Journal of General Virology (2009), 90, 1781–1794 DOI 10.1099/vir.0.011437-0

Review -borne encephalitis – 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 1Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency, Woodham Lane, New Haw, T. Solomon Surrey, UK [email protected] 2Health Protection Agency, London, UK 3Brain 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 (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 incidence of TBE cases in the world (10 298 cases in 1996), although the apparent yearly decrease to 3098 cases in 2007 Tick-borne encephalitis virus (TBEV) is the causative agent cannot be attributed to vaccination alone (Su¨ss, 2008). In of tick-borne encephalitis (TBE), a potentially fatal terms of morbidity of neurotropic infections, neurological infection affecting humans in Europe and these figures are second only to those for Japanese Asia (Gritsun et al., 2003a). From 1974 to 2003, a 400 % encephalitis (Lindquist & Vapalahti, 2008). Although increase in TBE morbidity had been observed in Europe Russia has by far the greatest number of confirmed TBE (Su¨ss, 2008), and TBEV can now be found in regions that cases (Su¨ss, 2008), the Czech Republic has one of the were previously unaffected (Charrel et al., 2004). TBE is highest incidence rates in Europe, with 400–1000 clinical now a notifiable disease in 16 European countries (Su¨ss, cases reported annually (Ru˚zˇek et al., 2008). Virulence of 2008) and cases have been confirmed in areas where it has TBEV for humans has been shown to decrease with its not been previously reported, for example Norway (Csa´ngo´ westward spread from Japan/far-eastern Russia, through et al., 2004). Between 1990 and 2007 there were an average central Europe to western Europe (Gritsun et al., 2003a), of 8755 reported cases of TBE per year in Europe and with the far-eastern subtype having a case fatality rate as Russia, in comparison with an average of 2755 per year high as 40 % (Mandl, 2005). between 1976 and 1989 (Su¨ss, 2008). This increase may have been caused by an expanding tick population, promoted by factors including climate change, social and Virology and epidemiology of TBEV political changes and changes in land use (Su¨ss, 2008). However, a number of European countries have observed a TBEV is a member of the genus Flavivirus, within the family reduction in the number of cases in recent years, including Flaviviridae (Mandl et al., 1997). It is a member of the Austria, which ran a successful vaccination campaign mammalian tick-borne flavivirus group, which, along with introduced in 1981 (Fig. 1) (Su¨ss, 2008). The overall other genetically and antigenically related , includes figures for Russia also suggest a similar trend, although the Omsk hemorrhagic fever virus (OHFV), Langat virus numbers of cases are still in the thousands (Fig. 1) and in (LGTV), Alkhurma hemorrhagic fever virus (AHFV), one region of Russia alone (Eikatherinburg, Ural region), Kyasanur Forest disease virus (KFDV), Powassan virus there are 300–500 reported cases every year (Toporkova (POWV), Royal Farm virus (RFV), Karshi virus (KSIV), et al., 2008). Within Russia, western Siberia has the highest Gadgets Gully virus (GGYV) and Louping ill virus (LIV)

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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 cells (Mandl et al., 1997). Many studies have focused on the are also known as the TBEV serocomplex (Clarke, 1964; TBEV E protein, which is known to exist as flat dimers Calisher 1988) (Table 1, Fig. 2). AHFV is a subtype of KFDV extending in a direction parallel to the viral membrane, (Charrel et al., 2001). Of these viruses, TBEV, LIV and with residues important for antibody binding exposed on POWV are all known to cause encephalitis in humans and the outer surface of the protein (Rey et al., 1995). The animals, whereas OHFV, KFDV and AHFV cause hemor- second transmembrane region of the E protein is known to rhagic fever (Gritsun et al., 2003b; Grard et al., 2007). LGTV be important for virion formation (Orlinger et al., 2006). is a naturally occurring non-pathogenic virus (Smith, 1956) The E protein also has the ability to form enveloped and there have been no reports of human disease caused by recombinant subviral particles (RSPs) with prM (Allison et KSIV, RFV or GGYV (Gritsun et al., 2003b). al., 1995a), which assemble in the endoplasmic reticulum , including TBEV, are small, lipid-enveloped (ER) and are transported through the secretory pathway in viruses with a spherical structure and a diameter of 40– the same way as whole virus particles (Lorenz et al., 2003). 60 nm (Lindenbach & Rice, 2001). The flavivirus genome Cryoelectron microscopy has shown that mature TBEV comprises a single-stranded, positive-sense RNA of RSPs have an icosahedral molecular organization approximately 11 kb in length, containing a 59 cap and (Ferlenghi et al., 2001). The flavivirus NS3 protein is lacking a polyadenylate tail (Wengler et al., 1978). The 59 multifunctional, including the C terminus region which cap is important for mRNA stability and translation possesses both nucleoside triphosphatase (NTPase) and (Furuichi & Shatkin, 2000). The single open reading frame helicase activities (Wengler & Wengler, 1991). The N- (ORF) encodes three structural proteins – capsid protein C, terminal region of the NS3 protein possesses protease membrane protein M (formed by cleavage from it’s activity, which is necessary for the post-translational precursor prM) and the large envelope glycoprotein (E) – cleavage of the viral polyprotein precurser (Chambers et along with seven non-structural proteins – NS1 (glycopro- al., 1990b). Studies with West Nile virus (WNV) have tein), NS2A, NS2B (protease component), NS3 (protease, suggested that these activities are all necessary for viral helicase and NTPase activity), NS4A, NS4B and NS5 replication (Brinton, 2002). The NS5 protein has two (RNA-dependent polymerase). The 59 and 39 ends of the domains, a C-terminal RNA-dependent RNA polymerase genome are non-coding regions (reviewed by Chambers et (RdRp) and the N-terminal methyltransferase core, which al., 1990a). The viral genome RNA is itself infectious and enables NS5 to exhibit the (nucleoside-29-O-)-methyl- would produce virus progeny if introduced into susceptible transferase activity required for methylation of the cap

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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 Principal tick vector Geographical distribution

