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Tick-Borne Encephalitis Viruses
Tick-Borne Encephalitis Viruses Peterson JO, Waltenbaugh C, and Miller SD (1992) IgG subclass responses to Theiler’s murine encephalomyelitis virus infection and immunization suggest a dominant role for Th1 cells in susceptible mouse strains. Immunology 75: 652–658. Roussarie J-P, Ruffie C, and Brahic M (2007) The role of myelin in Theiler’s virus persistence in the central nervous system. PLoS Pathogens 3(2): e23(doi:10.1371/journal.ppat.0030023). Simas JP and Fazakerley JK (1996) The course of disease and persistence of virus in the central nervous system varies between individual CBA mice infected with the BeAn strain of
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Theiler’s murine encephalomyelitis virus. Journal of General Virology 77: 2701–2711. Trottier M, Wang W, and Lipton HL (2001) High numbers of viral RNA copies in the central nervous system during persistent infection with Theiler’s virus. Journal of Virology 75: 7420–7428. Yauch RL, Palma JP, Yahikozawa H, Chang-Sung K, and Kim BS (1998) Role of individual T-cell epitopes of Theiler’s virus in the pathogenesis of demyelination correlates with the ability to induce a Th1 response. Journal of Virology 72: 6169–6174.
Tick-Borne Encephalitis Viruses T S Gritsun and E A Gould, University of Reading, Reading, UK ã 2008 Elsevier Ltd. All rights reserved.
Taxonomy, Nomenclature, and Phylogenetic Relationships ‘Tick-borne encephalitis antigenic complex’ was the original term for viruses now classified as the mammalian tick-borne flaviviruses. Together with the seabirdassociated tick-borne flaviviruses they comprise one ecological group in the genus Flavivirus, family Flaviviridae. The genus contains two other groups, namely, the mosquito-borne flaviviruses and the flaviviruses with no known arthropod vectors. Other flaviviruses are referred to as nonclassified flaviviruses. The prototype TBEV is a human and animal pathogen. The earliest TBEV isolate, known as Russian Spring and Summer Encephalitis virus, was isolated in 1937 in far-East Asia. Currently, the TBEV (currently classified as virus species in the group of mammalian tick-borne flaviviruses) is subdivided into three subtypes, far-Eastern, Siberian, and West European reflecting their antigenic, phylogenetic, and geographic relationships. Other antigenically related but distinct tickborne flaviviruses include Omsk hemorrhagic fever virus, Powassan virus (POWV), Langat virus, Kyasanur Forest disease virus, Alkhurma virus, Louping ill virus, Spanish sheep encephalomyelitis virus, Turkish sheep encephalomyelitis virus, and Greek goat encephalomyelitis virus. Each of them may cause encephalitis and/or hemorrhagic disease in humans, farmed, domestic, and wild animals. Other related mammalian tick-borne flaviviruses, not known to cause human or animal disease, include Royal Farm virus, Karshi virus, Gadgets Gully virus, and Kadam virus. The viruses associated primarily with seabirds and their ticks are Saumarez Reef virus, Meaban virus, and Tyuleniy (Three Arch) virus. They are not recognized pathogens but they show interesting geographic dispersion that reflects the flight patterns of the birds with which they are associated. The phylogenetic relationships between the TBEVs are illustrated in Figure 1.
Virions Infectious (mature) virions are spherical particles (50 nm) with a relatively smooth surface and no distinct projections. They have an electron-dense core (30 nm) surrounded by a lipid membrane. The core consists of positive-polarity genomic RNA (11 kbp) and capsid protein C (12K). The lipid membrane incorporates an envelope glycoprotein (E, 53K) and a membrane glycoprotein (M, 8K). The immature (intracellular) virions contain a precursor membrane protein (prM, 18K), the cleavage of which occurs in the secretory pathway during egress of virions from infected cells. Virions sediment at about 200S and their buoyant density is 1.19 g ml–1, although there is significant heterogeneity among them. Electron micrographs frequently revealed virions in association with cellular membranes, probably explaining this heterogeneity. They are most stable at pH 8.0, although TBEV virions remain infectious in normal human gastric juice at pH 1.49–1.80. As with all enveloped viruses, infectivity of TBEVs decays rapidly at temperatures above 40 C. Most flaviviruses agglutinate erythrocytes but a few nonagglutinating strains of TBEVs have been described. Most of the physical characteristics of the TBEVs were established using virus isolated from mammalian cells. However, after adaptation to ticks, they become less cytopathic for cultured cells and laboratory animals. These virions do not move toward the cathode in rocket immunoelectrophoresis and they have reduced hemagglutinating activity. These phenotypic characteristics may be reversed following re-adaptation to mammalian cells and host-selection of virions with a shifted net surface charge due to a single amino acid substitution in the E glycoprotein. The E glycoprotein mediates virus binding to cellular receptors and thereby directly affects virus host range, virulence, and immunological properties by inducing
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FETBE WTBE
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TSE SSE Ireland MA54 LI/I Wales 100 LI/31 SB/526 100 Norway LI/NOR LI/917 LI/261 100 PEN3 PEN6 Ireland IRE3 LI/369 British LI/G Isles INV14 LI/K THO1 99 THO2 97 INV6 INV1 Southwest LI/A 92 DEV4 England
Figure 1 Maximum likelihood phylogenetic tree of the E gene from 24 tick-borne flaviviruses. Branch lengths are drawn to scale and all nodes supported by more than 75% bootstrap support are indicated. The tree is rooted with the sequence from FETBE virus, Sofjin (RSSE) strain. The three main populations of virus in the British Isles are indicated, along with those viruses secondarily introduced into Ireland and Norway, and the viruses found in the south-west of England. Reproduced from McGuire K, Holmes EC, Gao GF, Reid HW, and Gould EA (1998) Tracing the origins of Louping ill virus by molecular phylogenetic analysis. Journal of General Virology 79: 981–988.
protective antibodies. X-ray crystallography has revealed the E-protein ectodomain (N-terminal 395 amino acids) as homodimers folded in a ‘head-to-tail’ manner (Figure 2(a)) and orientated parallel to the membrane surface. It contains three structural domains, each based on b-sheets: the central domain I, the dimerization (fusion) domain II, and receptor domain III (dI, dII, and dIII in Figure 2). The C-terminal 101 residues of the E protein form a stemanchor region consisting of two stem a-helices and two transmembrane a-helices that anchor the E protein into the lipid bilayer (Figure 3). Domain II contains a hydrophobic fusion peptide consisting of 13 residues that is highly conserved between all flaviviruses. It is located on the tip of domain II and plays a central role in fusion of the virion membrane to cellular endosomal membranes resulting in release of virion RNA into the cytoplasm (Figure 2). A three-dimensional image reconstruction of the virion surface reveals a protein shell composed of 90 E-dimers organized into a ‘herringbone’ configuration; three quasiparallel E-dimer molecules make up the main structural asymmetric unit of the shell (Figures 4(b) and 4(c)). The fivefold symmetry axes that appear as holes are generated by appropriate positioning of five domain IIIs and their lateral surface is accessible to cellular receptors and neutralizing antibodies. The M protein protrudes through holes formed between dimerization domains of E molecules. The transmembrane regions of the M and E proteins traverse the external but not the internal layer of the lipid membrane and there is no direct contact with the nucleocapsid (Figure 3). In contrast with those of many other enveloped viruses, the flavivirus capsid is poorly organized and appears to be
positioned randomly in the core. In solution, the capsid protein appears as dimer molecules with four helices (Figure 5). Two C-terminal helices fold into the positively charged interface interacting with viral RNA; the residues on the opposite side of these helices are hydrophobic and support dimerization. Two of the internal a-helices form capsid-dimers, are hydrophobic, and are required for contact with the virion membrane.
TBEV Life Cycle Virus Entry into the Cells Cellular receptors for tick-borne flaviviruses The virus life cycle commences with the attachment of virions to specific receptors on clathrin-coated pits on the cell surface. Flaviviruses infect a wide variety of primary and continuous cells, derived from mammalian, avian, and arthropod tissues. It is not yet clear if the different flaviviruses use similar or different cell surface receptors. Cellular heparan sulfate molecules are involved in TBEV attachment to cells. Nevertheless, cells that lack heparan sulfate are still sensitive to virus infection. For TBEV, a number of different proteins have been associated with cell receptor activity, including the highaffinity laminin receptor and a1b3-integrin that also recognizes laminin as a natural ligand. The E protein mediates attachment and entry of virions into cells
The immunoglobulin-like folding of the E-protein domain III implies that it is involved in cell attachment,
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Figure 2 Three-dimensional analysis of TBEV envelope (E) glycoprotein (a) at neutral pH (in mature virion) and (b) acidic pH (postfusion conformation). The view from above and lateral view correspond to the top and bottom rows. Protein domains dI, dII, and dIII are colored in red, yellow, and blue, respectively. Ribbon diagram corresponds to the lateral view of the E protein. Reproduced from Bressanelli S, Stiasny K, Allison SL, et al. (2004) Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO Journal 23: 728–738.
