Download Tick-borne encephalitis virus – a review of an emerging

Journal of General Virology (2009), 90, 1781–1794
Review
DOI 10.1099/vir.0.011437-0
Tick-borne encephalitis virus – a review of an
emerging zoonosis
K. L. Mansfield,1 N. Johnson,1 L. P. Phipps,1 J. R. Stephenson,2
A. R. Fooks1 and T. Solomon3
Correspondence
T. Solomon
[email protected]
1
Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency, Woodham Lane, New Haw,
Surrey, UK
2
Health Protection Agency, London, UK
3
Brain Infections Group, Divisions of Neurological Science and Medical Microbiology,
University of Liverpool, Liverpool, UK
During the last 30 years, there has been a continued increase in human cases of tick-borne
encephalitis (TBE) in Europe, a disease caused by tick-borne encephalitis virus (TBEV). TBEV is
endemic in an area ranging from northern China and Japan, through far-eastern Russia to Europe,
and is maintained in cycles involving Ixodid ticks (Ixodes ricinus and Ixodes persulcatus) and wild
vertebrate hosts. The virus causes a potentially fatal neurological infection, with thousands of
cases reported annually throughout Europe. TBE has a significant mortality rate depending upon
the strain of virus or may cause long-term neurological/neuropsychiatric sequelae in people
affected. In this review, we comprehensively reviewed TBEV, its epidemiology and pathogenesis,
the clinical manifestations of TBE, along with vaccination and prevention. We also discuss the
factors which may have influenced an apparent increase in the number of reported human cases
each year, despite the availability of effective vaccines.
Introduction
Tick-borne encephalitis virus (TBEV) is the causative agent
of tick-borne encephalitis (TBE), a potentially fatal
neurological infection affecting humans in Europe and
Asia (Gritsun et al., 2003a). From 1974 to 2003, a 400 %
increase in TBE morbidity had been observed in Europe
(Süss, 2008), and TBEV can now be found in regions that
were previously unaffected (Charrel et al., 2004). TBE is
now a notifiable disease in 16 European countries (Süss,
2008) and cases have been confirmed in areas where it has
not been previously reported, for example Norway (Csángó
et al., 2004). Between 1990 and 2007 there were an average
of 8755 reported cases of TBE per year in Europe and
Russia, in comparison with an average of 2755 per year
between 1976 and 1989 (Süss, 2008). This increase may
have been caused by an expanding tick population,
promoted by factors including climate change, social and
political changes and changes in land use (Süss, 2008).
However, a number of European countries have observed a
reduction in the number of cases in recent years, including
Austria, which ran a successful vaccination campaign
introduced in 1981 (Fig. 1) (Süss, 2008). The overall
figures for Russia also suggest a similar trend, although the
numbers of cases are still in the thousands (Fig. 1) and in
one region of Russia alone (Eikatherinburg, Ural region),
there are 300–500 reported cases every year (Toporkova
et al., 2008). Within Russia, western Siberia has the highest
011437 G 2009 Crown Copyright
Printed in Great Britain
incidence of TBE cases in the world (10 298 cases in 1996),
although the apparent yearly decrease to 3098 cases in 2007
cannot be attributed to vaccination alone (Süss, 2008). In
terms of morbidity of neurotropic flavivirus infections,
these figures are second only to those for Japanese
encephalitis (Lindquist & Vapalahti, 2008). Although
Russia has by far the greatest number of confirmed TBE
cases (Süss, 2008), the Czech Republic has one of the
highest incidence rates in Europe, with 400–1000 clinical
cases reported annually (Růžek et al., 2008). Virulence of
TBEV for humans has been shown to decrease with its
westward spread from Japan/far-eastern Russia, through
central Europe to western Europe (Gritsun et al., 2003a),
with the far-eastern subtype having a case fatality rate as
high as 40 % (Mandl, 2005).
Virology and epidemiology of TBEV
TBEV is a member of the genus Flavivirus, within the family
Flaviviridae (Mandl et al., 1997). It is a member of the
mammalian tick-borne flavivirus group, which, along with
other genetically and antigenically related viruses, includes
Omsk hemorrhagic fever virus (OHFV), Langat virus
(LGTV), Alkhurma hemorrhagic fever virus (AHFV),
Kyasanur Forest disease virus (KFDV), Powassan virus
(POWV), Royal Farm virus (RFV), Karshi virus (KSIV),
Gadgets Gully virus (GGYV) and Louping ill virus (LIV)
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K. L. Mansfield and others
Fig. 1. Reported number of cases of tick-borne
encephalitis in selected western European
countries and Russia, between 1990 and
2007. Data are taken from the International
Scientific Working Group on Tick-borne
Encephalitis website: http://www.tbe-info.com.
(Gritsun et al., 2003a; Calisher 1988). This group of viruses
are also known as the TBEV serocomplex (Clarke, 1964;
Calisher 1988) (Table 1, Fig. 2). AHFV is a subtype of KFDV
(Charrel et al., 2001). Of these viruses, TBEV, LIV and
POWV are all known to cause encephalitis in humans and
animals, whereas OHFV, KFDV and AHFV cause hemorrhagic fever (Gritsun et al., 2003b; Grard et al., 2007). LGTV
is a naturally occurring non-pathogenic virus (Smith, 1956)
and there have been no reports of human disease caused by
KSIV, RFV or GGYV (Gritsun et al., 2003b).
Flaviviruses, including TBEV, are small, lipid-enveloped
viruses with a spherical structure and a diameter of 40–
60 nm (Lindenbach & Rice, 2001). The flavivirus genome
comprises a single-stranded, positive-sense RNA of
approximately 11 kb in length, containing a 59 cap and
lacking a polyadenylate tail (Wengler et al., 1978). The 59
cap is important for mRNA stability and translation
(Furuichi & Shatkin, 2000). The single open reading frame
(ORF) encodes three structural proteins – capsid protein C,
membrane protein M (formed by cleavage from it’s
precursor prM) and the large envelope glycoprotein (E) –
along with seven non-structural proteins – NS1 (glycoprotein), NS2A, NS2B (protease component), NS3 (protease,
helicase and NTPase activity), NS4A, NS4B and NS5
(RNA-dependent polymerase). The 59 and 39 ends of the
genome are non-coding regions (reviewed by Chambers et
al., 1990a). The viral genome RNA is itself infectious and
would produce virus progeny if introduced into susceptible
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cells (Mandl et al., 1997). Many studies have focused on the
TBEV E protein, which is known to exist as flat dimers
extending in a direction parallel to the viral membrane,
with residues important for antibody binding exposed on
the outer surface of the protein (Rey et al., 1995). The
second transmembrane region of the E protein is known to
be important for virion formation (Orlinger et al., 2006).