Tick-borne encephalitis virus TBEV-Eu I. ricinus Central/western Europe, (European subtype) Scandinavia, Korea Tick-borne encephalitis virus TBEV-Sib I. persulcatus Russia, Finland (Siberian subtype) Tick-borne encephalitis virus TBEV-Fe I. persulcatus Russia, Far East (China, Japan) (far-eastern subtype) Louping ill virus LIV I. ricinus UK, Ireland, Norway Spanish sheep encephalomyelitis virus SSEV I. ricinus Spain Turkish sheep encephalitis virus TSEV I. ricinus Turkey Greek goat encephalitis virus GGEV I. ricinus Greece Powassan virus POWV Ixodes cookei, Ixodes marxi USA, Canada, far-eastern Russia Kadam virus KADV Rhipicephalus pravus Uganda, Saudi Arabia Omsk hemorrhagic fever virus OHFV Dermacentor reticulatus (Dermacentor Western Siberia marginatus) Kyasanur Forest disease virus KFDV Haemaphysalis spinigera (Ixodes spp., India Dermacentor spp., Haemaphysalis spp.) Alkhurma hemorrhagic fever virus AHFV Ornithodorus savignyi Saudi Arabia Langat virus LGTV Ixodes granulatus Malaysia, Thailand, Siberia Karshi virus KSIV Ornithodorus papillipes Uzbekistan Royal Farm virus RFV Argas hermanni Afganistan Gadgets Gully virus GGYV Ixodes uriae Macquarie Island (Southern Ocean) structure at the 59 end of the RNA genome (Egloff et al., It has been demonstrated that low pH plays an essential 2002). Furthermore, the NS5 protein has recently been role in both the entry and the release stages of the flavivirus identified as an interferon antagonist due to it’s ability to life cycle (Stadler et al., 1997). During the first stage of the inhibit STAT1 phosphorylation in response to interferon viral life cycle, the virions bind to the surface of the host and its association with interferon receptor complexes cell, mediated by the viral surface E protein and with (Best et al., 2005). 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 flavi- viruses are as follows: SREV, Saumarez Reef virus; MEAV, Meaban virus; TYUV, Tyuleniy virus; CFAV, cell fusing agent virus.

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(Kroschewski et al., 2003). Work with WNV suggests that useful experimental system for the specific mutagenesis of the virions are then transported by receptor-mediated TBEV, exhibiting identical biological properties to the endocytosis through clathrin-coated pits into prelysosomal parent viruses (Mandl et al., 1997). endocytic compartments of the host cell (Chu & Ng, 2004). TBEV complex viruses cause limited disease in indigenous Recently, it has been demonstrated that the low pH within forest animals but have the potential to emerge as the endosome causes the protonation of a conserved pathogens if they infect introduced species. During the histidine residue (His323) at the interface between last few thousand years, this group of viruses has evolved domains I and III of the E protein (Fritz et al., 2008), and spread westwards throughout Asian and European inducing a change in conformation where the E protein is forests (Gould et al., 2006). TBEV is now endemic in an reorganized from dimers into trimers (Allison et al., area ranging from northern China and Japan, through far- 1995b). The fusion peptide is now exposed and fusion of eastern Russia to Europe (Dumpis et al., 1999; Hou et al., the viral membrane with the endosomal membrane is 1997; Takashima et al., 1997). The European subtype of induced, releasing the viral nucleocapsid into the host cell TBEV is predominantly found throughout Europe and cytoplasm, where translation of the genome RNA occurs Russia, with Russia also having the Siberian subtype of (reviewed by Lindenbach & Rice, 2001). Uncoating of the TBEV. The far-eastern subtype of TBEV is endemic in viral RNA genome occurs and the viral polyprotein is northern regions of China and is also present in western processed to yield individual viral proteins (reviewed by and south-western China (Lu et al., 2008). This subtype has Chambers et al., 1990a). This leads to initiation of viral also been shown to be endemic in Japan (Takashima et al., genome replication, where full-length negative-strand 1997) and is present in far-eastern Russia. copies of the genome act as templates for the production of new positive-strand RNAs (Chu & Westaway, 1985). Phylogenetic studies of a number of TBEV genes over the During polyprotein synthesis, the surface proteins prM and last few years have all generated trees with a similar pattern E are translocated into the lumen of the ER and their (Gould et al., 2004). Studies have been based upon a amino-termini are released through proteolytic cleavage by number of nucleotide sequences, including the E, NS3 and host cell signallase (Nowak et al., 1989). The highly basic C NS5 sequences, and it has been shown that NS3 generates a protein packages the RNA genome into nucleocapsids on robust phylogenetic analysis, which is very similar to that the cytoplasmic side of the ER membrane and, at the same obtained from complete sequences (Cook & Holmes, 2006; time, assembly of the viral envelope containing prM and E Grard et al., 2007). Recent studies have shown that the far- occurs through budding of the nucleocapsid into the ER eastern and Siberian subtypes are phylogenetically more lumen (Chambers et al., 1990a). This assembly yields non- closely related to each other than to the European subtype, infectious immature virions, where proteins prM and E are which appears to be more closely related to LIV (Grard in a heterodimeric association on the viral surface (Lorenz et al., 2007) (Fig. 2). LIV is the only tick-borne flavivirus et al., 2002; Elshuber et al., 2003) and are transported currently found in the UK; it is transmitted by Ixodes through the host secretory pathway. In the acidic vesicles of ricinus and causes disease primarily in sheep and red the late trans-Golgi network, cleavage of prM protein by grouse, predominantly distributed on the sheep-rearing the host cell protease furin leads to virus maturation hillsides of Scotland, England, Wales and Ireland (McGuire (Stadler et al., 1997). This final activation cleavage leads to et al., 1998). In comparison, TBEV is generally found in production of protein M and the reorganization of E forests throughout Europe and Asia (Gritsun et al., 2003a). protein into fusion-competent homodimers; the infectious Unlike TBEV, LIV causes viraemia and fatal encephalo- mature virions are released from the cell through fusion of myelitis in domesticated