Tick-borne Encephalitis Virus RNA Research Articles

Epidemiological Study of Tick-Borne Encephalitis Virus in Northern Greece

Serum samples from 921 apparently healthy individuals living in different prefectures of Northern Greece were investigated for the presence of antibodies against tick-borne encephalitis (TBE) virus. In addition, serum and cerebrospinal fluid samples of 302 patients with central nervous system (CNS) infection were tested for the presence of specific IgG and IgM antibodies and TBE virus RNA. Two percent of the general population was found to have antibodies to the virus, with no significant differences among the age groups. Most of the seropositive individuals were male farmers, while seroprevalence varied among different prefectures (0%-5.8%). TBE was not confirmed by laboratory findings in any of the patients with CNS infection. Results of this study revealed that a flavivirus of the TBE serocomplex is circulating in Greece, yet is not a major public health problem.

The importance of tick-borne encephalitis virus RNA detection for early differential diagnosis of tick-borne encephalitis

Tick-borne encephalitis virus (TBEV) is one of the most important causes of human viral infections of the central nervous system in Europe. Currently, the diagnosis of TBE is based on the demonstration of specific antibodies in patient's serum, which appear only several weeks after the infection. To determine how successfully can viral RNA be detected by RT-PCR in the samples of body fluids of patients with TBE prior to and after the appearance of antibodies. Serum, whole blood and CSF samples from 34 patients with a serologically confirmed TBE were collected. Samples were tested for the presence of TBEV RNA by using RT-PCR method. Viral RNA was detected in all blood and serum samples collected before the development of antibodies. After the appearance of IgM antibodies, the number of positive samples dropped by at least one third. After the development of IgG antibodies, only 3% of serum and 16% of blood samples tested positive for viral RNA. Samples of cerebrospinal fluid were shown to be inappropriate for the molecular diagnosis of TBE using this assay, since only one sample (10%) that was collected in the sero-negative phase of disease was found positive by the PCR assay. RT-PCR is an efficient method for an early detection of TBEV in blood and serum samples collected prior to the appearance of antibodies. This method can be of valuable use for a differential diagnosis of TBEV infection in patients with febrile illness after a tick bite, particularly in regions where more than one tick-transmitted diseases are endemic.

Attempt to Detect Evidence for Tick-Borne Encephalitis Virus in Ticks and Mammalian Wildlife in the Netherlands

To investigate if tick-borne encephalitis virus (TBEV) is present in mammalian wildlife species or ticks in The Netherlands, serum samples and ticks were tested for TBEV antibodies and TBEV RNA, respectively. Serum samples were collected from wild boar (666), deer (13), fox (399), and rodents (90), and were tested for TBEV antibodies, using ELISA, and SN test or HI test. Over a period of 4 years, a total of 906 ticks was collected from seven regions in The Netherlands. In four different regions, this was done on a monthly basis and during four consecutive summers. All ticks were tested for TBEV RNA by RT-PCR. TBEV antibody was detected by ELISA in two (0.5%) sera of foxes and 49 (7%) sera of wild boar, but not confirmed by HI or SNT. TBEV RNA was not detected in any of 906 ticks. It was concluded that there is no real evidence for a TBEV reservoir in ticks or wildlife in The Netherlands.

Tick-borne encephalitis virus infections in Lithuanian domestic animals and ticks

In order to obtain information about the distribution of tick-borne encephalitis virus (TBEV) in Lithuania, sera of domestic animals were screened for TBEV antibodies by haemagglutination inhibition test. Samples were collected in 2001 from 423 cows, 561 goats and 118 sheep during a prophylactic examination or vaccination by a local veterinary specialist. In addition, a total of 3234 Ixodes ricinus ticks in 436 pools were collected and tested by RT-PCR for the presence of TBEV RNA (detailed analysis with genetic characterization is published separately [Han et al, J Med Virol 2005 (in press)]). Domestic animal sera from 8/18 districts were positive with an overall seropositivity of 1.7% with considerable regional differences. Sheep from the Radviliskis region had the highest seropositivity rate (16%). In comparison, the proportion of tick pools positive for TBEV-RNA was 1.38%, ranging from 1.03% in Panavezys, 3.33% in Siauliai to 16% in Radviliskis.