The E protein also has the ability to form enveloped
recombinant subviral particles (RSPs) with prM (Allison et
al., 1995a), which assemble in the endoplasmic reticulum
(ER) and are transported through the secretory pathway in
the same way as whole virus particles (Lorenz et al., 2003).
Cryoelectron microscopy has shown that mature TBEV
RSPs have an icosahedral molecular organization
(Ferlenghi et al., 2001). The flavivirus NS3 protein is
multifunctional, including the C terminus region which
possesses both nucleoside triphosphatase (NTPase) and
helicase activities (Wengler & Wengler, 1991). The Nterminal region of the NS3 protein possesses protease
activity, which is necessary for the post-translational
cleavage of the viral polyprotein precurser (Chambers et
al., 1990b). Studies with West Nile virus (WNV) have
suggested that these activities are all necessary for viral
replication (Brinton, 2002). The NS5 protein has two
domains, a C-terminal RNA-dependent RNA polymerase
(RdRp) and the N-terminal methyltransferase core, which
enables NS5 to exhibit the (nucleoside-29-O-)-methyltransferase activity required for methylation of the cap
Journal of General Virology 90
Tick-borne encephalitis virus – a review
Table 1. Mammalian tick-borne flavivirus group
Data are adapted from the following references: Calisher & Gould (2003); Gritsun et al. (2003b); Gould & Solomon (2008); Grard et al. (2007).
Virus name
Abbreviation
Tick-borne encephalitis virus
(European subtype)
Tick-borne encephalitis virus
(Siberian subtype)
Tick-borne encephalitis virus
(far-eastern subtype)
Louping ill virus
Spanish sheep encephalomyelitis virus
Turkish sheep encephalitis virus
Greek goat encephalitis virus
Powassan virus
Kadam virus
Omsk hemorrhagic fever virus
Kyasanur Forest disease virus
KFDV
Alkhurma hemorrhagic fever virus
Langat virus
Karshi virus
Royal Farm virus
Gadgets Gully virus
AHFV
LGTV
KSIV
RFV
GGYV
Principal tick vector
Geographical distribution
TBEV-Eu
I. ricinus
TBEV-Sib
I. persulcatus
Central/western Europe,
Scandinavia, Korea
Russia, Finland
TBEV-Fe
I. persulcatus
Russia, Far East (China, Japan)
LIV
SSEV
TSEV
GGEV
POWV
KADV
OHFV
I. ricinus
I. ricinus
I. ricinus
I. ricinus
Ixodes cookei, Ixodes marxi
Rhipicephalus pravus
Dermacentor reticulatus (Dermacentor
marginatus)
Haemaphysalis spinigera (Ixodes spp.,
Dermacentor spp., Haemaphysalis spp.)
Ornithodorus savignyi
Ixodes granulatus
Ornithodorus papillipes
Argas hermanni
Ixodes uriae
UK, Ireland, Norway
Spain
Turkey
Greece
USA, Canada, far-eastern Russia
Uganda, Saudi Arabia
Western Siberia
structure at the 59 end of the RNA genome (Egloff et al.,
2002). Furthermore, the NS5 protein has recently been
identified as an interferon antagonist due to it’s ability to
inhibit STAT1 phosphorylation in response to interferon
and its association with interferon receptor complexes
(Best et al., 2005).
India
Saudi Arabia
Malaysia, Thailand, Siberia
Uzbekistan
Afganistan
Macquarie Island (Southern
Ocean)
It has been demonstrated that low pH plays an essential
role in both the entry and the release stages of the flavivirus
life cycle (Stadler et al., 1997). During the first stage of the
viral life cycle, the virions bind to the surface of the host
cell, mediated by the viral surface E protein and with
heparan sulfate as the major host cell receptor
Fig. 2. Phylogenetic analysis of tick-borne
flaviviruses based upon the NS3 protein,
adapted from the report by Grard et al.
(2007) (condensed tree; length of horizontal
lines is not proportional to genetic distance).
Abbreviations for the mammalian tick-borne
flaviviruses are detailed in Table 1.
Abbreviations for the seabird tick-borne flaviviruses are as follows: SREV, Saumarez Reef
virus; MEAV, Meaban virus; TYUV, Tyuleniy
virus; CFAV, cell fusing agent virus.
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K. L. Mansfield and others
(Kroschewski et al., 2003). Work with WNV suggests that
the virions are then transported by receptor-mediated
endocytosis through clathrin-coated pits into prelysosomal
endocytic compartments of the host cell (Chu & Ng, 2004).
Recently, it has been demonstrated that the low pH within
the endosome causes the protonation of a conserved
histidine residue (His323) at the interface between
domains I and III of the E protein (Fritz et al., 2008),
inducing a change in conformation where the E protein is
reorganized from dimers into trimers (Allison et al.,
1995b). The fusion peptide is now exposed and fusion of
the viral membrane with the endosomal membrane is
induced, releasing the viral nucleocapsid into the host cell
cytoplasm, where translation of the genome RNA occurs
(reviewed by Lindenbach & Rice, 2001). Uncoating of the
viral RNA genome occurs and the viral polyprotein is
processed to yield individual viral proteins (reviewed by
Chambers et al., 1990a). This leads to initiation of viral
genome replication, where full-length negative-strand
copies of the genome act as templates for the production
of new positive-strand RNAs (Chu & Westaway, 1985).
