Jpn. J. Infect. Dis., 54, I‑ll, 2001

Review

Epidemiology of Tick‑Bone Encephalitis (TBE) and Phylogenetic●

AnalysIS OfTBE Ⅵmses in Japan and Far Eastem Russia

lkuo Takashima*, Daisuke Hayasaka, Akiko Goto, Hiroaki Kariwa and Tetsuya Mizutani
LabwatoヮofPublic Health. Departmen i ofEn viT10nmen tat Veterinayy Sciences.

Graduate School of Veterinary Medicine. Hokkaido Universio!.
Kita‑18, Nishi‑9, Kita‑ku, Sapporo 060‑0818, Japan(Received January 30, 2001. Accepted March I, 2001)
CONTENTS:
Summarylntroduction

Epidemiology ofTBE and isolation of TBE virus in Hokkaido, Japanl. Climical findings and virus isolation
2. Genetic characterization ofthevirus isolates
Phylogenetic analysis ofTBE viruses from Japan and Far Eastem Russia
l. Relatedness ofTBE virus, Hokkaido strain and Far Eastem Russian strains

2. Divergence ofTBE virus, Hokkaido strain from Khabarovsk strains

Pathogenicityof TBE virus, Hokkaido strain and protection from infection by Europeanvaccinel ･ Pathogenicityof the Hokkaido isolates
2. Evaluation or European T良E vaccine

SUMMARY: In Oshima, the southem part of Hokkaido, a tick‑bone encephalitis (TBE) patient was found in
1993; in addition TBE virus was isolated from the blood samples of sentinel dogs, ticks pools, and rodents

spleens in 1 995 and 1 996 by suckling mice･ To identifywhen these TBE viruses emerged in Hokkaido, the timesof divergence of TBE vims strains isolated in Oshima and Far Eastem Russla Were estimated. TBE vims was

isolated in Khabarovsk in 1998, and the nucleotide sequences of viral envelope protein genes of isolatesfrom

Oshima and Khabarovsk were compared. Based on the synonymous substitution rates of these virus E‑protein
genes, the lineage‑divergence times of these TBE virus strains were predicted phylogenetically to be approximately

260‑430 years ago. Furthemore, the virulence ofTBE virus isolatesfrom Oshima and Khabarovsk were comparedin a mouse model. The results showed that the isolates possessed very slmilarvirulence in mice. European TBE

vaccine was found to be effective in TBE virus, Hokkaido strain. This review provides evidence that the Oshima
strains ofTBE virus in Hokkaido emerged丘om the Far Eastem Russia a few hundred years ago, which explains

why the vimlence of these strains is similar to that ofTBE vinlSeS isolated in Russia. Practical application of thevaccine should be considered in J叩an.

Introduction

Virions are 40150 nm in diameter, spherical in sh叩e, andcontain a core and an envelope. The genome is a slngle positive‑

Tick‑borne encephalitis (TBE) is a zoonotic arbovirus

strand RNA (approximately I 1 ‑Kb in the length) encoding

infection prevalent over a wide area on the EurasianContinent

three structural proteins (capsid protein C, membrane precursor

including many European countries, Russia, Far EastemAsia,
and Japan (6,15)･ TBE virus causes sever.e encephalitis inprotein prM, and envelope protein E)and seven non‑structuralproteins (NSl, NS2A, NS28, NS3, NS4A, NS4B, NS5)

humans with serious sequelae and has a slgnificant impact

Within a single long open reading frame. It is believed thaton public health in these endemic reglOnS.

the 5'‑ and 3 '‑ termini of the genome (5'‑ and 3'‑non‑coding

TBE virus is a member of the family Flaviviridae, genus
FIavivirus. The genus Flavivirus consists pnmarily ofarbo‑

regions 【NCR】) Consist of a predicted RNA fわlding pattem

viruses and includes several important human pathogens

tion, and packaging of the gen0.me (3, 14)･ The viral genome

endemic throughout the world such as dengue viruses (typeRNA is infectious andgives rise tO the production of viral

1 to 4), yellow fever virus, Japanese encephalitis (JE) virus,

particles when introduced into susceptible cells.

and contain important elements for viral replication, transla‑

West Nile virus, and TBE viruses (Table 1) (2, 15, 26). On

TBE was血st described by an Austrian physician in 1931

the basis of cross‑neutralization tests and evolutionary tree(21). In 1937, a virus was isolated from the brain of ananalyses based onviral genome sequences, these viruses haveencephalitis patient in the southem far‑east region by former

been divided into several groups (Table I). Theflavivirus

Soviet scientist Zilber (28)I lt was shown that the virus.istransmitted to humans by tick bites ( 17). The main reservolrS*Corresponding author: E‑mail: takasima@vetmed.hokudai.ac.jp,
Tel/Fax: +81‑I 1‑706‑521 1

ofTBE viruSare small rodents, with ticks actlng aS a Vector.

Although several species of ticks can be infected with TBE

Table 1. List of major navivirus groups and viruses

Group

Viruses

Tick‑bone encephalitis(TBE) complex

VectorGeographic

Russianspnng‑surrmer encephalitis

dionAsia, JapanlFar Eastern subtype TBE]

Tick Central and Eastem

Central European encephalitis

Eumpe【European subtyp￠ TBE】

Louplng ill

Tick British Isles, Ireland

LangatTick

Omsk hcmorrhagic fcvcr

Tick Asia

Kyasanur Forest disease

Tick

Powas samTick NorthAmerica, Russia

Asia

Asia

Far Eastem Russia andJapanese enccpllalitis

complcx

stributi

Tick FarEastcrn Russia andJapancsc cnccphalitis

MosquitoSoutheast Asia, Japan,

St. Louis cncephalitis

Mosqul tO

America

Murray Valley encephalitis

Mosquito

Australia, New Guinea

Kunj inMosqul tO

Australia

West Nile

Mosquito

A舟ica, Europe,Asia,

Korea, China, India

America
DengucDenguetypc I

Tropical areas of Asia,

Dengue type 2

Mosquito AtlStralia, Occanla,
Africa, America

Dengue type 3

Denguetype 4
Yellow fever Yellow fever MosqultO Afhca, South America

Noknown vector

Rio

Bravo

Apoi

Unknown

Unknown

J

Americaapan

virus, Ixodes persulcatus and I. ricinus account for essential

rate is 0.5‑2.0%.

