Tickborne Encephalitis Virus

Introduction to Tickborne Encephalitis Virus and Its History

Tickborne encephalitis virus (TBEV) is one of the most important tickborne arboviruses in Central and Eastern European countries, Russia, and parts of Asia, infecting at least 13,000 people annually despite the fact that it is not a reportable disease in China. There has been a 300% increase in human infections during the 10 years preceding 2018. At least three groups of this virus exist: TBEV-European (TBEV-Eur), TBEV-Siberian (TBEV-Sib), and TBEV-Far Eastern (TBEV-FE). The viruses’ range is increasing and now includes parts of France, Sweden, Finland, Norway, Italy, and the Netherlands. This increase may be due to the growing numbers and spread of its tick vectors, which may reflect climatic changes as well as changes in the land usage. TBEV has been placed into category C of the CDCs list of potential bioterrorism agents due to its availability, ease of production and dissemination, and potential for high morbidity and mortality.

Social and political factors contribute to disease incidence. For example, reported tickborne encephalitis (TBE) incidence increased from 2- to 30-fold in many Central and Eastern European countries as they gained their independence from Soviet rule. This led to the subsequent breakdown of medical and public health institutions in addition to decreased income and availability of food. In Latvia, high-risk behavior included harvesting food from tick-infested forests and decreased vaccination. In eight Eastern European countries, national socioeconomic conditions, such as poverty and household expenditure on food, strongly correlate with the extent of TBE incidence.

In many endemic regions, TBEV distribution is highly focal. The spatiotemporal heterogeneities in TBE epidemiology in these countries appear to better explain the differences in national disease incidence patterns than those detected by climate change alone, since it is uniform across the affected areas. The apparent increases in TBEV incidence may also reflect more accurate reporting of current disease incidence in humans than was the case when these countries were part of the former Soviet Union.

A study of TBEV-FE in China between 2003 and 2016 found that the disease is reemerging and that its range is expanding. Approximately 99% of the cases in humans are reported in the forested areas of the northeastern part of China and 93% of them occur from May to July. The spatial distribution of human TBE in this area of China is associated with land use and the amounts of broad-leaved forest and mixed broadleaf-conifer forests, distribution of Ixodes persulcatus ticks, altitudes between 400 and 600 m, and climate. In contrast, disease incidence in northwestern China is highest in areas with altitudes between 1400 and 1700 m, while it is found between altitudes of 2000–3000 m in the southwestern endemic areas of the country. The differences in altitude correspond to Ix. persulcatus distribution. In a natural focus of TBEV-FE in southwestern China, 8.3% of the regional adult Ixodes ovatus ticks are infected. Temperature, relative humidity, rainfall, and sunshine hours also potentially affect the tick life cycles and viral replication in the tick. Relative humidity of 75%–90% is also important in increasing the activity of Ixodes ricinus nymphs and the subsequent TBEV-Eur incidence in humans in southern Poland.

In addition to recent increases in the range of TBEV-FE, changes in pathogenicity have been occurring in the populations of TBEV. A TBEV-FE strain that causes high morbidity and mortality in the large Japanese field mouse was isolated in Japan in 2008. A far less virulent strain with 36 nucleoside differences and 12 amino acid changes in the gene encoding the NS5 protein had been previously isolated from wild rodents in 1995. The more pathogenic strain 2008 strain underwent greater viral multiplication and inflammatory responses in the brains of mice than the earlier strain.

The reemergence of human infection in China may have at least partially resulted from the “Greening” Program, instituted in the mid-1900s that returned agricultural lands to forests and grasslands. While forestry workers are at high risk of infection, an increasing proportion of disease is occurring among farmers and domestic workers who perform pot herb-picking activities in the spring and summer. The latter two groups are less experienced in avoiding tick bites and also have a much lower rate of TBEV vaccination than do forestry workers. It should be noted, however, that TBE is not a notifiable disease in China, which may result in underreporting, especially in the more remote regions where tick-human contact is greater than found in urban areas.

