AD-751 205

STUDIES ON TICK-BORNE ENCEPHALITIS ANDOTHER ARTHROPOD-BORNE VIRUS DISEASES

Christian Kunz, et al

Vienna University

Prepared for:

V Army Research and Development Group (Europe)

o° July 1972

DISTRIBUTED BY:

ooiI•o! Technical Infor 3uCk1 SorvicoU. S. DEPARTMoE•dT OF CV2E5CE

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"`0 °DIST'BUTE' . . - - ,

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S.STUDIES ON TICK-aORNE ENCEPHALITIS

AND OTHER ARTHROPOD-BQRNE VIRVIJ1 DISEASES

-Final Technical Report

By

Ch. Kuiz, M. D., H. Aspbck, Ph. D., W. Frisch-Niggemeyer, Ph. D.,

"H. Hofmann, M. D., A. Radda, Ph. D.

- ,July 1972

European Research Office

Contract Number DAJA37-71-C-3718Reproduced by

NATIONAL TECHNICAL0, o00 INFORMATION SERVICE

U S Department of CommerceSpringfield VA 22151

, ;Lehrkanzel fMr Virologle der Universitdt Wlen

Kirderspitalgasse 15 - D D C "A-1095 Wien, Austria.

~~~ U U LL- / £ o-•A for public reo•moc; B

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. s~ewultv Classifl~eatinn

DOCUMENT CONTROL DATA - R & DiStcu.rL-1 Claaeulicersan o1 atitl. bodl, of mostract and inaoarnmq annotation enust 6. antered w1ier the orlbraI: reol to CIaD-Sled)

ORIGINA TING ACTIVITY (Cftorpegcl suthct) =0.REPORT SECURITY CLASSIFICATIONLehrkanzel fUr Virologie der Universit~t Wien Unclassified4A-1095 WIEN 2b. j2OUPKinders~itElgasse 15, Austria

3. 3-REPORT TITLEO~Studies on Tick-Borne Encephalitis and other Arthropod-Borne

VirUs Diseases

i4. DESCRIPTIVE NOTWQ MTpg of repot and Incluwsive date#)FinalTechnicalReport________

OýO J"q !1 AU THORIVI (Pitat nanie, mIdde9 ff11141 loest namel)Christian Kunz, Hurst ApekWatrFsh-Niggemeyer,Hanns Hofmann, and Alfred Radda

S. 6 REPORT OATEC 7a. TOTAL NO. OF PAGES 17.NO. OF REPSJuly 1972 81 7*23

1141. CONTRACT OR GRANT NO. 94. ORIGINATOR'S REPORT NUMBER(S)

DAJA37-71-C-37l8b. PRojKCCT NO.

C. 9b. OTHER REPORT NO(S) (Any oilitnzMb~f8 81tAtIRY 6. .eelg01edthis Ieport)

* 0d. ERG Project E-1400~ 7pprvedforpblic release, distribution unlimited. wt

* d13. ABSTRACT* .Distribution of tick-borne encephalitis virus (TBEV): six new foci

Shave been discovered in Austria. TBEV is widespread in Switzerland.2.TBEV control program: control of ticks and small miammals has mret wtýsome success. 3.Purification of arboviruses: accomplished with porous1glass exclusion chromatographj. 4.,Synthesis of reý.eptor substarce TPI:Ten steps out of 22 have been coimpi,.ted. S.Clinical studies: 288 casesof TBE have been diagnosed, 6.~Virus infections,: Langat virus causeschronic infection in mice which is not eliminated by Poly-T:C~. 7.Sero-

0 ~logical studies: Antibodies against Tribec virus were dete.:ted occasion-ally in sara of cattle from Carinthia., Antibodies against Uiikuniemi andother viruses were detected in birds. In Turkey, positivf.- human andanimal sera were found against TBEV, West Nile, and a grsup A virus.Among residents of West Cameroon positive human z-era we.e fcuni againstgroup A and B viruses'iiicludir.g O~nfrongnyong, Chikunguriya, Uganda S,rika, Yellow Ftevef. and.De~~u II. 8.An unidentified vifus was isolatedtram a mirtng 1,ird in As U na._________Key *ords: Arboviruses in Austria; tick-borne encephalitis eradication;tick-borne encephalitis ecology; virus receptor substanc~e synthesis;arboviruses, cocntaio nd purification; arbovirus infection, per-(sistent; Langat virus; tick borne excpaii nSitzrad roiuin Turkey; arbo~riruses in Cameroon; arinovirus hosts, birds.

for UNCLASSI FIEDDD 1.1.141

I I AD

] STUDIES ON TICK-BORNE ENCEPHALITIS ANDI ~OTHER ARTHROPOD.-BORNC VIRUS DISEASES

Fle Tehncl epr

00

0. July 1972

-~ European Research Off?16

Contract Number DA~JA37-71-C..3718

ov Lahrkanzal f~r Virologie der Univereittit Wien

Kidreialaoo1

411

"4.'

00 '° A b a t r a c t

Q+ \

(I) Field studies of TBE led to the detection of0 6 new TBE virus (TBEV) foci in Austria. Tick

and mammal control program appear to be effec-tive against virus cycle in nature, Surveywith sere of Carnivora showed wide distribU-tion of TBEV in Switzerland.*

(II) Ten of 22 steps necesi&;!y for chemical synthe-sis of TBEV receptor TPIs have been completed.,By means of exclusion chromatography with

S" porous glass arboviruaes can be purified and

concentrated. TBEV has no immunosuppressive

[ effect in mice. Langat virus, wh...ch belongsto TeE complex, causes chronic infection in