Molecular epidemiology of tick-borne encephalitis virus inIxodes ricinus ticks in Lithuania

In Lithuania, 171-645 serologically confirmed cases of tick-borne encephalitis occurred annually [Mickiene et al. (2001): Eur J Clin Microbiol Infect Dis 20:886-888] in 1993-1999, and the tick-borne encephalitis virus (TBEV) seroprevalence in the general population was found previously to be 3.0% [Juceviciene et al. (2002): J Clin Virol 25:23-27]. To assess the risk for TBEV virus infection in Lithuania and to characterize the agent a panel of 3,234 ticks combined into 436 pools [Juceviciene et al., 2005] were tested for presence of TBEV RNA by a nested RT-PCR targeting at the NS5 gene. Six pools were confirmed positive and the prevalence of the infected ticks was 0.2% (if one tick per pool [Juceviciene et al., 2005] was considered positive) and the proportion of positive tick pools was 1.4%. The prevalence of the infected ticks in the Panevezys, Siauliai, and Radviliskis regions (in central Lithuania) was 0.1%, 0.4%, and 1.7% corresponding with a higher TBE disease burden in these regions. The 252-nucleotide NS5-region amplicons, and a longer sequence (737 nucleotides) obtained from one sample from the PrM-E gene region, were sequenced. Phylogenetic analysis of the latter showed that all western type TBEV PrM-E sequences, including the Lithuanian strains, were monophyletic, showed no clustering and had very little variation. The NS5 sequences, although identical within one locality, did not show any mutations common to strains from the two Lithuanian regions, nor could any geographical clustering be found among western type TBEV strains from other areas.

Prevalence of tick-borne encephalitis virus in Ixodes ricinus ticks in Finland.

Approximately 20 cases of tick-borne encephalitis (TBE) occur annually in Finland. The known endemic areas are situated mainly in the archipelago and coastal regions of Finland, with highest incidence in Aland islands. Ixodes ricinus panels collected in 1996-1997 from two endemic areas were screened for the presence of RNA. Two distinct RT-PCR methods were applied, and were shown to have an approximate detection limit of 10 focus forming doses (FFD)/100 microl. One out of 20 pools (a total of 139 ticks) from Helsinki Isosaari Island and one out of 48 pools (a total of 450 ticks) from Aland were positive with both methods, whereas the remaining pools were negative. The observed overall frequency (0.34%) in ticks in endemic areas of Finland, was similar to the low incidence found by virus isolation in mice in the 1960s (0.5%). Viral RNA was detectable in a diluted sample representing 0.005% of a positive pool of ten nymphs suggesting that the viral RNA load within an infected tick pool was approximately equivalent to 20,000-200,000 FFD. Sequence analysis did not show geographical clustering of the Finnish strains, suggesting an independent emergence of different TBE foci from the south. TBE virus RNA positive ticks were not found in I. ricinus panels consisting of 130 pools (726 ticks) from Helsinki city parks or 41 pools (197 ticks) from Võrmsi Island in Estonia.