Domain II Domain I
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Figure 3 Schematic presentation of flavivirus structural glycoproteins E and M on the membrane surface. The coloring of the domains I, II, and III is as on Figure 2. Two a-helices of the stem-anchor region and two a-helices of the transmembrane domain of the E protein are depicted as blue- and colored cylinders. One a-helix of the stem region and two a-helices of the transmembrane domains of the M protein are depicted as orange-colored cylinders. Reproduced from Mukhopadhyay S, Kuhn RJ, and Rossmann MG (2005) A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3: 13–22, with permission from Macmillan Magazines Limited.
mediated by high-affinity cellular receptors. The receptors on the surface of vertebrate and invertebrate cells are different and probably recognized by different residues on domain III. However, domains I and II on the surface of the E protein also appear to play a role in virus–cell interactions. Domain I carries N-linked carbohydrates, which are recognized by cell surface lectins. Domain II is enriched with patches of basic positively charged surface residues that have been shown to mediate virion attachment to the cellular, negatively charged, heparan sulfate; domains I and III might also contribute to this type of interaction. Virus attachment to the cell receptors initiates receptormediated endocytosis, followed by fusion of virion and
endosomal membranes (Figures 4(c)–4(e)). Exposure of the attached virus to the endosomal acid pH converts the herringbone configuration into a fusogenic structure resulting in particle expansion and eventually in the re-assembly of the E dimers into vertical trimers (Figures 2 and 4). It is believed that during this process domains I and II flex relative to each other, allowing domain II and the stem region to move toward the endosomal membrane facilitating trimerization of domain II; the fusion tripeptide is embedded into the target membrane followed by formation of a pre-fusion intermediate (Figure 6). Trimerization spreads from fusion peptides to domain I; meanwhile domain III rotates relative to domain II pushing the stem region back toward the fusion peptide. This refolding
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Tick-Borne Encephalitis Viruses
Maturation
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Figure 4 Different conformations of E protein during the transition from immature (a) into the mature (b) particles and during pH-dependent fusion of mature particles (c), through the putative intermediates T ¼ 3 (d and e). Domains I, II, and III are depicted with the same colors as those on the Figure 2. Reproduced from Ma L, Jones CT, Groesch TD, Kuhn RJ, and Post CB (2004) Solution structure of Dengue virus capsid protein reveals another fold. Proceedings of the National Academy of Sciences, USA 101: 3414–3419, with permission from National Academy of Sciences USA.
of the distal lipid outlets and formation of a fusion pore. In the final low-pH conformation, domains I and III appear to move to the tip of the vertical trimer whereas the fusion peptide becomes embedded in the membrane eventually being juxtaposed to the transmembrane domains (Figures 2(b) and 6). Five trimers form the fusion pore; the stem region appears to play an essential role in promoting and stabilizing trimer assembly. Although the E-protein domains shift and rotate during exposure to acidic pH, each retains the neutral-pH conformation, the essential feature of class II fusion molecules that do not require major molecular rearrangements at acidic pH.
Membrane
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Figure 5 Model for the molecular interactions of flavivirus capsid dimers with genome RNA and virion membrane. Reproduced from Ma L, Jones CT, Groesch TD, Kuhn RJ, and Post CB (2004) Solution structure of Dengue virus capsid protein reveals another fold. Proceedings of the National Academy of Sciences, USA 101: 3414–3419, with permission from National Academy of Sciences USA.
brings two membranes together, resulting in the formation of the ‘hemifusion stalk’ with only the proximal lipid leaflets fused. The ‘zipping’ up of stems followed by the migration of transmembrane domains eventually leads to fusion
Translation and processing of virus proteins After uncoating, the RNA is translated into a polyprotein of 3400 residues from one open reading frame (ORF) that is co-translationally translocated and anchored in the endoplasmic reticulum (Figure 7). It is then processed by cellular signalases and viral serine protease producing three structural (capsid, prM, and E) and seven nonstructural (NS1 through NS5) proteins. The gene order, protein molecular masses, and their membrane localization are shown in Figure 7. Viral protease activity is provided by the N-terminal domain (180 residues) of the NS3 protein in association with cofactor activity of the NS2B protein that probably anchors the NS3 into
Tick-Borne Encephalitis Viruses
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Figure 6 Tentative mechanism for the fusion of viral (brown) and endosomal (green) membranes mediated by the flavivirus E protein following interaction with cellular receptor (gray) (a). Acidic pH triggers the movement of domain II (with fusion peptide on the top) toward the endosomal membrane (b) with its subsequent trimerization and insertion into the endosomal membrane (c). Trimerization spread toward domains I and III (d) causing the C-terminal part of E protein to fold backwards toward the fusion peptide. The trimerization between stem-anchor and domain II (e) results in partial fusion (e) and eventually in the formation of a fusion pore (f). Reproduced with permission from Modis Y, Ogata S, Clements D, and Harrison SC (2005) Variable surface epitopes in the crystal structure of Dengue virus type 3 envelope glycoprotein. Journal of Virology 79: 1223–1231.
the membrane. The ORF is flanked by 50 - and 30 -untranslated regions (UTRs; 130 and 700 bp, respectively) that contain signals essential to initiate translation and replication. The 50 -UTR preceding the ORF is capped by m7GpppAmpN2 and initiates virus translation in vitro although an interactive effect from the 30 -UTR has also been demonstrated. Computer-assisted prediction has demonstrated folding of the 50 -UTR into a Y-shaped structure, conserved between all flaviviruses; mutations in this structure appear to impact on virus translation and replication. The translation of flavivirus proteins is probably carried out in specialized smooth membrane structures, called convoluted membranes, derived from proliferation of the rough endoplasmic reticulum in response to flavivirus infections. Replication of viral RNA
The initiation of viral replication (synthesis of negative strand on positive strand from the 30 -UTR) requires direct interaction between the 50 - and 30 -UTRs resulting in the formation of a double-stranded RNA (dsRNA) stem and circularization of the virus genome. A terminal 30 long stable hairpin (30 LSH) structure has been revealed in the 30 -UTR that, together with the 50 -Y-shaped-structure and dsRNA circularization stem, forms a complex promoter required to initiate virus RNA replication. Additional predicted conserved secondary RNA structures in the 30 -UTR might function as replication enhancers,
essential for efficient RNA synthesis and virus transmission. The synthesis of genome-size dsRNA (replicative form, RF) accomplishes the first stage of viral replication. Flavivirus RNA replication is semiconservative and asymmetric, with an average of one nascent positivesense single-stranded RNA (ssRNA) molecule (genomic) per negative-sense template, with no free minus-strands and with 10–100 times excess of positive-strand relative to negative-strand RNA synthesis. The replicative intermediate presents a partially double-stranded RNA with nascent, and displaced, plus-sense ssRNA molecules undergoing elongation. Viral RNA capping of the 50 -UTR occurs on the displaced plus-strand RNA; the trifunctional NS3 and NS5 proteins provide nucleoside triphosphatase and guanylyl/methyltransferase activities, respectively. Formation of the replication complex involves nonstructural proteins (Figure 8) most of which are bi- and even trifunctional. The C-terminal domain of NS5 protein acts as a viral RNA polymerase and the C-terminal domain of NS3 as a helicase; both NS5 and NS3 proteins interact with 30 -LSH to initiate virus replication. The functions of other nonstructural proteins are less precisely identified. The NS1 glycoprotein forms membrane-associated hexamers that dissociate into dimers. This protein is translocated in the endoplasmic reticulum and secreted together with virions in mammalian but not in mosquito cells; its anchoring into the membranes is mediated by
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Tick-Borne Encephalitis Viruses
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(c) Figure 7 Genome strategy of TBEV. (a) Genomic RNA is presented as a solid line on the top; the 50 - and 30 -UTR are depicted in the predicted conformations according to Gritsun TS, Venugopal K, Zanotto PM, et al. (1997) Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: Analysis and significance of the 50 - and 30 -UTRs. Virus Research 49: 27–39. (b) Translation and co-translational processing of flavivirus polyprotein. The flavivirus polyprotein is depicted as a bar, with specified individual proteins and their molecular masses (numbers below the bar). (c) Membrane topology of viral proteins in relation to the lumen of the ER and cytoplasm after the completion of co-translational processing and translocation. Adapted from Westaway EG, Mackenzie JM, and Khromykh AA (2003) Kunjin RNA replication and applications of Kunjin replicons. Advances in Virus Research 59: 99–140. Transmembrane domains are shown as cylinders. Glycosylation is indicated as (*). The polyprotein is processed by ER signalases (s) and viral-specific protease NS2B-NS3 (V). The cleavage of M from prM is carried out by the Golgi protease, furin (f) and cleavage between NS1 and NS2A is by an unknown (?) ER protease.
