Genetic analysis of tick-borne encephalitis virus strains from West Siberia

Tick-borne encephalitis (TBE) virus strains were isolated in West Siberia in the forest-steppe region near the Ob river in 1981-1992. Hybridization of genome RNA of 46 TBE strains with [32P]cDNA of TBE Sofyin strain revealed essential differences in the genomes of West-Siberian and Far-Eastern Sofyin strains of TBE virus. Nucleotide sequences of 6 TBE strains (1348-1503 n.) have been determined. A 89-98% homology of Siberian TBE strains has been shown, while the similarity of the respective fragment of E gene for West Siberian and Sofyin strains was no more than 81%. No significant changes in E gene of TBE strains have been detected over a 12-year period.     

Monitoring of tick-borne encephalitis virus populations and etiological structure of morbidity over 60 years

The Siberian subtype of the virus of tick-borne encephalitis (TBE), which is predominant in Russia, constantly circulated in its eastern European regions in 1943-2003 and in the Urals and West and East Siberia in 1960-2003. This subtype is transmitted by two types of ticks: Ixodes persulcatus and I. ricinis. Changes were not found in the structure of viral populations at the peak and drop of the incidence of TBE. There was new evidence on the genetic heterogenicity of the Siberian subtype: in addition to the strains containing histidine (H) or glutamine (Q) in the position of 234 of protein E gene, there were strains having tyrosine (V). There were differences in the eastern European and Asian populations of the Siberian subtype. The strains with labeled amino acids of H and Q amounted to 87.1 and 3.2% in the eastern European population and 60 and 40% in the Asian population, respectively. The eastern European strains with labeled amino acid of H differed from the same Asian strains in the level of nucleotide replacements in the studied E gene fragment. The strains containing tyrosine in position 234 were found only in the eastern European population. Sixty-two cases of TBE were analyzed, which showed a significantly established role of a certain subtype. The Siberian and Far Eastern subtypes in the area of joint circulation were found to cause the whole spectrum of infection manifestations from unapparent to severe focal forms with a fatal outcome. There were no differences in the location of the virus and the topography of CNS morphological changes in patients who had died after infection with the Siberian or Far Eastern subtypes of the virus of TBE. The chronic forms of TBE are mainly associated with the Siberian subtype. These three subtypes (European, Far Eastern, and Siberian) may cause the disease via unpasteurized milk.     

Analysis of genetic variability of strains of tick-borne encephalitis virus by primary structure of a fragment of the membrane protein E gene

Primary structures of gene fragments of E protein (160 n.b.) have been determined for 29 tick-borne encephalitis (TBE) strains isolated from different parts of a territory. Analysis of homology of nucleotide sequences of these strains and data on 6 TBE strains published by other authors showed that they can be divided into 6 groups (genotypes) by the following gene typing criteria: strain structure within the genotypes differing by no more than 9%, differences between strains of different genotypes are at least 12%. Based on these criteria, the prototype strains of the Far Eastern antigenic variant (Sofyin), Central European antigenic variant (Neudoerfle), and Vergina strain form different genotypes 1, 2, and 6, respectively. East Siberian strain Aina and Ural Siberian strain Lesopark-II belong to the same TBE virus genotype 3; two-thirds of analyzed strains belong to this genotype. Genotype 4 is represented by one strain 178-79, and genotype 5 by strain 886-84, both isolated in East Siberia.     

Tick-borne encephalitis virus in a highly endemic area in Kemerovo (Western Siberia,Russia)

Western Siberia is the region with the highest known incidence of tick-borne encephalitis (TBE) in the world, with 40 to >80 cases/100,000 population. Few data are available on the circulation of TBE virus (TBEV) strains in the region. In the present study, a total of 468 pooled ticks (Ixodes persulcatus) collected in 7 areas around Kemerovo, Western Siberia, were tested for the presence of TBEV RNA by real-time RT-PCR. Positive tick pools were further investigated by conventional PCR and the nucleotide sequences of the partial TBEV E protein genes were compared to known nucleotide sequences of (Siberian) TBEV strains. In 4 of the 7 areas tested, TBEV RNA-positive ticks were found. Seven out of 28 tick pools were positive in real-time RT-PCR. Assuming only one tick of each pool to be positive, the overall minimal infection rate (MIR) was 1.5% (7/468), ranging from 0% up to 4% for positive regions. Molecular characterization of the E protein of 6 of the 7 positive pools exhibited a sequence variability of 1.4–2.6% in comparison to the nucleotide (nt) sequence of the Aina strain of the Siberian subtype of TBEV. The phylogenetic analysis of the nt sequences clearly indicates that two clusters of the Siberian subtype of TBEV seem to circulate simultaneously in the Kemerovo region. The pathogenicity of the respective virus variants, however, warrants further examination.     

