Laboratory of Public Health, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan1
Center for Information Biology, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan2
Plague Control Station of Khabarovsk, Khabarovsk 680311, Russia3
Public Health Interregional Association of Economic Interaction in Far East and Transbaical Area, Khabarovsk 680002, Russia4
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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It has been proposed that the TBE complex viruses evolved in a cline across the northern hemisphere from east to west (Zanotto et al., 1995 ). Using rates of nucleotide substitution estimated for these viruses, it has been suggested that the TBE complex viruses evolved during the last few thousand years (Zanotto et al., 1996
), reaching western Europe about 10002000 years ago. Louping ill virus, the most-recently emerged virus, appeared in the British Isles less than 800 years ago (McGuire et al., 1998
).
Until recently, no confirmed cases of TBE had been reported in Japan. However, in 1993 in Hokkaido, the northern-most island of Japan, a severe case of encephalitis was diagnosed as TBE. In addition, three TBE virus strains were isolated from the blood of sentinel dogs in the area and identified as far-eastern (RSSE) subtype virus (Takashima et al., 1997 ). Furthermore, four TBE virus strains were isolated from ticks and rodents (Takeda et al., 1998
, 1999
). These data show that TBE is endemic in a specific area of Hokkaido, Japan.
These data prompted further investigation of when these TBE viruses emerged in Hokkaido. To infer the time of virus emergence, we estimated the nucleotide substitution rate of far-eastern subtype TBE virus strains from Hokkaido and far-eastern Russia using viral envelope (E) protein gene nucleotide sequences and predicted the lineage divergence times of these virus strains. The flavivirus E gene sequence has been shown to be a good indicator for predicting phylogenetic lineage divergence times (Zanotto et al., 1996 ; McGuire et al., 1998
). First, we isolated TBE virus from ticks collected in Khabarovsk, far-eastern Russia, in 1998. Then we determined the nucleotide sequences of the E protein genes of TBE virus strains that included the newly isolated Khabarovsk strains and Hokkaido strains isolated previously from the blood of dogs, ticks and rodents in Oshima, Hokkaido, in 1995 and 1996. For phylogenetic comparison and estimation of lineage divergence times, the E protein gene sequences of these strains were compared with the prototype RSSE virus strain Sofjin, isolated in 1937 in the far east of Russia. Furthermore, the virulence of TBE virus isolates from different hosts in Hokkaido and Khabarovsk was compared by using mice to estimate their pathogenic potential.
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Methods |
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Virus isolates were identified by indirect immunofluorescent antibody (IFA) test with MAbs as described previously (Guirakhoo et al., 1989 ; Holzmann et al., 1992
; Takashima et al., 1997
). Briefly, samples of suckling mouse brain were made into a 10% suspension. The suspension was inoculated onto BHK cell monolayers and incubated at 37 °C for 3 days. The cell monolayer was trypsinized and the cell suspension was mounted on a multiwell slide. After incubation at 37 °C for 1 h, the slides were fixed with cold acetone and used as antigen slides.
Virus and cells.
Virus strains used in this study are shown in Table 1. The geographical origin, year of isolation and source of isolate of each strain is also described. Six strains were isolated previously from different sources in the area where a TBE patient was found, in Oshima district, Hokkaido (Fig. 1
). The other four strains newly isolated from Khabarovsk are described in this study. Virus strains other than Sofjin were passaged three times or fewer through suckling mice. The Sofjin strain used in this study was an isolate obtained in Khabarovsk in 1937 (Zilber & Soloviev, 1946
; G. Leonova, personal communication). The Sofjin strain has been kept lyophilized for over 30 years and has undergone about 10 suckling-mouse passages.
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Virulence comparison of TBE virus strains.
The virulence of virus strains was compared in 8-week-old male ICR mice. Eight mice in each group received 1000 focus-forming units (f.f.u.) of virus subcutaneously or 10 f.f.u. of virus intracerebrally and were observed for 28 days to obtain the survival curve.
Determination of the sequence of the viral E protein gene.
The nucleic acid sequence of the viral E protein gene was determined from 11 strains of TBE virus, six from Hokkaido and five from Khabarovsk. The sequence of strain Sofjin has already been determined (Pletnev et al., 1990 ; GenBank accession no. X07755). However, the virus used for that study had been passaged about 100 times in mice and cells. In order to obtain a more accurate sequence of the original Sofjin strain, we determined the sequence of the Sofjin-HO strain kept in our laboratory, with a 10-suckling-mouse passage history.
Viral RNA was extracted from brains of virus-inoculated suckling mice or virus-infected BHK cells by using the Isogen Kit (Nippon Gene). For cDNA synthesis, PCR was performed as described previously (Takashima et al., 1997 ). The cycle sequencing reaction was performed by using a DNA sequencing kit (ABI PRISM) and the sequence was determined by fluorescence autosequencer (ABI PRISM 310 Genetic Analyser). The nucleotide sequence data have been deposited in the DDBJ, EMBL and GenBank nucleotide sequence databases under the accession numbers shown in Table 1
.
