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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Characterization of tick-borne encephalitis virus from Estonia

Tick-borne encephalitis virus (TBEV) is a severe problem in Estonia. In the present article the first genetic analysis of Estonian TBEV strains is described. In total, seven TBEV strains were isolated from ticks (Ixodes ricinus and I. persulcaus), rodents (Apodemus agrarius and Cletrionomys glareolus), and serum from a tick-borne encephalitis (TBE) patient. The nucleic acid sequences of the viral genome encoding almost the complete E protein (nt 41-1250) and the 3'-NCR-termini of the Estonian TBEV strains were determined by direct sequencing of RT-PCR products. The results showed that all three known TBEV subtypes, Western TBEV (W-TBEV), Far-Eastern TBEV (FE-TBEV), and Siberian TBEV (S-TBEV), co-circulate in Estonia. The Estonian TBEV strains of the S-TBEV and W-TBEV subtypes clustered with the previously reported strains from Latvia and Lithuania. Within the FE-TBEV subtype, however, the Estonian strain Est2546 clustered together with the strain Sofjin, originating from the Far-East of Russia, but not with the strain RK1424, isolated in the neighboring Latvia. This suggests a different evolutionary history for the Estonian and the Latvian strains in the FE-TBEV subtype. The Estonian TBEV strain (Est3535), which belonged to the S-TBEV subtype, had an organization of the 3'-NCR similar to that of strains from the Far-East of Russia (Irkutsk). The 3'-NCRs of Estonian strains of the W-TBEV subtype (Est3051, Est3053, Est3476, and Est3509) were very similar to those of the strain Ljubljana I from the Balkans. In the 3'-NCR sequence of the Estonian strain Est2546, which belonged to the FE-TBEV subtype, a deletion from position 10461 to 10810 extending approximately 10 nucleotides into the core element, was detected.     

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.     

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).     

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.     

Tick-borne Encephalitis Virus in Lithuania

Tick-borne encephalitis (TBE) is a severe problem in Lithuania, indicated by the 171 to 645 serologically confirmed cases that occurred each year between 1993 and 1999. In the present report, the first isolation and partial genetic analysis of a Lithuanian TBE virus (TBEV) strain isolated from a patient's serum sample is described. The patient was bitten by a tick while visiting the Lazdijai district (Veisiejai forest) in the southernmost part of Lithuania, a geographical area where Ixodes ricinus but not Ixodes persulcatus ticks are known to be present. The E protein-encoding viral gene sequence (nt 74–1273) recovered from the TBEV isolate showed the closest similarity to previously characterized European strains of the Western TBEV subtype, including the prototype TBEV strain Neudoerfl and those from neighbouring Latvia. Accordingly, the Lithuanian isolate was placed within the Western genetic lineage of TBEV in phylogenetic trees. Electronic Publication     

Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk,non-risk and former risk areas of TBE,and in ticks removed from humans in Germany

Tick-borne encephalitis (TBE) is recognized as the most important viral tick-borne zoonosis in 27 countries in Europe. In this study, ticks were collected in Germany from two non-risk areas in the states of Saxony-Anhalt and Mecklenburg-Western Pomerania, where several single human TBE cases have occurred in recent years. Ticks were also collected from a region in Thuringia, known to be a former risk area for TBE virus (TBEV), where numerous human cases were reported between 1960 and 1975. Detection of TBEV RNA was conducted by real-time RT-PCR. No TBEV was detected in any field-collected ticks. However, ticks were also collected from volunteers living in Bavaria. Three of 239 ticks from this collection were positive for TBEV genome and two genetically distinct TBEV strains were detected and characterized.     

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.     

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.