TBEV Neurological Disease and Viral Structure

In experimentally infected bank voles, all three TBEV subtypes cause viremia. They are also highly neurotropic, infecting only neurons and not neuroglia, first in the cortex and hippocampus, and later appearing in Purkinje cells of the cerebellum, similar to viral distribution in experimentally infected mice. Mice infected with low doses of TBEV by the subcutaneous route may also develop systemic inflammatory thymic and splenic atrophy as well as stress responses, including increased serum levels of corticosterone (a stress hormone that downregulates some parts of the immune response) and tumor necrosis factor-α (TNF-α) (a powerful inflammatory cytokine). Humans develop nonspecific, microscopic lesions throughout the gray matter of the brainstem, cerebrum, cerebellar cortex, pons, cerebellum, thalamus, and anterior horns of the spinal cord. Degenerative changes in the cerebral cortex are confined to the pyramidal cells of the motor area where lymphocytes accumulate and glial cells proliferate near the surface of the cerebrum. Viral RNA, but not viral antigen, remains in the brain for up to 168 days after infection, but may or may not be infective throughout this time period. TBEV-Eur causes a biphasic disease in most regions outside of China, initially presenting with flu-like symptoms. In 20%–30% of cases, patients enter into the second phase of infection. This phase is characterized by central nervous system (CNS) involvement, including ataxia, paralysis of the limbs, meningitis, encephalitis, meningoencephalitis, or polioencephalomyelitis, primarily in the spinal cord, brainstem, and cerebellum, associated with infiltration of inflammatory cells into the CNS. Epilepsia partialis continua is a rare variant of simple focal motor status epilepticus and is reported in people infected by either TBEV-FE or TBEV-Eur. Neurons may be damaged by a combination of viral infection, infiltrating CD8 ⁺ T cells, proinflammatory cytokines, and activated microglial cells. Neighboring cells may also be damaged, presumably due to bystander effects. The virus is present in serum in the first, febrile phase, but only rarely in the second, neurological phase.

During infection with TBEV-FE, 31%–64% of patients develop focal encephalitic symptoms, 26% develop meningeal disease, 14%–16% become febrile, and only 3%–8% develop biphasic disease. The fatality rate is as high as 35%, and complete recovery only occurs in 25% of the patients; however, less than 0.5% of those infected develop chronic disease. Focal encephalitic symptoms are found in 5% of patients infected by TBEV-Sib: 47% develop meningeal disease, 40% become febrile, and 21% develop biphasic disease. The fatality rate is 2%, and complete recovery from this virus type occurs in 80% of the patients. During TBEV-Eur infection, 72%–87% of patients develop biphasic disease. In adults, the fatality rate is generally less than 2%, but a focus of highly virulent TBEV-Eur has been reported in southern Germany that had a fatality rate of 33%.

Variations in the E and nucleocapsid proteins affect virulence. The strain responsible for the high fatality rate in Germany is a TBEV-Eur variant. It has two unique amino acid substitutions in the E protein that are not found in typical TBEV-Eur strains. Additionally, as many as 50% of TBEV-Eur patients develop residual sequelae, even in people infected by normal, low-mortality strains. The risk of developing severe disease, higher mortality rates, and long-lasting sequelae increases with age. Permanent sequelae are seen in as many as 46% of the patients and include cognitive and neuropsychiatric complaints, balance disorders, headache, dysphasia, hearing defects, and spinal paralysis. Infection with TBEV-Eur is generally more common in adults than children. Children typically develop milder disease, although severe illness and permanent sequelae occasionally occur. Prognosis is favorable in children.