Phylogenetic analysis.
Phylogenetic analysis was carried out on the complete E gene sequences of TBE virus subgroup strains including strains isolated in Hokkaido and Khabarovsk and other virus strains (accession numbers of these sequences are shown in Fig. 4). OHF virus was used as the outgroup. The complete nucleotide sequences (1488 bp) of the E protein genes of TBE virus strains were aligned and synonymous distances between these sequences were estimated by the method of Nei & Gojobori (1986)
. A phylogenetic tree was constructed by using the neighbour-joining method (Saitou & Nei, 1987
) and bootstrap resampling (10000 replications) was used to assess the robustness of the groupings obtained (Felsenstein, 1985
).
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Results |
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The amino acid alignment also revealed four amino acids that are unique to western European TBE virus isolates, supporting the clinal concept of tick-borne virus evolution across Eurasia (Zanotto et al., 1995 ). Their respective positions are 88 (G), 206 (V), 317 (A) and 407 (K).
Phylogenetic analysis
A phylogenetic tree constructed from synonymous distances of TBE serocomplex viruses is shown in Fig. 4. Virus strains isolated in Hokkaido and Khabarovsk, including Sofjin-HO virus, form a cluster with 100% bootstrap support. Thus, these virus strains were classified as far-eastern subtype TBE virus, since strain Sofjin has been proposed as the prototype of the far-eastern subtype (Ecker et al., 1999
). The branching pattern of the phylogenetic tree (Fig. 4
) clearly distinguishes the far-eastern subtype TBE complex viruses from the Siberian subtype Vasilchenko and also the European and UK TBE complex viruses. The tree also shows that two independent but closely related lineages have developed following the introduction of virus into far-eastern Russia: the Khabarovsk lineages, including Sofjin virus, which became established in the far-eastern part of Russia, and the Hokkaido lineage, consisting of the Oshima strains, which was subsequently introduced into Japan. In view of the fact that the far-eastern subtype is distributed over a very wide geographical area of Russia and is distinguishable from Siberian subtype TBE viruses such as Vasilchenko (Ecker et al., 1999
), it seems reasonable to assume that the new Oshima viruses dispersed in an eastward direction from Russia to Japan rather than from Japan to Russia.
Times of divergence of far-eastern subtype TBE virus
A phylogenetic tree constructed from synonymous distances of KH98-2, -5 and -8, Sofjin-HO and Oshima strains and the Vasilchenko strain as the outgroup is shown in Fig. 5. The tree shows that strains from far-eastern Russia and strains from Hokkaido form distinct clusters with good bootstrap support, 89 and 95%, respectively. We estimated the synonymous substitution rates of these strains. The average synonymous substitution rate of these strains was estimated to be 2·9x10-4 per site per year. The time of divergence of the Khabarovsk and Hokkaido strains was calculated by using the average substitution rate and synonymous distances shown in Fig. 5
. The result showed that these virus strains had diverged approximately 260430 years ago. Therefore, since the Hokkaido strains are genetically distinct from the far-eastern subtype TBE viruses, they may have dispersed quite soon after diverging from the far-eastern subtype viruses. By using the above synonymous substitution rates and synonymous distances shown in Fig. 4
, the time of divergence of the Vasilchenko strain was estimated. The result showed that the Vasilchenko Siberian subtype TBE virus and the far-eastern subtype TBE virus diverged approximately 17002100 years ago. It must be acknowledged that the estimates of virus emergence times can only be approximate: nevertheless, they are consistent with previous estimates for these viruses.
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Discussion |
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The next question was the means by which the virus was introduced into Hokkaido. One possibility is that migratory birds may have transported infected ticks from far-eastern Russia to Hokkaido. I. persulcatus, the major vector of far-eastern subtype TBE virus (Korenberg, 1976 ; Korenberg et al., 1992
), was detected on several migratory bird species captured on the sea-coast located along the migratory route in Hokkaido (Miyamoto et al., 1993
). A second possibility is that small reservoir mammals infected with TBE virus might have emigrated from the Eurasian continent to Hokkaido. The Japanese islands, which were once connected to the Asian continent, have been separated for at least 10000 years. Therefore, if the Hokkaido TBE complex viruses diverged from the far-eastern strains over the last 200400 years, the reservoir animals would need to have been transported to Japan.
