Microevolution of tick-borne encephalitis virus in course of host alternation

Two tick-borne encephalitis (TBE) virus variants were studied: mouse brain-adapted strain EK-328 and its derivate adapted to Hyalomma marginatum ticks. The tick-adapted virus exhibited small-plaque phenotype and slower replication in PEK cells, higher yield in ticks, decreased neuroinvasiveness in mice, increased binding to heparin-sepharose. A total of 15 nucleotide substitutions distinguished genomes of these variants, six substitutions resulted in protein sequence alterations, and two were in 5'NTR. Two amino acid substitutions in E protein were responsible for the observed phenotypic differences. Data obtained during reverse passaging of the tick-adapted virus in vivo and in vitro suggest that TBE virus exists as a heterogeneous population that contains virus variants most adapted to reproduction in either ticks or mammals. Host switch results in a change in the ratio of these variants in the population. Plaque purification of the tick-adapted virus resulted in the prompt emergence of new mutants with different virulence for mammals.     

Analysis of the complete genome of the tick-borne flavivirus Omsk hemorrhagic fever virus

Omsk hemorrhagic fever virus (OHF) is a tick-borne flavivirus endemic to Western Siberia. This virus is the only known tick-borne flavivirus to cause hemorrhagic disease in humans in the absence of encephalitis. OHF virus circulates within a small, defined niche in which other tick-borne complex flaviviruses are also present. The objectives of this study were to genetically classify OHF virus based on its complete genome and to identify genetic determinants that might be involved in tissue tropism and viral replication leading to the disease state caused by this virus. The OHF virus genome was sequenced and phylogenetic analysis demonstrated that OHF virus falls within the tick-borne encephalitis serocomplex of flaviviruses, yet is distinct from other members of the complex, including those closely associated geographically. OHF is also distinct from Alkhurma (ALK) and Kyasanur forest disease (KFD) viruses, both of which cause disease that includes hemorrhagic and encephalitic manifestations. Several amino acid residues were found to be distinct among OHF, KFD, and ALK viruses; these residues include E-76, which is closely associated with the viral envelope protein fusion peptide. In addition, variation between the viral 5'-untranslated region of OHF and other tick-borne flaviviruses suggests potential variability in viral replication. These data demonstrate that OHF is a unique virus among the tick-borne flaviviruses and also provide insight to viral biodiversity and tropism.     

GAG-binding variants of tick-borne encephalitis virus

Previously different authors described various flavivirus mutants with high affinity to cell glycosaminoglycans and low neuroinvasiveness in mice that were obtained consequently passages in cell cultures or in ticks. In present study the analysis of TBEV isolates has shown existence of GAG-binding variants in natural virus population. Affinity to GAG has been evaluated by sorption on heparin-Sepharose. GAG-binding phenotype corresponds to such virus properties, like small plaque phenotype in PEK cells, absence of hemagglutination at pH 6.4, and low neuroinvasiveness in mice. Mutations increasing charge of E protein were necessary but not sufficient for acquisition of GAG-binding phenotype. Molecular modeling and molecular dynamics simulation have shown that the flexibility of E protein molecule could bear influence on the phenotypic manifestation of substitutions increasing charge of the virions.     

The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3′ non-coding region of the genome

Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E₃₁₅) and NS5 (NS5_654,655) proteins, and into the 3′ non-coding region (Δ30) of TBEV/DEN4. The variant that contained all three mutations (vΔ30/E₃₁₅/NS5_654,655) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vΔ30/E₃₁₅/NS5_654,655 should be further evaluated as a TBEV vaccine.     

Role of heparan sulfate for attachment and entry of tick-borne encephalitis virus

Attachment of the flavivirus tick-borne encephalitis (TBE) virus to different permissive cell lines was investigated by a newly established quantitative assay using fluorescence-labeled virus. Previous work had shown that BHK-21 cell-adapted mutants of TBE virus had acquired potential heparan sulfate (HS) binding sites on the outer surface of protein E. Quantitative analysis of one of these mutants indicated that it attached to HS-expressing cell lines with a 10- to 13-fold higher affinity than wild-type TBE virus strain Neudoerfl. CHO cells deficient in HS synthesis bound less than 5% of the amount of wild-type or mutant virus that could attach to HS-containing CHO cells but were nevertheless found to be highly susceptible to infection with both viruses. Thus, even though HS is a major determinant of TBE virus attachment on HS-expressing cells, our findings suggest the existence of an alternative host cell receptor that is less abundant than HS.     

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.     

Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed.     