transmissions to humanS. TBE virus canchronically infectticks
throughout their lives and can be transmitted transovariallyIn the 1990S, total annual TBE case numbers were 6,0001
10,000, and most cases were from Russia. In Russia, TBE

and sexually (6, 10). Human infections mainly occur by tickcase numbers in recent years have been increaslng, Wi血5,486

bite in the spnng and early summer, Corresponding to periods

cases in 1990, 7,893 cases in 1993, and 9,548 cases in 1996.

of tick activity. On the other hand, humanscan also be infected

Such 乱血astic increase occu汀ed because many city dwellers

by raw milk or cheese from TBE virus‑infected goats, sheep,came into contact with ticks during visits to gardens or dachas

located near big cities. TBE cases have also occurred inrural

Or COWS.

TBE virus has been subdivided into two subtypes; the Far

areas in the 1950s and 1960S (10).

Eastem subtype known as Russian sprlng‑Sumer enCepha‑1itis (RSSE) virus, and the Westem European subtypeknOYn

immunogloblin Injection is used to protect against outcomes

There is no curative therapy for TBE. ln Russia, specificas Central European encephalitis (CEE) virus. Their malれofTBE disease. Vaccination appears to be the most effective

vectorticks are different; the Far Eastem subtype is transmitted

means of preventing TBE. Cu汀ently, several vaccines are usedby I･ persulcatus and the WesternEuropean subtype by I･

in Europe and Russia. The most widely used vaccine in Europe

ricinus. They also show different clinical manifestations andis FSME‑Immune Inject (Immune AG, Vienna, Austria),

mortality rates in human. The Far Eastem subtype disease iswhich consists of purified and formaldehyde‑inactivatedregarded as more severe and prolonged than the Western

whole virus. In Austria, mass vaccination and active surveil‑

European subtype disease. The incubation period of TBE islance resulted in a dramatic decline in the incidence ofTBE

usually 7‑14 days. In the Far Eastem subtype disease, cases

virus infection (9,1 1‑13).

show a monophasic course. Onset is generally abmpt, withIn Japan, no confirmed TBE cases had been reported forfever, headache,flushing of the face and neck, conJunCtival

many years. However, in 1 993 in Oshima district, Hokkaido,

Injection, Somnolence, nausea, vomltlng, dizziness, anda severe encephalitis casewith neurologlCal symptoms andmyalgia. The fbllowlng are the encephalitic syn血omes with

convulsions was diagnosed as viral encephalitis based on

severe palm in the ms and legs, back, hyperesthesia, asym‑

cerebrosplnalfluid examinations at acute and convalescentmetrical paresis of cramial nerves, tremor, ataxia, sensory

phases of the patient. We describe the epidemiology of this

disturbances, and unconsciousness. Permanent paresis occurs

case and TBE virus isolation from sentinel dogs in Hokkaido,in 5‑30% of cases, and the case‑fatality rate is 5120%. On theother hand, in the Westem European subtype disease, cases

Japan. Tb obtain the preventive measures f♭r TBE vinュs inof menlngOenCePhalitis in the first phase. ln approximately

Japan, We evaluated European TBE vaccine to TBE virus,
Hokkaido strain. Because of their geographical closeness, it
was considered that the ancestor virus of Hokkaido strain had

one･third ofpatlentS,the second phase of the disease develops

been diverged from far eastem reg10n OfRussia, where TBE iswith signs and symptoms of meningoencephalitis (6). Per‑

prevalent. Therefore, the divergence time ofTBE viruses fromshow a biphasic course and there are no slgnS Or Symptoms

manent paresis occurs in 2‑ 1 0% of cases, andthe case‑fatality

Hokkaido and Far Eastem Russia was estimated. Fur仙emore,

2

Table 3. Monitoringvirus activity and virus isolation using sentineldogs in 1995
NTl) antibody liter to TBE (Langat) vinュs

Dog No.
April 221 2 1 4 5 6 7 8 9̲ 0

May 4

May 13

May 20

May 27
<20

<20

く20

<20

<20

<20

<20

<20

<20

<20

<2

<2

<20

<20

<20

0

<20

0

1 60

<20

<20 *

<20

80

<20

<20

<20

<20

<20

40

80

320

1 60

80
<20

<20

<20

<20

80
640

<20

<20

<20

80
<2

0

<20*

<20*
<20

80
<2

0

1 60

1

60
<20

りNeutralization test (NT).

*; Viruses were isolated from blood of sentinel dogs by suckling mouse
inoculation.

(ref.

22)

Fig, 1. Geogr叩hicaHocation ora case‑S山dy area, Kamiiso (▲).

another navivirus infection was suspected as the cause of
encephalitis in this case, and a more specific neutralization

the virulence ofTBE virus strains recently isolated from various

test (NT) was performed including JE virus strain JaGAr‑0 1 ,hosts in Hokkaido and Far Eastem Russia were also compared.

Negishivirus isolatedin1948inJapan(I), Apoi virus isolated
from rodents in Hokkaido in 1954 (20), and RSSE virus strain

Sofjin. NT antibody titers increased significantly to RSSE

EpidemioLogy of TBE and isolation of TBE virus

virus at acute and convalescent phases amongflavivirusesin Hokkaido, Japan

tested ( I 6). The results indicated that the patient was infected

1. Clinical findiTtgS and virus isolation
In the town of Kamiis0, Oshima, Japan, a dairy farmer's
wife (37 years old) suddenly suffered a febrile illness with atemperature of 390C on October, 1 993. She was hospitalized onwith a TBE･related vims.

the 3rd血y a鮎r the onset ofillness. Double vision叩pearedparalysis Of the arms and neck was present 3 years later.

were determined, and virus isolation was carried out (Table3). On April 22, all 10 dogs had a negative antibody to Langat
vims. Two animals converted to positive on May 4th, threeon May 13th, and five on May 27th. The results clearly showed

Cerebrosplnalのuid was tested at the acute and convalescentthat the virus was prevalent in the area in 1995. The bloodphases, and clinical data fわr the respective phases showed

was inoculated into suckling mice via an intracerebral route.