TBEV-FE variants differ in their pathogenic potential, with many seropositive, yet healthy people found in China. Nevertheless, TBEV-FE produces a more prolonged viremia in comparison with the other TBEV variants. In addition to nervous system disease, severe cases of TBE in China report paralysis, disturbance of consciousness, difficulties in swallowing and verbal communication, myocardial damage, and liver dysfunction. The wide range of differences in pathogenicity appears to be at least partially due to single amino acid substitutions in the E protein that are associated with low levels of neuroinvasion, limited peripheral replication, and lessened pathogenicity in mice. Differences in the host's major histocompatibility complex (MHC) molecules or regional human activities, including occupational variation within a given area, may also contribute to disease severity and neuroinvasiveness. At least three specific amino acid substitutions in TBEV-Eur’s E protein are known to result in decreased disease in vivo. One of these substitutions (Glu122 → Gly), found in both TBEV-FE and TBEV-Eur, is associated with binding to host heparan sulfate during viral entry into its target cells.

Isolating and sequencing of strains of TBEV-FE from patients with varying degrees of severity (encephalitic, febrile, and subclinical) indicate that the nucleocapsid protein also influences pathogenicity. The nucleocapsid is involved in viral budding during transit of virus between the endomembranous components (endosomes, the endoplasmic reticulum, and Golgi apparatus) of the host cell. Several amino acid substitutions in the viral nonstructural NS3 or NS5 proteins decrease TBEV-FE virulence. Additionally, four amino acids in the C-terminus of NS5’s polymerase are important in neuronal degeneration as well as neuronal dysfunction, including decreased neurite differentiation and outgrowth. These NS5 alterations do not directly affect viral replication or the host inflammatory response. They may do so, however, by altering interactions between NS5 and host PDZ domain-containing proteins, effecting synaptic plasticity and axonal degeneration in the nervous system.

TBEV’s NS5 contains 2 PDZ binding motifs. PDZ domains are often present in host multidomain scaffolding proteins in the CNS. These scaffolds are important for the regulation of synaptic plasticity and synaptic vesicle dynamics. The NS5 methyltransferase and RNA polymerase domains inhibit neuronal differentiation by binding to the host PDZ domain protein Scribble and outcompete the binding of the host Rho GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1) and the guanine nucleotide exchange factor, PAK-interacting guanine nucleotide exchange factor (βPIX). Rac1 is critical in regulating cytoskeletal actin rearrangements in cells, including axon guidance and neurite outgrowth. Its activity is controlled by βPIX. NS5 is able to bind to several other host proteins as well, including those regulating synaptic membrane exocytosis-2, zonula occludens-2, GIAP glutamate receptor-interacting protein 2, GIAP C-terminus-interacting protein, the calcium/calmodulin-dependent serine protein kinase, and interleukin-16 (IL-16).

The 3′-untranslated region (UTR) of TBEV-FE is also involved in pathogenesis. Partial deletions and the addition of poly A’s in the variable 5′-terminal of the 3′-UTR increase virulence in mouse brains, while decreasing viral multiplication in the periphery, without effecting the induction of interferon (IFN) or IFN-stimulated genes (ISGs). Such alterations in the 3′-UTR’s variable region are also present in TBEV strains passaged in mammalian cell cultures in vitro as well as in TBEV-Eur strains isolated from humans. Moreover, deletions in this region are associated with disease severity in the highly virulent TBEV-FE strain Sofjin-HO and the low virulent strain Oshima 5-10 in mice. This region of Sofjin-HO contains fewer stem-loop structures than Oshima 5-10. Host cell proteins, including La, p100, fructose-bisphosphatase 1 (FBP1), and Mov34, bind to the viral 3′-UTRs as well.

Infiltrating inflammatory cells cross the blood–brain barrier (BBB) as they enter the CNS. TBEV entry into the brain precedes BBB disruption. BBB permeability increases during later stages of infection in the presence of high virus loads in the brain. Among patients with neuroinvasive infection, 25%–40% of the survivors develop long-lasting neurological sequelae, which include severe headaches and decreased quality of life. Although only about 5% of primary human microvascular endothelial cell cultures are infected in vitro, the infection is persistent and yields high TBEV titers (over 10 ⁶ plaque-forming units/mL), without causing cytopathic effect. The low infection rate in the brain microvascular endothelial cells may partially explain why only some TBEV-infected individuals develop CNS infection. Astrocytes, glial cells which help to maintain the BBB, may be infected by TBEV as well.

