Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan1
Plaque Control Station of Khabarovsk, Ministry of Health of the Russian Federation, Chabarovsk 680311, Russia2
Institute of Epidemiology and Microbiology, Academy of Medical Sciences, Siberian Branch, Vladivostok 690028, Russia3
Author for correspondence: Ikuo Takashima. Fax +81 11 706 5211. e-mail takasima{at}vetmed.hokudai.ac.jp
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Abstract |
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Introduction |
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Based on geographical origin and antigenic characteristics, TBE viruses were originally subdivided into two subtypes, far-eastern and European. The far-eastern subtype virus is known as Russian springsummer encephalitis (RSSE) virus and its main vector is the tick Ixodes persulcatus. The Sofjin strain, isolated in Primorsky, is regarded as the prototype virus of the far-eastern TBE virus subtype. The European subtype virus is known as Central European encephalitis (CEE) virus and the tick Ixodes ricinus is its main vector. The Neudoerfl strain, isolated in Austria, is regarded as the prototype virus of the European subtype. It is known that in far-eastern Russia, fatality rates of RSSE cases range from 5 to 20%, whereas fatality rates of CEE cases range from 0·5 to 2·0% in western European countries (Dumpis et al., 1999 ).
Based on phylogenetic analysis, a third subtype was identified recently in Siberia (Gritsun et al., 1993 ; Ecker et al., 1999
; Heinz et al., 2000
). The Vasilchenko strain, isolated in Novosibirsk from a human with non-paralytic febrile illness, is regarded as the prototype virus of this subtype. Other than strain Vasilchenko, only two other strains, Aina and Latvia-1-96, were classified as Siberian subtype viruses (Ecker et al., 1999
; Mavtchoutko et al., 2000
). This information prompted our study into the different TBE virus strains, the natural foci of which are in Siberia and far-eastern Russia.
The TBE virus genome (single-stranded positive-sense RNA of approximately 11 kb) encodes three structural proteins (capsid protein C, membrane precursor protein prM and envelope protein E) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) within a single long open reading frame (Chambers et al., 1990 ). The 5' and 3' non-coding regions (NCRs) have a predicted secondary structure that contains elements important for virus replication, translation and packaging of the genome (Chambers et al., 1990
; Gritsun et al., 1997
; Mandl et al., 1998
).
With respect to strain characterization, the various lengths of the 3'NCRs of TBE virus strains have been reported. Variation in the length of the 3'NCR does not appear to correspond with subtype, geographical origin, isolation source or isolation year (Wallner et al., 1995 ). It was demonstrated that spontaneous deletions in the 3'NCR occurred during propagation in either cell lines or suckling mice (Mandl et al., 1998
). Therefore, these deletions may have occurred during passage in the laboratory. To verify this hypothesis, it is important to examine the 3'NCR of virus isolates that have a short passage history in the laboratory.
In this study, we isolated TBE viruses from Siberia (Irkutsk) and far-eastern Russia (Vladivostok and Khabarovsk) in 1999 to determine virus subtype distribution. TBE virus isolates were classified into subtypes by phylogenetic analysis and antigenic characteristics were examined using monoclonal antibodies (MAbs). Furthermore, the virulence of these isolates was compared in a mouse model. TBE virus strains used in this study, including the Japanese and Khabarovsk isolates reported in a previous study (Hayasaka et al., 1999 ), were passaged only a few times after their isolation in the field. We sequenced the 3'NCR of these new isolates to ascertain the characteristics of the 3'NCR in viruses circulating in natural foci.
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Methods |
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Virus and cells.
Virus strains used in this study are shown in Table 1. The year of isolation, geographical origin and source of isolation of each strain are described. Strains VL99-m11, KH99-m9, D1283 and IR99 are newly isolated strains from this study. Oshima, KH98 and Sofjin (Sofjin-HO) strains were described in previous studies (Takashima et al., 1997
; Hayasaka et al., 1999
). The Hochosterwitz strain of TBE virus was provided by Franz Heinz, University of Vienna, Austria (Heinz & Kunz, 1981
). Sequence data from the Sofjin and Vasilchenko strains were referred to in previous studies (Pletnev et al., 1990
; Gritsun et al., 1993
, 1997
).
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Indirect immunofluorescent antibody (IFA) test.