During polyprotein synthesis, the surface proteins prM and
E are translocated into the lumen of the ER and their
amino-termini are released through proteolytic cleavage by
host cell signallase (Nowak et al., 1989). The highly basic C
protein packages the RNA genome into nucleocapsids on
the cytoplasmic side of the ER membrane and, at the same
time, assembly of the viral envelope containing prM and E
occurs through budding of the nucleocapsid into the ER
lumen (Chambers et al., 1990a). This assembly yields noninfectious immature virions, where proteins prM and E are
in a heterodimeric association on the viral surface (Lorenz
et al., 2002; Elshuber et al., 2003) and are transported
through the host secretory pathway. In the acidic vesicles of
the late trans-Golgi network, cleavage of prM protein by
the host cell protease furin leads to virus maturation
(Stadler et al., 1997). This final activation cleavage leads to
production of protein M and the reorganization of E
protein into fusion-competent homodimers; the infectious
mature virions are released from the cell through fusion of
the transport vesicles with the host cell plasma membrane
(Wengler & Wengler, 1989).
The three main subtypes of TBEV are the European,
Siberian and far-eastern subtypes (Ecker et al., 1999),
which are all closely related both genetically and antigenically. Studies with European TBEV isolates suggest that
TBEV is quite stable under natural ecological conditions
and does not undergo significant antigenic variation.
Indeed, there is known to be a high degree of antigenic
homogeneity between different strains of TBEV
(Holzmann et al., 1992). Culturing TBEV in the laboratory
is known to affect both the genotypic and the phenotypic
characteristics of the virus, and phenotypic characteristics
are also thought to change following mammal-to-tick
transmission of the virus (Kaluzová et al., 1994; Romanova
et al., 2007). Infectious cDNA clones composed of two
different strains of TBEV have been demonstrated as a
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useful experimental system for the specific mutagenesis of
TBEV, exhibiting identical biological properties to the
parent viruses (Mandl et al., 1997).
TBEV complex viruses cause limited disease in indigenous
forest animals but have the potential to emerge as
pathogens if they infect introduced species. During the
last few thousand years, this group of viruses has evolved
and spread westwards throughout Asian and European
forests (Gould et al., 2006). TBEV is now endemic in an
area ranging from northern China and Japan, through fareastern Russia to Europe (Dumpis et al., 1999; Hou et al.,
1997; Takashima et al., 1997). The European subtype of
TBEV is predominantly found throughout Europe and
Russia, with Russia also having the Siberian subtype of
TBEV. The far-eastern subtype of TBEV is endemic in
northern regions of China and is also present in western
and south-western China (Lu et al., 2008). This subtype has
also been shown to be endemic in Japan (Takashima et al.,
1997) and is present in far-eastern Russia.
Phylogenetic studies of a number of TBEV genes over the
last few years have all generated trees with a similar pattern
(Gould et al., 2004). Studies have been based upon a
number of nucleotide sequences, including the E, NS3 and
NS5 sequences, and it has been shown that NS3 generates a
robust phylogenetic analysis, which is very similar to that
obtained from complete sequences (Cook & Holmes, 2006;
Grard et al., 2007). Recent studies have shown that the fareastern and Siberian subtypes are phylogenetically more
closely related to each other than to the European subtype,
which appears to be more closely related to LIV (Grard
et al., 2007) (Fig. 2). LIV is the only tick-borne flavivirus
currently found in the UK; it is transmitted by Ixodes
ricinus and causes disease primarily in sheep and red
grouse, predominantly distributed on the sheep-rearing
hillsides of Scotland, England, Wales and Ireland (McGuire
et al., 1998). In comparison, TBEV is generally found in
forests throughout Europe and Asia (Gritsun et al., 2003a).
Unlike TBEV, LIV causes viraemia and fatal encephalomyelitis in domesticated animals when infected ticks
feed on them (McGuire et al., 1998). Although LIV is
potentially a threat to humans in the UK, natural exposure
is rare and there have been very few reports of cases in
humans (Gritsun et al., 2003a). The few reported human
cases of LIV have mainly been laboratory-acquired
(Davidson et al., 1991) and the disease course is similar
to that observed with TBEV (Gritsun et al., 2003a).
However, although the disease is often subclinical in
humans, the meningoencephalitis caused can be severe
(Davidson et al., 1991). A disease similar to that caused by
LIV has been reported in sheep and goats in other
European countries, including Spain, Greece and Turkey
(Gritsun et al., 2003a). The viruses causing disease are
genetically distinguishable from LIV and have been named
according to the country they were first isolated in, for
example Spanish sheep encephalomyelitis, Greek goat
encephalitis and Turkish sheep encephalitis (Table 1)
(Grard et al., 2007). Although domestic animals infected
Journal of General Virology 90
Tick-borne encephalitis virus – a review
with TBEV are generally asymptomatic, many studies have
demonstrated the presence of circulating antibodies to
TBEV in both animal and human populations in countries
throughout Europe, including Lithuania, Norway and
Sweden (Juceviciene et al., 2005; Csángó et al., 2004;
Stjernberg et al., 2008). In Denmark, it was found that
8.7 % of roe deer were seropositive (Skarphédinsson et al.,
2005) and a recent study of a small endemic island in
south-east Sweden has found human seropositivity to be
2 % (Stjernberg et al., 2008). Similarly, a human study in
south-eastern Turkey found circulating TBEV-specific IgG
(10.5 %) and IgM (23 %) antibodies, suggesting possible
human cases despite there being no reported cases of TBE
in Turkey to date. However, these are ELISA-derived data
and they have not been confirmed by neutralization tests
(Ergunay et al., 2007). Often, there appears to be a
correlation between seropositivity in domestic animals, the
number of infected ticks and the number of human clinical
cases of TBE in a particular region (Juceviciene et al.,
2005). A recent study has shown that a significant number
of domestic dogs in the Aust-Agder region of southern
Norway had circulating antibodies to TBEV, even though
TBE had not previously been observed in this region
(Csángó et al., 2004). These data correlated with the first
cases of human TBE in Norway and provide further
evidence that TBEV can be considered an emerging
pathogen. Furthermore, strains of the Siberian subtype of
TBEV were isolated from Ixodes persulcatus ticks in Finland
in 2004, suggesting that I. persulcatus and Siberian TBEV
are found significantly further north-west of the previously
known range in eastern Europe and Siberia (Jääskeläinen
et al., 2006). Indeed, all three known subtypes of TBEV are
capable of co-circulating at the same time in the same area,
as is currently the situation in Estonia (Golovljova et al.,
2004).