In 1995, we kept 10 sentinel dogsinthe area in afree‑living
environmentand collected serum samples once a week from
April to July (22). NT titers of the dog sera to Langat vims

on the 2nd hospital day, convulsions on the 5th day. She wasunder artificial ventilationfrom days 5 to 20. Remaining motor

cell counts of468 and 6 cells/mm3, and glucose concentrations

Threeviral strains were isolated from the blood of sentinel

76 and 69 mg/dl (25). The results showed that she suffereddogs No. 5, 8, and 9, all of which were obtained 7 days priorfrom encephalitis or encephalomyelitis of viral orlgln.

to the sero‑converted day. The virus strains from dogs No. 5,

The geographical location of the case‑study area is shown

8, and 9 were designated as Oshima‑3‑6, ‑5‑10, and ‑5‑ll,(Fig. I). There were four farms within a 1.0‑km radius. The

respectively. The antlgenicities of the virus isolates werearea was deforested 50 years ago. The sera and cerebrosplnal

examined by indirect immunofluorescent‑antibody assay

fluid were examined by ELISA for JE virus (Table 2). The

EuSA lgG titer for JE virus increased significantly &om the(IFA) test using monoclonalantibodies supplied by Dr. F. X.
Heinz at the University of Vienna, Austria (22). Three of theacute phase to the convalescent phase. However, the ELISA

isolates showed high titers to TBE‑complex‑specific 2E7 andIgM titer essential to JE diagnosis was negative. Therefore,

TBE‑type‑specific 7G7 monoclonal antibodies (Table 4). The

Table 2. ScrologlCalexamination on sera and cerebrosplnal fluid ofthc patient
Anti‑JEl ) antibody Anti‑navi NT21 antibody(days)

S erum1gG‑ELISA

IgM‑ELISA

JE

RSSE】I

1,600

<100

I

16,000

<100

20

1,600

<10

0

Negishi

640

40

Apoi
10

(6 days)

S erum

2,560

320

10

(43 days)

CSF41

nt5)

nt

nt

nt

(52 days)
1リapanesc eヮcephalitis (JE)･(ref. 16)

21 NeutralizatlOn test (NT),31 Russianspring‑summer enccphalitis (RSSE).
41 ccrebrospinal fluid (CSF).
5) Not tested (nt).

3

Table 4. Identification of virus isolates by IFA test uslng mOnOClonal antibodies
IFA2) liters toSpecificities (MAb)17JE3)Group‑rcactivc (6E2)

Langat>6400

TBE‑complexISPeCific (2E7)< 1 00Type‑specirIC

<

(7G7)

I

00

Oshima‑5‑100shimal5‑1

10shima‑3‑6

1 600

>6400

>6400

>6400

>6400

>6400

>6400

>6400

1

1

<

I

00

1

600

600

りMonoclonal antibody (MAb).

600(ref. 22)2) Indirect immunonuorcsccnt antibody assay (IPA).
･1) Japanese cncephalitis (JE).

viral strainsisolated were identified as TBE‑related virus. In

2, ‑5, and Ilo) isolated in Kbabarovsk (8),five strains isolated

1995 and 1996, TBE virus strainswere isolated from 1. ovatus

in Hokkaido from dog blood (Oshima 5‑1 I, 3‑6), ). ovatus

ticks and rodents (23, 24).

(Oshima I‑1), Apodemus speciosus (Oshima A‑1) andClethrionomys rufocanus (Oshima C‑ 1 ) and the low‑passage

2. Genetic characterization of the virus isolates

Sofjin virus (Sofjin‑HO) (22‑24). The data of Sofjin‑HO
showed 37 nucleotide and two amino acid differences fromthe data of Sofjinvirus strain determined by Pletnev et al.

The E‑protein gene sequence of one of the isolates, Oshima‑

5‑ 10 strain, was detemined. The whole E‑protein gene wasamplified by RT‑PCR and directly sequenced. The sequenceof Oshima vims was compared with those of TBE complex
viruses (Table 5). The nucleotide identities of Oshima virus(1 8). Identities of the E‑protein gene nucleotide sequenceswere 95.7% and 84.3%, respectively.Amino acid sequences

between the Khabarovsk isolates were 98.8‑99.5%, between
the Oshima isolates were 99.0‑99.9%, and between the
Khabarovsk isolates and the Oshima isolates were 95.2‑95.7%

of Oshima virus showed extremely highidentities with those(data not shown)･ All pairs amPng these virus isolates showedwith RSSE (FarEastem subtype) and Westem TBE (WTBE)

very highidentities, and palrS Of isolates from the sameofRSSE (99.0%)and WTBE (95.8%).
Phylogenetic analysis of the E‑protein gene was carriedgeographical area had extrePely highidentities･ The positionout to study the genetic relationship between Oshima virus
and other TBE complex viruses (22). Oshima virus showed

and variety of deduced amlnO acid changes among Oshima

血e closest relationship with RSSE vims among other TBE

tions were found only at 14 different positions, which shows

and K血abarovsk strains are shown in Fig. 3. Amino acid varia‑

complex viruses by the unweighted pair group method withthe highdegree of conservation ofamino acid sequences aTtOng

arithmetic means (UPGMA) method (Fig. 2) and a similar

these viruses. Differences in three of the 1 4 different posltlOnS

result was also obtained by the Neighbor‑Joining method(results not shown). The high reliability of the tree wasascertained by Majority‑ruleand strict consensustrec program;
RSSE versus Oshima was 1 ,000/I ,000.