Annual and seasonal variation of tick-borne encephalitis virus (TBEV) prevalence in ticks in selected hot spot areas in Germany using a nRT-PCR: Results from 1997 and 1998

The prevalence of tick-borne encephalitis virus (TBEV) in Ixodes ricinus tick populations in endemic areas of Germany with the highest TBE risk is unknown. Annual and seasonal differences in TBEV prevalence have also not been studied. Against this background, in May 1997 we started a systematic virus surveillance programme in ticks collected in locations known to have a high incidence of autochthonous TBE cases. These were 5 locations in Baden-Württemberg (Black Forest) and 8 locations in Bavaria (surrounding Passau). Field-collected ticks were randomly assigned to pools of 10 adults or 20 nymphs, respectively. The tick pools were tested for the presence of TBEV-RNA using a newly developed, sensitive nested reverse transcriptase polymerase chain reaction assay (nRT-PCR). The primer pairs were selected from the 5'-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences, as well as by sequencing of the first and the second amplificates, by Southern blot hybridisation with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive, with a detection limit of 20 fg of TBEV RNA per PCR run, or a single positive tick. Based on biostatistical considerations a sample size of at least 1000 ticks per estimation point was chosen. The estimated TBEV prevalence and confidence intervals (CI) were calculated from the nRT-PCR results of pooled samples (10 adults or 20 nymphs) using appropriate formulae for pooled testing. In order to identify the estimated TBEV prevalence as well as to assess the influence of annual and seasonal factors on TBEV prevalence, ticks were sampled twice a year (May and September) in 1997 and 1998 at exactly identical sites. These sites were selected because they were known to have had the highest incidence of autochthonous TBE cases during the previous 10 years. On sampling days, relevant local meteorological data were also noted. In total, 8500 I. ricinus ticks were investigated in this study, 4270 (3540 nymphs, 730 adults) from the Black Forest habitats, and 4230 (3680 nymphs, 550 adults) from the Bavarian locations. In the foci near Freiburg (Black Forest), the estimated virus prevalence was relatively high in the whole tick population, during 1997 with only slight seasonal differences [3.4% (confidence interval, CI, 2.3-4.8%) in May and 2.9% (CI 1.7-4.5%) in September]. In contrast, in 1998, in the same foci the estimated TBEV prevalence was considerably lower [1.1% (CI 0.5-2.0%) in May and 0.6% (CI 0.2-1.4%) in September]. Thus, while the seasonal differences again remained low, the annual variation was marked. In the Bavarian foci in 1997, the estimated virus prevalence of the whole tick population studied was lower than in the Black Forest foci and the seasonal fluctuations were low: in May 1997 0.9% (CI 0.4-1.8%) of the ticks were positive, in September 1.1% (CI 0.5-1.9%). In 1998, in May 2.0% (CI 1.1-3.3%) of the ticks were positive, and in September 1.1% (CI 0.5-2.1%). For the whole study period, every 50th to 100th I. ricinus nymph or adult in the Passau region was calculated to give a positive signal in the nRT-PCR. The TBEV prevalence data indicate that residents and visitors of areas in Germany known for high endemic activity take a significant risk of contracting TBEV infection, if bitten by ticks. In addition, the data suggest that annual fluctuations may exist in the whole tick population studied. Seasonal fluctuations of the virus prevalence in ticks were small.

ChemInform Abstract: Synthesis of New Photocross‐Linking 5‐C‐Base‐Substituted UTP Analogues and Their Application in Highly Selective Affinity Labeling of the Tick—Borne Encephalitis Virus RNA Replicase Proteins

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

A nested RT-PCR for the detection of Tick-borne Encephalitis Virus (TBEV) in ticks in natural foci

We have developed a sensitive nested reverse-transcriptase polymerase chain reaction assay (n RT-PCR) for the detection of the tick-borne encephalitis virus (TBEV) RNA, especially in ticks. The primer pairs were selected from the 5'-terminal noncoding region, a highly conserved part of the virus. The specificity was tested by computer homology searches of sequences as well as by the sequencing of the first and second amplificate, by Southern blot hybridization with a DIG-labelled oligonucleotide probe, and by restriction enzyme analysis. The method has proved to be very sensitive. The detection limit is about 20 fg of TBEV RNA per PCR run (25 microliters), or a single positive tick, i.e. (adult or nymph). The method can be used for comparative studies of the epidemiological situation, as well as for the screening of natural foci for the presence and circulation of TBEV or for the detection of TBEV-genome-sequences in clinical materials.