glycosyl-phosphatidylinositol. The NS1 protein induces the production of protective antibodies that also participate in complement-mediated lysis of infected cells, implying a role in immunopathology. The NS2A protein is found in association with the NS3 helicase domain, the NS5 protein, and the 30 -LSH. It may be involved in trafficking of viral RNA between the translation, replication, and virion assembly sites. The NS2B protein is a membrane-anchored cofactor of the serine proteinase, NS3. It also has membranepermeability modulating activity possibly related to replication. The hydrophobic NS4A protein in conjunction with the NS1 protein probably anchors the polymerase complex to cell membranes and is involved in virus-induced membrane rearrangements that compartmentalize virus translation, replication, and assembly processes (Figure 8). Replication of flavivirus RNA is associated with host membrane 75–100 nm vesicular packets enclosed in the second outer membrane and connected to convoluted membranes, the sites of virus translation. The vesicular
packets proliferate in the perinuclear region in response to virus infection and are probably derived from the transGolgi network. The architecture of the replication complex in relation to vesicular packets and convoluted membranes is illustrated in Figure 9. The replicative forms and replication complexes are enclosed within vesicular packets, whereas nascent ssRNA is externally located. In addition, a variety of cellular proteins associates with the 50 -UTR, 30 -UTR, and the RNA polymerase; they probably facilitate assembly of the replication complex and trafficking of the viral RNA/polymerase into the appropriate cellular compartment for replication. Assembly, Maturation, and Release of Virions The sequence of molecular events during virion morphogenesis is not completely understood. On completion of post-translational processing, the prM and E proteins rapidly form heterodimeric complexes; the
Tick-Borne Encephalitis Viruses
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3⬘ Figure 8 Representation of the assembly of the membrane-anchored polymerase complex. The conformations of 30 LSH and 50 Y-shaped structure are shown. This stage probably precedes the cyclization of the virus genome mediated by direct interaction between the inverted complementary 50 - and 30 -cyclization sequences (thick lines); this is followed by the initiation of minus-strand RNA synthesis. Adapted from Westaway EG, Mackenzie JM, and Khromykh AA (2002) Replication and gene function in Kunjin virus. Current Topics in Microbiology and Immunology 267: 323–351.
chaperone-like function of the prM protein is essential for correct folding of E protein. Heterodimers are rapidly assembled into immature particles or, in the absence of capsid, into virus-like particles that also accumulate during infection. Virion packaging is coupled with RNA replication; only actively replicating RNA is encapsidated. The orientation of structural proteins in the lipid membrane suggests that assembly is mediated by capsid budding through the endoplasmic reticulum. However, assembled virus capsids have never been visualized, neither have budding particles nor specific release mechanisms been identified. Assembly of structural proteins in the viral lipid membrane initially results in the formation of immature (intracellular) virions that are noninfectious, resistant to acidic pH, and unable to be structurally rearranged prior to fusion. Image reconstruction has defined the different organization of E/prM proteins compared with E/M proteins on the surface of immature and mature virions, respectively (Figure 4). Sixty projections were identified on the surface of immature virions making them look larger (60 nm) in comparison with smooth 50 nm mature virus particles. Each spike is formed by three heterodimeric E/M molecules. In immature particles the E protein
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points away from the membrane with the fusion peptide at its extremity. Virion maturation is accompanied by cleavage of prM to M, mediated by the cellular protease furin in the trans-Golgi network at acidic pH, during the transportation of virions to the cell surface. PrM-to-M cleavage results in the dissociation of E/M trimeric spikes with the formation of E/E homodimers, probably following shift of the stem-transmembrane domain relative to the ectodomain. In mature particles, the E ectodomain lies on the virus membrane, with the fusion peptide embedded in the cavity between domains I and III of the neighboring dimer. Formation of the herringbone building unit and release of the glycosylated pre-peptide then follows. Eventually, trans-Golgi-derived vesicles packed with mature virions follow the host secretory pathway, fuse with the plasma membrane, and release mature virions.
Pathogenesis of TBEV Incidence of Tick-Borne Encephalitis Classical tick-borne encephalitis (TBE) is contracted by humans when they are fed upon by infected Ixodes spp. ticks. Forested areas across the Northern Hemisphere, with thick moist undergrowth and abundance of small wild animals, provide ideal habitats for ticks and for TBEV. Long-term survival of the virus occurs mainly in ticks, which remain infected throughout their 2–5-year life cycle. Efficient virus transmission between infected nymphs and noninfected larvae occurs on the forest animals when these ticks co-feed. There is no requirement for viremia, since the virus can be transmitted to the larvae in animals that are not susceptible to the virus. Moreover, nonviremic transmission between the ticks can occur even on immune animals. Cells that migrate between the skin surface and local lymph nodes (e.g., dendritic cells) become infected and are then imbibed by the feeding noninfected larvae. Nonviremic transmission provides an efficient mechanism for long-term virus survival in the absence of overt disease. At the same time, susceptible animals such as sheep, goats, horses, pigs, dogs, grouse, and even humans may inadvertently become infected by ticks either in the forests or by ticks carried away from the forests. Under these circumstances the vertebrate host may become viremic and the virus may then be transmitted to a noninfected tick that subsequently transmits the virus to another vertebrate host when the infected tick feeds for a second time. Such situations arise on the sheep-rearing moorlands in the British Isles, Norway, Spain, Turkey, Greece, and other parts of Europe. In some situations sheep/goat and grouse populations may be severely affected. The incidence of human TBE varies from year to year in different areas. The Urals and Siberia annually
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Protein synthesis
CM/PC Polyprotein processing
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1. Naked vRNA 2. Formation of viral core 3. Immature virions
TX100 sensitive membrane TX100 resistant membrane
4. Processing of prM in Golgi 5. Mature virus in endosomes 6. Release of virus by fusion with the plasma membrane
Figure 9 Proposed model of flavivirus replication in connection with cellular membranous structures. Translation of viral proteins is associated with CMs whereas replication is carried out within VPs. The dsRNA of RF in association with proteins of the polymerase complex (RC) is anchored to the internal leaflet of VP membrane whereas newly synthesized plus ssRNA is released from the RC. Reproduced with permission from Uchil PD and Satchidanandam V (2003) Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. Journal of Biological Chemistry 278: 24388–24398.
record the highest number of hospitalized cases. These numbers have risen from 700 to 1200 cases per year in the 1950s and 1960s to greater than 11 000 per year in the 1990s, following Perestroika and initial breakdown of the infrastructure. The incidence in Europe is lower but nonetheless significant, with about 3000 cases per year. Although it can affect people of all ages, the highest incidence occurs among the most active groups, that is, 17–40 year olds. The incidence of clinical disease in endemic regions is dependent on the frequency of forest visits, the density of ticks in different years, the concentration of virus in ticks, and the virulence of circulating strains. Taking all these factors into account, it was estimated that one clinical case must occur for every 100 people bitten by ticks, and this correlates with the observation that in regions where more than 60% of ticks carry virus, about 1.4% of people develop TBE after being bitten. Variety of Clinical Manifestations TBEV is recognized as a dangerous human pathogen, causing a variety of clinical manifestations, although asymptomatic infections constitute about 70–95% of all TBEV infections. Symptoms include (1) mild or moderate
fever with complete recovery of patients (about 90% of all clinical manifestations), (2) subacute encephalitis with nearly complete recovery or residual symptoms that may or may not disappear with time, (3) severe encephalitis associated with irreversible damage to the central nervous system (CNS), resulting in disability or death (about 4–8%), and (4) slow (months to decades) progressive or chronic encephalitis (1–2%). Initially in the 1940–50s it was believed that the same virus produced encephalitis over the whole of Europe and Asia. However, with the improvement of serological diagnosis and the advent of phylogenetic analysis, three subtypes of TBEV were identified. Human infections with far-Eastern TBEV subtype viruses produce the most severe form of CNS disorder, with a tendency for the patient to develop focal meningoencephalitis or polyencephalitis, and case–fatality rates between 20% and 40%. Siberian strains are often associated with high prevalence of the nonparalytic febrile form, with case–fatality rates rarely exceeding 6–8%. There is a tendency for some patients to develop chronic TBE, predominantly in association with Siberian TBEV strains. The disease produced by West European TBEV strains is biphasic, with fever during the first phase and neurological disorders of differing severity during the
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second phase, which occurs in 20–30% of patients, with a case–fatality rate of 1–2%. While Louping ill virus and the related Spanish, and Turkish/Greek viruses produce fatal encephalitis in animals, they are only rarely associated with disease in humans, probably primarily because of the lower (in comparison with TBEV) rates of human exposure to infected ticks. Biphasic milk fever is an unusual form of TBE originally observed in western Russia mainly associated with the consumption of goat’s milk. West European strains of TBEV were isolated from unpasteurized goat’s milk and from patients. Later, similar outbreaks of biphasic milk fever were reported in central Europe and Siberia. The apparent difference in clinical manifestations of TBE contracted by tick-bite or by the alimentary route may be explained by differences in the type of immunological response. Human Disease Produced by Other Tick-Borne Flaviviruses POWV also produces encephalitic infections in humans although not on an epidemic scale. POWV circulates in Russia, the USA, and Canada, where it may cause human encephalitis with a high incidence of neurological sequelae and up to a 60% case–fatality rate. In North America POWV has diverged to produce a closely related deer tick virus (DTV) that has a predilection for different rodent species. Langat virus has been isolated in Malaysia and Thailand; there are no registered cases of human disease associated with this virus. However, when Langat virus was used as a live, attenuated vaccine in human trials in Russia, one in 10 000 patients developed encephalitis. Three tick-borne flaviviruses cause hemorrhagic disease rather than encephalitis in humans, namely Omsk hemorrhagic fever virus, Kyasanur Forest disease virus, and Alkhurma virus. As yet there is no explanation for these differences in virulence characteristics. Epidemic foci of Omsk hemorrhagic fever virus in the Omsk and Novosibirsk regions of western Siberia usually follow epizootics in muskrats (Ondatra zibethica) from which local hunters become infected by the virus when they handle infected animals. The most marked pathological signs of the disease are focal visceral hemorrhages in the mucus of the nose, gums, uterus, and lungs. Convalescence is usually uneventful without residual effects; fatal cases have been recorded, but rarely (0.5–3%). Kyasanur Forest disease virus was first recognized in 1957 in India where it caused hemorrhagic disease among monkeys and humans, frequently with fatal outcome. It is believed that perturbation of regions of the forest for land development led to increased exposure of monkeys and humans to ticks carrying the virus. Subsequently, it has become evident that this virus circulates throughout western India and this may explain the close antigenic and genetic link with Alkhurma virus, which was isolated
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from fatal human cases of hemorrhagic fever in Saudi Arabia. Recent evidence has demonstrated the presence of Alkhurma virus in ticks associated with camels. Whether or not this or a closely related virus circulates in Africa remains to be determined.