Localization of tick-borne encephalitis virus protein E antigenic determinant recognized by antihemagglutinating monoclonal antibodies using a phage-display peptide library

Bacteriophages bearing peptides reacting with antihemagglutinating monoclonal antibodies (MAb) 10H10 to tick-borne encephalitis (TBE) virus protein E were selected from a phage-display peptide library by affinity selection and enzyme immunoassay. The library contained randomized peptides that are 6 amino acids long, fused with protein pIII and exposed on the surface of the bacteriophage. No significant homology between the sequences of selected peptides and TBE virus protein E was detected. Computer software was created to locate the conformation epitopes on the surface of protein E. Amino acids R73, C74, T76, M77, N103, C105, L107, and S112 were found to form a discontinuous epitope recognized by MAb 10H10. These amino acids are remote in the protein sequence but close in the tertiary structure and form a whole epitope located in the structural domain II of protein E. Presumably the localized amino acids bind to the cellular receptor for TBE virus.     

Characterization of tick-borne encephalitis virus from Latvia: evidence for co-circulation of three distinct subtypes.

Viruses of the tick-borne encephalitis (TBE) antigenic complex within the family Flaviviridae cause a variety of diseases, including uncomplicated febrile illness, meningoencephalitis, and hemorrhagic fever. Different domesticated animals or wildlife species often act as reservoir hosts and ixodid ticks serve as vectors. Although TBE is a serious problem in Latvia, the knowledge concerning TBE virus (TBEV) strains circulating in the country is most limited. Only two strains (Latvia-1-96 isolated from a TBE patient, and RK1424 originating from an Ixodes persulcatus tick), which belonged to the Siberian and the Far Eastern subtypes of TBEV, respectively, have previously been characterized. In the present study, we concentrated on the western and central regions of Latvia, with predominantly Ixodes ricinus ticks. Five virus strains were isolated from serum samples of patients with clinical symptoms of an acute TBE infection. Nucleotide sequences encoding the envelope (E) protein of TBEV, which were recovered from the five TBEV isolates, showed the highest level of identity to the corresponding sequences of the prototype strain Neudoerfl and other European strains of the Western TBEV subtype characterized previously. Accordingly, phylogenetic analysis placed the new Latvian isolates within the Western genetic lineage of TBEV. Taken together with earlier observations, the results proved that all three TBEV subtypes are co-circulating in Latvia and indicated that the genetic diversity of TBEV within certain geographical areas is much more complex than previously believed.     

Polytypic strains in the genofund of tick-borne encephalitis virus

Eighteen polytypic tick-borne encephalitis virus (TBEV) strains containing the fragments of E and NS1 protein genes of Siberian and Far Eastern, occasionally Siberian and European subtypes were isolated in the European and Asian parts of the tick-borne encephalitis (TBE) area. They were identified using real-time polymerase chain reaction, hybridization-fluorescence detection with genotype-specific probes, restriction fragment length polymorphism analysis, and E protein sequencing. The polytypic strains were isolated from individual Ixodes persulcatus ticks, their pools, from the blood of patients and the brain of dead patients. The isolation rates of the polytypic strains in the sympathry area of different TBEV subtypes ranged from 4.4% (the Irkutsk Region) to 15.1% (the Yaroslavl Region). In addition to 2 polytypic strains, a strain similar to the TBEV 886-84 strain was isolated. The TBEV subtypes entering into the composition of the polytypic strains show nongenetic interactions, such as neutral replication or competition. The polytypic strains are stable during passages in the cultured pig embryo kidney epithelial cells and on cloning. Mouse brain passage promotes dissociation of polytypic strains. The conditions for the formation of polytypic strains and their role in the etiology of TBE are discussed.     