It has been shown that nymphs and larvae co-feed together
on the same rodent host, and the extent to which this
occurs depends upon the patterns of seasonal activity
which is influenced by temperature (Randolph et al., 2000).
The apparent increase in incidence of TBEV over the last
two decades has been attributed to climate change
(affecting both tick and rodent population dynamics) in
many reports, although in reality, it is likely that a number
of factors have simultaneously influenced this (Šumilo et
al., 2007). Apart from changing climate conditions, social,
political, ecological, economic and demographic factors all
appear to play a role in aiding the spread of tick-borne
disease (Süss, 2008). These include changes in land usage
(such as increased forestation or newly created gardens)
and the growing popularity of outdoor pursuits such as
hill-walking and fishing. In particular, socio-economic
conditions have been shown to have an impact on the
incidence of TBE, as people who exist in poverty [for
example, through unemployment, or political upheaval
(such as the break-up of the Soviet Union)] are less likely to
be vaccinated against TBEV and more likely to go foraging
for food such as wild fruit and mushrooms in the forest,
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thus increasing the risk of a tick bite and of contracting
TBE (Šumilo et al., 2008a). However, this does not explain
the increasing incidence in countries such as Germany,
Italy, Finland and Sweden (Süss, 2008). Other factors that
may have contributed towards the increase in incidence of
TBEV in central and Eastern Europe include an increase in
the amount of land cultivated and a decrease in both the
use of pesticides and the amount of industrial pollution
(Šumilo et al., 2008b). These factors would have had a
positive effect on the habitat and rodent host populations
for I. ricinus ticks. Changes in hunting practices have also
led to an increase in larger mammal populations, such as
roe deer and wild boar, providing a greater feeding
opportunity for questing ticks (Šumilo et al., 2008b).
Alternatively, there has been an improvement in the quality
of epidemiological surveillance systems and diagnostics,
which may also influence the reporting of TBEV cases
(Süss, 2003). Furthermore, in an age when travel is
increasingly popular, and previously remote areas of the
planet are now more accessible, the risk of a traveller
becoming infected through a tick bite is greatly increased.
In a recent study, birds migrating from western Russia and
Fennoscandia to Sweden have been shown to carry TBEVinfected I. ricinus ticks (Waldenström et al., 2007). This
leads to the possibility that migrating birds may play a role
in the dispersal of TBEV-infected ticks, although the
importance of birds as a reservoir for TBEV has yet to be
determined. Indeed, migratory birds have been suggested
as one of the routes by which far-eastern strains have been
reported as far west as Latvia and Estonia (Golovljova et al.,
2004; Gould & Solomon, 2008). The ecological and
phylogenetic characteristics of TBEV and other flaviviruses
(both mammalian and seabird-borne) suggest that ticks
that feed on both mammals and seabirds may form the
evolutionary bridge between these different lineages (Grard
et al., 2007).
Vector ecology
In western Europe, TBEV is transmitted primarily by the I.
ricinus tick (Fig. 3a), whereas the vector for the Siberian
and far-Eastern subtypes is I. persulcatus (Gritsun et al.,
2003b). I. ricinus, the principal vector for the European
subtype of TBEV, is a three-host tick, where each parasitic
stage (larva, nymph and adult females) feed for a period of
a few days on a different host from a range of species (Gray,
1991) (Fig. 4). Each stage of the life cycle takes
approximately 1 year to develop to the next, so the entire
life cycle is generally completed in 3 years, although this
can vary from 2 to 6 years, depending upon the
geographical location (Gray, 1991). If infected with
TBEV, the ticks remain infected throughout their life cycle
(Gritsun et al., 2003a); nymphs are thought to be the most
important stage in the transmission of TBEV as they are
more numerous than adults (Süss, 2003). Throughout the
majority of its geographical range, I. ricinus becomes active
and starts feeding on a range of hosts in spring and early
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K. L. Mansfield and others
Fig. 4. Transmission of tick-borne encephalitis virus within the life
cycle of I. ricinus (tick stages are highlighted with grey shading).
Fig. 3. I. ricinus nymphs on herbage (a) and on a woodmouse (b).
summer, with ticks being observed on vegetation and
animals from late March (Gray, 1991). A study in the UK
has shown that the tick will parasitize any mammal or bird
that it meets, although adults only feed successfully on
larger animals such as deer or livestock (Milne, 1949).
Generally, throughout Europe, one major peak of tick
activity is seen, where high numbers of larvae and nymphs
occur together, peaking around April/May, or as late as
July in cooler Scandinavia (Randolph et al., 2000). It is
during this peak that there is sufficient transmission to
allow TBEV to persist within TBE foci, where coincident
feeding by larvae and nymphs on rodent hosts is essential
for the maintenance of TBEV transmission (Randolph et al.,
1999). The tick is infected with TBEV during feeding; virus
replication commences after entry into cells of the midgut
wall, and infection of the salivary glands occurs prior to
transmission in the tick saliva during the next blood meal
(Nuttall et al., 1994). The infected tick can transmit the
virus to a vertebrate host during feeding and is also able to
pass on the virus to a non-infected tick during co-feeding
at the same site on the host. Studies into the transmission
of TBEV between co-feeding ticks have shown that
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vertebrate hosts can play an important role in TBEV
transmission in the absence of a detectable level of viraemia
(Labuda et al., 1993). Natural hosts that have detectable
levels of neutralizing antibodies and no detectable viraemia
can still support transmission of virus between infected and
uninfected ticks feeding closely (Labuda et al., 1997). The
process of virus transmission between ticks during cofeeding is thought to occur via cellular infiltration of tick
feeding sites and the migration of cells from these sites
(Labuda et al., 1996). Studies have shown that mass cofeeding of larvae alone is also important in the persistent
circulation of TBEV, as well as the feeding of infected
nymphs alongside infectable larvae (Danielová et al., 2002).