Prevention and Control of TBE Vaccination is the most efficient method available for preventing TBE in enzootic regions. Currently two inactivated vaccines are commercially available. The European vaccine has significantly reduced the annual incidence of TBE in Austria and Germany. A similar vaccine, based on a far-Eastern strain of TBEV, has been used successfully in Russia to immunize at-risk laboratory personnel. Other preventive measures routinely employed in TBE endemic areas are (1) education of residents in methods of avoiding tick-bites (when visiting tick-infested areas, wearing appropriate clothing, regularly inspect for feeding ticks, report tick-bite to medical authorities); (2) treat cats and dogs with acaricides; (3) clear thick, moist vegetation areas from around houses; and (4) spray acaricides in forested areas, close to habitation. Human trials of a live, attenuated vaccine for Langat virus produced an unacceptably high incidence of TBE (1/10 000). However, these trials demonstrated the higher protection efficiency of live, attenuated vaccines in comparison with inactivated vaccines. Currently safer strategies to produce live, attenuated vaccine are being developed such as (1) RNA- or DNA-based vaccines; (2) engineering TBEV mutants with multiple attenuating mutations or large deletions within their genomes, resulting in the loss of neuroinvasiveness; and (3) engineering chimeric yellow fever virus vaccine containing substituted TBEV E and M proteins. Currently there are no safe, effective antivirals for TBEV infections, but there is promising progress in tests with small-interfering RNAs (siRNAs). These short molecules, 21 nt long, bind to homologous regions of viral mRNA, thus interfering with the replication cycle. Another antiviral strategy with which rapid progress is being made is based on the design of molecules that can target specific regions within viral replicative enzymes. If the twentieth century was significant for its development of effective vaccines, the twenty-first century might be recognized for its development of effective antivirals. See also: Japanese Encephalitis Virus; West Nile Virus.
Further Reading Aberle JH, Aberle SW, Kofler RM, and Mandl CW (2005) Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus. Journal of Virology 79: 15107–15113.
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Anderson R (2003) Manipulation of cell surface macromolecules by flaviviruses. Advances in Virus Research 59: 229–274. Bressanelli S, Stiasny K, Allison SL, et al. (2004) Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO Journal 23: 728–738. Gelpi E, Preusser M, Garzuly F, Holzmann H, Heinz FX, and Budka H (2005) Visualization of Central European tick-borne encephalitis infection in fatal human cases. Journal of Neuropathology and Experimental Neurology 64: 506–512. Gritsun TS, Lashkevich VA, and Gould EA (2003) Tick-borne encephalitis. Antiviral Research 57: 129–146. Gritsun TS, Nuttall PA, and Gould EA (2003) Tick-borne flaviviruses. Advances in Virus Research 61: 317–371. Gritsun TS, Tuplin AK, and Gould EA (2006) Origin, evolution and function of flavivirus RNA in untranslated and coding regions: Implications for virus transmission. In: Kalitzky M and Borowski P (eds.) Flaviviridae: Pathogenesis, Molecular Biology and Genetics, pp. 47–99. Norwich, UK: Horizon Scientific Press. Heinz FX and Allison SL (2003) Flavivirus structure and membrane fusion. Advances in Virus Research 59: 63–97.
Heinz FX, Stiasny K, and Allison SL (2004) The entry machinery of flaviviruses. Archives of Virology Supplement 18: 133–137. Lindenbach BD and Rice CM (2001) Flaviviridae: The viruses and their replication. In: Knipe DM and Howley PM (eds.) Fields Virology, 4th edn., vol. 1, pp. 991–1042. London: Lippincott Williams and Wilkins. Markoff L (2003) 50 - and 30 -noncoding regions in flavivirus RNA. Advances in Virus Research 59: 177–228. Mukhopadhyay S, Kuhn RJ, and Rossmann MG (2005) A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3: 13–22. Rey FA, Heinz FX, Mandl C, Kunz C, and Harrison SC (1995) The envelope glycoprotein from tick-borne encephalitis virus at 2 A˚ resolution. Nature 375: 291–398. Uchil PD and Sachidanandam V (2003) Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. Journal of Biological Chemistry 278: 24388–24398. Westaway EG, Mackenzie JM, and Khromykh AA (2003) Kunjin RNA replication and applications of Kunjin replicons. Advances in Virus Research 59: 99–140.
Tobacco Mosaic Virus M H V Van Regenmortel, CNRS, Illkirch, France ã 2008 Elsevier Ltd. All rights reserved.
The Beginnings of Virology Tobacco mosaic virus occupies a unique place in the history of virology and was in the forefront of virus research since the end of the nineteenth century. It was the German Adolf Mayer, working in the Netherlands, who in 1882 first described an important disease of tobacco which he called tobacco mosaic disease. He showed that the disease was infectious and could be transmitted to healthy tobacco plants by inoculation with capillary glass tubes containing sap from diseased plants. Although Mayer could not isolate a germ as the cause of the disease, he did not question the then prevailing view that all infectious diseases were caused by microbes and he remained convinced that he was dealing with a bacterial disease. About the same time, in St. Petersburg, Dmitri Ivanovsky was studying the same disease and he reported in 1892 that when sap from a diseased tobacco plant was passed through a bacteria-retaining Chamberland filter, the filtrate remained infectious and could be used to infect healthy tobacco plants. Ivanovsky was the first person to show that the agent causing the tobacco mosaic disease passed through a sterilizing filter and this gave rise to the subsequent characterization of viruses as filterable agents. A virology conference was held in 1992 in St. Petersburg to celebrate the centenary of this discovery. Although Ivanovsky is often considered one of the fathers of
virology, the significance of his work for the development of virology remains somewhat controversial because all his publications show that he did not really grasp the significance of his filtration experiments. He remained convinced that he was dealing with either a small bacterium or with bacterial spores and never appreciated that he had discovered a new type of infectious agent. Following in the footsteps of Mayer, Beijerinck in Delft, Holland, again showed in 1898 that sap from tobacco plants infected with the mosaic disease was still infectious after filtration through porcelain filters. He also demonstrated that the causative agent was able to diffuse through several millimeters of an agar gel and he concluded that the infection was not caused by a microbe. Beijerinck called the agent causing the tobacco mosaic disease a contagium vivum fluidum (a contagious living liquid), in opposition to a contagium vivum fixum. In those days the term contagium was used to refer to any contagious, disease-causing agent, while the term fixum meant that the agent was a solid particle or a cellular microbe. On the basis of his filtration and agar diffusion experiments, Beyerinck was convinced that the agent causing tobacco mosaic was neither a microbe nor a small particle or corpuscle (meaning a small body or particle from the Latin corpus for body). He proposed instead that the disease-causing agent,which he called a virus, was a living liquid containing a dissolved, nonparticular and noncorpuscular entity.