Phylogenetic analysis of TBE viruses from Japanand Far Eastern Russia

I. Relatedness OfTBE virus, Hokkaido strain and Far
EasterA Russian strains
To reveal the relationship among TBE viruses from Japanand Far Eastem Russia, we isolated TBE viruses舟om ticks

collected in Khabarovsk, Russia in 1998 by the sucklingmouse inoculation method and geneticallyanalyzedthese andHokkaido isolates. We detemined the nucleotide sequenceof the 1 ,488 nucleotide long E gene丘omthree strains (KH98‑

Table 5. Identities of the nuclcotidc and deduced amino acid sequencesofOshima viruSwiththose ofTBE complex viruses
Identity (%)
Nucleotide Amino acid
*Russianspnng‑summer encephalitis

9 0 8 8 8 8 7 7 6 5 4 L L L o 5 2 9 7 tJ Oノ 0 0 つJ tJ 2 (XU

9 0ノ 9 9 9 0ノ 0 0 7 9 5 L 2 3 4 8 L 8 0 8 3 9 5 0 7 2 ｣Westem tick‑bomモ enc￠phalitis

Fig. 2. Phylogcnetic tree ofTBE complex viruses. The nucleotidc

Louping ill

sequencc of the E‑protein gene ofOshima virus obtained inthc study

Spanish sheep encephalitis

was comparedwiththose of other TBE complex viruses available in

Turkish sheep encephalitis

Omsk hemorrhagic fever

DNA databases. The tree shovm above was obtained by the UPGMA
mcthod. Virus strains used for the analysts arc aSfoIlows; Russian

Langatspring summer cncephalitis (RSSE), Oshima 51 1 0 (Oshima), louping

Kyasanur Forest discasc

ill (Louping), Spanish sheep cnccphalitis (Spanish), Wcstcm TBE

Powassan(Wcstem), Turkish sheep encephalitis (Turkish), Omsk hemorrhagic'Far Eastem tick‑bomc encephalitis.

fevcr (Omsk), Lamgat (Langat), Kyasanur Forest disease (Kyasanur),

E

人●▲.

nlHu

22

and

4

Powassan(Powassan)･

(ref,

22)

Os h ind 5 I 10

1 : SRCTHLE NRDFVTGTQmRVTLVLE LGG〔VTITAE GKPSNDWLDSI YQE NPAmE Y( LHAKLS DTKVAARCPTMGPATLAE E HQSGTVCKRDQSDRCWGNH( G LF GKGSIVTCVKAS 120

Oshinu1

120

0shiJTd 3‑6

11200shirru I‑1

1120

0shiITK) A11

1

120

0shink) (‑1 1120

KH98 ‑ 2

1

120

KH98 ‑5

1120

KH98‑ 10

1

leo

5‑11

Sofj in‑HO 1

120

Vosi lcherlko 1
Neudoe r･fl 1
Hyllr

1

263

1

Spdnt

Sh

‥..A‥‥A 120‥HA.‥.A 120

A.‥‥‥q‥HA‥‥▲ 120

1A"..S.日.日日.日日.E日日.日.日日.A...日.T.

‖‥A.‥.A 120

1

V.....A....S..............E..........日.A.日.A.I.

‥‥A‥‥A 120

LI/G

1

A日.GS.日日.日...."E......"日."V.T..A.I.

‖‥A.‥.A 120

KydSdnLJrl

1:T‥.S.......̲S......日..L日,....V.....I).H.日..日.........ANS....S..A...日.I.日.G.

.B‥A,A.F. 】20

LI/31

PcMdSSdn

1:T‥

‥.Q.

‥

‥.

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120

Oshtlm 5‑ 10 121 : CEAKKKATGHVYDANKIVYTVKVEPHTGDYVAANETHSGRKTASFTVSSEKTI LTNGDYGDVSLLCRVASGVDLAQTVI LE LDKTSEMLPTAWQVHR抑FNDLALFMKHEGAQNMNAER 240

Oshind 5‑ll 121
0shtlTK) 316

ユ21

0shiITu I‑1】21

0shind A‑1

121

D‥‥‥‥‥

240

240

0shilM C‑1 121
KH98 ‑ 2

KH9&

‑

121

5

121

KH98 ‑ 10

121

Sofj in‑HO IZIV4Silchertko 】21
Neudoe rf t

121

Hypr'

121

263

121

Sprlish 121:.........Y.

.K.....T.日日."日.i....‥‥‥.‥.R‥‥‥

LI/31

121:‥‥‥‥.Y.

.K.‖‥T..‥‥‥‥.し.E‥.‥...‥‥‥‥‥.Ⅰ..‥‥.A....‥.‥......‥‥‥NPM‥.V‥240

121:日日.‥‥γ.

.K.....T.日.....日.L.(.......日..日..日..I.."日.A日.H‥R.

...‥..‥.D.NPH‥....

240

LI/G

KydSdnur

121:..T‥‥‥Y‥.V‥.T.V‥‥‥‥目し‥‥S‥N.‥‥‥TQ..‥‥.L‥‥.Ⅰ‥T‥.T‥..P‥‥V‥‥..A‥‥K.

Po仰SSdnlZl:HEA‥.V‥...ST‥T.V‥.日...‥L‖‥.〜.N..S.Q‥.A‥.V..RL‥‥‥‥T.K‥"I.V‥HVMS‥SSKD‥.S.

.E‥S‥.R...‥E‥‥D. 240.E‥.‥‥.KDN.0..SV.K

240

306
Oshlnd 5‑10

241 : LVEFGAPHAVKNDVYNLG町GVLLKSLAGVPVAHIDGTKYHLKSGHVTCEV(;LEKLKNKGLTYTPlDKTKFIYKRIPTDSGHDTVVNEVAFSGT‑KP⊂RIPVRAVAHGSPDVNVAMLIT 359

OshtrrK) 5‑11241‥... 359

0shill旧316

241.‥‥ 】59

0shinq I‑1

241

‥.‥

0shiw A‑1

241..‥. 359

0shimO C‑1

241

.し.‥

KH98 ‑2

241

359

359

‥‥. 359〜.‥‥‥‥‥.Y.