Furthermore, the isolation of TBEV from unfed larvae
provides evidence for transovarial transmission of TBEV
(Danielová et al., 2002). In the UK, it has been shown that
I. ricinus becomes biologically active at temperatures of
11 uC and above, and that the lower limit of humidity for
survival is represented by a relative humidity value of
between 70 and 80 % (MacLeod, 1935), hence the forests of
Europe and Asia provide an ideal tick habitat, with high
humidity in the dense undergrowth that prevents desiccation (Gritsun et al., 2003a). Furthermore, it has been
shown that both larval–nymph synchrony, where nymphs
and larvae co-feed together on the same rodent host, and
TBE foci are statistically significantly associated with
temperature, characterized by a rapid fall in ground level
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Tick-borne encephalitis virus – a review
temperatures from August to October (Randolph et al.,
2000).
Studies have shown a variable prevalence of infected I.
ricinus ticks; up to 1.7 % in areas of Lithuania (Han et al.,
2005) and as high as 14.3 % in a recognized TBEV focus in
Switzerland (Casati et al., 2006). Recently, it has been
reported that the prevalence of TBEV in ticks removed
from humans was significantly higher than in unfed, freeliving I. ricinus ticks in the same area, possibly associated
with different replication strategies of the virus in tick and
mammalian cells (Süss et al., 2006). Furthermore, ticks are
capable of maintaining a double infection, being able to
transmit both TBEV and Borrelia to humans, as demonstrated by I. persulcatus ticks in Russia (Korenberg, 1994).
TBEV is maintained in cycles involving Ixodid ticks and
wild mammalian hosts (Fig. 3b), particularly rodents
(Charrel et al., 2004). The rodent host acts as both
maintenance and amplifying host and also as a reservoir
host (Süss, 2003), and it has been suggested that small
mammals may maintain a persistent infection with TBEV
throughout the year (Bakhvalova et al., 2006). Although
larger animals (such as birds, deer and horses) may act as
hosts for ticks, they are not thought to have an important
role in virus transmission between ticks (Gritsun et al.,
2003a). Indeed, studies have shown that a localized absence
of deer increases tick feeding on rodents, with the potential
to cause tick-borne disease hotspots (Perkins et al., 2006).
In western Slovakia, yellow-necked field mice and bank
voles comprise around 75 % of the rodent population, of
which approximately 15 % are seropositive for TBEV
(Kozuch et al., 1990). A recent study in Korea demonstrated molecular detection of European subtype TBEV
genes in 20 % of wild rodents sampled, and in 10 % of
Ixodes nipponensis ticks sampled (Kim et al., 2008). This
was the first report of TBEV in Korea, where the number of
patients presenting with encephalitis of unknown origin
was seen to be increasing annually. It is interesting that it
was a European rather than a far-eastern strain of TBEV
that was detected in this study, as one would have expected
a far-eastern strain similar to those found in Japan and
China. Furthermore, I. nipponensis is not the usual tick
vector for European TBEV and may provide further
indications that TBEV can be considered an emerging
pathogen. Similarly, although the predominant vector in
China is I. persulcatus, a number of additional vectors are
thought to be involved in the transmission of TBEV,
including Ixodes ovatus in Yunnan province (south-western
China), a region where I. persulcatus is not detected (Lu
et al., 2008).
Pathogenesis
The majority of TBEV infections occur through a tick bite,
although a small number of infections occur through
consuming infected unpasteurized milk (Dumpis et al.,
1999). Following infection with tick-borne flaviviruses
(either naturally or experimentally via intradermal or
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subcutaneous routes), the virus is known to first replicate
at the site of inoculation and then in the lymph nodes that
drain the inoculation site (Albrecht, 1968). TBEV has been
found in the Langerhans cells of the skin before reaching
the regional lymphatic nodules via the lymphatic system
(Haglund & Günther, 2003). Virus replication in the
draining lymph nodes is followed by development of
plasma viraemia (Málková & Fraňková, 1959). During the
viraemic phase, many extra-neural tissues are infected and
the release of virus from these tissues enables the viraemia
to continue for several days (Albrecht, 1968; Monath &
Heinz, 1996). Haematogenic spread allows different organs
to be infected, especially the reticulo-endothelial system
(spleen, liver and bone marrow) (Haglund & Günther,
2003), and it is during this phase that the virus also crosses
the blood–brain barrier to invade the central nervous
system (CNS), where viral replication causes inflammation,
lysis and cellular dysfunction (Dumpis et al., 1999).
Neuroinvasion of TBEV in humans has been well reported,
although the mechanisms by which an acute, non-fatal
febrile infection develops into a severe, possibly fatal CNS
disease are not clearly understood (Toporkova et al., 2008).
There are a number of animal models regularly employed
to examine the neuropathogenicity of TBEV; the mouse is
the most commonly used model as it is susceptible to
TBEV-induced disease, unlike other wild and domestic
animals (Mandl, 2005). Wild-type strains of TBEV are
generally neuropathogenic when experimentally inoculated
into mice (intracranially or peripherally) usually resulting
in a lethal infection (Mandl, 2005), although this is very
much dependent upon the age of the mice (Andzhaparidze
et al., 1978). An apparent feature of TBEV is the ability to
cause persistent infections in experimental animals and
humans (Monath & Heinz, 1996), and a number of animal
models have been used to demonstrate degenerative
changes in the CNS following infection with TBEV.
Intracranial inoculation of hamsters with the Soph-K
strain of TBEV induced clinical disease in 14 % of animals,
although pathological lesions characterized as meningoencephalitis were found in the CNS of all the animals
(Andzhaparidze et al., 1978). Histological examination
45 days post-inoculation demonstrated signs of neuronophagy and marked glial proliferation. In the same study,
subcutaneous inoculation of hamsters induced meningoencephalitis in the 4 % of animals that were exhibiting
clinical disease, although lesions in the brain were observed
in the majority of animals, including perivascular infiltration and encephalitis ‘granulomas’. In an earlier study,
when monkeys were infected intranasally or intracerebrally
with European TBEV, they were shown to develop chronic
encephalitis with degenerative spongiform lesions and
astrocytic proliferation (Zlontnik et al., 1976).