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Theiler’s murine encephalomyelitis virus. Journal of General Virology 77: 2701–2711. Trottier M, Wang W, and Lipton HL (2001) High numbers of viral RNA copies in the central nervous system during persistent infection with Theiler’s virus. Journal of Virology 75: 7420–7428. Yauch RL, Palma JP, Yahikozawa H, Chang-Sung K, and Kim BS (1998) Role of individual T-cell epitopes of Theiler’s virus in the pathogenesis of demyelination correlates with the ability to induce a Th1 response. Journal of Virology 72: 6169–6174.
Tick-Borne Encephalitis Viruses T S Gritsun and E A Gould, University of Reading, Reading, UK ã 2008 Elsevier Ltd. All rights reserved.
Taxonomy, Nomenclature, and Phylogenetic Relationships ‘Tick-borne encephalitis antigenic complex’ was the original term for viruses now classified as the mammalian tick-borne flaviviruses. Together with the seabirdassociated tick-borne flaviviruses they comprise one ecological group in the genus Flavivirus, family Flaviviridae. The genus contains two other groups, namely, the mosquito-borne flaviviruses and the flaviviruses with no known arthropod vectors. Other flaviviruses are referred to as nonclassified flaviviruses. The prototype TBEV is a human and animal pathogen. The earliest TBEV isolate, known as Russian Spring and Summer Encephalitis virus, was isolated in 1937 in far-East Asia. Currently, the TBEV (currently classified as virus species in the group of mammalian tick-borne flaviviruses) is subdivided into three subtypes, far-Eastern, Siberian, and West European reflecting their antigenic, phylogenetic, and geographic relationships. Other antigenically related but distinct tickborne flaviviruses include Omsk hemorrhagic fever virus, Powassan virus (POWV), Langat virus, Kyasanur Forest disease virus, Alkhurma virus, Louping ill virus, Spanish sheep encephalomyelitis virus, Turkish sheep encephalomyelitis virus, and Greek goat encephalomyelitis virus. Each of them may cause encephalitis and/or hemorrhagic disease in humans, farmed, domestic, and wild animals. Other related mammalian tick-borne flaviviruses, not known to cause human or animal disease, include Royal Farm virus, Karshi virus, Gadgets Gully virus, and Kadam virus. The viruses associated primarily with seabirds and their ticks are Saumarez Reef virus, Meaban virus, and Tyuleniy (Three Arch) virus. They are not recognized pathogens but they show interesting geographic dispersion that reflects the flight patterns of the birds with which they are associated. The phylogenetic relationships between the TBEVs are illustrated in Figure 1.
Virions Infectious (mature) virions are spherical particles (50 nm) with a relatively smooth surface and no distinct projections. They have an electron-dense core (30 nm) surrounded by a lipid membrane. The core consists of positive-polarity genomic RNA (11 kbp) and capsid protein C (12K). The lipid membrane incorporates an envelope glycoprotein (E, 53K) and a membrane glycoprotein (M, 8K). The immature (intracellular) virions contain a precursor membrane protein (prM, 18K), the cleavage of which occurs in the secretory pathway during egress of virions from infected cells. Virions sediment at about 200S and their buoyant density is 1.19 g ml–1, although there is significant heterogeneity among them. Electron micrographs frequently revealed virions in association with cellular membranes, probably explaining this heterogeneity. They are most stable at pH 8.0, although TBEV virions remain infectious in normal human gastric juice at pH 1.49–1.80. As with all enveloped viruses, infectivity of TBEVs decays rapidly at temperatures above 40 C. Most flaviviruses agglutinate erythrocytes but a few nonagglutinating strains of TBEVs have been described. Most of the physical characteristics of the TBEVs were established using virus isolated from mammalian cells. However, after adaptation to ticks, they become less cytopathic for cultured cells and laboratory animals. These virions do not move toward the cathode in rocket immunoelectrophoresis and they have reduced hemagglutinating activity. These phenotypic characteristics may be reversed following re-adaptation to mammalian cells and host-selection of virions with a shifted net surface charge due to a single amino acid substitution in the E glycoprotein. The E glycoprotein mediates virus binding to cellular receptors and thereby directly affects virus host range, virulence, and immunological properties by inducing
46
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FETBE WTBE
88 95
10% divergence
TSE SSE Ireland MA54 LI/I Wales 100 LI/31 SB/526 100 Norway LI/NOR LI/917 LI/261 100 PEN3 PEN6 Ireland IRE3 LI/369 British LI/G Isles INV14 LI/K THO1 99 THO2 97 INV6 INV1 Southwest LI/A 92 DEV4 England
Figure 1 Maximum likelihood phylogenetic tree of the E gene from 24 tick-borne flaviviruses. Branch lengths are drawn to scale and all nodes supported by more than 75% bootstrap support are indicated. The tree is rooted with the sequence from FETBE virus, Sofjin (RSSE) strain. The three main populations of virus in the British Isles are indicated, along with those viruses secondarily introduced into Ireland and Norway, and the viruses found in the south-west of England. Reproduced from McGuire K, Holmes EC, Gao GF, Reid HW, and Gould EA (1998) Tracing the origins of Louping ill virus by molecular phylogenetic analysis. Journal of General Virology 79: 981–988.
protective antibodies. X-ray crystallography has revealed the E-protein ectodomain (N-terminal 395 amino acids) as homodimers folded in a ‘head-to-tail’ manner (Figure 2(a)) and orientated parallel to the membrane surface. It contains three structural domains, each based on b-sheets: the central domain I, the dimerization (fusion) domain II, and receptor domain III (dI, dII, and dIII in Figure 2). The C-terminal 101 residues of the E protein form a stemanchor region consisting of two stem a-helices and two transmembrane a-helices that anchor the E protein into the lipid bilayer (Figure 3). Domain II contains a hydrophobic fusion peptide consisting of 13 residues that is highly conserved between all flaviviruses. It is located on the tip of domain II and plays a central role in fusion of the virion membrane to cellular endosomal membranes resulting in release of virion RNA into the cytoplasm (Figure 2). A three-dimensional image reconstruction of the virion surface reveals a protein shell composed of 90 E-dimers organized into a ‘herringbone’ configuration; three quasiparallel E-dimer molecules make up the main structural asymmetric unit of the shell (Figures 4(b) and 4(c)). The fivefold symmetry axes that appear as holes are generated by appropriate positioning of five domain IIIs and their lateral surface is accessible to cellular receptors and neutralizing antibodies. The M protein protrudes through holes formed between dimerization domains of E molecules. The transmembrane regions of the M and E proteins traverse the external but not the internal layer of the lipid membrane and there is no direct contact with the nucleocapsid (Figure 3). In contrast with those of many other enveloped viruses, the flavivirus capsid is poorly organized and appears to be
positioned randomly in the core. In solution, the capsid protein appears as dimer molecules with four helices (Figure 5). Two C-terminal helices fold into the positively charged interface interacting with viral RNA; the residues on the opposite side of these helices are hydrophobic and support dimerization. Two of the internal a-helices form capsid-dimers, are hydrophobic, and are required for contact with the virion membrane.
TBEV Life Cycle Virus Entry into the Cells Cellular receptors for tick-borne flaviviruses The virus life cycle commences with the attachment of virions to specific receptors on clathrin-coated pits on the cell surface. Flaviviruses infect a wide variety of primary and continuous cells, derived from mammalian, avian, and arthropod tissues. It is not yet clear if the different flaviviruses use similar or different cell surface receptors. Cellular heparan sulfate molecules are involved in TBEV attachment to cells. Nevertheless, cells that lack heparan sulfate are still sensitive to virus infection. For TBEV, a number of different proteins have been associated with cell receptor activity, including the highaffinity laminin receptor and a1b3-integrin that also recognizes laminin as a natural ligand. The E protein mediates attachment and entry of virions into cells
The immunoglobulin-like folding of the E-protein domain III implies that it is involved in cell attachment,
Tick-Borne Encephalitis Viruses
dIII
CHO
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dl/dIll linker dl top E0F0 sheet Loop
47
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Figure 2 Three-dimensional analysis of TBEV envelope (E) glycoprotein (a) at neutral pH (in mature virion) and (b) acidic pH (postfusion conformation). The view from above and lateral view correspond to the top and bottom rows. Protein domains dI, dII, and dIII are colored in red, yellow, and blue, respectively. Ribbon diagram corresponds to the lateral view of the E protein. Reproduced from Bressanelli S, Stiasny K, Allison SL, et al. (2004) Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO Journal 23: 728–738.