animals when infected ticks the transport vesicles with the host cell plasma membrane feed on them (McGuire et al., 1998). Although LIV is (Wengler & Wengler, 1989). potentially a threat to humans in the UK, natural exposure is rare and there have been very few reports of cases in The three main subtypes of TBEV are the European, humans (Gritsun et al., 2003a). The few reported human Siberian and far-eastern subtypes (Ecker et al., 1999), cases of LIV have mainly been laboratory-acquired which are all closely related both genetically and anti- (Davidson et al., 1991) and the disease course is similar genically. Studies with European TBEV isolates suggest that to that observed with TBEV (Gritsun et al., 2003a). TBEV is quite stable under natural ecological conditions However, although the disease is often subclinical in and does not undergo significant antigenic variation. humans, the meningoencephalitis caused can be severe Indeed, there is known to be a high degree of antigenic (Davidson et al., 1991). A disease similar to that caused by homogeneity between different strains of TBEV LIV has been reported in sheep and goats in other (Holzmann et al., 1992). Culturing TBEV in the laboratory European countries, including Spain, Greece and Turkey is known to affect both the genotypic and the phenotypic (Gritsun et al., 2003a). The viruses causing disease are characteristics of the virus, and phenotypic characteristics genetically distinguishable from LIV and have been named are also thought to change following mammal-to-tick according to the country they were first isolated in, for transmission of the virus (Kaluzova´ et al., 1994; Romanova example Spanish sheep encephalomyelitis, Greek goat et al., 2007). Infectious cDNA clones composed of two encephalitis and Turkish sheep encephalitis (Table 1) different strains of TBEV have been demonstrated as a (Grard et al., 2007). Although domestic animals infected

Downloaded from www.microbiologyresearch.org by 1784 Journal of General Virology 90 IP: 54.70.40.11 On: Wed, 03 Apr 2019 19:51:37 Tick-borne encephalitis virus – a review with TBEV are generally asymptomatic, many studies have thus increasing the risk of a tick bite and of contracting demonstrated the presence of circulating antibodies to TBE (Sˇumilo et al., 2008a). However, this does not explain TBEV in both animal and human populations in countries the increasing incidence in countries such as Germany, throughout Europe, including Lithuania, Norway and Italy, Finland and Sweden (Su¨ss, 2008). Other factors that Sweden (Juceviciene et al., 2005; Csa´ngo´ et al., 2004; may have contributed towards the increase in incidence of Stjernberg et al., 2008). In Denmark, it was found that TBEV in central and Eastern Europe include an increase in 8.7 % of roe deer were seropositive (Skarphe´dinsson et al., the amount of land cultivated and a decrease in both the 2005) and a recent study of a small endemic island in use of pesticides and the amount of industrial pollution south-east Sweden has found human seropositivity to be (Sˇumilo et al., 2008b). These factors would have had a 2 % (Stjernberg et al., 2008). Similarly, a human study in positive effect on the habitat and rodent host populations south-eastern Turkey found circulating TBEV-specific IgG for I. ricinus ticks. Changes in hunting practices have also (10.5 %) and IgM (23 %) antibodies, suggesting possible led to an increase in larger mammal populations, such as human cases despite there being no reported cases of TBE roe deer and wild boar, providing a greater feeding in Turkey to date. However, these are ELISA-derived data opportunity for questing ticks (Sˇumilo et al., 2008b). and they have not been confirmed by neutralization tests Alternatively, there has been an improvement in the quality (Ergunay et al., 2007). Often, there appears to be a of epidemiological surveillance systems and diagnostics, correlation between seropositivity in domestic animals, the which may also influence the reporting of TBEV cases number of infected ticks and the number of human clinical (Su¨ss, 2003). Furthermore, in an age when travel is cases of TBE in a particular region (Juceviciene et al., increasingly popular, and previously remote areas of the 2005). A recent study has shown that a significant number planet are now more accessible, the risk of a traveller of domestic dogs in the Aust-Agder region of southern becoming infected through a tick bite is greatly increased. Norway had circulating antibodies to TBEV, even though In a recent study, birds migrating from western Russia and TBE had not previously been observed in this region Fennoscandia to Sweden have been shown to carry TBEV- (Csa´ngo´ et al., 2004). These data correlated with the first infected I. ricinus ticks (Waldenstro¨m et al., 2007). This cases of human TBE in Norway and provide further leads to the possibility that migrating birds may play a role evidence that TBEV can be considered an emerging in the dispersal of TBEV-infected ticks, although the pathogen. Furthermore, strains of the Siberian subtype of importance of birds as a reservoir for TBEV has yet to be TBEV were isolated from Ixodes persulcatus ticks in Finland determined. Indeed, migratory birds have been suggested in 2004, suggesting that I. persulcatus and Siberian TBEV as one of the routes by which far-eastern strains have been are found significantly further north-west of the previously reported as far west as Latvia and Estonia (Golovljova et al., known range in eastern Europe and Siberia (Ja¨a¨skela¨inen 2004; Gould & Solomon, 2008). The ecological and et al., 2006). Indeed, all three known subtypes of TBEV are phylogenetic characteristics of TBEV and other flaviviruses capable of co-circulating at the same time in the same area, (both mammalian and seabird-borne) suggest that ticks as is currently the situation in Estonia (Golovljova et al., that feed on both mammals and seabirds may form the 2004). evolutionary bridge between these different lineages (Grard It has been shown that nymphs and larvae co-feed together et al., 2007). on the same rodent host, and the extent to which this occurs depends upon the patterns of seasonal activity Vector ecology which is influenced by temperature (Randolph et al., 2000). The apparent increase in incidence of TBEV over the last In western Europe, TBEV is transmitted primarily by the I. two decades has been attributed to climate change ricinus tick (Fig. 3a), whereas the vector