Deoxyoligonucleotide probes that differentiate antigenic and pathogenetic variants of the tick-borne encephalitis virus]

Experiments on molecular hybridization were carried out using a panel of 11 deoxyoligonucleotide probes complementary to different parts of tick-borne encephalitis (TBE) virus, strain Sophyin, genome. Under study were the TBE virus strains differing by 3 criteria: (1) source of isolation (patients with acute and chronic TBE, Ixodes persulcatus and D. nuttalli ticks, small mammals); (2) serotype (eastern and Siberian Aina/1448), (3) virulence for Syrian hamsters. RNA of all the strains was hybridized with kDNA, 90% of strains with probe Sh5 complementary to protein E gene, nucleotide positions 1285-1311. The highest differentiating capacity was observed with probes P131 and Sh3 complementary to genes of proteins ns2b and M. These probes reacted with RNA of 100% of highly virulent strains of the eastern serotype and only with 20-30% of strains of the Aina/1448 serotype of lower virulence. A certain differentiating capacity was demonstrated by probes Sh2 and P10 complementary to genes of prm and C proteins: they hybridized with RNA of 80% of eastern serotype strains highly virulent for hamsters and with only 20% of Aina/1448 serotype strains of low virulence. The panel of probes used revealed no significant differences among strains in relation to their isolation source, with the exception of a strain isolated from D. nuttalli ticks which reacted only with kDNA and probe P2 complementary to nsI protein gene, but not with other probes. The TBE virus strains isolated from patients with chronic TBE were shown to represent a genetically heterogeneous group.     

Unique signature amino acid substitution in Baltic tick-borne encephalitis virus (TBEV) strains within the Siberian TBEV subtype

Tick-borne encephalitis virus (TBEV) is an arthropod-borne virus, which is transmitted to vertebrates by the bite of infected ticks. TBEV plays an important role in human morbidity in Europe and in Estonia in particular. All three known TBEV subtypes, Western (W-TBEV), Far-Eastern (FE-TBEV), and Siberian (S-TBEV), co-circulate in Estonia. In the present study, we collected ticks in the eastern part of the country where one of the TBEV vectors, Ixodes persulcatus, is prevalent. In total, 8 TBEV strains were isolated and characterized by partial sequencing of the surface E glycoprotein gene. Phylogenetic analysis showed that all 8 strains belonged to the S-TBEV subtype and clustered geographically with Baltic TBEV strains from Estonia, Latvia, and Finland. Analysis of amino acid sequences revealed a new signature amino acid, Asn, at position 175 for Baltic strains from Estonia, Latvia, Finland, and the European part of Russia, and Ala at position 313 for Siberian strains from Novosibirsk, Tomsk, and Irkutsk within the S-TBEV subtype. According to these findings, discrimination of Baltic and Siberian lineages within the S-TBEV subtype is possible. These data support geographic clustering of Baltic TBEV strains within the S-TBEV subtype in contrast to the previous postulation that TBEV strains could not be distinguished according to place and time of isolation. Both signature amino acids, 175 and 313, are located close to each other at one side of the E protein dimer molecule. Protein structure modeling showed that at position 175, the Baltic strains of S-TBEV had lost one hydrogen bond with Asp181, thus making the nearby 177–179 loop more flexible at the molecule surface. At position 313, the Siberian strains of S-TBEV had a substitution of non-polar Thr to polar Ala. Geometrical analysis of the molecular surface around amino acid 313 hinted at the presence of a cleft between this residue and a loop formed by residues 308–311, which has been suggested as a putative flavivirus receptor-binding site. This substitution may influence the binding properties of the cleft formed by signature amino acid 313 and the receptor-binding loop.     

Evolution of tick-borne encephalitis and a problem of evolution of its causative agent

The evolution of tick-borne encephalitis (TBE) is marked by the expanded nosological area, the transformation of landscapes, the formation of anthropurgic foci, the change of environmental systems, the increase of mortality rate mainly among urban dwellers, as well as pathomorphism. The evolution of natural TBE virus (TBEV) populations was studied in Eastern and Western Siberia, Middle Urals, and the European part of the nosological area. The paper first describes the types of evolutionary transformations of viral populations under the conditions of a varying environmental and epidemiological situation. These include: 1) the change of TBEV subtypes over 50-60 years; substitution of the Far-Eastern subtype for its Siberian subtype (the Sverdlovsk and Kemerovo regions); 2) the steady-state circulation of one Siberian subtype with mutanttypes being accumulated (the Vologda region); 3) co-existence of the Far-Eastern and Siberian subtypes with the common vector Ixodes persulcatus (the Yaroslavl and Irkutsk regions, etc.); 4) original mixed TBEV strains including the gene sites of proteins E and NSI of two subtypes. There is new evidence that the Siberian subtype is able to induce focal TBE forms, leading to death.     