Domain II Domain I
Domain I
4
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-12 1 -1
A4
A4
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Figure 3 Schematic presentation of flavivirus structural glycoproteins E and M on the membrane surface. The coloring of the domains I, II, and III is as on Figure 2. Two a-helices of the stem-anchor region and two a-helices of the transmembrane domain of the E protein are depicted as blue- and colored cylinders. One a-helix of the stem region and two a-helices of the transmembrane domains of the M protein are depicted as orange-colored cylinders. Reproduced from Mukhopadhyay S, Kuhn RJ, and Rossmann MG (2005) A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3: 13–22, with permission from Macmillan Magazines Limited.
mediated by high-affinity cellular receptors. The receptors on the surface of vertebrate and invertebrate cells are different and probably recognized by different residues on domain III. However, domains I and II on the surface of the E protein also appear to play a role in virus–cell interactions. Domain I carries N-linked carbohydrates, which are recognized by cell surface lectins. Domain II is enriched with patches of basic positively charged surface residues that have been shown to mediate virion attachment to the cellular, negatively charged, heparan sulfate; domains I and III might also contribute to this type of interaction. Virus attachment to the cell receptors initiates receptormediated endocytosis, followed by fusion of virion and
endosomal membranes (Figures 4(c)–4(e)). Exposure of the attached virus to the endosomal acid pH converts the herringbone configuration into a fusogenic structure resulting in particle expansion and eventually in the re-assembly of the E dimers into vertical trimers (Figures 2 and 4). It is believed that during this process domains I and II flex relative to each other, allowing domain II and the stem region to move toward the endosomal membrane facilitating trimerization of domain II; the fusion tripeptide is embedded into the target membrane followed by formation of a pre-fusion intermediate (Figure 6). Trimerization spreads from fusion peptides to domain I; meanwhile domain III rotates relative to domain II pushing the stem region back toward the fusion peptide. This refolding
48
Tick-Borne Encephalitis Viruses
Maturation
(a)
(b)
5
5
3
3
3
(c)
(d)
5
3
3
3
(e)
Figure 4 Different conformations of E protein during the transition from immature (a) into the mature (b) particles and during pH-dependent fusion of mature particles (c), through the putative intermediates T ¼ 3 (d and e). Domains I, II, and III are depicted with the same colors as those on the Figure 2. Reproduced from Ma L, Jones CT, Groesch TD, Kuhn RJ, and Post CB (2004) Solution structure of Dengue virus capsid protein reveals another fold. Proceedings of the National Academy of Sciences, USA 101: 3414–3419, with permission from National Academy of Sciences USA.
of the distal lipid outlets and formation of a fusion pore. In the final low-pH conformation, domains I and III appear to move to the tip of the vertical trimer whereas the fusion peptide becomes embedded in the membrane eventually being juxtaposed to the transmembrane domains (Figures 2(b) and 6). Five trimers form the fusion pore; the stem region appears to play an essential role in promoting and stabilizing trimer assembly. Although the E-protein domains shift and rotate during exposure to acidic pH, each retains the neutral-pH conformation, the essential feature of class II fusion molecules that do not require major molecular rearrangements at acidic pH.
Membrane
Hyd
+
t rophobic clef
+
+
− − −
−
−
−
− −
+
+
+
−
− −
vRNA
−
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−
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Strategy of the TBEV Genome −
Figure 5 Model for the molecular interactions of flavivirus capsid dimers with genome RNA and virion membrane. Reproduced from Ma L, Jones CT, Groesch TD, Kuhn RJ, and Post CB (2004) Solution structure of Dengue virus capsid protein reveals another fold. Proceedings of the National Academy of Sciences, USA 101: 3414–3419, with permission from National Academy of Sciences USA.
brings two membranes together, resulting in the formation of the ‘hemifusion stalk’ with only the proximal lipid leaflets fused. The ‘zipping’ up of stems followed by the migration of transmembrane domains eventually leads to fusion
Translation and processing of virus proteins After uncoating, the RNA is translated into a polyprotein of 3400 residues from one open reading frame (ORF) that is co-translationally translocated and anchored in the endoplasmic reticulum (Figure 7). It is then processed by cellular signalases and viral serine protease producing three structural (capsid, prM, and E) and seven nonstructural (NS1 through NS5) proteins. The gene order, protein molecular masses, and their membrane localization are shown in Figure 7. Viral protease activity is provided by the N-terminal domain (180 residues) of the NS3 protein in association with cofactor activity of the NS2B protein that probably anchors the NS3 into
Tick-Borne Encephalitis Viruses
(a)
(b)
(c)
(d)
(e)
(f)
49
Figure 6 Tentative mechanism for the fusion of viral (brown) and endosomal (green) membranes mediated by the flavivirus E protein following interaction with cellular receptor (gray) (a). Acidic pH triggers the movement of domain II (with fusion peptide on the top) toward the endosomal membrane (b) with its subsequent trimerization and insertion into the endosomal membrane (c). Trimerization spread toward domains I and III (d) causing the C-terminal part of E protein to fold backwards toward the fusion peptide. The trimerization between stem-anchor and domain II (e) results in partial fusion (e) and eventually in the formation of a fusion pore (f). Reproduced with permission from Modis Y, Ogata S, Clements D, and Harrison SC (2005) Variable surface epitopes in the crystal structure of Dengue virus type 3 envelope glycoprotein. Journal of Virology 79: 1223–1231.
the membrane. The ORF is flanked by 50 - and 30 -untranslated regions (UTRs; 130 and 700 bp, respectively) that contain signals essential to initiate translation and replication. The 50 -UTR preceding the ORF is capped by m7GpppAmpN2 and initiates virus translation in vitro although an interactive effect from the 30 -UTR has also been demonstrated. Computer-assisted prediction has demonstrated folding of the 50 -UTR into a Y-shaped structure, conserved between all flaviviruses; mutations in this structure appear to impact on virus translation and replication. The translation of flavivirus proteins is probably carried out in specialized smooth membrane structures, called convoluted membranes, derived from proliferation of the rough endoplasmic reticulum in response to flavivirus infections. Replication of viral RNA
The initiation of viral replication (synthesis of negative strand on positive strand from the 30 -UTR) requires direct interaction between the 50 - and 30 -UTRs resulting in the formation of a double-stranded RNA (dsRNA) stem and circularization of the virus genome. A terminal 30 long stable hairpin (30 LSH) structure has been revealed in the 30 -UTR that, together with the 50 -Y-shaped-structure and dsRNA circularization stem, forms a complex promoter required to initiate virus RNA replication. Additional predicted conserved secondary RNA structures in the 30 -UTR might function as replication enhancers,
essential for efficient RNA synthesis and virus transmission. The synthesis of genome-size dsRNA (replicative form, RF) accomplishes the first stage of viral replication. Flavivirus RNA replication is semiconservative and asymmetric, with an average of one nascent positivesense single-stranded RNA (ssRNA) molecule (genomic) per negative-sense template, with no free minus-strands and with 10–100 times excess of positive-strand relative to negative-strand RNA synthesis. The replicative intermediate presents a partially double-stranded RNA with nascent, and displaced, plus-sense ssRNA molecules undergoing elongation. Viral RNA capping of the 50 -UTR occurs on the displaced plus-strand RNA; the trifunctional NS3 and NS5 proteins provide nucleoside triphosphatase and guanylyl/methyltransferase activities, respectively. Formation of the replication complex involves nonstructural proteins (Figure 8) most of which are bi- and even trifunctional. The C-terminal domain of NS5 protein acts as a viral RNA polymerase and the C-terminal domain of NS3 as a helicase; both NS5 and NS3 proteins interact with 30 -LSH to initiate virus replication. The functions of other nonstructural proteins are less precisely identified. The NS1 glycoprotein forms membrane-associated hexamers that dissociate into dimers. This protein is translocated in the endoplasmic reticulum and secreted together with virions in mammalian but not in mosquito cells; its anchoring into the membranes is mediated by
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Tick-Borne Encephalitis Viruses
3⬘-UTR
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Polyprotein 3400 amino acids vs C
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(c) Figure 7 Genome strategy of TBEV. (a) Genomic RNA is presented as a solid line on the top; the 50 - and 30 -UTR are depicted in the predicted conformations according to Gritsun TS, Venugopal K, Zanotto PM, et al. (1997) Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: Analysis and significance of the 50 - and 30 -UTRs. Virus Research 49: 27–39. (b) Translation and co-translational processing of flavivirus polyprotein. The flavivirus polyprotein is depicted as a bar, with specified individual proteins and their molecular masses (numbers below the bar). (c) Membrane topology of viral proteins in relation to the lumen of the ER and cytoplasm after the completion of co-translational processing and translocation. Adapted from Westaway EG, Mackenzie JM, and Khromykh AA (2003) Kunjin RNA replication and applications of Kunjin replicons. Advances in Virus Research 59: 99–140. Transmembrane domains are shown as cylinders. Glycosylation is indicated as (*). The polyprotein is processed by ER signalases (s) and viral-specific protease NS2B-NS3 (V). The cleavage of M from prM is carried out by the Golgi protease, furin (f) and cleavage between NS1 and NS2A is by an unknown (?) ER protease.