for the Siberian (affecting both tick and rodent population dynamics) in and far-Eastern subtypes is I. persulcatus (Gritsun et al., many reports, although in reality, it is likely that a number 2003b). I. ricinus, the principal vector for the European of factors have simultaneously influenced this (Sˇumilo et subtype of TBEV, is a three-host tick, where each parasitic al., 2007). Apart from changing climate conditions, social, stage (larva, nymph and adult females) feed for a period of political, ecological, economic and demographic factors all a few days on a different host from a range of species (Gray, appear to play a role in aiding the spread of tick-borne 1991) (Fig. 4). Each stage of the life cycle takes disease (Su¨ss, 2008). These include changes in land usage approximately 1 year to develop to the next, so the entire (such as increased forestation or newly created gardens) life cycle is generally completed in 3 years, although this and the growing popularity of outdoor pursuits such as can vary from 2 to 6 years, depending upon the hill-walking and fishing. In particular, socio-economic geographical location (Gray, 1991). If infected with conditions have been shown to have an impact on the TBEV, the ticks remain infected throughout their life cycle incidence of TBE, as people who exist in poverty [for (Gritsun et al., 2003a); nymphs are thought to be the most example, through unemployment, or political upheaval important stage in the transmission of TBEV as they are (such as the break-up of the Soviet Union)] are less likely to more numerous than adults (Su¨ss, 2003). Throughout the be vaccinated against TBEV and more likely to go foraging majority of its geographical range, I. ricinus becomes active for food such as wild fruit and mushrooms in the forest, and starts feeding on a range of hosts in spring and early

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Fig. 4. Transmission of tick-borne encephalitis virus within the life cycle of I. ricinus (tick stages are highlighted with grey shading).

vertebrate hosts can play an important role in TBEV transmission in the absence of a detectable level of viraemia Fig. 3. I. ricinus nymphs on herbage (a) and on a woodmouse (b). (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 summer, with ticks being observed on vegetation and uninfected ticks feeding closely (Labuda et al., 1997). The animals from late March (Gray, 1991). A study in the UK process of virus transmission between ticks during co- has shown that the tick will parasitize any mammal or bird feeding is thought to occur via cellular infiltration of tick that it meets, although adults only feed successfully on feeding sites and the migration of cells from these sites larger animals such as deer or livestock (Milne, 1949). (Labuda et al., 1996). Studies have shown that mass co- Generally, throughout Europe, one major peak of tick feeding of larvae alone is also important in the persistent activity is seen, where high numbers of larvae and nymphs circulation of TBEV, as well as the feeding of infected occur together, peaking around April/May, or as late as nymphs alongside infectable larvae (Danielova´ et al., 2002). July in cooler Scandinavia (Randolph et al., 2000). It is Furthermore, the isolation of TBEV from unfed larvae during this peak that there is sufficient transmission to provides evidence for transovarial transmission of TBEV allow TBEV to persist within TBE foci, where coincident (Danielova´ et al., 2002). In the UK, it has been shown that feeding by larvae and nymphs on rodent hosts is essential I. ricinus becomes biologically active at temperatures of for the maintenance of TBEV transmission (Randolph et al., 11 uC and above, and that the lower limit of humidity for 1999). The tick is infected with TBEV during feeding; virus survival is represented by a relative humidity value of replication commences after entry into cells of the midgut between 70 and 80 % (MacLeod, 1935), hence the forests of wall, and infection of the salivary glands occurs prior to Europe and Asia provide an ideal tick habitat, with high transmission in the tick saliva during the next blood meal humidity in the dense undergrowth that prevents desic- (Nuttall et al., 1994). The infected tick can transmit the cation (Gritsun et al., 2003a). Furthermore, it has been virus to a vertebrate host during feeding and is also able to shown that both larval–nymph synchrony, where nymphs pass on the virus to a non-infected tick during co-feeding and larvae co-feed together on the same rodent host, and at the same site on the host. Studies into the transmission TBE foci are statistically significantly associated with of TBEV between co-feeding ticks have shown that temperature, characterized by a rapid fall in ground level

Downloaded from www.microbiologyresearch.org by 1786 Journal of General Virology 90 IP: 54.70.40.11 On: Wed, 03 Apr 2019 19:51:37 Tick-borne encephalitis virus – a review temperatures from August to October (Randolph et al., subcutaneous routes), the virus is known to first replicate 2000). at the site of inoculation and then in the lymph nodes that drain the inoculation site (Albrecht, 1968). TBEV has been Studies have shown a variable prevalence of infected I. found in the Langerhans cells of the skin before reaching ricinus ticks; up to 1.7 % in areas of Lithuania (Han et al., the regional lymphatic nodules via the lymphatic system 2005) and as high as 14.3 % in a recognized TBEV focus in (Haglund & Gu¨nther, 2003). Virus replication in the Switzerland (Casati et al., 2006). Recently, it has been reported that the prevalence of TBEV in ticks removed draining lymph nodes is followed by development of ´ ´ ˇ ´ from humans was significantly higher than in unfed, free- plasma viraemia (Malkova & Frankova, 1959). During the living I. ricinus ticks in the same area, possibly associated viraemic phase, many extra-neural tissues are infected and with different replication strategies of the virus in tick and the release of virus from these tissues enables the viraemia mammalian cells (Su¨ss et al., 2006). Furthermore, ticks are to continue for several days (Albrecht, 1968; Monath & capable of maintaining a double infection, being able to Heinz, 1996). Haematogenic spread allows different organs transmit both TBEV and Borrelia to humans, as demon- to be infected, especially the reticulo-endothelial system strated by I. persulcatus ticks in Russia (Korenberg, 1994). (spleen, liver