j° . mice and cannot be eliminated by interferon[ inducer Poly I:C.

(III) 'Clinical studies: 288 cases of TOE werediaGnosed.

(IV.)' Surveys with sera of cattle ans birds showedincidence of the tick-borne viruses "Tribeo"and "Uukuniemi" in Austria.

(V) Unidentified virus was isolated from migratingbird after arrival in hustria.

(VI) Survey with human and animal sera indicatedactivity of at least one group A virus and ofTBE and West Nile viruses in Turkay. Sara fromresidents of Cameroon had antibodies to se-

', veral group A and £ arboviruses includingU'nyongnyong, Chikungunya, Uganda S, Zika, Yel-

,low Fever and Dangue 2.

PI

V.

V

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C C ONT E N T S

Page

TICK-BORNE ENCEPHALITIS (TOE) AND OTHER GROUP 8ARBOVIRUSES .... ..............................

Field Studies e.. .. °........V°........o...& . .1

I3U Detection of new foci and attempts toeradicate the virus I.°°....°°..° 1

S(091) Introduction 1

(143) Result i ................... 2

S(1#,4) Discussion and Conclusions ... ,..., 4

(It), Summary ...... o..°.. ... .e..° 4

00 •2 Survey in Lower Austria with sera offorest workers

(2,1) Summary ................... 5

r> 2j Survey with sera of Carnivora from

Swi tzerland ..... 5...............

(3,1) Summary f V .* 4 a 0* 4. 7

o perimental Laboratory investigations *..., 7

o'" Studies on the receptor suostance (RS)

for TOE virus and other arboviruess ofV group B , ....... *...............,.. 7

(1,1) Introduction and earlier results ... 7

(1,2) Preparation of receptor substance .. 7"J (1,3) Chemical composition of TPI-Ca prepa-

r a t i o n s a, . , . . .0.. . . ° , , . . . . . . . o o * . . 8

(1,4) Chemical synthesis of intermediate pro-ducts on the way for a total synthesiso

LII

Page

L2 Concentration and purification ofarboviruses ......... ............. 9

(2,1) Concentration of TBE virus by an aqueoustwo phase system ................. 9

(2,2) Purification of arboviruses by chromato-graphy on porous glass ........... 0,

(2p3) Summary 11.................. ...... 11

j Chronic infection of mice with LaIcqatvi rus ... °. ..... o.oooo..•°. 13

(3,1) Persistence of Langat virus in thebrains of mice after s.c.infection .. 12

(3,2) Behaviour of Langat virus after ic.1

infection and of TBE virus after s.c.Sinfection .......... ......... ,.,, 12

(3,3) Susceptibility of chronically infectedanimals for a challenge infection go. 13

(3,4) Influence of the interferon inducerPoly I:C on "zhe chronic Langat virus• •'infection 13

(3,5) Discussion ....... ,..°.... .... ..... 14

(3,6) Summary ..... .06000000.0.... ... oo.. s 14

j Study on the possible immunosuppressive(1 effect of T8E virus .. ............ 15

(4,1 Summery ...... o. ............... 16

Studies onp.atients ..... ,......•.........* 16

(,j Diagnostic studies ................ 16• (II) Summarcy . o . . .. ......o,...... o .-o ... 16

TRIBEC VIRUS .. o. ....... .. .. .... • .... .. o.....1SQ• Survey for antibodies against Tribec

virus with sera of cattle .... ,... 17'0 (1,I) Summary oo .... 17

C,,O@ ° O O0 O ° ° °° °° °

S.'

S•SURVEY ON THE INCIDENCE OF ARBOVIRUSES ......... 18

o• • • Investigations on the zole of birds asS•hosts of arboviru3es in Austria 00.,,. is

°(11) Introduction 18

• (1,2) Methods .... 9

,+"(1,0) Virological and serological studies withSa of starlings 20

• °(1.,4) Virological and serological studies withS• sera of 21 migrating and non-migrating

• • ~bird species captured in autumA ... 20

> °•'• Ill(1,S) Serological survey on 6 bird species,• . during winter ........... . 21

";• "(196) Virological and serological studies on> "• i•Imigrating bird spacies just at arrival

• '[ ~in Austria in spring ..... .. .. 21(197) Discussion 21

S-(1,8) Summary ..... 0............... .e.e.s... 23

~ U

QI InvStudies on the and ecology of

Shbovit usso in Turkey s 990 8u6tria 6.... 0. 24

• ,( 2 # 1 ) , S u m m a r y . . . . . . . . . . . 6 eo T v,, . ao o. . . . . . , . . . 2 6

'° • • Survey with human sera from West Cameroon 26%3,1) Summary od t on ....................... 17

LITERATgRE CITED .................... 28

• 'TABLES 32

J•FIGURES 73

(13)Viologicalodo erolo gica studieeoo woeth~

(£4 iooia n erlgclsuiswt

s-ao 1mge~~ n a-irtn

2 Z2:

IC.K-.0RNE ENCEPHALITIS'(TBE) AN "OTHER GROUP B ARBO -VIRUSES

14 Field Studies

(1) Detection of new foci and attempts to eradicate•,•, th~e virus.