glycosyl-phosphatidylinositol. The NS1 protein induces the production of protective antibodies that also participate in complement-mediated lysis of infected cells, implying a role in immunopathology. The NS2A protein is found in association with the NS3 helicase domain, the NS5 protein, and the 30 -LSH. It may be involved in trafficking of viral RNA between the translation, replication, and virion assembly sites. The NS2B protein is a membrane-anchored cofactor of the serine proteinase, NS3. It also has membranepermeability modulating activity possibly related to replication. The hydrophobic NS4A protein in conjunction with the NS1 protein probably anchors the polymerase complex to cell membranes and is involved in virus-induced membrane rearrangements that compartmentalize virus translation, replication, and assembly processes (Figure 8). Replication of flavivirus RNA is associated with host membrane 75–100 nm vesicular packets enclosed in the second outer membrane and connected to convoluted membranes, the sites of virus translation. The vesicular
packets proliferate in the perinuclear region in response to virus infection and are probably derived from the transGolgi network. The architecture of the replication complex in relation to vesicular packets and convoluted membranes is illustrated in Figure 9. The replicative forms and replication complexes are enclosed within vesicular packets, whereas nascent ssRNA is externally located. In addition, a variety of cellular proteins associates with the 50 -UTR, 30 -UTR, and the RNA polymerase; they probably facilitate assembly of the replication complex and trafficking of the viral RNA/polymerase into the appropriate cellular compartment for replication. Assembly, Maturation, and Release of Virions The sequence of molecular events during virion morphogenesis is not completely understood. On completion of post-translational processing, the prM and E proteins rapidly form heterodimeric complexes; the
Tick-Borne Encephalitis Viruses
Lumen of ER
Cytosol
5⬘Y-shaped structure
?
NS5 NS4A NS1
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5⬘ 3⬘cyclization sequences
5⬘cyclization sequences
3⬘LSH
3⬘ Figure 8 Representation of the assembly of the membrane-anchored polymerase complex. The conformations of 30 LSH and 50 Y-shaped structure are shown. This stage probably precedes the cyclization of the virus genome mediated by direct interaction between the inverted complementary 50 - and 30 -cyclization sequences (thick lines); this is followed by the initiation of minus-strand RNA synthesis. Adapted from Westaway EG, Mackenzie JM, and Khromykh AA (2002) Replication and gene function in Kunjin virus. Current Topics in Microbiology and Immunology 267: 323–351.
chaperone-like function of the prM protein is essential for correct folding of E protein. Heterodimers are rapidly assembled into immature particles or, in the absence of capsid, into virus-like particles that also accumulate during infection. Virion packaging is coupled with RNA replication; only actively replicating RNA is encapsidated. The orientation of structural proteins in the lipid membrane suggests that assembly is mediated by capsid budding through the endoplasmic reticulum. However, assembled virus capsids have never been visualized, neither have budding particles nor specific release mechanisms been identified. Assembly of structural proteins in the viral lipid membrane initially results in the formation of immature (intracellular) virions that are noninfectious, resistant to acidic pH, and unable to be structurally rearranged prior to fusion. Image reconstruction has defined the different organization of E/prM proteins compared with E/M proteins on the surface of immature and mature virions, respectively (Figure 4). Sixty projections were identified on the surface of immature virions making them look larger (60 nm) in comparison with smooth 50 nm mature virus particles. Each spike is formed by three heterodimeric E/M molecules. In immature particles the E protein
51
points away from the membrane with the fusion peptide at its extremity. Virion maturation is accompanied by cleavage of prM to M, mediated by the cellular protease furin in the trans-Golgi network at acidic pH, during the transportation of virions to the cell surface. PrM-to-M cleavage results in the dissociation of E/M trimeric spikes with the formation of E/E homodimers, probably following shift of the stem-transmembrane domain relative to the ectodomain. In mature particles, the E ectodomain lies on the virus membrane, with the fusion peptide embedded in the cavity between domains I and III of the neighboring dimer. Formation of the herringbone building unit and release of the glycosylated pre-peptide then follows. Eventually, trans-Golgi-derived vesicles packed with mature virions follow the host secretory pathway, fuse with the plasma membrane, and release mature virions.
Pathogenesis of TBEV Incidence of Tick-Borne Encephalitis Classical tick-borne encephalitis (TBE) is contracted by humans when they are fed upon by infected Ixodes spp. ticks. Forested areas across the Northern Hemisphere, with thick moist undergrowth and abundance of small wild animals, provide ideal habitats for ticks and for TBEV. Long-term survival of the virus occurs mainly in ticks, which remain infected throughout their 2–5-year life cycle. Efficient virus transmission between infected nymphs and noninfected larvae occurs on the forest animals when these ticks co-feed. There is no requirement for viremia, since the virus can be transmitted to the larvae in animals that are not susceptible to the virus. Moreover, nonviremic transmission between the ticks can occur even on immune animals. Cells that migrate between the skin surface and local lymph nodes (e.g., dendritic cells) become infected and are then imbibed by the feeding noninfected larvae. Nonviremic transmission provides an efficient mechanism for long-term virus survival in the absence of overt disease. At the same time, susceptible animals such as sheep, goats, horses, pigs, dogs, grouse, and even humans may inadvertently become infected by ticks either in the forests or by ticks carried away from the forests. Under these circumstances the vertebrate host may become viremic and the virus may then be transmitted to a noninfected tick that subsequently transmits the virus to another vertebrate host when the infected tick feeds for a second time. Such situations arise on the sheep-rearing moorlands in the British Isles, Norway, Spain, Turkey, Greece, and other parts of Europe. In some situations sheep/goat and grouse populations may be severely affected. The incidence of human TBE varies from year to year in different areas. The Urals and Siberia annually
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Protein synthesis
CM/PC Polyprotein processing
Release of vRNA into cytosol???
ER connection between VP and CM
1. Naked vRNA 2. Formation of viral core 3. Immature virions
TX100 sensitive membrane TX100 resistant membrane
4. Processing of prM in Golgi 5. Mature virus in endosomes 6. Release of virus by fusion with the plasma membrane
Figure 9 Proposed model of flavivirus replication in connection with cellular membranous structures. Translation of viral proteins is associated with CMs whereas replication is carried out within VPs. The dsRNA of RF in association with proteins of the polymerase complex (RC) is anchored to the internal leaflet of VP membrane whereas newly synthesized plus ssRNA is released from the RC. Reproduced with permission from Uchil PD and Satchidanandam V (2003) Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. Journal of Biological Chemistry 278: 24388–24398.
record the highest number of hospitalized cases. These numbers have risen from 700 to 1200 cases per year in the 1950s and 1960s to greater than 11 000 per year in the 1990s, following Perestroika and initial breakdown of the infrastructure. The incidence in Europe is lower but nonetheless significant, with about 3000 cases per year. Although it can affect people of all ages, the highest incidence occurs among the most active groups, that is, 17–40 year olds. The incidence of clinical disease in endemic regions is dependent on the frequency of forest visits, the density of ticks in different years, the concentration of virus in ticks, and the virulence of circulating strains. Taking all these factors into account, it was estimated that one clinical case must occur for every 100 people bitten by ticks, and this correlates with the observation that in regions where more than 60% of ticks carry virus, about 1.4% of people develop TBE after being bitten. Variety of Clinical Manifestations TBEV is recognized as a dangerous human pathogen, causing a variety of clinical manifestations, although asymptomatic infections constitute about 70–95% of all TBEV infections. Symptoms include (1) mild or moderate
fever with complete recovery of patients (about 90% of all clinical manifestations), (2) subacute encephalitis with nearly complete recovery or residual symptoms that may or may not disappear with time, (3) severe encephalitis associated with irreversible damage to the central nervous system (CNS), resulting in disability or death (about 4–8%), and (4) slow (months to decades) progressive or chronic encephalitis (1–2%). Initially in the 1940–50s it was believed that the same virus produced encephalitis over the whole of Europe and Asia. However, with the improvement of serological diagnosis and the advent of phylogenetic analysis, three subtypes of TBEV were identified. Human infections with far-Eastern TBEV subtype viruses produce the most severe form of CNS disorder, with a tendency for the patient to develop focal meningoencephalitis or polyencephalitis, and case–fatality rates between 20% and 40%. Siberian strains are often associated with high prevalence of the nonparalytic febrile form, with case–fatality rates rarely exceeding 6–8%. There is a tendency for some patients to develop chronic TBE, predominantly in association with Siberian TBEV strains. The disease produced by West European TBEV strains is biphasic, with fever during the first phase and neurological disorders of differing severity during the
Tick-Borne Encephalitis Viruses
second phase, which occurs in 20–30% of patients, with a case–fatality rate of 1–2%. While Louping ill virus and the related Spanish, and Turkish/Greek viruses produce fatal encephalitis in animals, they are only rarely associated with disease in humans, probably primarily because of the lower (in comparison with TBEV) rates of human exposure to infected ticks. Biphasic milk fever is an unusual form of TBE originally observed in western Russia mainly associated with the consumption of goat’s milk. West European strains of TBEV were isolated from unpasteurized goat’s milk and from patients. Later, similar outbreaks of biphasic milk fever were reported in central Europe and Siberia. The apparent difference in clinical manifestations of TBE contracted by tick-bite or by the alimentary route may be explained by differences in the type of immunological response. Human Disease Produced by Other Tick-Borne Flaviviruses POWV also produces encephalitic infections in humans although not on an epidemic scale. POWV circulates in Russia, the USA, and Canada, where it may cause human encephalitis with a high incidence of neurological sequelae and up to a 60% case–fatality rate. In North America POWV has diverged to produce a closely related deer tick virus (DTV) that has a predilection for different rodent species. Langat virus has been isolated in Malaysia and Thailand; there are no registered cases of human disease associated with this virus. However, when Langat virus was used as a live, attenuated vaccine in human trials in Russia, one in 10 000 patients developed encephalitis. Three tick-borne flaviviruses cause hemorrhagic disease rather than encephalitis in humans, namely Omsk hemorrhagic fever virus, Kyasanur Forest disease virus, and Alkhurma virus. As yet there is no explanation for these differences in virulence characteristics. Epidemic foci of Omsk hemorrhagic fever virus in the Omsk and Novosibirsk regions of western Siberia usually follow epizootics in muskrats (Ondatra zibethica) from which local hunters become infected by the virus when they handle infected animals. The most marked pathological signs of the disease are focal visceral hemorrhages in the mucus of the nose, gums, uterus, and lungs. Convalescence is usually uneventful without residual effects; fatal cases have been recorded, but rarely (0.5–3%). Kyasanur Forest disease virus was first recognized in 1957 in India where it caused hemorrhagic disease among monkeys and humans, frequently with fatal outcome. It is believed that perturbation of regions of the forest for land development led to increased exposure of monkeys and humans to ticks carrying the virus. Subsequently, it has become evident that this virus circulates throughout western India and this may explain the close antigenic and genetic link with Alkhurma virus, which was isolated
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from fatal human cases of hemorrhagic fever in Saudi Arabia. Recent evidence has demonstrated the presence of Alkhurma virus in ticks associated with camels. Whether or not this or a closely related virus circulates in Africa remains to be determined.