It is also of interest how the TBE virus has adapted to the natural environment and formed a transmission cycle in Hokkaido. Our past epidemiological survey combined with the results of this report suggest that the virus can switch vector tick species easily and adapt to different ecological niches within only a few hundred years. The vector tick species of TBE virus was revealed to be I. ovatus by virus isolation results at Oshima in Hokkaido, where a TBE patient was found. In far-eastern Russia, I. persulcatus is the main tick vector of TBE virus and Haemaphisalis concinnae is a second important tick vector (Korenberg, 1976 ; Korenberg et al., 1992
). In the Oshima district, I. ovatus is a major tick species and I. persulcatus has rarely been collected (Takeda et al., 1998
).
TBE virus Oshima strains isolated from different hosts (dog, tick and rodent) showed similar neuroinvasiveness when tested in the mouse model. In contrast, Khabarovsk strains exhibited different virulence: strain KH98-5 was more neuroinvasive than strain KH98-10. Variations in virulence were reported among TBE virus strains isolated from I. persulcatus at different localities in Russia. Homogeneity of neuroinvasiveness among the Oshima strains might be due to the very limited range of the study area for collection of virus. The Sofjin strain was reportedly isolated from the brain of a patient in far-eastern Russia and our previous study showed higher virulence of this strain than of Oshima 5-10 (Chiba et al., 1999 ). By bringing the previous and present results together, the degree of neuroinvasiveness of TBE strains is Sofjin>Oshima=KH98-5>KH98-10. It is conceivable that various virus strains of different neuroinvasiveness are circulating in the natural virus foci and that highly neuroinvasive virus strains may cause severe encephalitis in humans.
The E protein of flaviviruses appears to be an important determinant of neurovirulence and neuroinvasiveness and to have a major role in the pathogenesis of encephalitis (Holzmann et al., 1990 , 1997
; McMinn, 1997
). However, differences in neuroinvasiveness observed between the Khabarovsk isolates KH98-5 and KH98-10 could not be attributed to amino acid changes in the E proteins of the two isolates. There were only two amino acid differences (positions 153 and 205) between the E proteins of these isolates and both changes (valine to alanine, arginine to lysine) are considered to be conservative (Wallner et al., 1996
). Furthermore, the amino acid substitutions in KH98-10 (position 153, alanine; 205, lysine) were also identified in the Oshima strains, which showed similar virulence to KH98-5. Therefore, other regions of the genome than the E protein may influence virulence.
The average synonymous substitution rate of far-eastern TBE viruses from Hokkaido and far-eastern Russia was estimated to be 2·9x10-4 per site per year, which is similar to the rate estimated previously for louping ill virus (5·68±2·54x10-4 per site per year; McGuire et al., 1998 ). It is considered that TBE viruses evolve at a lower substitution rate than other RNA viruses such as influenza virus (13·10x10-3 per site per year), human immunodeficiency virus (13·08x10-3 per site per year) and hepatitis C virus (6·29x10-3 per site per year), which evolve without arthropod vectors (Gojobori et al., 1990
; Ina et al., 1994
). The proposed reason for this is that TBE virus evolves within the constraining effects imposed by the two-host system. Furthermore, it has been suggested that tick-borne flaviviruses evolve at 0·56 times the rate of mosquito-borne flaviviruses, because of the long life-cycle of the ticks and low virus titres in ticks as compared with mosquitoes (Zanotto et al., 1996
; Gould et al., 1997
).
It has been suggested that TBE virus has evolved in a cline across the Eurasian continent in a westerly direction in the last few thousand years (Zanotto et al., 1996 ). The divergence of far-eastern TBE virus strains and the Vasilchenko strain was estimated at approximately 17002100 years ago. From Fig. 4
, it was recognized that western TBE virus strains and far-eastern TBE virus strains had diverged before the far-eastern and Siberian subtype TBE virus strains diverged. Consequently, TBE virus might have evolved in far-eastern Russia earlier than 17002100 years ago, which agrees with a previous suggestion that TBE virus has diverged continuously over the last 2000 years.
Finally, an interesting observation was made from the amino acid alignment. The presence of the amino acid signature GVAK for the western European TBE viruses is significant for two independent reasons. Firstly, this group of four amino acids identifies and distinguishes western European TBE viruses from all other flaviviruses. Secondly, the amino acid signature GVAK implies a clonal evolutionary introduction of western European TBE viruses into this geographical region.
This report provides evidence that the Oshima and Khabarovsk TBE virus strains emerged a few hundred years ago in far-eastern Russia. It seems likely that the Oshima strains were then dispersed to Japan either by birds carrying infected ticks or by rodents or other animals transported on ships. The similarity in virulence for mice between the Oshima and Khabarovsk strains supports this suggestion. Further studies on viruses isolated at different localities in Hokkaido and far-eastern Russia, together with analysis of phylogeny and associated environmental factors, should define more precisely the origin of the Oshima TBE complex viruses.
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Acknowledgments |
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Footnotes |
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References |
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Received 7 May 1999;
accepted 2 August 1999.