Furthermore, monkeys inoculated intracerebrally with the
Soph-K strain of TBEV showed an asymptomatic infection
with subacute disseminated meningoencephalitis, with a
progradient course for the 3 months of observation; this
chronic infection provides a model of progressive degen1787
K. L. Mansfield and others
erative disease of the CNS (Andzhaparidze et al., 1978).
The first case of TBE in a monkey (Macaca sylvanus) after
natural exposure (tick bite) in a TBE risk area has recently
been described (Süss et al., 2007). TBEV was present in the
brain and was identified as the European subtype, closely
related to the Neudoerfl strain; clinical illness similar to
that observed in a typical severe human TBE case was
observed (Süss et al., 2007, 2008). Indeed, chronic
progressive human encephalitis and seizure disorders
(Kozhevnikov’s epilepsy) have been associated with
infection with Siberian and far-eastern TBEV (Zlontnik
et al., 1976).
Host immune response
Shortly after the onset of neurological symptoms in
humans, the mononuclear cells in the cerebrospinal fluid
(CSF) are predominantly composed of CD4+ T lymphocytes and less numerous CD8+ T lymphocytes, with
limited natural killer cells and B-lymphocytes (Holub et al.,
2002). Levels of TBEV-specific antibodies have been shown
to increase in both serum and CSF; maximum IgM levels
were seen during early disease and persisted after 6 weeks,
whereas peak IgG levels were observed in late convalescent
samples (around 6 weeks) (Günther et al., 1997). However,
although IgM antibodies can be detected for a few months
after infection, IgG antibodies persist for a lifetime and
provide a level of immunity that prevents reinfection
(Holzmann, 2003).
Recent work has investigated the role of viral proteins in
the immune response to TBEV infection. The NS5 protein
of TBEV has been shown to act as an interferon antagonist
and is able to inhibit interferon-stimulated JAK–STAT
signalling by blocking the phosphorylation of STAT1, thus
inhibiting the expression of antiviral genes (Best et al.,
2005; Werme et al., 2008). However, there are limited data
on the role of cytokines and chemokines in the pathogenesis of TBEV. Recently, a number of studies have
investigated the level of certain cytokines and chemokines
in the serum and CSF of patients with TBE. A study in
Russia found that on admission to hospital, TBE patients
had elevated serum levels of tumour necrosis factor (TNF)a, interleukin (IL)-1a and IL-6, where IL-1a and TNF-a
were acting synergistically to initiate the cascade of
inflammatory mediators by targeting the endothelium;
levels of these cytokines then declined during the first week
of hospitalization, which was accompanied by an increase
in levels of IL-10 (an inhibitor of cytokine synthesis)
(Atrasheuskaya et al., 2003). Another study found that
during TBEV infection, the concentration of CXCL10 was
higher in CSF than in serum, suggesting a role in the
recruitment of CXCR3-expressing T cells into the CSF
(Lepej et al., 2007). Similarly, another study found that the
concentration of CCL5 (another lymphocyte attractant)
was increased in the CSF but not the serum of TBE
patients, and that this increase was sustained after the
disappearance of clinical symptoms (Grygorczuk et al.,
1788
2006a). Conversely, it has been reported that although
synthesis of CCL3 was increased during TBE infection, its
concentration was much lower in CSF than in serum,
suggesting that its major role is not as a chemoattractant of
leukocytes into the CNS (Grygorczuk et al., 2006b).
However, it has been shown that both sPECAM-1 (a
glycoprotein involved in the transendothelial migration of
leukocytes) and CCL2 (involved in the activation of certain
leukocytes) were elevated in the CSF of TBE patients
(Michałowska-Wender et al., 2006). Only a modest
increase in interferon (IFN)-c in CSF has been documented
during infection with TBEV, in comparison with other
viral infections, such as Varicella–Zoster virus, where it is
highly upregulated (Glimåker et al., 1994).
A recent study has demonstrated that TBE is an
immunopathological disease, where the inflammatory
reaction, mediated by CD8+ T cells, contributes to
neuronal damage and could lead to a fatal outcome
(Růžek et al., 2009). Unlike humans, however, when
animals are naturally infected with TBEV, the host immune
response is clearly capable of preventing the development
of disease, and this is an area that needs further
investigation. Paradoxically, the reverse happens during
infection with LIV; although LIV is closely related to TBEV,
infected animals do exhibit clinical signs, whereas humaninfections are often asymptomatic (Davidson et al., 1991).
Clinical manifestations
The clinical manifestations in human cases have been welldocumented and there is a range of symptoms that can be
observed.
Although the far-eastern subtype of TBEV causes a
monophasic course of illness, infection with a western
European subtype usually produces a biphasic course of
illness (Dumpis et al., 1999; Gritsun et al., 2003a). The
incubation period is generally 7–14 days and during a
typical biphasic infection, symptoms during the initial
short febrile period can include fatigue, headache and pain
in the neck, shoulders and lower back, accompanied by
high fever and vomiting (Gritsun et al., 2003a). This is
often followed by an asymptomatic period lasting 2–
10 days and if the disease progresses to neurological
involvement, this leads to the second phase, characterized
by acute CNS symptoms with a high fever. CNS infection
can manifest in the meninges (where inflammation causes
meningitis), the brain parenchyma (to cause encephalitis),
the spinal cord (myelitis), the nerve roots (radiculitis) or
indeed any combination of these.
Acute TBE is characterized by encephalitic symptoms in
45–56 % of patients (Haglund & Günther, 2003).
Symptoms range from mild meningitis to severe meningoencephalomyelitis, which is characterized by muscular
weakness (paresis) which develops 5–10 days after
remission of the fever. Severely affected patients may
demonstrate altered consciousness and a poliomyelitis-like
Journal of General Virology 90
Tick-borne encephalitis virus – a review
syndrome that may lead to long-term disability (Dumpis et
al., 1999; Kleiter et al., 2007; Gritsun et al., 2003a). The
acute febrile period of illness correlates with the presence of
viraemia. Following the asymptomatic phase, the second
stage of illness correlates with virus invasion into the CNS,
where viral replication is associated with inflammation,
lysis and dysfunction of the cells (Dumpis et al., 1999).