and bone marrow) (Haglund & Gu¨nther, 2003), and it is during this phase that the virus also crosses TBEV is maintained in cycles involving Ixodid ticks and the blood–brain barrier to invade the central nervous wild mammalian hosts (Fig. 3b), particularly rodents system (CNS), where viral replication causes inflammation, (Charrel et al., 2004). The rodent host acts as both lysis and cellular dysfunction (Dumpis et al., 1999). maintenance and amplifying host and also as a reservoir Neuroinvasion of TBEV in humans has been well reported, host (Su¨ss, 2003), and it has been suggested that small although the mechanisms by which an acute, non-fatal mammals may maintain a persistent infection with TBEV febrile infection develops into a severe, possibly fatal CNS throughout the year (Bakhvalova et al., 2006). Although disease are not clearly understood (Toporkova et al., 2008). larger animals (such as birds, deer and horses) may act as hosts for ticks, they are not thought to have an important There are a number of animal models regularly employed role in virus transmission between ticks (Gritsun et al., to examine the neuropathogenicity of TBEV; the mouse is 2003a). Indeed, studies have shown that a localized absence the most commonly used model as it is susceptible to of deer increases tick feeding on rodents, with the potential TBEV-induced disease, unlike other wild and domestic to cause tick-borne disease hotspots (Perkins et al., 2006). animals (Mandl, 2005). Wild-type strains of TBEV are In western Slovakia, yellow-necked field mice and bank generally neuropathogenic when experimentally inoculated voles comprise around 75 % of the rodent population, of into mice (intracranially or peripherally) usually resulting which approximately 15 % are seropositive for TBEV in a lethal infection (Mandl, 2005), although this is very (Kozuch et al., 1990). A recent study in Korea demon- much dependent upon the age of the mice (Andzhaparidze strated molecular detection of European subtype TBEV et al., 1978). An apparent feature of TBEV is the ability to genes in 20 % of wild rodents sampled, and in 10 % of cause persistent infections in experimental animals and Ixodes nipponensis ticks sampled (Kim et al., 2008). This humans (Monath & Heinz, 1996), and a number of animal was the first report of TBEV in Korea, where the number of models have been used to demonstrate degenerative patients presenting with encephalitis of unknown origin changes in the CNS following infection with TBEV. was seen to be increasing annually. It is interesting that it Intracranial inoculation of hamsters with the Soph-K was a European rather than a far-eastern strain of TBEV strain of TBEV induced clinical disease in 14 % of animals, that was detected in this study, as one would have expected although pathological lesions characterized as meningoen- a far-eastern strain similar to those found in Japan and cephalitis were found in the CNS of all the animals China. Furthermore, I. nipponensis is not the usual tick (Andzhaparidze et al., 1978). Histological examination vector for European TBEV and may provide further 45 days post-inoculation demonstrated signs of neurono- indications that TBEV can be considered an emerging phagy and marked glial proliferation. In the same study, pathogen. Similarly, although the predominant vector in subcutaneous inoculation of hamsters induced menin- China is I. persulcatus, a number of additional vectors are goencephalitis in the 4 % of animals that were exhibiting thought to be involved in the transmission of TBEV, clinical disease, although lesions in the brain were observed including Ixodes ovatus in Yunnan province (south-western in the majority of animals, including perivascular infiltra- China), a region where I. persulcatus is not detected (Lu tion and encephalitis ‘granulomas’. In an earlier study, et al., 2008). when monkeys were infected intranasally or intracerebrally with European TBEV, they were shown to develop chronic encephalitis with degenerative spongiform lesions and Pathogenesis astrocytic proliferation (Zlontnik et al., 1976). The majority of TBEV infections occur through a tick bite, Furthermore, monkeys inoculated intracerebrally with the although a small number of infections occur through Soph-K strain of TBEV showed an asymptomatic infection consuming infected unpasteurized milk (Dumpis et al., with subacute disseminated meningoencephalitis, with a 1999). Following infection with tick-borne flaviviruses progradient course for the 3 months of observation; this (either naturally or experimentally via intradermal or chronic infection provides a model of progressive degen-

Downloaded from www.microbiologyresearch.org by http://vir.sgmjournals.org 1787 IP: 54.70.40.11 On: Wed, 03 Apr 2019 19:51:37 K. L. Mansfield and others erative disease of the CNS (Andzhaparidze et al., 1978). 2006a). Conversely, it has been reported that although The first case of TBE in a monkey (Macaca sylvanus) after synthesis of CCL3 was increased during TBE infection, its natural exposure (tick bite) in a TBE risk area has recently concentration was much lower in CSF than in serum, been described (Su¨ss et al., 2007). TBEV was present in the suggesting that its major role is not as a chemoattractant of brain and was identified as the European subtype, closely leukocytes into the CNS (Grygorczuk et al., 2006b). related to the Neudoerfl strain; clinical illness similar to However, it has been shown that both sPECAM-1 (a that observed in a typical severe human TBE case was glycoprotein involved in the transendothelial migration of observed (Su¨ss et al., 2007, 2008). Indeed, chronic leukocytes) and CCL2 (involved in the activation of certain progressive human encephalitis and seizure disorders leukocytes) were elevated in the CSF of TBE patients (Kozhevnikov’s epilepsy) have been associated with (Michałowska-Wender et al., 2006). Only a modest infection with Siberian and far-eastern TBEV (Zlontnik increase in interferon (IFN)-c in CSF has been documented et al., 1976). during infection with TBEV, in comparison with other viral infections, such as Varicella–Zoster virus, where it is highly upregulated (Glima˚ker et al., 1994). Host immune response A recent study has demonstrated that TBE is an Shortly after the onset of neurological symptoms in immunopathological disease, where the inflammatory humans, the mononuclear cells in the cerebrospinal fluid + + reaction, mediated by CD8 T cells, contributes