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.     

Molecular epidemiology of tick-borne encephalitis virus in Ixodes 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.     

Acute West Nile virus neuroinvasive infections: cross-reactivity with dengue virus and tick-borne encephalitis virus

Cross-reactions in serology are common among flaviviruses. During the outbreak of West Nile virus (WNV) infections in Greece in 2010, WNV IgM-positive serum and cerebrospinal fluid samples were tested for the presence of IgM and IgG antibodies against Dengue virus (DENV) and tick-borne encephalitis virus. Higher cross-reactivity was observed in IgM antibodies between WNV and DENV; however, the index of the WNV antibodies was in all cases higher than that of the DENV antibodies. There is a need for caution when evaluating serologic results of flaviviral infections, while efforts have to be focused on the development of diagnostic assays with increased specificity.     

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.     

Phylogenetic and virulence analysis of tick-borne encephalitis virus field isolates from Switzerland

Tick-borne encephalitis (TBE) is an endemic disease in Switzerland, with about 110-120 reported human cases each year. Endemic areas are found throughout the country. However, the viruses circulating in Switzerland have not been characterized so far. In this study, the complete envelope (E) protein sequences and phylogenetic classification of 72 TBE viruses found in Ixodes ricinus ticks sampled at 39 foci throughout Switzerland were analyzed. All isolates belonged to the European subtype and were highly related (mean pairwise sequence identity of 97.8% at the nucleotide and 99.6% at the amino acid level of the E protein). Sixty-four isolates were characterized in vitro with respect to their plaque phenotype. More than half (57.8%) of isolates produced a mixture of plaques of different sizes, reflecting a heterogeneous population of virus variants. Isolates consistently forming plaques of small size were associated with recently detected endemic foci with no or only sporadic reports of clinical cases. All of six virus isolates investigated in an in vivo mouse model were highly neurovirulent (100% mortality) but exhibited a relatively low level of neuroinvasiveness, with mouse survival rates ranging from 50% to 100%. Therefore, TBE viruses circulating in Switzerland belong to the European subtype and are closely related. In vitro and in vivo surrogates suggest a high proportion of isolates with a relatively low level of virulence, which is in agreement with a hypothesized high proportion of subclinical or mild TBE infections.     

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.     

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.     

Changes in immune parameters and their correction in human cases of tick-borne encephalitis

Tick-Borne Encephalitis virus (TBEV) causes dangerous central nervous system diseases in humans. General infection leads to the development of meningitis or encephalitis, which is characterized by swelling of the brain due to inflammation. Tetracyclines may act locally to moderate inflammation in the CNS. In this study, we investigated the potential clinical benefits of administering tetracycline hydrochloride to patients hospitalized due to suspected TBEV infection presenting with fever and evidence of a recent tick bite. We also characterized an acute immune response to TBEV by profiling certain cytokines and soluble receptors in Tetracycline-treated and untreated patients. Increased serum levels of TNF-alpha, IL-1 alpha and IL-6 were found in all patients at admission. Soluble receptors presented in the serum of patients in a magnitude higher levels than the corresponding cytokines and were increasing during first weak of hospitalization. Levels of IL-10 were also rising during that period. In our study tetracycline hydrochloride acted as an immunomodulator, which was able to reduce manifestations of inflammation response during TBE course; this action led to quicker improvement of symptoms and, consequently, to a faster clinical recovery. The positive result of tetracycline hydrochloride treatment was accompanied by certain particularities in the dynamics of studied cytokines and receptors: the concentrations of IL-6, IL-1 beta, TNF-alpha dropped quicker and reached lower levels, and the concentrations of sIL-6R, IL-1RA, sTNFR1 increased faster and reached higher maximum levels in the tetracycline-treated groups. Children had the highest levels of IL-6, which were not neurotoxic.     

Prevalence of tick‐borne encephalitis virus in Ixodes ricinus ticks from three islands in north‐western Norway

Tick‐borne encephalitis (TBE) is the most important viral tick‐borne disease in Europe and can cause severe disease in humans. In Norway, human cases have been reported only from the southern coast. The aim of this study was to investigate the prevalence of tick‐borne encephalitis virus (TBEV) in questing Ixodes ricinus ticks from the north‐western part of Norway. A total of 4509 ticks were collected by flagging in May and June 2014. A subpopulation of 2220 nymphs and 162 adult ticks were analysed by real‐time PCR and positive samples were confirmed by pyrosequencing. The estimated prevalence of TBEV was 3.08% among adult ticks from Sekken in Møre og Romsdal County and 0.41% among nymphs from both Hitra and Frøya in Sør‐Trøndelag County. This study indicates that TBEV might be more widespread than the distribution of reported human cases suggests.     