241..... 359

KH98‑ 10241‥‥, 359

Sofj in‑HO

241

Vqsil⊂henko

241

KH98 ‑5‥‥,A.榊T.

.ド.

.丁..‥‑.

...‥

359

‥...

359

359

Neudoer‑f1

241:..‥‥‥‥.‥‥"..,..‥.A‥‥‥‥.∈‥‥‥‥‥.‥‥

日日.....A.

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.‥‥

Hypr

241:..‥‥‥‥‥‥.‥‥‥‥.

.A‥‥‥‥.ど.‥‥‥‥.‥.‥‥‥日.‥人.

.T....‑.

‥‥. 359

241:.‥‥.‥‥.‥‥.‥‥‥‥.A‥‥‥.‥∈‥‥‥‥‥‥.‥‥‥‥‥.A.

.〜.‥.一.

241:‥‖‖."日.‥‥‥‥‥H.▲‥日.‥‥E‥.‥日‥‥‥‥‥S‥A‥.V.

.T...S‑.
.T.‥S‑.

263

SpdnishLI′31

241:...‥..‥‥‥‥‥..‥‥.

.A..日.....E.N....日.......

‥S‥A‥.T.

しⅠ′G

241:‥‥‥‥‥‥‥.‥.‥‥‥.A....日日.E.N..."日日.."日SHA...T.

KyaSdnur

241:‥‥･E‥‥‥.IF‥R..‥Ⅰ.

日..日...N.E.i..日Q日....D.

poWdSSdn

241:..‥.I...…‥F‥‥‥A‥‥‥..‥し.SVE.Q...‥‥‥‥D.‥‥..し..T‥S払‥A..K日.V.V‥‥..‥‥.SYT.SD･.〜....

EGS..A...P.

‥‥. 359.I.

.T...S‑.
.TYT.S‑.

359.‥‥‥‥...

359.E.N‥‥S‥. 359.V.A....".. 360
462 ,463

oshind 5‑10471
NS360: PNPTIENNGGGFIEMQLPPGONIIYVGE LSM騨FQKGSSIGRVFQKTRKGIERLTVIGEHAVVDFGSTGGFLTSVGKALHTVLGGAFNSLFGGVGFLPKILVG

OshirrK) 5‑110shillW 3‑6

360

360

0shirrK) A‑1

360

0shlmd C‑1

360

479

.. 479.. 479.. 479360

0shimd I‑1‥‥. 359.‥‥‥‥‥.

.. 479

G‥‥‥‥‥....

.. 479

KH98 ‑ 2360‥

KH98‑ 5

360

‥

479

KH98 ‑ 10

360

‥

479

360

.. 479360.. 479

Sofj i n‑HO

VdSilChenk0
Neudoe r f 1

360.▲‥‥S.Ⅰ‥.V. .‥‥‥‥Ⅰ‥‥.‥‥L.し.

Nypr

360

.A.日.S.I..". ‥‥‥.‥Ⅰ‥.‥‥‥し.し.

‥

479

479

., 479･T T T一ゝ.A.A日..S.I...V. .‥‥..‥Ⅰ..‥‥.‥し.し.

‥

479

263

360

Spdrtish360:....‥.E‥‥..‥‥‥..‥.し.T‥‥‥‥‥‥.V.".S.I".V. .........I..日日.HL.〜.

しⅠ/31

360:‥..‥.D‥‥…..

.T.‥‥.‥.T‥.‥‥‥‥.

.A...FS.I...V. .日......I.........L.〜.

LI/6

360:.‥.‥.D‥‥‥‥.T‥‥‥‥.T‥‥‥‥.‥.

.▲‥.FS‥.‥V. 日.......I.....日..L.M.

日..T.日.LE...A.......V.

.V..〜.S.."". .AF.A‥.TI‥‥‥‥R‥し.

479....T...M.E.HR.L...S.V.

.V‥V.S‥‥.I. ‥‥.‥.丁目.‥‥Ⅰ‥H.し.

480

KydSdnur

360;. ‥SNHT‥ HV.L.

PoけdSSdn361;....‥T.‥‥..‥.Ⅰ.

.D..Q.

Oshi伽5‑10

480:FLLAGGLVLANTLGVGA 496

OshilⅦ 5‑11

480:

496

Osh irrK) 3‑6

480:

496

0sh ind エー1

480:

496

OshiW A1148○:

Oshi巾O 亡一1

480:

496

KH98 ‑ 2

480:

496

48◎:

496

480:

496

Sofjirl‑HO

480: ‥ ‥...

496

480;‥... ‥496

480; ‥ ‥ ‥.

496

480: ‥ ‥.V.

496

480:‥..‥.

496

480:..... ‥

496

LI′31

480: ‥.T. ‥

496

LI′G

480:.‥ ‥‥4%

VdSil⊂herlko
Neudoerf1
HylIr
263

Spdnish･ ･G T 479

479
･･V

479

496

KH98‑ 10

KH98 ‑S･･ ･∨ ･

KydSdhLJr

48l:‥IT..‥.T.. ‥‥. 496

PtMdSSdn481:..AV.A.T.NHN‥.. 497

Fig･ 3･ Compadson of the amino acid sequences deduced丘omthc E‑gene scqucnccs ofvariousTBE complexviruses. u, Louplng ill virus.

(ref.8)5

Russian isolates have lost this genetic marker.