Prevention and Control of TBE Vaccination is the most efficient method available for preventing TBE in enzootic regions. Currently two inactivated vaccines are commercially available. The European vaccine has significantly reduced the annual incidence of TBE in Austria and Germany. A similar vaccine, based on a far-Eastern strain of TBEV, has been used successfully in Russia to immunize at-risk laboratory personnel. Other preventive measures routinely employed in TBE endemic areas are (1) education of residents in methods of avoiding tick-bites (when visiting tick-infested areas, wearing appropriate clothing, regularly inspect for feeding ticks, report tick-bite to medical authorities); (2) treat cats and dogs with acaricides; (3) clear thick, moist vegetation areas from around houses; and (4) spray acaricides in forested areas, close to habitation. Human trials of a live, attenuated vaccine for Langat virus produced an unacceptably high incidence of TBE (1/10 000). However, these trials demonstrated the higher protection efficiency of live, attenuated vaccines in comparison with inactivated vaccines. Currently safer strategies to produce live, attenuated vaccine are being developed such as (1) RNA- or DNA-based vaccines; (2) engineering TBEV mutants with multiple attenuating mutations or large deletions within their genomes, resulting in the loss of neuroinvasiveness; and (3) engineering chimeric yellow fever virus vaccine containing substituted TBEV E and M proteins. Currently there are no safe, effective antivirals for TBEV infections, but there is promising progress in tests with small-interfering RNAs (siRNAs). These short molecules, 21 nt long, bind to homologous regions of viral mRNA, thus interfering with the replication cycle. Another antiviral strategy with which rapid progress is being made is based on the design of molecules that can target specific regions within viral replicative enzymes. If the twentieth century was significant for its development of effective vaccines, the twenty-first century might be recognized for its development of effective antivirals. See also: Japanese Encephalitis Virus; West Nile Virus.
Further Reading Aberle JH, Aberle SW, Kofler RM, and Mandl CW (2005) Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus. Journal of Virology 79: 15107–15113.
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Anderson R (2003) Manipulation of cell surface macromolecules by flaviviruses. Advances in Virus Research 59: 229–274. Bressanelli S, Stiasny K, Allison SL, et al. (2004) Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. EMBO Journal 23: 728–738. Gelpi E, Preusser M, Garzuly F, Holzmann H, Heinz FX, and Budka H (2005) Visualization of Central European tick-borne encephalitis infection in fatal human cases. Journal of Neuropathology and Experimental Neurology 64: 506–512. Gritsun TS, Lashkevich VA, and Gould EA (2003) Tick-borne encephalitis. Antiviral Research 57: 129–146. Gritsun TS, Nuttall PA, and Gould EA (2003) Tick-borne flaviviruses. Advances in Virus Research 61: 317–371. Gritsun TS, Tuplin AK, and Gould EA (2006) Origin, evolution and function of flavivirus RNA in untranslated and coding regions: Implications for virus transmission. In: Kalitzky M and Borowski P (eds.) Flaviviridae: Pathogenesis, Molecular Biology and Genetics, pp. 47–99. Norwich, UK: Horizon Scientific Press. Heinz FX and Allison SL (2003) Flavivirus structure and membrane fusion. Advances in Virus Research 59: 63–97.
Heinz FX, Stiasny K, and Allison SL (2004) The entry machinery of flaviviruses. Archives of Virology Supplement 18: 133–137. Lindenbach BD and Rice CM (2001) Flaviviridae: The viruses and their replication. In: Knipe DM and Howley PM (eds.) Fields Virology, 4th edn., vol. 1, pp. 991–1042. London: Lippincott Williams and Wilkins. Markoff L (2003) 50 - and 30 -noncoding regions in flavivirus RNA. Advances in Virus Research 59: 177–228. Mukhopadhyay S, Kuhn RJ, and Rossmann MG (2005) A structural perspective of the flavivirus life cycle. Nature Reviews Microbiology 3: 13–22. Rey FA, Heinz FX, Mandl C, Kunz C, and Harrison SC (1995) The envelope glycoprotein from tick-borne encephalitis virus at 2 A˚ resolution. Nature 375: 291–398. Uchil PD and Sachidanandam V (2003) Architecture of the flaviviral replication complex. Protease, nuclease, and detergents reveal encasement within double-layered membrane compartments. Journal of Biological Chemistry 278: 24388–24398. Westaway EG, Mackenzie JM, and Khromykh AA (2003) Kunjin RNA replication and applications of Kunjin replicons. Advances in Virus Research 59: 99–140.
Tobacco Mosaic Virus M H V Van Regenmortel, CNRS, Illkirch, France ã 2008 Elsevier Ltd. All rights reserved.
The Beginnings of Virology Tobacco mosaic virus occupies a unique place in the history of virology and was in the forefront of virus research since the end of the nineteenth century. It was the German Adolf Mayer, working in the Netherlands, who in 1882 first described an important disease of tobacco which he called tobacco mosaic disease. He showed that the disease was infectious and could be transmitted to healthy tobacco plants by inoculation with capillary glass tubes containing sap from diseased plants. Although Mayer could not isolate a germ as the cause of the disease, he did not question the then prevailing view that all infectious diseases were caused by microbes and he remained convinced that he was dealing with a bacterial disease. About the same time, in St. Petersburg, Dmitri Ivanovsky was studying the same disease and he reported in 1892 that when sap from a diseased tobacco plant was passed through a bacteria-retaining Chamberland filter, the filtrate remained infectious and could be used to infect healthy tobacco plants. Ivanovsky was the first person to show that the agent causing the tobacco mosaic disease passed through a sterilizing filter and this gave rise to the subsequent characterization of viruses as filterable agents. A virology conference was held in 1992 in St. Petersburg to celebrate the centenary of this discovery. Although Ivanovsky is often considered one of the fathers of
virology, the significance of his work for the development of virology remains somewhat controversial because all his publications show that he did not really grasp the significance of his filtration experiments. He remained convinced that he was dealing with either a small bacterium or with bacterial spores and never appreciated that he had discovered a new type of infectious agent. Following in the footsteps of Mayer, Beijerinck in Delft, Holland, again showed in 1898 that sap from tobacco plants infected with the mosaic disease was still infectious after filtration through porcelain filters. He also demonstrated that the causative agent was able to diffuse through several millimeters of an agar gel and he concluded that the infection was not caused by a microbe. Beijerinck called the agent causing the tobacco mosaic disease a contagium vivum fluidum (a contagious living liquid), in opposition to a contagium vivum fixum. In those days the term contagium was used to refer to any contagious, disease-causing agent, while the term fixum meant that the agent was a solid particle or a cellular microbe. On the basis of his filtration and agar diffusion experiments, Beyerinck was convinced that the agent causing tobacco mosaic was neither a microbe nor a small particle or corpuscle (meaning a small body or particle from the Latin corpus for body). He proposed instead that the disease-causing agent,which he called a virus, was a living liquid containing a dissolved, nonparticular and noncorpuscular entity.