Growth of tick-borne encephalitis virus (European subtype) in cell lines from vector and non-vector ticks

We undertook a comparative study of the susceptibility of different tick cell lines to infection with the European subtype of tick-borne encephalitis virus (TBEV), prototype strain Neudoerfl. The growth of TBEV was investigated in lines derived from vector Ixodes ricinus L. ticks (IRE/CTVM18, 19, and 20), as well as non-vector ticks, namely Ixodes scapularis Say (IDE2), Boophilus microplus Canestrini (BME/CTVM2), Hyalomma anatolicum anatolicum Koch (HAE/CTVM9), Rhipicephalus appendiculatus Neumann (RA-257) and recently established and herein described lines from the argasid tick Ornithodoros moubata Murray (OME/CTVM21 and 22). All the tick cell lines tested were susceptible to infection by TBEV and the virus caused productive infection without any cytopathic effect. However, there was a clear difference between the TBEV growth in vector and non-vector cell lines, since I. ricinus cell lines produced 100-1000-fold higher virus yield than the non-vector cell lines. The lowest virus production was observed in O. moubata and R. appendiculatus cell lines.     

Vaccinia virus recombinant expressing gene of tick-borne encephalitis virus non-structural NS1 protein elicits protective activity in mice

A vaccinia virus recombinant containing the non-structural tick-borne encephalitis virus (TBEV) NS1 gene was developed. The recombinant expressed native dimeric form of the NS1 protein in infected cells and protected mice against lethal infection with TBEV.     

Expression of cloned envelope protein genes from the flavivirus tick-borne encephalitis virus in mammalian cells and random mutagenesis by PCR

The structural membrane proteins prM and E of the flavivirus tick-borne encephalitis (TBE) virus were expressed in mammalian cells for the purpose of probing the structure and molecular interactions of these proteins. Advantage was taken of the natural error frequency of the Taq polymerase used in the PCR amplification to generate a randomly mutated population of genes that were then cloned directly into plasmid expression vectors under the control of an SV40 promoter. Analysis of the mutation frequency by direct sequencing of 22 separate clones showed that the PCR produced mutations at a rate yielding an average of one to two amino acid changes per clone in the 496 amino acid long protein E. This is an ideal rate for assessing the importance of individual amino acid residues within protein domains, thus demonstrating the potential value of the PCR as a random mutagenesis method. Clones encoding wild-type prM and E proteins, and a truncated form of E, were also constructed by recombining portions of selected PCR clones. Transfection of COS-1 cells with these constructs resulted in expression of the prM and E proteins, which was demonstrated by indirect immunofluorescence using monoclonal antibodies (Mabs). The intracellular level of TBE virus antigen, measured in lysates of transfected cells by ELISA, reached approximately 25% of that found in virusinfected COS cells. Furthermore, it was shown by immunofluoresence using a panel of 19 anti-E Mabs that the antigenic structure of the expressed E proteins was nearly identical to that of E protein in infected cells, thus confirming the suitability of this model system as a tool for studying flavivirus protein structure.     

Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication

We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severely defective as templates for either polymerase. We also evaluated the infectivity of substitution mutant WNV genome-length RNAs. All WNV RNAs containing the DV2 3'SL were unable to replicate. However, WNV RNAs containing substitutions of the 5'NCR, the capsid gene, and/or 3'NCR nt sequences upstream from the WNV 3'SL, by the analogous DV2 nt sequences, were infectious. Combined results suggested that replication was not dependent upon species homology between the 3'SL and NS5.     

Genetic characterization of tick-borne flaviviruses: new insights into evolution, pathogenetic determinants and taxonomy

Here, we analyze the complete coding sequences of all recognized tick-borne flavivirus species, including Gadgets Gully, Royal Farm and Karshi virus, seabird-associated flaviviruses, Kadam virus and previously uncharacterized isolates of Kyasanur Forest disease virus and Omsk hemorrhagic fever virus. Significant taxonomic improvements are proposed, e.g. the identification of three major groups (mammalian, seabird and Kadam tick-borne flavivirus groups), the creation of a new species (Karshi virus) and the assignment of Tick-borne encephalitis and Louping ill viruses to a unique species (Tick-borne encephalitis virus) including four viral types (i.e. Western Tick-borne encephalitis virus, Eastern Tick-borne encephalitis virus, Turkish sheep Tick-borne encephalitis virus and Louping ill Tick-borne encephalitis virus). The analyses also suggest a complex relationship between viruses infecting birds and those infecting mammals. Ticks that feed on both categories of vertebrates may constitute the evolutionary bridge between the three distinct identified lineages.     

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.     