to (CSF) are predominantly composed of CD4 T lympho- + neuronal damage and could lead to a fatal outcome cytes and less numerous CD8 T lymphocytes, with (Ru˚zˇek et al., 2009). Unlike humans, however, when limited natural killer cells and B-lymphocytes (Holub et al., animals are naturally infected with TBEV, the host immune 2002). Levels of TBEV-specific antibodies have been shown response is clearly capable of preventing the development to increase in both serum and CSF; maximum IgM levels of disease, and this is an area that needs further were seen during early disease and persisted after 6 weeks, investigation. Paradoxically, the reverse happens during whereas peak IgG levels were observed in late convalescent infection with LIV; although LIV is closely related to TBEV, samples (around 6 weeks) (Gu¨nther et al., 1997). However, infected animals do exhibit clinical signs, whereas huma- although IgM antibodies can be detected for a few months ninfections are often asymptomatic (Davidson et al., 1991). after infection, IgG antibodies persist for a lifetime and provide a level of immunity that prevents reinfection (Holzmann, 2003). Clinical manifestations Recent work has investigated the role of viral proteins in The clinical manifestations in human cases have been well- the immune response to TBEV infection. The NS5 protein documented and there is a range of symptoms that can be of TBEV has been shown to act as an interferon antagonist observed. and is able to inhibit interferon-stimulated JAK–STAT Although the far-eastern subtype of TBEV causes a signalling by blocking the phosphorylation of STAT1, thus monophasic course of illness, infection with a western inhibiting the expression of antiviral genes (Best et al., European subtype usually produces a biphasic course of 2005; Werme et al., 2008). However, there are limited data illness (Dumpis et al., 1999; Gritsun et al., 2003a). The on the role of cytokines and chemokines in the pathogen- incubation period is generally 7–14 days and during a esis of TBEV. Recently, a number of studies have typical biphasic infection, symptoms during the initial investigated the level of certain cytokines and chemokines short febrile period can include fatigue, headache and pain in the serum and CSF of patients with TBE. A study in in the neck, shoulders and lower back, accompanied by Russia found that on admission to hospital, TBE patients high fever and vomiting (Gritsun et al., 2003a). This is had elevated serum levels of tumour necrosis factor (TNF)- often followed by an asymptomatic period lasting 2– a, interleukin (IL)-1a and IL-6, where IL-1a and TNF-a 10 days and if the disease progresses to neurological were acting synergistically to initiate the cascade of involvement, this leads to the second phase, characterized inflammatory mediators by targeting the endothelium; by acute CNS symptoms with a high fever. CNS infection levels of these cytokines then declined during the first week can manifest in the meninges (where inflammation causes of hospitalization, which was accompanied by an increase meningitis), the brain parenchyma (to cause encephalitis), in levels of IL-10 (an inhibitor of cytokine synthesis) the spinal cord (myelitis), the nerve roots (radiculitis) or (Atrasheuskaya et al., 2003). Another study found that indeed any combination of these. during TBEV infection, the concentration of CXCL10 was higher in CSF than in serum, suggesting a role in the Acute TBE is characterized by encephalitic symptoms in recruitment of CXCR3-expressing T cells into the CSF 45–56 % of patients (Haglund & Gu¨nther, 2003). (Lepej et al., 2007). Similarly, another study found that the Symptoms range from mild meningitis to severe menin- concentration of CCL5 (another lymphocyte attractant) goencephalomyelitis, which is characterized by muscular was increased in the CSF but not the serum of TBE weakness (paresis) which develops 5–10 days after patients, and that this increase was sustained after the remission of the fever. Severely affected patients may disappearance of clinical symptoms (Grygorczuk et al., demonstrate altered consciousness and a poliomyelitis-like

Downloaded from www.microbiologyresearch.org by 1788 Journal of General Virology 90 IP: 54.70.40.11 On: Wed, 03 Apr 2019 19:51:37 Tick-borne encephalitis virus – a review syndrome that may lead to long-term disability (Dumpis et hyperkinesias and epileptoid syndrome. Hyperkinesia al., 1999; Kleiter et al., 2007; Gritsun et al., 2003a). The occurs frequently and may arise during the acute phase acute febrile period of illness correlates with the presence of of TBE or persist as Kozshevnikov’s epilepsy. viraemia. Following the asymptomatic phase, the second The incidence of the different forms of TBE is variable stage of illness correlates with virus invasion into the CNS, according to the region (reviewed by Gritsun et al., 2003a). where viral replication is associated with inflammation, In Siberia, approximately 80 % of TBE cases present with a lysis and dysfunction of the cells (Dumpis et al., 1999). fever but without neurological sequelae. Paralytic forms are Although magnetic resonance imaging (MRI) is usually observed in approximately 7–8 % of cases and normal, abnormalities have been shown, including pro- Kozshevnikov’s epilepsy in about 4–5 % of patients. In nounced bilateral lesions in the thalamus, cerebral general, the case fatality rate is approximately 1–2 % peduncles and the left caudate nucleus (Lorenzl et al., following European subtype infection, but can be as high as 1996). However, it has been shown that the abnormalities 20–40 % following infection with a far-eastern subtype detected in TBE patients depend upon the MRI technique (Mandl, 2005). Infection with the Siberian subtype used, as T2-weighted and turbo FLAIR images demon- produces a mortality rate of no more than 2–3 % strated more abnormalities than T1-weighted images (Atrasheuskaya et al., 2003). However, it is possible that (Marjelund et al., 2004). the high mortality figures for the far-eastern subtype may A study of TBEV patients in Germany reported that the be due to the lack of detection of mild cases. A study of 62 average incubation period between tick bite and onset of cases of TBE in an area of Russia where the Siberian and symptoms was 11 days (with a range of 4–28 days) (Kaiser, far-eastern subtypes of TBEV co-exist has shown a large 1999). This study showed that 74 % of patients experienced variation