The Siberian and Far-Eastern subtypes of tick-borne encephalitis virus registered in Russia's Asian regions: genetic and antigen characteristics of the strains

Agar gel precipitation test with cross-adsorbed immune sera was used for the antigenic differentiation of strains of tick-borne encephalitis virus (TBEV). Fifty strains of the Far East TBEV serotype and 46 strains of the Siberian (Aina) TBEV serotype were isolated from Ixodes persulcatus, which is the main vector of the above TBEV subtypes in the Asian and European parts of Russia. The fragment of the envelope protein gene was sequenced for TBEV strains. Sequences of new-group strains of the Siberian subtypes isolated from 3 patients with chronic TBE and from brain tissues of 4 deceased patients were determined. Lethal TBE outcomes were registered in Siberia (Irkutsk Region and Krasnoyarsk Territory) and in Russia's European part (Yaroslavl Region).     

Interaction of tick-borne encephalitis virus RNA with proteins

Binding of tick-borne encephalitis (TBE) virus RNA to proteins was studied by 3 methods: gel retardation, RNA-protein cross-linking under UV light, and RNA-protein blotting. Two cellular proteins with molecular weights about 30 and 22 kDa were detected in the nuclear fraction of two cell strains (PS and RH) and in blood mononuclear cells of patients with TBE. Weak interaction between the studied RNA and viral proteins can result from trace amounts of TBE virus proteins in infected cells and by lack of additional binding factors. Bacterial super-products and affinity chromatography for isolation of native glycoproteins were used to increase the amount of viral proteins. Purified E and NS1 proteins did not react with viral RNA, while incubation with recombinant nonstructural TBE virus NS3 protein led to a shift in the mobility of TBE virus RNA in gel in the absence of cross-linking under conditions of UV exposure after addition of heparin. Poor binding of nonstructural TBE virus NS3 protein is based on electrostatic binding. Oligoribonucleotide 14 nucleotide residues long corresponding to 5'-terminal of TBE virus genome did not react with NS3 protein, probably because of its small size or absence of certain sequences.     

Comparative analysis of virulence of the Siberian and Far-East subtypes of the tick-born encephalitis virus

The Siberian subtype of the tick-borne encephalitis virus (TEV) is different from the Far-East subtype by a moderate virulence observed in Siberian hamsters and by a low infection development rate (100 strains were compared). No differences were found in neuro-invasiveness. Clinical findings and experiments with monkeys denote the ability of the Siberian subtype to provoke severe forms of tick-borne encephalitis (TBE). The inflammation-and-degenerative changes were localized in the brain cortex, subcortical ganglions, nuclei of medulla oblongata, in the cortex and nuclei of the cerebellum as well as in the anterior horns of the spinal cord. 18 disease cases triggered by the Siberian TEV subtypes in residents of the Western and Eastern Siberia and of Central Russia (Yaroslavl Region), including 7 acute TBE cases (5 lethal outcomes), as well as 11 chronic TBE cases are analyzed. The viral RNA was found in the cortex, medulla oblongata, horn and in the cervical part of the spinal cord of those diseased of acute TBE. Sequences of genotyped strains were presented to Gen Bank, NCBI (AY363846-AY363865).     

Biological and molecular genetic characteristics of a Far-Eastern tick-borne encephalitis virus population and its pathogenetic implication

The authors have got an idea of the structure of the tick-borne encephalitis (TBE) virus population forming in the human body after tick bite in the south of the Far East. A hundred and forty-five antigen-positive samples were virologically studied in enzyme immunoassays. Human blood leukocytic virus isolation on the first day of tick suction testified to the capacity of the virus to adsorb and multiply just in the peripheral blood immunocompetent cells. The bulk (as high as 70%) of the TBE virus population was non-neuroinvasive strains, most of which could rapidly eliminate from man and albino mice. The neuroinvasive strains (as high as 30%) caused encephalitis in albino mice and different TBE forms (inapparent, feverish, focal). The sequences of 160 bp fragment of glycoprotein E gene of 24 strains have shown that they belong to one Far Eastern subtype of TVE virus.     

