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.     

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.     

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).     

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).     

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.     

Tick-borne encephalitis virus strains of Western Siberia

Tick-borne encephalitis virus (TBEV) strains were isolated from ticks in Western Siberia for 12 years. Molecular hybridization of the 46 viral RNA with the TBEV cDNA and oligonucleotide probes revealed differences between the Siberian and Far Eastern strains. A comparison of the viral E gene fragment nucleotide sequence showed 89–98% homology between Siberian TBEV strains, whereas their similarity with strains from other populations was less than 83%. However, the viral E and NS1 glycoprotein antigenic structures appeared to be conservative because of the degenerate genetic code. This was shown by enzyme-linked immunosorbent assay with the corresponding monoclonal antibodies (MAb). The single exception was the MAb 17C3 against nonstructural glycoprotein NS1, which could distinguish Siberian from Far Eastern strains. Moreover, the neurovirulence differed between strains from the two natural populations. Lower neuroinvasiveness of the Siberian strains in comparison with Far Eastern Sofyin strain might be caused by both E and NS1 glycoprotein mutations.     

Genetic typing of tick-borne encephalitis virus based on an analysis of the levels of homology of a membrane protein gene fragment

All heretofore known genomic structures of tick-borne encephalitis (TBE) virus are analyzed. The authors prove the adequacy of using short fragment of E protein gene for characterization of philogenetic relationships between TBE strains. Three main genotypes of the virus are distinguished, one corresponding to Far Eastern variant, one to West, and the third includes strains belonging to Ural Siberian and Central Siberian and Transbaikal variants. Results of genetic typing by nucleotide sequences are confirmed by analysis of amino acid sequences of E protein fragments, specific marker amino acids in definite positions being determined for each genotype.     

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.     

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.     

External quality assurance studies for the serological and PCR diagnostics of tick-borne encephalitis virus infections

Improved diagnostics are critical for optimally detecting and managing tick-borne encephalitis (TBE) infections, and therefore quality control measures are essential for both serological and molecular diagnostics. Two external quality assurance (EQA) studies were performed to assess the quality of serological and molecular diagnostics of TBE infections. For the EQA of serological diagnostics, each participating laboratory received a proficiency panel of freeze-dried human sera containing 7 TBE-positive samples, 3 negative samples, and 2 samples positive for heterologous flaviviruses. For the EQA of molecular diagnostics, panels of prepared human plasma samples comprising 8 samples spiked with RNA from different TBE virus strains of European, Siberian, and Far Eastern subtypes, 2 specificity controls with heterologous flaviviruses, and two negative control samples were distributed. A total of 46 invited expert laboratories from 28 European and non-European countries participated at least in one of these studies. Applying proficiency criteria, the number of participating laboratories who passed the minimum requirements for successful participation was 60% for the EQA of serological (both IgM and IgG detection) and 48% for the EQA of PCR diagnostics. The EQA studies provide information on the diagnostic quality of the participating laboratories and indicate that most of them need to improve their assays.     

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.     

Rapid subtyping of tick-borne encephalitis virus isolates using multiplex RT-PCR

Tick-borne encephalitis virus, an emerging pathogen in several countries in Europe and Asia, has been divided into three subtypes (European, Siberian and Far Eastern). These subtypes are associated with different severities of the disease. For that reason, early determination of the subtype in a clinical sample or in ticks removed from a patient in areas of co-circulation of two or three subtypes is of high importance. The development of a simple method of multiplex RT-PCR for rapid and easy subtyping of tick-borne encephalitis virus isolates is reported to fill this requirement. The method is based on the unique combination of oligonucleotide primers hybridizing with subtype-specific "signature" positions of the sequence encoding the viral envelope protein. The developed multiplex RT-PCR also appears to be a useful method in studies focused on the molecular-epidemiology of tick-borne encephalitis virus.     

Variability in the 3′ untranslated regions of the genomes of the different tick-borne encephalitis virus subtypes

Virus Genes - Tick-borne encephalitis viruses (TBEVs) are usually divided into three major subtypes: European (TBEV-Eu), Siberian (TBEV-Sib) and Far Eastern (TBEV-FE). The TBEV-Eu strains have the...     

The geographical distribution of genetic variants of the tick-borne encephalitis virus]

Geographic distribution of 185 tick-borne encephalitis (TBE) virus strains isolated in 8 physico-geographic areas and classified into six genetic variants was analysed. The strains of genetic variant I homologous to the Sophyin prototype strain were found to occur predominantly in the Far East and also frequently found in Western and North-Western parts of the East European plain. The vast territories from lake Baikal in the East to Ukraine in the West harbor mostly the strains significantly different from the Far-Eastern Sophyin strain. Hybridization experiments with oligonucleotide probes specific for the Neudorffle strain showed that the strains genetically similar to the virus of central European encephalitis occurred also in Eastern Europe and Western Siberia. It is concluded that a relationship exists between genetic types of TBE virus and their geographic origin.     