Although magnetic resonance imaging (MRI) is usually
normal, abnormalities have been shown, including pronounced bilateral lesions in the thalamus, cerebral
peduncles and the left caudate nucleus (Lorenzl et al.,
1996). However, it has been shown that the abnormalities
detected in TBE patients depend upon the MRI technique
used, as T2-weighted and turbo FLAIR images demonstrated more abnormalities than T1-weighted images
(Marjelund et al., 2004).
A study of TBEV patients in Germany reported that the
average incubation period between tick bite and onset of
symptoms was 11 days (with a range of 4–28 days) (Kaiser,
1999). This study showed that 74 % of patients experienced
a biphasic course of illness, typically with the first stage
lasting between 1 and 7 days, and consisting of fever,
headache, occasional malaise and upper respiratory and/or
abdominal symptoms. The intermediate asymptomatic
phase lasted between 3 and 21 days, followed by a
prodromal period which was reported less often in patients
with meningoencephalomyelitis (inflammation of the brain
and spinal cord) than in patients with meningoencephalitis
(inflammation of the brain and meninges) or isolated
meningitis. Of the 656 patients, 49 % presented with
meningitis, 41 % with meningoencephalitis and 10 % with
meningoencephalomyelitis. In patients with meningoencephalomyelitis, the most common feature was flaccid paresis
of the extremities, whereas this was less common with
meningoencephalitis, and was usually transitory. Similarly,
paresis of the cranial nerves was more frequent and severe
in patients with meningoencephalomyelitis. Eight patients
(1.2 %) were ventilated until death (1–52 weeks after onset
of disease). This study also showed that the severity of
illness increased with the age of the patient. An earlier
study in Sweden reported similar findings, with spinal
nerve paralysis observed in 13 % of patients; only 60 % of
patients were considered to be recovered after 1 year
(Günther et al., 1997).
A chronic form of TBE has been observed in patients from
Siberia and far-eastern Russia and is thought to be
associated with the Siberian subtype of TBEV (reviewed
by Gritsun et al., 2003a). There are two forms of chronic
TBE, the first being long-term sequelae of any of the acute
forms of TBE, where the development of neurological
symptoms may take years following the bite from the
infected tick. Clinical symptoms include Kozshevnikov’s
epilepsy, progressive neuritis of the shoulder plexus, lateral
and dispersed sclerosis, a Parkinson’s-like disease and
progressive muscle atrophy. Often the physical deterioration is accompanied by mental deterioration and even
death. A second chronic form of TBE is associated with
http://vir.sgmjournals.org
hyperkinesias and epileptoid syndrome. Hyperkinesia
occurs frequently and may arise during the acute phase
of TBE or persist as Kozshevnikov’s epilepsy.
The incidence of the different forms of TBE is variable
according to the region (reviewed by Gritsun et al., 2003a).
In Siberia, approximately 80 % of TBE cases present with a
fever but without neurological sequelae. Paralytic forms are
observed in approximately 7–8 % of cases and
Kozshevnikov’s epilepsy in about 4–5 % of patients. In
general, the case fatality rate is approximately 1–2 %
following European subtype infection, but can be as high as
20–40 % following infection with a far-eastern subtype
(Mandl, 2005). Infection with the Siberian subtype
produces a mortality rate of no more than 2–3 %
(Atrasheuskaya et al., 2003). However, it is possible that
the high mortality figures for the far-eastern subtype may
be due to the lack of detection of mild cases. A study of 62
cases of TBE in an area of Russia where the Siberian and
far-eastern subtypes of TBEV co-exist has shown a large
variation in the clinical symptoms observed, ranging from
unapparent to severe (Pogodina, 2005). Hence, an increase
in detection of milder cases would lead to an overall
decrease in mortality rate figures.
In comparison with humans, domestic animals infected
with TBEV are generally asymptomatic. Indeed, recent data
suggest that wild small mammals are able to maintain
TBEV as a persistent infection throughout the year
(Bakhvalova et al., 2006).
Diagnosis and treatment
The clinical symptoms observed with TBE are shared with
other neurological disorders such as herpes encephalitis;
therefore, differential diagnosis of a TBEV infection must
be established in the laboratory (Holzmann, 2003).
Diagnosis of infection with TBEV is now possible with a
variety of techniques, from both ante-mortem (serum/
CSF) and post-mortem (tissue) samples. Along with virus
isolation, these techniques include RT-PCR, which is able
to detect viral RNA in serum and CSF prior to the
appearance of antibodies (Saksida et al., 2005), multiplex
PCR to differentiate between TBEV subtypes (Růžek et al.,
2007) and quantitative PCR on ante-mortem CSF samples
(Schwaiger & Cassinotti, 2003). Most patients present at
the hospital during the non-viraemic second phase of
disease, when virus has been cleared from the blood and
neurological symptoms have commenced (Holzmann,
2003). Development of the humoral immune response
enables diagnosis of TBE by ELISA detection of antibodies
in the blood, such as TBEV-specific IgM and IgG
antibodies (Holzmann, 2003). It has been shown that
between 0 and 6 days after onset of encephalitic symptoms,
TBEV-specific IgM activity increased in serum, declining
after 6 weeks (Günther et al., 1997). In CSF, IgM activity
peaked between 9 days and 6 weeks after onset. In
comparison, maximum IgG activity in both serum and
CSF was in 6 week samples. In countries such as Japan,
1789
K. L. Mansfield and others
human cases of TBE may have been underreported due to
the cross-reactivity between TBEV and JEV in serological
assays and the similarity of clinical symptoms that are
observed with each infection (Takashima et al., 1997).
There is no curative therapy for TBE, so supportive
treatment includes paracetamol, aspirin and other nonsteroidal anti-inflammatory drugs. In severe cases, some
clinicians administer corticosteroids, although their use has
not been validated. Patients with severe CNS symptoms
have to be closely monitored as coma or neuromuscular
paralysis may develop rapidly, in which case intubation and
ventilation is necessary (Dumpis et al., 1999).