(positions 306, 462, and 463) were fわund only among

The amino acid alignment also revealedfour amino acids

Kbabarovsk isolates, including So勺in‑HO. Other variations

unlque tO Westem European TBE virus isolates, supportlng
the clinal concept of tick‑bone virus evolution across Eurasia

were fわund randomly, and a consistent distribution was notfound. With reference to the three‑dimensional structure of

the E protein ofTBE virus strain Neudoerfl(19), most amino(27). Their respective positions are 88 (G), 206 (V), 317 (A),and 407 (K).

acid changes were located on the upper or lateral surface (datanot shown). No specific amino acid changes in the E protein

A phylogenetic tree constructed from symonymousdistanceswere found in KH98‑10, even though this strain showed aofTBE serocomplex vimses is shown in Fig. 4. Ⅵms strains

lower vimlence than other strains.

isolated in Hokkaido and Kもabarovsk, including Sofjin‑HO

Amino acid position 47 1 is valine in the Khabarovsk isolates,

virus, fom a cluster with lOO% bootstrap support. Thus, thesewhich is unlque among TBE complex viruses, even whenvirus strains were classified as Far Eastem subtype TBE virus,

other sequencedflaviviruses are compared (Fig. 3). This

since strain Sofiin has been proposed as a prototype of theimplies that the lnabarovsk strains have evolved independently

Far Eastem subtype (7). The branching pattem of the phyloge‑of the standard Far Eastem TBE vims strains and also inde‑

netic tree (Fig･ 4) clearly distinguishes the Far Eastem subtype

pendently of the Japanese isolates. Amino acid position 462
is alanine inthe Hokkaido isolates but valine in the Khabarovsk

TBE complex viruses from the Siberian subtype Vasilchenko

and Sofjin viruses. Alanine is also found in Westem European
TBE viruses, louplng ill virus and Kyasanur Forest disease

tree also shows that two independent but closely related(KFD) and Powassan (POW) viruses. This again implies

into Far Eastem Russia: the Khabarovsk lineages, including

and also the European and UK TBE complex viruses. The
lin°ages have developed fbllowlng Introduction of the virus

independent evolution between the Japanese and Russian TBE

Sofjin virus, which became established in the far eastem partviruses. Moreover, since the alanine residue is common toof Russia,and the Hokkaido lineage, consistlng Ofthe Oshima

most of the other tick‑bomeflaviviruses, this implies that thestrains, which was subsequently introduced into Japan. In view

Fig･ 4･ Phylogcnetic tree ofTBEvirus strains containing the Far Eastem subtype, Siberian subtype and Wcstem subtypevirus

strains･ The tree was rooted using I 488 nucleotides ofviral E‑gene sequcncesand Omsk haemorrhagic fever (OHF) virus as
the outgroup viruS･ Ⅵrus strains are indicated as accession no. Bom DDBJ, EMBL, and Gene Bank nucleotidc sequence
databases･ The scale barindicates the number of substitutions per nucleotide site.

6(ref. 8)

Fig. 5. Phylogenetic tree of the Far Eastern subtype TBE virus strains isolated in Oshimaand K血abarovsk.

of the Far Eastem subtype being distributed over a very wide(ref. 8)

Pathogenicity of TBE virus, Hokkaido strain andgeographical area of Russia and being distinguishable fromprotection from infection by European vaccine

the Siberian subtype TBE viruses such as Vasilchenko (7), it
seems reasonable to assume that the new Oshima viruses

I. Pathogenicity of the Hokkaido isohtes
We compared the pathogenic characteristics among Hokkaido

dispersed inaneastward direction from Russia to Japan rather
than from Japan to Russia.

isolates and other strains of TBE virus (4). The degrees of

2･ Divergence of TBE virus, Hokkaido strain from
Khabarovsk strains

neuroinvasiveness of the viruses were examined by subcu‑

tane?us (S･C･) inoculation into the axilla ormice･ Mice wereA phylogenetic tree constructed from synonymousdistances

also ln?Culated intracerebrally (i･C･) to evaluate the degree ofofKH98‑2, ‑5,and 18, So巧in‑HO and Oshima strains and theneurovlmlence. In addition, intraperitoneal inoculation was

Vasilchenko strain aP an outgroup is shown in Fig･ 5 (8)･ The
tree shows that strains from Far Eastem Russia and strainsfrom Hokkaido form distinct clusters with good bootstrap

employed to assess the neuroinvasiveness of the vims and tosupport, 89% and 95%, respectively. We estimated the

The differences in neurovirulence and neuroinvasivenessamong TBE virus, Oshima, Sofiin and Hochosterwitz strains,establish a mouse model for vaccine evaluation by determina‑tion of 50% lethal dose (LD50) values.

synonymous substitution rates or these strains. The averagesynonymous substitution rate of these strains was estimatedand TBE complex virus TP‑21 strain are shown in Fig. 6,

to be 2.9 × 10‑l per site per year. The time of divergence ofFig. 7, and Table 6.

the Khabarovsk and Hokkaido strains was calculated byuslng

Mice were inoculated s･C･ with 10,000 focus‑formlng units

the average substitution rate and the synonymous distances(FFU) of each strain (Fig. 6a) and significant differences inshown in Fig. 5. The result showed that these virus strains

neuroinvasiveness were revealed among the strains. Seventy‑

had diverged approximately 260‑430 years ago. Therefわre,five percent of the mice inoculated with TPl2 1 strain survived,since the Hokkaido strainsare genetically distinct from the Far

but only 12.5, 0, and O% of those inoculated with Oshima,

Eastem subtype TBE viruses, they may have dispersed qulte

Sofjin, and Hochosterwitz strains survived, respectively.

soon after diverglng from the Far Eastem subtype viruses.

Moreover, the survival timesL of mice inoculated with Sofiin
and Hochosterwitz strains were slgnificantly shorter thanthose with Oshima strain (mean survival times 9.5, 1 1.6, and

14･ 1 days, respectively, P < 0.05; Oshima versus So8in and7

■ー]mL■､｣i

08642008642(
10FFU/mouse
au!u6u!^!^]⊃s IO.oN

ao!uJ6u!^ LnsIO.oN'H

一一一'ー‥ヽヽ…,,,,,,

0o
5

JL

10

(b)1.000FFU/mouse

15

20

25

30

Days post‑inoc山ation

a3ニヒBu!^!^Lns IO'oN

86420

ここここここここ墓石売Fig. 7. Survival ofadultmice intraccrebrally inoculatcdwith TBE virus
strains. Mice were inoculated with10 FFU ofTBEvirus strains Oshima
5‑10 (●), Sofiin (■), Hochostcrwitz (▲), or TPl21 (▼). Survival wasrecordedfor

‑I‑

28

days

post‑inoculation.