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.     

Molecular evidence for potential transovarial transmission of Jingmen tick virus in Haemaphysalis longicornis fed on cattle from Yunnan Province,China

Jingmen tick virus (JMTV) is a novel tick-borne virus first identified from Jingmen city, Hubei Province of China in 2010. It has been proved that JMTV can cause human diseases and is widely distributed both inside and outside of China. However, the survival mode and transmission characteristics of JMTV still need further research, particularly in terms of transovarial transmission. In this study, an investigation was conducted to explore the presence of JMTV from engorged female ticks to their offspring. All engorged female adult ticks were collected from domestic cattle and allowed to lay eggs in appropriate humidity and temperature conditions. Maternal ticks, eggs and larvae were screened for JMTV RNA through real-time polymerase chain reaction (RT-PCR) and nested PCR methods. The results revealed the positive rate of 10.53% (10/95) in engorged ticks, 9.09% (2/22) in eggs and 8% (4/50) in larvae pools, respectively. Phylogenetic analysis confirmed that sequences from eggs and larvae had closer relationship with those isolates from maternal engorged ticks with more than 99.7% homology and JMTV manifested with evolutional conservatism. Our study has identified for the first time that JMTV could be transmitted from mother generation to offspring of Haemaphysalis Longicornis. Nonetheless, the efficiency of transovarial transmission in JMTV and the significance of ticks as amplification hosts still need to be further illustrated.     

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.     

Mutation analysis of the fusion domain region of St. Louis encephalitis virus envelope protein

The immune response to flavivirus infections produces both species-specific and flavivirus cross-reactive antibodies. The presence of cross-reactive antibodies complicates serodiagnosis of flavivirus infections, especially secondary infections caused by a heterologous virus. A successful public health response to the growing global threat posed by flaviviruses necessitates the development of virus-specific diagnostic antigens. The flavivirus envelope (E) glycoprotein is the principle antigen stimulating protective immunity during infection. Using recombinant St. Louis encephalitis virus-like particles (VLPs), we have identified amino acid residues involved in flavivirus cross-reactive epitope determinants. Most significant among the residues studied are three highly conserved amino acids in the fusion peptide: Gly104, Gly106, and Leu107. Substitutions of these residues dramatically influenced VLP secretion and cross-reactive monoclonal antibody reactivity. These results provide critical insight into the antigenic structure of the flaviviral E protein and toward development of species-specific diagnostic antigens that should improve both flavivirus diagnosis and estimates of disease burden.     

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.     

Phylogenetic analysis of North American West Nile virus isolates, 2001-2004: evidence for the emergence of a dominant genotype

The distribution of West Nile virus has expanded in the past 6 years to include the 48 contiguous United States and seven Canadian provinces, as well as Mexico, the Caribbean islands, and Colombia. The suggestion of the emergence of a dominant genetic variant has led to an intensive analysis of isolates made across North America. We have sequenced the pre-membrane and envelope genes of 74 isolates and the complete genomes of 25 isolates in order to determine if a dominant genotype has arisen and to better understand how the virus has evolved as its distribution has expanded. Phylogenetic analyses revealed the continued presence of genetic variants that group in a temporally and geographically dependent manner and provide evidence that a dominant variant has emerged across much of North America. The implications of these findings are discussed as they relate to transmission and spread of the virus in the Western Hemisphere.     

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.     

Genomic sequencing of Highlands J virus: A comparison to western and eastern equine encephalitis viruses

Highlands J virus (HJV) is a member of the genus Alphavirus, family Togaviridae. HJV is the sole representative of the western equine encephalitis (WEE) serocomplex found in the eastern United States, and circulates in nature in an apparently identical transmission cycle as eastern equine encephalitis virus (EEEV). North American representatives of the WEE serocomplex [HJV, WEE virus (WEEV), and Fort Morgan virus (FMV)] are believed to be derived from a recombination event involving EEEV and a Sindbis (SIN)-like virus, such that the nonstructural polyprotein, the capsid, and the terminal end of the 3′ UTR are derived from EEEV, while the surface glycoproteins (E1 and E2) and small peptides (E3 and 6K) encoded in the subgenomic RNA are derived from the SIN-like virus. In this report, the complete nucleotide sequence of HJV is described, along with a comparative analysis of the HJV nonstructural polyprotein to WEEV and EEEV.