Vaccination and prevention
Active vaccination is the most effective method for
preventing TBE, with modern vaccines shown to be safe
and between 95 and 99 % effective (Heinz et al., 2007).
Currently, there are at least four vaccines available against
TBEV infection, which are manufactured from purified,
formaldehyde-inactivated virus. These include: Austrian
‘FMSE-Immun 0.5 ml’ and ‘FSME-Immun 0.25 ml
Junior’, German ‘Encepur Adult and Children’ and two
Russian vaccines manufactured in Moscow and Tomsk
(Leonova et al., 2007). The European vaccines are
produced using the Neudorfl (Austria) and K23
(Germany) strains of the European subtype (Charrel et al.,
2004), whereas the Russian vaccines are produced from the
far-eastern strains 205 and Sofjin (Leonova et al., 2007).
An annual TBE vaccination campaign was introduced in
Austria in 1981. Despite once having the highest incidence
rate in Europe (up to 700 hospitalised cases annually), this
campaign caused a steady decline in the number of cases of
TBE (Süss, 2008). However, with the exception of Austria,
vaccination campaigns have had varying degrees of success,
since the vaccines are relatively expensive and must have
repeated administrations in order to maintain protective
immunity (Juceviciene et al., 2005). Post-exposure injection with specific immunoglobulin, when given within
96 h of exposure, has been shown to prevent disease in
approximately 60 % of patients (Dumpis et al., 1999).
However, post-exposure prophylaxis should be discouraged, as it has been suggested that the administration of
post-exposure passive immunization may actually exacerbate the disease through antibody-dependent enhancement
of infection (Arras et al., 1996; Phillpotts et al., 1985).
Studies in mice have demonstrated a high level of crossprotection between European and far-eastern subtypes of
TBEV, where immunization with the European subtype
vaccine protected the mice against infection with fareastern strains (Holzmann et al., 1992). Furthermore, a
recent study in human volunteers has shown that following
vaccination with Encepur Adult (based upon the European
subtype), they were able to produce a humoral response
towards strains of the far-eastern TBEV subtype (Leonova
et al., 2007). This neutralization assay data demonstrated
1790
that the human sera contained neutralizing antibodies
capable of neutralizing far-Eastern TBEV strains, suggesting that this vaccine may offer protection in humans
against a viral infection caused by a far-eastern strain of
TBEV.
A recent study in Lithuania demonstrated that antibodies
to TBEV were found most often in people who frequently
spent time in the countryside or who had consumed
unpasteurized goats’ milk (Juceviciene et al., 2002) and
there have been many reports of the transmission of TBEV
to humans through the consumption of unpasteurized
goats’ milk (causing ‘biphasic milk fever’). This oral route
of infection is possible, since TBEV has been shown to
retain infectivity within the low pH of gastric juices
(Pogodina, 1958). Similarly, the consumption of unpasteurized cows’ milk is common in a number of countries,
including Lithuania, and has been suggested as a minor
route of infection to humans (Juceviciene et al., 2005).
Therefore, campaigns encouraging people to pasteurize all
milk, particularly goats’ milk, before consumption may
help to reduce the risk of exposure.
Vaccination of those individuals who are most at risk
would therefore be advantageous, although the risk of
infection is dependent upon factors such as location,
season and activity. As recent data from Latvia confirm, it
is clear that people who visit forests (for work, food
collection or leisure) are four to five times more likely to
encounter ticks than people who do not visit forests
(Šumilo et al., 2008a). With the growing popularity for
people to spend their leisure time in the countryside,
further measures alongside vaccination could be encouraged in order to prevent infection through a tick bite.
These include the use of tick repellents in combination
with the wearing of appropriate clothing (for example, long
trousers) and avoidance of the tick habitat if possible
(Dumpis et al., 1999), although a recent study has shown
that tick repellents are only 20 % effective (Vázquez et al.,
2008). Indeed, a recent study has suggested that risk
avoidance through changing human behaviour (independent of the seasonal changes in tick activity) has led to a
decrease in incidence of TBE infection in a number of
Baltic countries in recent years (Šumilo et al., 2008a).
Similarly, vaccination of people travelling to areas known
to be endemic for TBEV is advisable.
The current situation
As an emerging zoonosis, there is currently increasing
interest in TBEV. In particular, this has prompted the
identification of particular regions of the genome that may
influence phenotypic characteristics of the virus. Recently,
certain mutations that influence the neuroinvasive capacity
of the virus in mice have been identified (Růžek et al.,
2008). This has suggested that non-virulent variants of
TBEV, which are highly adapted to ticks yet sustain human
population immunity by inducing subclinical infections,
are found in the natural environment. Furthermore, these
Journal of General Virology 90
Tick-borne encephalitis virus – a review
authors suggest that virulent and attenuated viruses may
co-exist as quasispecies in the same TBEV population and
that conversion of neurovirulence during tick/mammal
adaptation of the virus is mediated by selection from this
quasispecies population rather than random mutagenesis.
There is mounting speculation that TBEV has the potential
to move further westward towards the UK. Predictive
models suggest that the UK can potentially be considered
as an emerging disease ‘hotspot’, with the likelihood that
new emerging infectious diseases are likely to appear (Jones
et al., 2008). Increased temperatures have already allowed
the limit of I. ricinus to extend north- and westwards, thus
fuelling the prediction that TBEV may also extend to
previously unaffected areas (Randolph & Rogers, 2000).
Indeed, in South Korea it has been shown that western
TBEV is in a species of tick that is not normally associated
with this virus subtype, and the detection of I. persulcatus
ticks in Finland suggests a possible extension to the
previously known geographical range of this vector.
Conversely, in some areas, climate change may actually
disrupt the conditions required for enzootic cycles of TBEV
(Randolph & Rogers, 2000). However, in an age of climate
change, the increasing popularity of outdoor pursuits and
changes in land usage, this is a risk that cannot be ignored.
There are clearly a number of factors that may influence
the incidence and extent of TBEV throughout Europe and
will continue to do so in the years to come.
Acknowledgements
This work was funded by the Department for Environment, Food and
Rural Affairs (Defra) grants SE4106 and SCO213. T. S. is an MRC
senior clinical fellow.
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