Virus isolates were identified by IFA testing using MAbs 1H4, 4H8, 2F9, 5D10, 6E2, 2E7, 7G7 and 1C3 specific for the TBE virus E protein (Komoro et al., 2000 ; Guirakhoo et al., 1989
; Holzmann et al., 1993
). Briefly, the brains of infected suckling mice were removed and homogenized into a 10% suspension. The suspension was inoculated onto BHK cell monolayers and incubated at 37 °C for 3 days. The cell monolayer was then trypsinized and the cell suspension was mounted onto a multiwell slide. After incubation at 37 °C for 1 h, slides were fixed with cold acetone for 20 min. Slides were then incubated with MAbs at 37 °C for 1 h and washed with PBS. Fluorescein isothiocyanate-conjugated antibody to mouse IgG was added to the slides and incubated at 37 °C for 1 h. After washing with PBS, the slides were observed under a fluorescence microscope. The IFA titre was determined to be the highest dilution of the MAb that showed a positive fluorescent reaction.
Determination and analysis of the TBE viral genes.
The nucleic acid sequences of the viral gene encoding the E protein and the 3'NCR were determined by direct sequencing of RTPCR products. Viral RNA was extracted using the Isogen kit (Nippon Gene) from brains of infected suckling mice (one passage). RTPCR was performed using the Thermoscript RTPCR system (Gibco BRL) and the cycle sequencing reaction was performed by using a DNA Sequencing kit (ABI PRISM). The DNA sequence was determined with a fluorescence autosequencer (ABI PRISM 310 Genetic Analyzer). Primers HO1 for RT reaction and HO2 for PCR of the 3'NCR were designed according to a previous report (Wallner et al., 1995 ). All nucleotide sequence data generated from this study have been deposited in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession numbers shown in Table 1
.
Sequence alignment and construction of the phylogenetic tree were carried out with GENETYX-MAC version 10 (Software Development). The phylogenetic tree was constructed by using the neighbour-joining method and bootstrap resampling (10000 replications) on the complete nucleotide sequences (1488 bp) of the E protein gene of TBE virus strain sequences taken from the DDBJ/EMBL/GenBank databases.
Comparison of TBE virus strain virulence.
The virulence of virus strains was compared in 8-week-old male ICR mice (SLC) with body weights of about 3035 g. Ten mice in each group received either 1000 focus-forming units (f.f.u.) of virus subcutaneously or 10 f.f.u. of virus intracerebrally. The survival of mice was observed and recorded for 28 days post-infection (p.i.) to obtain the survival curve. Animals were infected and handled under P3 containment conditions.
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Results |
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Antigenic characterization of TBE virus isolates
The antigenicity of TBE virus strains was examined by IFA testing using various MAbs (Table 2). MAbs 4H8, 5D10 and 6E2 (flavivirus group-specific) reacted to all virus strains, including Langat virus TP-21 and Japanese encephalitis virus (JEV) JaGAr-01. MAbs 1H4 and 2E7 (tick-borne flavivirus complex-specific) reacted with all virus isolates in this study, including the Hochosterwitz (Heinz & Kunz, 1981
) and Langat virus TP-21 strains. MAb 2F9 was identified as TBE virus type-specific (Komoro et al., 2000
) and reacted with all isolates in this study, including the Hochosterwitz strain, isolated in Austria. Therefore, these isolates were all antigenically identified as TBE viruses. However, MAbs 7G7 and 1C3 did not react with the virus strains isolated in Irkutsk, implying that these strains have amino acid changes located in the epitopes for which these MAbs are specific.
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Discussion |
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From the phylogenetic tree (Fig. 2), it was noted that the Siberian subtype strains were more closely related to far-eastern subtype strains than to European subtype strains. Therefore, it was estimated that the Siberian and far-eastern subtype viruses had diverged later than the European subtype viruses and that their ancestor virus had also diverged. The divergence time of the Siberian and far-eastern subtypes was calculated to be approximately 17002100 years ago (Hayasaka et al., 1999
). It has been suggested that the TBE virus has evolved in a cline across the Eurasian continent (in a westerly direction) in the last few thousand years (Zanotto et al., 1995
). The result of this study is not contradictory to the theory that TBE virus has diverged continuously over the last 2000 years.
In the cluster of Siberian subtypes, strains have diverged further into two subclusters (Fig. 2). However, these subclusters did not reflect the virus isolation points. For example, two isolates from Irkutsk region isolation point 1 (IR99-1m1 and -1m4) were in different subclusters. Shifting of TBE viruses may occur between the points of TBE virus foci. In contrast, Oshima strains isolated in Hokkaido formed a subcluster that was clearly distinguishable from the Irkutsk strains.
In this study, Siberian subtype viruses (Irkutsk isolates) were isolated from I. persulcatus ticks, which suggests that I. persulcatus is a tick vector for TBE viruses in the Siberian region. Previous studies have not included vector tick information, as the Siberian subtypes (Vasilchenko, Aina and Latvia-1-96 strains) used in other studies were isolated from either human blood or brain tissue.