Experimental tick-borne encephalitis in golden hamsters treated with specific immunotherapy

Efficiency of various protocols of specific immunoglobulin treatment was evaluated in golden hamsters inoculated with two Far Eastern tick-borne encephalitis (TBE) strains. After a low therapeutic dose (0.1 ml) of immunoglobulin, corresponding to total dose (60 ml) per course, all parameters (survival, immunogenicity, pathomorphology of the brain) deteriorated in animals infected with both strains. A higher dose (0.2 ml) corresponding to total dose of 120 ml notably improved all the studied parameters. The efficiency of specific immunoglobulin depends on the clinical and pathogenetic characteristics of TBE, determined by the properties of TBE strains. The results validate therapy and prevention of TBE by high-titer immunoglobulin in adequate total dose, monitored by blood analyses for TBE antigen and evaluations of the time course of IgM antibodies.     

Molecular hybridization with cloned fragments of tick-borne encephalitis (TBE) virus cDNA in acute and chronic TBE infection.

Recombinant plasmid DNA was used as a probe to detect tick-borne encephalitis (TBE) virus RNA during incubation period, acute disease and persistent infection of syrian hamsters. Within the first three weeks post-infection the results of direct virus isolation and RNA detection in the brain agreed by a rate of 100%, the virus titre ranging between 10(1.9) to 10(10.5) LD50/ml and viral RNA concentration at 1-1000 pg. At the same time TBE virus RNA was detected in the spleen when the virus titre was greater than or equal to 10(6.5) LD50/ml. By 8 months post infection (p.i.) viral RNA was found in the brain, liver, and spleen in the absence of infectious TBE virus. No viral RNA was present in the thymus. In addition, electron microscopic findings in hamster brain confirmed the hypothesis that TBE virus persistence was accompanied by formation of virus-specific structures but impaired virion maturation.     

Test based on subtype-specific μ-capture IgM immunoassay can distinguish between infections of European and Siberian subtypes of tick-borne encephalitis virus

BackgroundIn many European countries (including Finland, Estonia, Latvia and Russia) two subtypes of tick-borne encephalitis virus (TBEV) occur with overlapping geographic distribution yet with apparently different severity and persistence of symptoms. However, it has not usually been possible to distinguish these infections in the laboratory, as TBEV RNA or sequences have rarely been retrieved from patients seeking medical care in the second phase of infection when the neurological symptoms occur, and serological tests have so far not been able to discriminate between the subtype-specific responses.ObjectivesThe aim of this study was to assess the applicability of a μ-capture enzyme immunoassay (EIA) based on TBEV prME subviral particles produced in mammalian cells from Semliki-Forest virus replicons (SFV-prME EIA) to distinguish reactivity to European and Siberian strains of TBEV.Study designAltogether 54 TBEV IgM positive acute human serum samples and 6 positive cerebrospinal fluid (CSF) samples from different regions of Finland were tested in EIA with subtype-specific antigens and TBEV-IgM subtype-specific index ratios were determined.ResultsAll 30 samples from patients whose transmission had occurred in foci where only Siberian subtype of TBEV is occurring had an index ratio of more than 1.8, whereas all 30 acute TBE samples from an area where only European subtype circulates had an index ratio below 1.5.ConclusionsWe conclude that the assay is a useful tool to distinguish between acute infections of European and Siberian strains of TBEV, and should help in further studies of the clinical outcome of these two subtypes.     

A case of tick-borne encephalitis in Japan and isolation of the the virus.

A case of tick-borne encephalitis (TBE) has not been reported for many years in Japan, although a serological survey of sera from domestic animals suggested the presence of TBE foci in Hokkaido, the northern island of Japan. Studies were conducted to prove the presence of an endemic focus of TBE virus in Japan by means of serology and virus isolation. In October 1993 in Hokkaido, a severe case of encephalitis in a dairy farmer's wife was diagnosed as TBE. Serological examination of paired serum specimens showed a rise in the neutralization antibody titer to Russian spring summer encephalitis virus. A seroepizootiological survey of dogs showed that the TBE-related virus was prevalent in the area. Three virus isolates were obtained from the blood of sentinel dogs, and antigenic analysis grouped the isolates into TBE-related viruses. Sequence analysis of the envelope protein gene identified one of the isolates as being of the same subtype as the Russian spring summer encephalitis (Far Eastern TBE) virus. The results provide evidence that TBE is endemic in a certain area of Japan.