Unlike some mosquito-borne flaviviruses, including WNV, no changes are found in the expression of key tight junction proteins, such as occludin and zonula occludens-1, cell adhesion molecules, or alterations in the intercellular junctions between these brain endothelial cells. Moreover, in in vitro models, TBEV crosses the BBB using a transcellular pathway that does not compromise the cellular monolayer integrity, is independent of TBEV strain virulence, and also is found during infection with several other flaviviruses, including WNV and JEV. CD8 ⁺ T killer cells, however, alter BBB tight junction proteins and increase CNS vascular permeability, but not in infected perforin-deficient mice. Increased BBB permeability, however, still occurs in the absence of CD8 ⁺ T killer cells, suggesting the involvement of additional factors, including proinflammatory cytokines, chemokines, and matrix-degrading metalloproteinase-9 (MMP-9).

Increased BBB permeability in mice additionally corresponds to high levels of mRNA for TNF-α, IL-6, IFN-γ, ICAM-1 (intercellular adhesion molecule), RANTES (regulated upon activation, normal T cell expressed, and secreted), MCP-1 (macrophage chemotactic protein-1), MIP-1α (migration inhibitory protein), MIP-1β, and IP-10 (interferon gamma-induced protein 10). In humans, serum levels of the proinflammatory cytokines TNF-α and IL-6 peak during the first week of hospitalization. Reducing levels of these cytokines and chemokines is associated with quicker improvement and faster recovery in vivo. Moreover, TBE patients’ CSF contains increased levels of MCP-1 and RANTES. In humans, high levels of MMP-9 are also present in the serum and CSF.

In vitro, TBEV infection of several types of cultured human neural cell lines (neuroblastoma, medulloblastoma, and glioblastoma) leads to very high viral titers. Proliferation of rough ER membranes, especially in the perinuclear region, and extensive rearrangement of the cytoskeleton occur in both medulloblastoma and glioblastoma, but not neuroblastoma, cell lines. A dense network of microtubulin is found in areas in which viral E protein accumulates, suggesting that the host cytoskeleton may be involved in the TBEV maturation process. While a large number of apoptotic signs are present in neuroblastoma cells, including condensation, margination, and fragmentation of chromatin; vacuolation of the cytoplasm; dilatation of the ER cisternae; and shrinkage of cells, the neuroblastoma cells primarily die by necrosis rather than by apoptosis.

Host cell genes affect disease severity as well. At least 140 genes are involved in the human cellular response to TBEV. The effects of any of these single genes upon disease severity in humans are difficult to determine since virulence may be multifactorial, with more than one host gene contributing to the overall extent of pathology. In Russia and Lithuania, a variety of innate immune system-related genes contain deletions or single-nucleotide polymorphisms that are associated with predisposition to TBEV infection or to the development of severe neurological disease. These virulence-associated nucleoside alterations are present in the following host cell genes: oligoadenylate synthase 2 (OAS2) and 3 (OAS3), the dendritic cell-specific intercellular adhesion molecule (ICAM)-3 grabbing nonintegrin (DC-SIGN), interleukin 28B/interferon lambda 3 (IL28B/IFNL3), IL-10, chemokine receptor 5 (CCR5), MMP9, and AT-rich DNA-interacting domain-containing protein. A polymorphism in the Toll-like receptor 3 (TLR3) gene correlates with more severe disease as well. MMPs degrade extracellular matrix proteins that are important in cellular development and morphogenesis, in addition to activating the production of cytokines and chemokines involved in tissue remodeling. High levels of these molecules are present during inflammation and in the serum and CSF of TBE patients. Increased activity of these proteinases may aid in the disruption of the BBB by degrading the barrier’s protein connections, allowing leukocytes to enter the CNS and cause local inflammatory reactions.