There was a minor difference in the antigenicity of the TBE virus isolates, as revealed by the MAb reactivities between the Irkutsk strains and other TBE viruses. MAbs 7G7 and 1C3 did not react with the Irkutsk isolates. These antibodies react to a synthetic peptide corresponding to amino acid positions 221240 of the TBE virus E protein (Holzmann et al., 1993 ). The domain spanned by this synthetic peptide covers the amino acids at positions 232234, a region that is proposed to be a flavivirus type-specific hypervariable domain. As the Irkutsk isolates (Siberian subtype) had an amino acid change at position 234, it is conceivable that this mutation resulted in the loss of MAb 7G7 and 1C3 reactivity. This result is in agreement with the previous observation that MAbs 7G7 and 1C3 do not react with the Hypr strain (European subtype), which has a mutation at amino acid position 233 of the E gene (Wallner et al., 1996
). Furthermore, amino acid alignment of the E gene showed that the amino acid at position 234 (Q or H) is a signature amino acid for Siberian subtype TBE viruses, which may be used to easily identify Siberian TBE virus subtypes.
Irkutsk isolates showed equivalent or somewhat stronger virulence compared with far-eastern isolates in the mouse model. It has been shown that Siberian subtype strains (Vasilchenko and Latvia-1-96) possess weaker pathogenicity compared with the far-eastern and western subtype viruses in animal model experiments (Asher, 1979 ; Frolova et al., 1982
; Mavtchoutko et al., 2000
). TBE viruses distributed in the Irkutsk region may be as virulent as the subtypes isolated in the far-eastern regions. The observation that the Vasilchenko and TBE-Latvia-1-96 strains were less virulent may be due to the difference in the animal species used for examination (rhesus monkey and Syrian hamster) or to the geographical difference in isolation points.
It is a unique and interesting observation that all new TBE virus isolates in this study had 3'NCRs of almost the same length. The 3'NCR includes the entire core element and a variable region of approximately 380 nucleotides (Fig. 5). The Neudoerfl strain had an extra long poly(A) sequence (Mandl et al., 1991
, 1998
). The isolates Oshima, KH, VL and IR used in this study were passaged only once or twice after isolation from field sources (ticks, rodent spleens, dog blood and human brains). Therefore, it is likely that deletions or additions to the genome did not occur during laboratory passage and that the sequences reflect the properties of the viruses in nature. The deletions that are observed in TBE virus strains have occurred in the variable region located between the open reading frame and the 3'-terminal core element. In fact, engineered mutants which lack the entire variable region did not exhibit any change in virus propagation characteristics or plaque morphology in cultured cells compared to the parent virus (Mandl et al., 1998
). It is considered that the variable region is not necessary for functions relevant to virus growth in such cell lines.
However, the results in this study suggest that TBE viruses in nature need a 3'NCR that includes both a core element and a variable region. It is thought that the 3'NCR RNA of flaviviruses forms the specific secondary structure of stems and loops (Proutski et al., 1997 ). The 3'NCR secondary structure plays an important role in viral RNA replication, behaving as the cis-acting signals for the initiation of transcription (You & Padmanabhan, 1999
) and the specific binding site recognized by viral and cellular proteins (Chambers et al., 1990
; Chen et al., 1997
; Blackwell & Brinton, 1995
, 1997
; Ta & Vrati, 2000
). Several cellular host proteins have been shown to interact with the 3'NCR of flaviviruses. Elongation factor-1
binds to the 3'NCR of West Nile virus (Blackwell & Brinton, 1995
, 1997
) and the Mov34 protein binds to the 3'NCR of JEV (Ta & Vrati, 2000
). The secondary structure in the 3'NCR core element of TBE viruses has been predicted and it was suggested that these structural elements may be needed for virus replication (Mandl et al., 1998
; Gritsun et al., 1997
; Proutski et al., 1997
, 1999
). Conversely, the variable region does not have any relevant functions for virus growth in laboratory passages. All of the TBE virus isolates in this study exhibited almost the same nucleotide sequence length in the 3'NCR, without deletion. This may suggest that both the core element and the variable region (without deletion at the 3'NCR) are needed to form the specific secondary structure that is essential for TBE virus growth in tick (vector) and/or rodent (reservoir) host cells.
In this study, it was shown that Siberian subtype TBE viruses are distributed in the Irkutsk region. Furthermore, it was observed that these viruses have the same virulence as the far-eastern subtype viruses in the mouse model and have only minor antigenic differences.
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Acknowledgments |
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References |
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Received 2 November 2000;
accepted 23 February 2001.