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.     

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.     

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.     

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).     

Molecular epidemiology of tick-borne encephalitis

The review presents information on the development of studies into the molecular epidemiology of tick-borne encephalitis (TBE) in Russia and foreign countries. The existence of three major virus genotypes has been established by various techniques, such as genomic fragment sequencing, molecular hybridization using genotype-specific probes, and restriction fragment length polymorphism test. Each of the genotypes prevails in different parts of a natural habitat; the Ural-Siberian genotype (a Siberian subtype) is most commonly encountered. The genetic differences between the strains belonging to different genotypes are great and comparable with differences between some mammalian flaviviruses transmitted by ticks (viruses of a TBE complex). Further studies of the molecular epidemiology of TBE are of importance in understanding the evolution of the causative agent, improving the taxonomy and the classification of flavivuruses, and designing highly effective methods for the specific diagnosis, prevention, and treatment of the disease.     

On the problem of pathomorphosis of modern tick-borne encephalitis in Ural

Pathology study of 32 patients who died of tick-borne encephalitis (TBE) in the Ural Region in 1990s revealed differences with this infection registered in 1940-1950s. Pathological changes in the central nervous system by their severity and location are like those observed in the Far East TBE. Apart from grave alterative changes in the nerve cells of motor nuclei of the spinal cord and brain, three types of pathological changes are observed: productive inflammation, exudative inflammation without pronounced inflammatory reaction. Differences in the organism reactions to the infectious process are explained by differences in the virus virulence and organism immunological status.     

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.     

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.     

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.     

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.     

Studies on the glycosylation of flavivirus E proteins and the role of carbohydrate in antigenic structure

The glycosylation pattern of several flavivirus E proteins as well as the role of carbohydrate in biological functions and the antigenic structure of tick-borne encephalitis (TBE) virus were investigated by the use of specific endoglycosidases. Endoglycosidase F digestion revealed the presence of a single asparagine-linked oligosaccharide side chain in TBE virus (Western and Far Eastern subtype), Louping III virus, Murray Valley encephalitis virus, and Rocio virus. Consistent with published sequence data, the E protein of West Nile virus apparently is not glycosylated at all. Evidence derived from digestion experiments using endoglycosidase H indicates that the tick-borne viruses contain high-mannose type N-linked oligosaccharide side chains, whereas that of the mosquito-borne Murray Valley encephalitis virus and Rocio virus is endoglycosidase H resistant. Complete deglycosylation of TBE virus by endoglycosidase F did not impair infectivity and HA activity. Carbohydrate does not seem to play a major role in the antigenic structure of the TBE virus glycoprotein since the reactivity of the native virus and the deglycosylated virus was identical when analyzed with monoclonal as well as polyclonal immune sera.     

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.     

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.     

The specific reactivity of cDNA and deoxyoligonucleotide probes, complementary to the tick-borne encephalitis virus genome, with the RNA of strains of different geographical origins]

Hybridization experiments with RNA of 143 tick-borne encephalitis (TBE) virus strains isolated in different parts of the distribution area were used to study the reactivity of kDNA- and a set of 10 synthetic deoxyoligonucleotide probes. The kDNA probe under certain conditions was shown to hybridize with RNA of all the strains under study, and under other (strict) hybridization conditions did so selectively with a small number of strains. The capacity of oligonucleotide probes for hybridization with RNA of TBE virus strains varied from 12% to 100%. The differences in the hybridization activity of kDNA- and oligonucleotide probes complementary to the genomes of the Sophyin strain (Far-Eastern subtype) and Neudorffle strain (Western subtype) with TBE virus strains were used for differentiation of the strains into six genetic variants. Comparison of the reactivity of molecular probes in experiments with RNA of TBE virus strains and viruses of the TBE complex showed that the differences of the strains belonging to different genetic variants from the prototype Sophyin strain were comparable to those of some members of the TBE complex, with the exception of Powassan virus. These data attest to the necessity of further studies dealing with specification of the taxonomy of TBE complex viruses.     

The differentiation of viruses of the tick-borne encephalitis complex by means of RNA-DNA hybridization]

Nucleic acid spot hybridization with cloned cDNA of tick-borne encephalitis (TBE) virus, strain Sofjin, was used to differentiate strains of TBE and other flaviviruses. The cDNA probe reacted with strains of TBE and flaviviruses of TBE subgroup with the exception of Powassan virus. The probe did not react with viruses of Japanese encephalitis and Gendue subgroups. The viruses of TBE subgroup and some strains of TBE virus were differentiated from TBE strain Sofjin by thermal stability of RNA-DNA hybrids. Negishi and Louping ill viruses were found to be most closely related to TBE strain Sofjin among viruses of the TBE subgroup.     

Characterisation of human tick-borne encephalitis virus from Sweden

Viruses of the tick-borne encephalitis (TBE) antigenic complex, within the family Flaviviridae, cause a variety of diseases including uncomplicated febrile illness, meningo-encephalitis and haemorrhagic fever. Different wildlife species act as reservoir hosts with ixodid tick species as vectors. TBE virus (TBEV) causes 40-130 cases confirmed serologically in Sweden each year. Characteristics of TBEV strains circulating in Sweden have not been investigated previously and no viral sequence data has been reported. In the present study, virus strains were isolated from serum of patients with clinical symptoms consistent with acute TBEV infection. Serologic characterisation, using a panel of E-specific monoclonal antibodies and cross-neutralisation tests, indicated that the Swedish strains of TBEV, isolated 1958-1994, all belonged to the Western TBEV subtype, which includes the Austrian vaccine strain Neudoerfl. Genetic analysis of a partial E-sequence confirmed this close relationship: all Swedish TBEV strains belonged to the European lineage of the Western TBEV subtype, which includes the previously characterised strains Neudoerfl, Hypr, and Kumlinge. Further, three Swedish strains showed partial E-sequences identical to that of the Finnish Kumlinge strain, ten Swedish strains formed a well-supported separate cluster, whereas four others did not show any real clustering. No apparent correlation was observed in comparison of clinical parameters with genetic data or geographic origin of the strains.     

Comparative study of the PrPBSE distribution in brains from BSE field cases using rapid tests

The distribution of PrP(BSE) in the brain of nine confirmed BSE field cases was analyzed using immunohistochemistry and compared to the levels of PrP(BSE) determined by two rapid tests (Prionics-Check WESTERN and Prionics-Check LIA). Each brain was dissected into 16 areas: spinal cord, medulla oblongata, pons, mesencephalon, thalamus, hippocampus, cerebellar vermis, cerebellar medulla, cerebellar hemispheres, occipital cortex, temporal cortex, parietal cortex, striatum, frontal cortex, piriform lobe and olfactory bulbs. The highest levels of PrP(BSE) were detected in the medulla oblongata, spinal cord and pons, and correspondingly both rapid tests showed 100% correlation with the immunohistochemistry with regard to sensitivity and specificity. Some inconsistencies between the levels of PrP(BSE) determined either by immunohistochemistry or by the rapid tests were found in brain areas with medium to low levels of PrP(BSE). These brain areas included the cerebellar hemisphere, olfactory bulb, and the temporal and parietal cortices. A brain PrP(BSE) distribution curve (BPDC) was designed by plotting the PrP(BSE) signals obtained from the two rapid tests versus the anatomical region along the caudal-rostral axis of the brain. Comparison of the BPDC of the nine BSE cases showed that all cases had a similar PrP(BSE) distribution in the brain but with variable intensities, which could be explained by different stages in the progression of the disease. We propose that the BPDC could be used as a tool to differentiate classical cases of BSE from the recently identified atypical BSE cases.