can be infected by TBEV but they are more likely to play a role in dispersal of TBEV-infected ticks. TBEV was found in birds or bird-infesting ticks in the Baltic region of Russia [139], Siberia [140,141], Slovakia [142], Sweden [143], and Latvia
[144]. Their role in virus circulation and dissemination is not clear and no outbreak was associated with this dispersal route but the involvement of birds in transport of TBEV-infected ticks from Russia to Japan was hypothesized [145].
In dogs, TBEV infection induces clinical signs similar to those observed in human cases, including fever, apathy and neurological signs such as paresis, seizures or hyperalgesia. The infection can be acute or chronic, and the outcome is often fatal
[146]. The neuropathology of TBE in dogs was found consistent with observations in humans and laboratory mice [147]. Seropositive dogs were also found in Spain [148], a country that is not endemic for TBEV and where no human case was recorded [107], suggesting they can be sentinels for TBEV risk [149,150].
Ungulates can be infected by TBEV and clinical signs were sparsely reported in infected horses, including poor general condition, anorexia, ataxia, cramps, seizures, and paralyses [151,152]. Roe deer develop a short and low-grade viremia but are non-receptive hosts for TBEV [150]. TBEV-neutralizing antibodies and TBEV RNA were found in Dutch roe deer [153]. They can hence be used as sentinel [154]. It is unclear whether they contribute positively to TBEV dissemination and amplification, through co-feeding and transport of infected ticks [155], or negatively, by diverting questing ticks from hosts able to act as reservoir, such as rodents [122,156]. Furthermore, wild ungulates could also be infected, as TBEV-neutralizing antibodies were found in Flemish wild boars [157].
In sheep, goats, and cows, TBEV infection rarely causes clinical signs, and blood viremia is not detectable (or for very short time periods) [158,159]. Infectious virus is found in milk [86], and neutralizing antibodies are produced, suggesting that they can be used as sentinels in non-endemic or low prevalence areas [159,160].
Non-human primates are susceptible to infection and symptoms similar to those observed in mild humans cases as well as chronic infection were described [161,162]. The main reference for experimental infections on non-human primates was published in Russian in the 1980s [163–165]. However, there is no description of natural TBEV infection of non-human primates.

Table of contents :

Chapter I: Introduction
Tick-borne encephalitis virus
Genomic and structural organization
Replication cycle
1.2. Transmission and epidemiology of tick-borne encephalitis virus
1.2.1. Risk areas and endemic zone
1.2.2. Amplifying and spreading hosts
1.2.3. Biology of the vector
1.3. Tick-borne encephalitis
1.3.1. Clinical presentation in humans
1.3.2. Vaccination against TBEV in humans
1.3.3. Viral pathogenesis
1.4. Cell response to TBEV infection
1.4.1. Intrinsic immune response
1.4.2. Cell death
Objectives
Chapter II: Results
2.1. TBEV infects human brain cells differentiated from fetal neural progenitors
2.2. TBEV infects human neurons, astrocytes, and oligodendrocytes
2.3. TBEV induces massive neuronal loss whereas it moderately affects glial cells viability
2.4. TBEV-induced cell death: apoptosis or autophagy?
2.5. Human neural cells develop a strong antiviral response to TBEV infection
2.6. TBEV induces an antiviral response in human neurons and human astrocytes
2.6.1. Experimental design for enrichment of neurons and astrocytes
2.6.2. Antiviral response to TBEV infection is weaker in human neurons than in human astrocytes
2.7. Complex interplay between neurons and astrocytes modulates TBEV infection in each cell type
2.7.1. Astrocytes modulate TBEV infection in neurons
2.7.2. Neurons modulate astrocytes fate in TBEV-infected cells
2.8. Preliminary data: siRNA transfections to downregulate PRRs
Chapter III: Discussion
Material and methods
Human neural progenitor cells culture
Neural stem cells differentiation
Virus and infection
Cell transfection
Intracellular RNA extraction
Viral RNA extraction
PCR array analysis
Reverse transcription and quantitative polymerase chain reaction analysis
Immunofluorescence and TUNEL analyses
Magnetic-activated cell sorting
Statistical analyses
References
Supplementary information

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