Synthesis of New Photocross-Linking 5-C-Base-Substituted UTP Analogs and Their Application in Highly Selective Affinity Labelling of the Tick-Borne Encephalitis Virus RNA Replicase Proteins

A new photocross-linking 5-C-base-substituted UTP analogs, carrying 4-azidoperfluorobenzoyl and 4-azidoaniline residues were synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation (λ > 300 nm) and subsequent [α-32P]NTP incorporation.

Affinity labelling of the tick-borne encephalitis virus RNA replicase proteins by 4-N-exo-base-substituted photoreactive CTP analogs.

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.

CDNA encoding tick-borne encephalitis virus RNA; useful for recombinant vaccine production and for genetic immunization : Immuno World 9630 521; 3 October 1996

cDNA encoding tick-borne encephalitis virus RNA; useful for recombinant vaccine production and for genetic immunization : Immuno World 9630 521; 3 October 1996

Detection of the tick-borne encephalitis virus (TBEV) in ticks in several federal "Länder" of Germany by means of the polymerase chain reaction (PCR)--characterization of the virus.

The aim of the present study was to analyse the current epidemiological situation with respect to TBE in the new federal "Länder" of Germany and in Saarland through detection of the TBEV genome in unengorged ticks using an RT-PCR technique. 22,273 ticks (Ixodes ricinus) were collected in the five new "Länder" (and some in Bavaria and Baden-Württemberg) and divided into 294 pools. It was possible to detect TBEV RNA in six pools of ticks from Mecklenburg Western-Pomerania [4], Brandenburg [1], Thuringia [1] (and in three pools from Bavaria and Baden-Württemberg). The nucleotide sequence data of the PCR products were analysed and compared. In Saarland 8,780 ticks were collected in 70 habitats from all the geographic regions and analysed using the PCR in 21 pools; two pools produced positive PCR signals (Saarlouis, Perl). We cannot as a result make a general recommendation that TBE-immunization be introduced in Saarland and in the new federal Länder of Germany. In Germany, however, TBE immunoprophylaxis in Bavaria and Baden-Württemberg is very important.

T7 DNA-dependent RNA polymerase can transcribe RNA from tick-borne encephalitis virus (TBEV) cDNA with SP6 promoter

T7 RNA polymerase is shown to recognize the SP6 promoter including 17 base pairs before the transcription start site and produce the 5′-end TBEV RNA. The yield of TBEV RNA synthesized by heterologous T7 RNA polymerase from cDNA construction with SP6 promoter is higher than the RNA production by homologous SP6 RNA polymerase. The addition of 1 pmol template DNA with SP6 17 bp promoter in transcription mixture for SP6 or T7 RNA polymerases resulted in 1–5 × 10 −2 pmol RNA production.

Identification of tick-borne encephalitis virus ribonucleic acid in tick suspensions and in clinical specimens by a reverse transcription-nested polymerase chain reaction assay

Background: Tick-borne encephalitis virus (TBEV) is a major human pathogenic flavivirus. Sensitive assays for the detection of viral RNA may be valuable both for the identification of virus in ticks as well as for diagnostic purposes. Objectives: (1) The development of a sensitive polymerase chain reaction (PCR) test system for the detection of TBEV-RNA and its application to the identification of infected ticks; and (2) evaluation of the PCR assay for diagnostic purposes, i.e., detection of TBE virus RNA in blood and in cerebrospinal fluid (CSF) of TBE patients. Study design: (1) Establishment of a TBEV-specific reverse transcription (RT)-nested PCR assay and evaluation of its sensitivity; (2) comparison of the PCR assay with that of virus isolation from tick suspensions; and (3) investigation of 105; serum and CSF samples from patients with serologically confirmed TBE by RT-nested PCR. Results: An RT-nested PCR assay was established with a detection limit of 100–1000 copies of TBEV RNA. All tick suspensions from which the virus could be isolated by inoculation of suckling mice also screened positive in the PCR assay. Of the 105 clinical samples investigated, only one serum and one CSF sample were positive by PCR assay, and these were both obtained very early in the course of the disease. Conclusions: The PCR assay described is valuable for the detection of TBEV in tick suspensions and can substitute for the usual virus isolation procedure in which suckling mice are inoculated. Its application for diagnostic purposes, however, does not seem to provide a significant improvement over serological diagnosis. Only in very rare cases, when a sample is drawn extremely early in the course of disease, may TBEV RNA be detected in serum or CSF before the appearance of specific IgM antibodies and thus allow an earlier diagnosis.