Ixodes species ticks are vectors for a number of neurological diseases of viral and bacterial origin, including TBE and neuroborreliosis caused by the Lyme disease spirochete. Levels of serum HMGB-1 (high-mobility group box 1) early during diseases aid in differentiation between these tickborne microbes and help to indicate appropriate treatment for people with neurological disease following tick bites.

The Virus

Viral Subtypes

According to the International Committee on Taxonomy of Viruses, TBEV is divided into three subtypes (TBEV-Eur, TBEV-FE, and TBEV-Sib), which differ in amino acid 206 of the E protein. European strains contain Val at this position, Siberian strains contain Leu, and Far-Eastern strains contain Ser. TBEV-Eur is primarily transmitted by Ixodes ricinus hard ticks. These ticks are found throughout Europe and transmit almost all of the TBEV-Eur in Europe. TBEV-Eur RNA is detectable by PCR in samples of both human clinical sera and urine, but not in the CSF. This type of TBEV may also be found when performing typical immunological tests for other conditions, suggesting a higher incidence of TBEV in humans than that which is being reported. Several other TBEV subtypes have also been proposed based on nucleoside and amino acid differences. One such potential new subtype, containing the 886-84-like virus strains, is proposed to be named TBEV-Baikalian (TBEV-Bkl) and is related to TBEV-Sib, with both TBEVs having a Leu at position 206 of the E protein. An additional proposed rodent subtype, subtype Himalayan TBEV, was detected in 200 marmots in Qinghai–Tibet Plateau in China. This subtype needs to be monitored for possible zoonotic transmission to humans, especially those handling muskrats, muskrat hides, or their bodily fluids.

TBEV-Eur appears to cause a milder disease than other TBEV subtypes. In the Republic of Korea, numbers of human encephalitis cases of unknown origin are being increasingly detected. While no confirmed human cases of TBE have been reported in that country, nevertheless, TBEV-Eur is present in its tick populations, including the primary vectors, Haemaphysalis longicornis ( Fig. 9.1 ) and Haemaphysalis flava , Ixodes nipponensis , Amblyomma testudinarium , and Haemaphysalis phasiana may also, rarely, act as vectors as well. TBEV-Eur incidence in humans is linked to host-seeking activity of nymphal Ix. ricinus . Interestingly, while the ratio of TBE cases to questing nymphs is highest during summer-autumn seasons, the numbers of questing nymphs are highest during the spring–summer period. Discrepancies between TBE incidence in humans and abundance of host-questing nymphs may be due to temperature-related differences of virus replication in their tick vectors. Extrinsic climatic conditions also affect the infection of the Ix. persulcatus vector of TBEV-FE.

TBEV-Sib and TBEV-FE are present in Ix. ricinus. Strains of TBEV-Sib and TBEV-FE have also been isolated from Aedes vexans nipponii mosquitoes in Russia, but their relevance as viral reservoirs or possible agents of zoonotic disease is unknown. Based on genetic comparison of five TBEV-Eur strains from humans with severe disease, the emergence of TBEV-Eur in Central Europe appears to have been due to a pool of viral strains whose nucleotide identity ranges from 97.5% to 99.6%, despite the fact that these strains were all isolated in 1953 in Central Bohemia, Czechoslovakia, after only a few passages in cell culture. The TBEVs isolated from this region vary greatly in length, from 445 to 751 nucleosides, and differ among themselves by three to nine unique amino acid substitutions. All of these sequenced strains have large deletions in their 3′ noncoding regions, an area associated with strain multiplication and virulence in mouse brains in TBEV-FE.

TBEV-Sib has a large range and is the dominant viral type in many parts of Russia and neighboring countries. It is, moreover, slowly replacing the two other subtypes in Northern Europe. It is less neuroinvasive than TBEV-FE, especially in children, and has a mortality rate of 2%–3%. TBEV-Sib is, however, more likely to cause recurrent infection or chronic disease than TBEV-Eur. TBEV-Sib has at least three lineages that infect humans: the Asian, South-Siberian, and Baltic lineages, plus a suggested East-Siberian lineage. Based upon sequences of their TBEV E genes, the first three of these subtypes are present in the Crimean peninsula in southeastern Europe. TBEV-Sib is typically transmitted by Ix. persulcatus , whose range extends from Eastern Europe to China and Japan.