(ref.

4)

､亡

and that of mice inoculated i.C. with Hochosterwitz strain ( 1 0.0

versus ll.1 days). All or almost all mice were killed by
Oshima, Sofiin, and Hochosterwitz strains, but 62.5 % of mice
5

10

15

20

25

30

inoculated with TP‑2 1 strain survived.

Days post‑inocu一ation

The mortalityratesand mean survival days of mice inocu‑

Fig. 6. Survival of adultmice subcutaneously inoculated withTBE
virus strains. Mice were inoculatedwith10,000 (a) or 1,000 FFU (b)lated intraperitoneally (i･p･) With Oshima, Hochosterwitz, Pr
Sofjin strain are Shovm in Table 6. The LD50 0fOshima straln

ofTBEvirus sbins Oshma 5‑10 (●), Sofiin (I), Hochosterwitz (▲),

was approximately 10‑ to 20‑fold higher than that ofor TP‑2 1 (▼), rcspectivcly, Survival was recorded for 28 days post‑
inoculation.

(ref.

Hochosterwitz and Sofiin strains, and the mean survival timefor each virus dilution of Oshima strain was slgnificantly

4)longer than that of the other two strains.

Oshima versus Hochosterwitz), however death from strain
TP‑21 was delayed (mean survival time 18.0 days). Similar
results were obtained when 1 ,000 FFU was injected s.C. (Fig.

s.C. inoculated was Sofiin ‑ Hochosterwitz > Oshima > TP‑6b), whereas none of the mice were killed by TP‑21 strain.

Oshima. Neurovimlence, as determined a鮎r intracerebral

When 10 FFU was Injected s.C., only one mouse was killed
by Sofjin strain, and no clear differencesamong TBE strains

inoculation, was Sofiin > Oshima ‑ Hochosterwitz > TPl2 1.
Virus replication in the brains were compared in the s.C.

were obseⅣed (data not shown).

and i･C･ iヮoculated mice with Oshima and Sofjin strains (Fig･In conclusion, the degree of neuroinvasiveness of the strains

21, and in mice inoculated i.p. was Sofiin ‑ Hochosterwitz >We examinedthe neurovirulence byintracerebralinoculation8)･ In mlCe iヮoculated s･C･ with Sofjin strain (10,000 FFU/

of 10 FFU, and the results are shown in Fig. 7･ Sofjin strain

mouse),the vlruS Was first detected in the brainon day 5 (1.1

was the most neurovimlent among the strains examined in

X I 05 FFU/g) , and the titer increased progressively, reaching

this experiment (mean survival time 6.5 days, P < 0･05;

a pla!imum titer (1･5 × 109 FFU/g) on day 8 (Fig･ 8a)･ ln

Oshima versus Soqin and Hochosterwitz v?rsus Sofjin)･As

mlCeチnOCulated s･C･ withOshima strain ( 1 0,000 FFU/mouse),pathogenicinits neuroinvasiveness thanHochosterwitz strain.

the vlruS Was first detected in the brain on day 6 (5 X 102
FFU/g),and thetiterincreased gradually, reaching its maximum

Whereas, no slgnificant difference was observed between the(3 X 105 FFU/g) on day 10･ Of7 mice in?culated with Sofjinmean survival time of mice inoculated i.C. withOshima strainstrain, 4 individuals in which the virus tlter exceeded 5.2 X

can be seen in Fig. 6, 0Shima strain was slgnificantly less

Table 6. Mortality ofmicc intraperitoneaIIy inoculated with TBE viruses
Dose

Oshima(FFU/mouse)

5‑

1

0

Sofi

in

Hoct10SterWitz

Mortalityu Survivaltime2) Mortality Survivaltime Mortality

1,000

9/10

100

6/10

10

6/10

1

4/10

14.3±2.93･47

10/10

13.3±2.43･4I 10/10
15.5±4.03･41
15.5±2.43･41

8.2±0.83･5)

8.8±0.63)

Survivaltime

10/10

9.6±

9/10

9. 1 ±1.24)
10. 1 ± 1.34)

10/10

9.6±0.731

10/10

6/10

10.5±1.931

9/10

1.34･51

r 10.6±1.547

0.1

0/10

0/10

0.01

0/10

0/10

0/10

0.68

0.36

1/10

11.0

LD5｡
(FFU/mouse)≧ 8. 1 3I) number of deaths / total number tcsted.

(ref. 4)2) Mean±SD (days).

3)p < 0.05 0shima vs Sofiin.
4)P < 0.05 0shima vs Hochostcrwitz.
5)P < 0,05 SoT]in vs HochosteTWitz at each dose.

8

1 08 FFU/g were dead, whereas only one mouse inoculatedwith

mouse), the vims was丘rst detected in the brain on day 2 (3.6

Oshima strain, which had a virus titer of I.2 X 106FFU/g, was

× 1 02 FFU/g); this titer r.epresentedanapproximately 1 0‑folddead on day 10.

increase over the origlnal inoculum (Fig･ 8b)･ The titer

ARer mice were inoculated i.C. with Sofjin strain (10 FFU/

Increased similarly as in the case of s.C. inoculation, reachingチmaximum (l･1 × 109 FFU/g) on day 6･ In mi?e inoculated.
I.C. With Oshima strain (10 FFU/mouse), the vlruS Was first10

detected in the brain on day 3 (2.2 X 103 FFU/g),and the titer(b) Lc,: 10 FFU/mouse(a) S･C･; 10.000 FFU/mouseincreased gradually betTeen 3 to 5 days post‑inoculation (p･i･),

followed by an abrupt Increase between 5 to 6 days p.i. Of

9mice inoculated i.C. with Sofjin strain, some mice died during

the observation period, and the vims titers in the brains of

8these mice exceeded 1.6 × 108 FFU/g. None of the miceinoculated i.C. with Oshima strain died during the experiment.