Experimental evidence that RNA recombination occurs in the Japanese encephalitis virus

Due to the lack of a proofreading function and error-repairing ability of genomic RNA, accumulated mutations are known to be a force driving viral evolution in the genus Flavivirus, including the Japanese encephalitis (JE) virus. Based on sequencing data, RNA recombination was recently postulated to be another factor associated with genomic variations in these viruses. We herein provide experimental evidence to demonstrate the occurrence of RNA recombination in the JE virus using two local pure clones (T1P1-S1 and CJN-S1) respectively derived from the local strains, T1P1 and CJN. Based on results from a restriction fragment length polymorphism (RFLP) assay on the C/preM junction comprising a fragment of 868 nucleotides (nt 10-877), the recombinant progeny virus was primarily formed in BHK-21 cells that had been co-infected with the two clones used in this study. Nine of 20 recombinant forms of the JE virus had a crossover in the nt 123-323 region. Sequencing data derived from these recombinants revealed that no nucleotide deletion or insertion occurred in this region favoring crossovers, indicating that precisely, not aberrantly, homologous recombination was involved. With site-directed mutagenesis, three stem-loop secondary structures were destabilized and re-stabilized in sequence, leading to changes in the frequency of recombination. This suggests that the conformation, not the free energy, of the secondary structure is important in modulating RNA recombination of the virus. It was concluded that because RNA recombination generates genetic diversity in the JE virus, this must be considered particularly in studies of viral evolution, epidemiology, and possible vaccine safety.     

CD8 T-cells mediate immunopathology in tick-borne encephalitis

Epidemics of tick-borne encephalitis involving thousands of humans occur annually in the forested regions of Europe and Asia. Despite the importance of this disease, the underlying basis for the development of encephalitis remains undefined. Here, we prove the key role of CD8⁺ T-cells in the immunopathology of tick-borne encephalitis, as demonstrated by prolonged survival of SCID or CD8^−/− mice, following infection, when compared with immunocompetent mice or mice with adoptively transferred CD8⁺ T-cells. The results imply that tick-borne encephalitis is an immunopathological disease and that the inflammatory reaction significantly contributes to the fatal outcome of the infection.     

Comparative mechanistic studies of de novo RNA synthesis by flavivirus RNA-dependent RNA polymerases

Flavivirus protein NS5 harbors the RNA-dependent RNA polymerase (RdRp) activity. In contrast to the RdRps of hepaci- and pestiviruses, which belong to the same family of Flaviviridae, NS5 carries two activities, a methyltransferase (MTase) and a RdRp. RdRp domains of Dengue virus (DV) and West Nile virus (WNV) NS5 were purified in high yield relative to full-length NS5 and showed full RdRp activity. Steady-state enzymatic parameters were determined on homopolymeric template poly(rC). The presence of the MTase domain does not affect the RdRp activity. Flavivirus RdRp domains might bear more than one GTP binding site displaying positive cooperativity. The kinetics of RNA synthesis by four Flaviviridae RdRps were compared. In comparison to Hepatitis C RdRp, DV and WNV as well as Bovine Viral Diarrhea virus RdRps show less rate limitation by early steps of short-product formation. This suggests that they display a higher conformational flexibility upon the transition from initiation to elongation.     

不同试剂盒对乙脑病毒RNA提取效能的比较

目的比较常见的3种商售RNA提取试剂对乙脑病毒检测的相对效率、耗时和成本,为实验室应用及检测结果的评价提供依据。方法对乙脑病毒参考株悬液作10^-1～10^-4系列稀释,选用QIAGEN公司的QIAamp Viral RNA Mini Kit、Sangon生物公司的RNA提取试剂盒和Invitrogen公司的Trizol试剂3种核酸提取方法提取上述样本RNA．用TaqMan实时荧光定量反转录聚合酶链反应对其提取效率进行评价,并比较3种试剂（盒）的耗费时间和价格。结果对不同稀释度乙脑病毒样本的检测,均以QIAGEN公司QIAamp Viral RNA Mini Kit检测的敏感度最高。以该试剂盒的提取效率为100％,则Invitrogen和Sangon生物公司RNA提取试剂（盒）的提取效率分别为77．4％～86．8％和64．2％～74．1％。3种试剂（盒）对cDNA起始模板量的估计也有明显的影响,差别达到1～3个数量级,以QIAGEN公司的试剂盒最敏感。3种试剂（盒）提取RNA耗时分别为60min、100min和70min,价格效率比分别为46元、23元和20元。结论RNA提取试剂可影响JEV荧光定量PCR检测的结果,各实验室应根据实验目的和实验室条件．合理选择病毒RNA提取试剂盒。