TBEV-FE is found predominantly in Far-Eastern Russia, China, and Japan. It is present in at least three species of ticks: Ix. persulcatus (its predominant vector), D. reticulatus , and Haemaphysalis punctate in Moldova. In addition to Ix. persulcatus (infection rate = 7.9%), a recent study revealed additional arthropod vectors for TBEV-FE in the Far East of Russia, Haemaphysalis concinna (infection rate = 5.6%), Haemaphysalis japonica douglasi (infection rate = 2.0%), and Dermacentor silvarum (infection rate = 1.3%), in addition to a pool of Aedes vexans mosquitoes. In the northern part of Hokkaido, the northernmost Japanese island, Ix. persulcatus is the primary vector for TBEV-FE, while Ix. ovatus serves this role in the southern part of the island. The range of TBEV-FE may thus be larger than reported, especially if the virus is present in as-yet-unknown tick species.

Among the tick vector species, Ix. ricinus may pose the greatest risk to animal and human health since they remain attached to the vertebrate hosts for up to 10 days and transmit the largest variety of pathogens, including other viruses, bacteria, and protozoa. They also take blood meals as larvae and nymphs, as well as adult females. Ix. ricinus feeds on almost all wild or domestic animals living in woods and pastures, while humans serve as incidental hosts upon entering tick habitats. In a study conducted in 2012 in France, half of all these ticks ( n = 267) carried one or more pathogens. Half of the infected ticks hosted at least two pathogens and, in some instances, were infected by as many as five pathogenic microbes, in addition to symbiotic organisms. Human infection by multiple tickborne pathogens might result in a synergistic increase in disease severity since some of the pathogens are immunosuppressive. While TBEV RNA was not detected in this study, future studies in ticks should be conducted in regions where TBE is endemic and in surrounding areas so as to take measures to prevent zoonotic spread over a larger region of Eurasia.

Intriguingly, TBEV-FE is found in a mosaic pattern in various European regions of the former Soviet Union, the Urals, and Siberia. It has been suggested that the odd distribution of pockets of TBEV-FE strains in Europe and Siberia, thousands of kilometers away from its endemic range, may be due to a massive westward redistribution of game animals, including roe deer, wild boars, and game fowl, in the former Soviet Union, especially since the western distribution of this subtype ends in European part of the former Soviet Union and it has not expanded into the neighboring European countries.

It has been suggested that migratory birds carrying infected ticks may spread various subtypes of TBEV over long distances. In support of this hypothesis, infected Ix. ricinus larvae are found on some migrating birds, including tree pipits and European robins. However, adult Ix. ricinus very seldom parasitize birds, except for large ground-dwelling birds, such as pheasants, so that birds appear to be incompetent hosts for transmitting TBEV to ticks. Additionally, bird migration patterns are typically along longitudinal, not latitudinal, directions. Moreover, in a Swedish study that tested 13,260 migrating birds, only 4 bore TBEV-infected Ix. ricinus nymphs. It is thus unlikely that birds from the Russian Far East or East Siberia would migrate to the Urals, the Ukraine, or Estonia or be the primary driver behind the development of the widely separated TBEV clines in these areas. This may not be the case in other areas where the virus is endemic. Based on phylogeny, TBEV-FE may have been exported to Japanese islands by migratory birds from Far-Eastern Russia. Similarly, the reemergence and spread of TBEV in northeastern Germany may be due to migrating birds or to a multiyear persistence of low levels of virus. After the establishment of new disease foci, TBEV persistence may rely on the presence of transmission-competent small mammals, especially mice, bank voles, and some hedgehogs.