7

2. Evaluation of European TJIE vaccine

BnS !76]⊃j 6oJ

We tested the efficacy of European TBE vaccine agalnStqB

TBE virus, Hokkaido strain (5). Neutralizing antibody titersln

4

agalnSt TBE virus strains and JE virus strain in sera fromhealthy male volunteers vaccinated twice with FSME‑
lMMUN twice at a I‑month interval are shown in Table 7.
Seroconversion rates to Oshima, Hochosterwitz, and Sofjin
8strains 4 weeks a鮎r the 2nd vaccination were 80, 100, and90%, respectively; 1n addition, those rates obtained a洗er the2

first vaccination were 60, 70,and 20%, respectively (data notshown). No significant differences were observed among thegeometric mean titers (GMTs) (Oshima; 1 :44, Hochosterwitz;
1:65, Sofjin; 1:43). Four of 10 persons were revaccinated I
0

2

4

6

8

10

12

0

2

4

6

8

year later. A洗er the 3rd vaccination, all persons showed two

to four‑fold increases in neutralizing antibody titer agalnSt at

Days post‑jnoculation

least some strains (Table 7). After the 2nd vaccination, two

Fig. 8. Replication ofTBE virus strains in mouse brain. Mice were
inoculated subcutaneously (S.C.) wittl I 0,000 F一u (a) or intracere‑

persons (Nos･ 1 and 3) showed higher neutralizing antibody

brally (i.C.)with10 FFU (b) ofTBE virus strains Oshima 5‑10 (+ ;tlterS agalnSt JE vims, JaGAト0 1 strain, than those against

surviving, lL ; dead) or Sofiin (○; sutviving, □; dead), respectively.

Oshima strain (Table 7).

I Groups of 1 0 mice were s.C. vaccinated with FSME‑IMMUN

Virus titers in each mouse are shown. Geometric mean titers arecalculatcd for each timeperiOd in Oshima 5‑ 1 0 (solid line)and Sofiin

and revaccinated 7 days later. GMTs against TBE virus strainsin the sera obtained from the mice are shownin Table 8. With

(dotted line) strains, respectively. There were significant di frerences(P < 0,05) in geometric mean liters between themice inoculatedwith

the except10n Ofone individual showlng anundetectable level

Oshima and Soqin strains onthe days indicated by an asterisk,of antibody agalnSt Sofjin strain, all of the mice seroconverted(ref. 4)

Table 7. Neutralizing antibodytiters in humans agalnSt TBE virus strains and JE virus strain aRer vaccination

NT liter agalnSt
TBE

Oshima 5‑ 1 0
after

日234567890

4 4 2 5 2 4 つJ 3 3 2

n7 2 9 3 4 5 ∠U 7 7 8

く く 2 つL一 2 4 4 4 4 4 〈X) 0 0 0 0 0 0 0 0 0 0

vinュs

JE

vinュs

Hochosterwitz So匂in JaGAト01'‑

2 4 2 4 8 8 ′0 6 0 ′b0 0 0 0 0 0 0 0 0 02md vaccination<20

1く くくくくくくく

KV 2 4 2 2 2 2 2 2 20000000000

20
20
20
80
80
40

l11

20
80

160

1

65±22
after

l‑ 2 4 0

4 4 5 つJ凸7 2 つJ 7

4 ′b OO AV 0 0 0 0

4 00 4 60 0 0 0

43±23
4 00 4 00 0 0 0 0

1くくく

上U 2 2 20000

3rd vac(:ination

1= りりPerson who had NT antibody against JE virus (JaGAr‑01) before vaccination (Not 1 ; 1 : 160, No･ 3; 1:20)I2)Each titcr was dctctmined asthe rccIPrOCal of the highest serum dilution that rcduccdthc virus focus cotlntS
by 50%.
37GMT; geometric mean titcr of positive sera (mcan± SD).

(ref: 5)9

Table 8. Neutralizing antibody liters in mice against TBE virus strains beforeanda氏er infection

Virus strain lmmunogen Pre‑infection Post‑infection n
Oshima 5‑10PBS

く20 (0/I 0)2)

320士16 (3/3)

Vaccine130±273) (10/10)

208±22 (8/8)<20 (0/10)<20 (0/1)

ⅥlCCi ne160士25 (10/10)

587± 19 (8/8)辛PBS<20 (0/10)

Vacci ne127±30 (9/10)

Hochosterwitz PBS

Soqin

n t41

80±17 (10/10)

nScra were collected舟om survivlng mice 21 days after infection.

(rcr. 5)21 ( ); number of positive sera/number of mice tested.
･11 Gcomctric mean titcr of positive sera (mean ± SD).

4)nt, not tcsted because none ofthemicc inoculated with So勺in strain survived.

* ; Significantly diffcrcnt from the pre‑infection value (P < 0.05).

with GMTs or 1:130, 1:160, and 1:127

to Oshima,

ACKNOWLEJ)GMENTS

Hochosterwitz, and Sofj in strains, respectively. No significant
differences were observed among GMTs similar to humanThis work was supported by grants‑in‑aid for Scientificvaccine trials.

Research (良) (Nos. 11460143 and 12556051) and a grant‑in‑aid for JSPS Fellows (No. 5814) from the Ministry of
Education, Culture, Sports, Science and Teclmology of Japan,

The percentages of mice survivlng after being vaccinatedand challenged with 100 LD50 0f each TBE virus strain are
showninFig. 9. Vaccinated mice can be significantly protected

and Health Sciences Grants for Research on Emerglng andagalnSt a Challenge of T古E virus strains of Oshima,

Re‑emerging InfectiousDiseasefrom the Ministry of Health,Hochosterwitz, and Sofjin. Notably, 1 00% protection wasLabour and We胞re of Japan.

We thank Dr. Franz X. Heinz of the University of Vienna

achieved a触r Sofiin infection. Neutralizing antibody titers

against homologousstrains were assayed 2 1 days aRer virus

for providing monoclonal antibodies.

challenge ln SurvlVlng mice. Although there were no slgmiGcant
differences in GMTs in sera of the vaccinated mice before or

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