Detection of tick-borne encephalitis virus RNA in ticks ( Ixodes ricinus) by the polymerase chain reaction

A polymerase chain reaction for the detection of tick-borne encephalitis virus (TBEV) RNA in ticks was developed. Two pairs of primers for nested PCR were selected from the 5'-NCR and the 5'-terminus of the C protein coding region, which are highly conserved among the TBEV isolates sequenced so far. The sensitivity of the nested PCR was tested by dilution experiments of a TBEV positive brain suspension. The specificity of the PCR products was confirmed by Southern blotting. In a pilot study, 60 homogenates of 7200 ticks ( I. ricinus) were examined by PCR. Two homogenates were found positive. The PCR for TBEV RNA appears to be a valuable method to define endemic areas of TBE.

Differentiation of strains of tick-borne encephalitis virus by means of RNA-DNA hybridization

Cloned cDNA and synthetic deoxyoligonucleotides, complementary to various parts of the genomic RNA of tick-borne encephalitis virus (TBEV), strain Sofjin, were used to distinguish between strains of TBEV and other flaviviruses. The cDNA probe hybridized with strains of TBEV and related flaviviruses of the TBE complex except for Powassan virus, and it did not react with flaviviruses of the Japanese encephalitis and dengue subgroups. Viruses of the TBE complex and some strains of TBEV were differentiated from TBEV strain Sofjin by the thermal stability of RNA-DNA hybrids. Negishi and louping-ill viruses were the most closely related to TBEV strain Sofjin, among viruses of the TBE complex. Eight strains of TBEV isolated in different geographical areas from different sources were tested by dot-hybridization with 11 deoxyoligonucleotide probes. The probes revealed genetic variations among strains of TBEV. The pattern of hybridization correlated with the source of virus strains: TBEV strains isolated from TBE patients reacted with more probes than strains isolated from ticks. Within a group of epidemic strains of TBEV there was a correlation between the geographical area of isolation and similarity to TBEV strain Sofjin.

Differences between translation products of tick-borne encephalitis virus RNA in cell-free systems from Krebs-2 cells and rabbit reticulocytes: Involvement of membranes in the processing of nascent precursors of flavivirus structural proteins

Tick-borne encephalitis virus (TBEV) RNA was translated in extracts from Krebs-2 cells and in rabbit reticulocyte lysates. In the former system, two polypeptides, p53 and p13, corresponding to envelope (E) and core (C) proteins of the virion, respectively, were synthesized preferentially. In contrast, the major product in reticulocyte lysates was represented by a heterogeneous set of high-molecular-weight polypeptides which did not appear to include p53 or p13. The reticulocyte lysates, however, acquired the ability to produce structural proteins (p53 at least) after addition of purified membranes isolated from the rough endoplasmic reticulum of Krebs-2 cells. On the other hand, the ability of Krebs-2 extracts to generate identifiable viral structural proteins was lost after degradation of membranes by the nonionic detergent Triton X-100. These findings strongly suggest that membrane-dependent processing of protein precursors is involved in the formation of TBEV structural proteins. Evidence has been obtained that only nascent precursor polypeptides can be processed efficiently into structural proteins in the membrane-dependent reaction.