Monoclonal antibodies to tick-borne encephalitis (TBE) virus: their use for differentiation of the TBE complex viruses.

Monoclonal antibodies (MoAbs) to Central European tick-borne encephalitis virus (strain Hypr) were used for differentiation of eight viruses of the TBE complex by indirect immunofluorescence. MoAb 11/B3 (in Western blot recognizing 52 and 70 kD polypeptides) reacted with five out of the eight TBE complex viruses, MoAb 13/E5 (anti-52 kD protein) reacted with the western or eastern subtype of TBE virus only, while MoAb 12/G4 (anti-70 kD protein) distinguished the western subtype of TBE virus from the rest of the TBE complex. These three MoAbs were able to differentiate the virulent strain Hypr from attenuated strains Skalica and Hy-HK-18-"3". MoAb 2/10C (anti-56 and 70 kD proteins) which reacted with all viruses of the TBE complex, recognized both virulent and attenuated strains of TBE virus.     

Variabilities in the antigenic structure of persisting tick-borne encephalitis virus strains

Antigenic variants in the E protein from persisting tick-borne encephalitis (TBE) virus strains were analyzed using monoclonal antibodies (MAbs) to an analogous protein of the reference Sofyin strain. MAbs to sequential epitopes demonstrated their ability to differentiate persisting TBE virus strains from Sofyin and from each other. Two MAbs (2H3 and 13D6) showed a higher neutralizing activity in the interaction with persisting TBE virus variants as compared to the Sofyin strain. Based on the obtained data, a comparison was made of topologically identical epitopes from the E protein of reference and persisting virus strains. The possibility of increasing the neutralizing activity of MAbs through alterations in the primary structure of sequential antigenic sites is discussed.     

Complete genome sequence analysis of tick-borne encephalitis viruses isolated in northeastern China

Tick-borne encephalitis virus (TBEV) causes lethal encephalitis in humans, posing a growing public-health problem in many European and Asian countries. TBEV is currently endemic in northeastern China, but the complete genome sequences of Chinese TBEV strains have not been reported. During a TBE outbreak in 2010 in Mudanjiang City, Heilongjiang Province, China, two TBEV strains were isolated from serum samples of two patients, and the complete sequences were determined and compared with other known TBEV strains. Both Mudanjiang isolates consisted of 10,774 nucleotides and encoded a single open reading frame coding for a polyprotein of 3414 amino acids, and a unique deletion of 364 nucleotides in the 3’ untranslated region (UTR) was recorded. Phylogenetic analysis based on the amino acid sequence of the E protein and the nucleotide sequence of the 3’UTR revealed that the Mudanjiang isolates are closely related to the Senzhang and Sofjin-HO strains and belong to the Far Eastern subtype of TBEV. These findings provide insight into the evolutionary relationships among Chinese TBEV isolates and are useful for laboratory diagnosis and vaccine development for TBEV.     

Ability of inactivated vaccines based on far-eastern tick-borne encephalitis virus strains to induce humoral immune response in originally seropositive and seronegative recipients

Tick-borne encephalitis (TBE) remains one of the major public health concerns in northern Eurasia, and its’ area is expanding. TBE virus (TBEV) includes three subtypes and several monophyletic groups, cocirculating in Russia. Five inactivated vaccines are used for TBE prophylaxis. The rising number of people subjected to vaccination brings up the issue of the impact of individual recipient characteristics on vaccination efficacy. The present work studies correlations among the vaccination scheme, sex, age, body mass index (BMI), chronic diseases, postvaccinal reaction, pre-existing anti-TBEV antibodies, and postvaccinal humoral immunity development. Sera were collected during clinical trials in the TBEV Siberian subtype endemic area. Adult recipients were vaccinated with Tick-E-Vac and EnceVir vaccines based on Far-Eastern TBEV strains. Vaccine ability to induce humoral immunity in different categories of recipients was estimated by seroconversion rates and the percentage of recipients with high neutralizing antibody titers (≥1:500). High immunogenicity of vaccines based on Far-Eastern TBEV strains in the TBEV Siberian subtype endemic area in all groups of recipients was demonstrated. Impact of pre-existing contact with the virus and high BMI on humoral immune response development 14 days after the first immunization was evidenced. Nevertheless, the difference was significantly less pronounced 30 days after the first vaccination and undetectable after the second one.