in the clinical symptoms observed, ranging from a biphasic course of illness, typically with the first stage unapparent to severe (Pogodina, 2005). Hence, an increase lasting between 1 and 7 days, and consisting of fever, in detection of milder cases would lead to an overall headache, occasional malaise and upper respiratory and/or decrease in mortality rate figures. abdominal symptoms. The intermediate asymptomatic In comparison with humans, domestic animals infected phase lasted between 3 and 21 days, followed by a with TBEV are generally asymptomatic. Indeed, recent data prodromal period which was reported less often in patients suggest that wild small mammals are able to maintain with meningoencephalomyelitis (inflammation of the brain TBEV as a persistent infection throughout the year and spinal cord) than in patients with meningoencephalitis (Bakhvalova et al., 2006). (inflammation of the brain and meninges) or isolated meningitis. Of the 656 patients, 49 % presented with meningitis, 41 % with meningoencephalitis and 10 % with Diagnosis and treatment meningoencephalomyelitis. In patients with meningoence- phalomyelitis, the most common feature was flaccid paresis The clinical symptoms observed with TBE are shared with of the extremities, whereas this was less common with other neurological disorders such as herpes encephalitis; meningoencephalitis, and was usually transitory. Similarly, therefore, differential diagnosis of a TBEV infection must paresis of the cranial nerves was more frequent and severe be established in the laboratory (Holzmann, 2003). in patients with meningoencephalomyelitis. Eight patients Diagnosis of infection with TBEV is now possible with a variety of techniques, from both ante-mortem (serum/ (1.2 %) were ventilated until death (1–52 weeks after onset CSF) and post-mortem (tissue) samples. Along with virus of disease). This study also showed that the severity of isolation, these techniques include RT-PCR, which is able illness increased with the age of the patient. An earlier to detect viral RNA in serum and CSF prior to the study in Sweden reported similar findings, with spinal appearance of antibodies (Saksida et al., 2005), multiplex nerve paralysis observed in 13 % of patients; only 60 % of PCR to differentiate between TBEV subtypes (Ru˚zˇek et al., patients were considered to be recovered after 1 year 2007) and quantitative PCR on ante-mortem CSF samples (Gu¨nther et al., 1997). (Schwaiger & Cassinotti, 2003). Most patients present at A chronic form of TBE has been observed in patients from the hospital during the non-viraemic second phase of Siberia and far-eastern Russia and is thought to be disease, when virus has been cleared from the blood and associated with the Siberian subtype of TBEV (reviewed neurological symptoms have commenced (Holzmann, by Gritsun et al., 2003a). There are two forms of chronic 2003). Development of the humoral immune response TBE, the first being long-term sequelae of any of the acute enables diagnosis of TBE by ELISA detection of antibodies forms of TBE, where the development of neurological in the blood, such as TBEV-specific IgM and IgG symptoms may take years following the bite from the antibodies (Holzmann, 2003). It has been shown that infected tick. Clinical symptoms include Kozshevnikov’s between 0 and 6 days after onset of encephalitic symptoms, epilepsy, progressive neuritis of the shoulder plexus, lateral TBEV-specific IgM activity increased in serum, declining and dispersed sclerosis, a Parkinson’s-like disease and after 6 weeks (Gu¨nther et al., 1997). In CSF, IgM activity progressive muscle atrophy. Often the physical deteriora- peaked between 9 days and 6 weeks after onset. In tion is accompanied by mental deterioration and even comparison, maximum IgG activity in both serum and death. A second chronic form of TBE is associated with CSF was in 6 week samples. In countries such as Japan,

Downloaded from www.microbiologyresearch.org by http://vir.sgmjournals.org 1789 IP: 54.70.40.11 On: Wed, 03 Apr 2019 19:51:37 K. L. Mansfield and others human cases of TBE may have been underreported due to that the human sera contained neutralizing antibodies the cross-reactivity between TBEV and JEV in serological capable of neutralizing far-Eastern TBEV strains, suggest- assays and the similarity of clinical symptoms that are ing that this vaccine may offer protection in humans observed with each infection (Takashima et al., 1997). against a viral infection caused by a far-eastern strain of TBEV. There is no curative therapy for TBE, so supportive treatment includes paracetamol, aspirin and other non- A recent study in Lithuania demonstrated that antibodies steroidal anti-inflammatory drugs. In severe cases, some to TBEV were found most often in people who frequently clinicians administer corticosteroids, although their use has spent time in the countryside or who had consumed not been validated. Patients with severe CNS symptoms unpasteurized goats’ milk (Juceviciene et al., 2002) and have to be closely monitored as coma or neuromuscular there have been many reports of the transmission of TBEV paralysis may develop rapidly, in which case intubation and to humans through the consumption of unpasteurized ventilation is necessary (Dumpis et al., 1999). 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 Vaccination and prevention (Pogodina, 1958). Similarly, the consumption of unpas- Active vaccination is the most effective method for teurized cows’ milk is common in a number of countries, preventing TBE, with modern vaccines shown to be safe including Lithuania, and has been suggested as a minor and between 95 and 99 % effective (Heinz et al., 2007). route of infection to humans (Juceviciene et al., 2005). Currently, there are at least four vaccines available against Therefore, campaigns encouraging people to pasteurize all TBEV infection, which are manufactured from purified, milk, particularly goats’ milk, before consumption may formaldehyde-inactivated virus. These include: Austrian help to reduce the risk of exposure. ‘FMSE-Immun 0.5 ml’ and ‘FSME-Immun 0.25 ml Vaccination of those individuals who are most at risk Junior’, German ‘Encepur Adult and Children’ and two would therefore be advantageous, although the risk of Russian vaccines manufactured in Moscow and Tomsk infection is dependent upon factors such as location, (Leonova et al., 2007). The European vaccines are season and activity. As recent data from Latvia confirm, it produced using the Neudorfl (Austria) and K23 is clear that people who visit forests (for work, food (Germany) strains of the European subtype (Charrel et al., collection or leisure) are four to five times more likely to 2004), whereas the Russian vaccines are produced from the encounter ticks than people who do not visit forests far-eastern strains 205 and Sofjin (Leonova et al., 2007). (Sˇumilo et al., 2008a). With the growing popularity for An annual TBE vaccination campaign was introduced in people to spend their leisure time in the countryside, Austria in 1981. Despite once having the highest incidence further measures alongside vaccination could be encour- rate in Europe (up to 700 hospitalised cases annually), this aged in order to prevent infection through a tick bite. campaign caused a steady decline in the number of cases of These include the use of tick repellents in combination TBE (Su¨ss, 2008). However, with the exception of Austria, with the wearing of appropriate clothing (for example, long trousers) and avoidance of the tick habitat if possible vaccination campaigns have had varying degrees of success, (Dumpis et al., 1999), although a recent study has shown since the vaccines are relatively expensive and must have that tick repellents are only 20 % effective (Va´zquez et al., repeated administrations in order to maintain protective 2008). Indeed, a recent study has suggested that risk immunity (Juceviciene et al., 2005). Post-exposure injec- avoidance through changing human behaviour (independ- tion with specific immunoglobulin, when given within ent of the seasonal changes in tick activity) has led to a 96 h of exposure, has been shown to prevent disease in decrease in incidence of TBE infection in a number of approximately 60 % of patients (Dumpis et al., 1999). Baltic countries in recent years (Sˇumilo et al., 2008a). However, post-exposure prophylaxis should be discour- Similarly, vaccination of people travelling to areas known aged, as it has been suggested that the administration of to be endemic for TBEV is advisable. post-exposure passive immunization may actually exacer- bate the disease through antibody-dependent enhancement of infection (Arras et al., 1996; Phillpotts et al., 1985). The current situation Studies in mice have demonstrated a high level of cross- As an emerging zoonosis, there is currently increasing protection between European and far-eastern subtypes of interest in TBEV. In particular, this has prompted the TBEV, where immunization with the European subtype identification of particular regions of the genome that may vaccine protected the mice against infection with far- influence phenotypic characteristics of the virus. Recently, eastern strains (Holzmann et al., 1992). Furthermore, a certain mutations that influence the neuroinvasive capacity recent study in human volunteers has shown that following of the virus in mice have been identified (Ru˚zˇek et al., vaccination with Encepur Adult (based upon the European 2008). This has suggested that non-virulent variants of subtype), they were able to produce a humoral response TBEV, which are highly adapted to ticks yet sustain human towards strains of the far-eastern TBEV subtype (Leonova population immunity by inducing subclinical infections, et al., 2007). This neutralization assay data demonstrated are found in the natural environment. Furthermore, these

Downloaded from www.microbiologyresearch.org by 1790 Journal of General Virology 90 IP: 54.70.40.11 On: Wed, 03 Apr 2019 19:51:37 Tick-borne encephalitis virus – a review authors suggest that virulent and attenuated viruses may encephalitis infection among wild small mammals in the southeastern co-exist as quasispecies in the same TBEV population and part of western Siberia, Russia. Vector Borne Zoonotic Dis 6, 32–41. that conversion of neurovirulence during tick/mammal Best, S. M., Morris, K. L., Shannon, J. G., Robertson, S. J., Mitzel, adaptation of the virus is mediated by selection from this D. N., Park, G. S., Boer, E., Wolfinbarger, J. B. & Bloom, M. E. (2005). quasispecies population rather than random mutagenesis. Inhibition of interferon-stimulated JAK–STAT signalling by a tick- borne flavivirus and identification of NS5 as an interferon antagonist. There is mounting speculation that TBEV has the potential J Virol 79, 12828–12839. to move further westward towards the UK. Predictive Brinton, M. A. (2002). The molecular biology of West Nile virus: a new models suggest that the UK can potentially be considered invader of the Western hemisphere. Annu Rev Microbiol 56, 371–402. as an emerging disease ‘hotspot’, with the likelihood that Calisher, C. H. (1988). Antigenic classification and taxonomy of new emerging infectious diseases are likely to appear (Jones flaviviruses (family Flaviviridae) emphasizing a universal system for et al., 2008). Increased temperatures have already allowed the taxonomy of viruses causing tick-borne encephalitis. Acta Virol the limit of I. ricinus to extend north- and westwards, thus 32, 469–478. fuelling the prediction that TBEV may also extend to Calisher, C. H. & Gould, E. A. (2003). Taxonomy of the virus family previously unaffected areas (Randolph & Rogers, 2000). Flaviviridae. Adv Virus Res 59, 1–19. Indeed, in South Korea it has been shown that western Casati, S., Gern, L. & Piffaretti, J.-C. (2006). 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Evidence that the N-terminal change, the increasing popularity of outdoor pursuits and domain of non-structural protein NS3 from yellow fever virus is a changes in land usage, this is a risk that cannot be ignored. serine protease responsible for site-specific cleavages in the viral There are clearly a number of factors that may influence polyprotein. Proc Natl Acad Sci U S A 87, 8898–8902. the incidence and extent of TBEV throughout Europe and Charrel, R. N., Zaki, A. M., Attoui, H., Fakeeh, M., Billoir, F., Yousef, will continue to do so in the years to come. A. I., de Chesse, R., de Mico, P., Gould, E. A. & de Lamballerie, X. (2001). Complete coding sequence of the , a tick- borne flavivirus causing severe hemorrhagic fever in humans in Saudi Acknowledgements Arabia. Biochem Biophys Res Commun 287, 455–461. Charrel, R. N., Attoui, H., Butenko, A. M., Clegg, J. C., Deubel, V., This work was funded by the Department for Environment, Food and Frolova, T. V., Gould, E. A., Gritsun, T. 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