"7• (4;1) Introduction.

ItFollowing t e.msthod cf sending out questionnairesto Patients with TBE we couldfind.o-ne.new foci of TOE"in Lower Austria in 1970 and l?71 (see last Annual Report A- 73' j

F • (16')).Then also studies on the effectiveness of the organo-phosporus Gardona against ticks were startedý Qri theother hand we tried to reduce the small mammal populations

'in a focus in Carinthia in order to intsrrupt ths naturalK • cycle of T8E virus. Under the present contract (July 1971-

Ju, U• 9 72) w6 continueo the searchihg for new foci inLweOr Austriat Burgenland, Carinthia and even in Styria

;- and for the f'irst time in the Tyrols. Fiold trials weredone with anothar insect-cide, namely idith Nlalathion mf,which is also an organophosporus compound. The small mammalreduction program in Taggenbrunn, Carinthia, was continued.

(1,2) Methods.

Ticks: Nyjaiha and adults of Xxodej ricinus werecollected -by Vlagdrapping at'd-trenspurted to thc labora-tcmy. The nymphs were homogenized in pools o0 1-50 indivi-duals, th& adults in pools rof 1-2C individuals#, reepective-ly. They were suspended in a medlum consisting of PBS andlD0% horse serum and inoculated intracerebrally into babymice. The animals were observed for 18 days.

Malathion(R): Two field trials were done in orderto develop a control program with this compound againstticks. In tho spring of 1972 in a wood near the Danube atIOhlleiten, a few miles southeast of Vienna, and in ap af-forestation near Heornstein we marked 8 fields of 25 m' each.Four of these fields were spraywd using the ULV (ij LowVolume) method at a concentration of JO g PlalathLon ow

"C. diluted in 5 liters of water per 25 m . Tick collectionswere made before spreying and on the 3rd and 8th daythereafter.

0•

0

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Gerdona. Two field trials with Gardona (H) werecarried out in 3uly 1971 and in Play 1972 in Hochooterwitz(Carinthia), ,•:ich has been showsi by us to be a permanentforis of TBE virus. The .zound and the.egetation bordering

"the road to the fortress (i..e. th:eA.e'6sibble part of themountain on which the forrzXýs situated) were sprayed

with1 lbperac_ý1adnar J. In autumn the area was

Small mammals: In the foctjs.of' Taggenbrunn, Carin-this, eradication of the vijus-i5y1rapping of small maremale was attemp1ded.,U-farpr Sexcursions were done in 1971and one. in * bout 120 small mammal traps were set up? for 3 nights.

. (10) Results.

New foci in different p arts of Austria: AoOblding to theinforinations obtained from patients with" TBE,oticks worecollected in. di? fergnt areas in Lower Austria, Bwgenlond,

0 Cgrinthkia and *Styria. For the first time investigationson the'possible occurrence of TBE virus in the Tyrole

°- were conducted althouyh'no cases of TBE have..been reportedinth's province." The rebu.ts can be seen in Tables 1-4.S~Altooethar 20-%Arus.qtrains ,qould be isolated -from ticks

collected at 98. diffr.erent plac.es. New foci wead"ft"nd inS, Lowex. Austria (•Enz6'efeld),. tauling/Enzoesfeld,.,Airtbnborg),Pouýgenland (Allhad), Carinthia (Nicheldorf) and Styria(Giaz) (see Fig-.l). So far ws failed to isolate viuo fromtiqks collbcted in the Tyrols.

S, areTh'e 3 foci found in the area of .Hir•'nbdrg-Enzes-feld are of special interest because $.n the.se foci mnnypersons contracted infection. The ef6re, in future studieswe will try. to'.Interz.upt the virus: cycle in nature byspzaying...Of these fOCi with insecticides.

'L -'

Plalathion(R): 'he results. of the field study withoOalathidn.r) are-presented in Table 5 and in Fig.2. A eta-tistical evaluatibn done with the )( 2-test revealed thatthe numbdr of ticks in the fields did not diff Ir signifi-

0 o, cantly prior to the treatment with Malathion . By con-trast, after spraying with the compound there was a verysignificant difference between the number of ticks collec-

0 o ted in the treated and in the nontreated fields. In MUhl-leiten the tick population was reduced by approximately95b seven days after the treatment. In Hernstein where

V the undergrowth is rather dense the. even distributionof Malathion was not very easy. Nevertheless we observed1 a reduction of the tick population of about 8S.

Gardona(R): The first field trial with Gardona(R)0 oin an established focus of TBE virus was carried out on

July 8,1971, in Hochosterwitz (Carinthia). In July beforethe spraying we ware able to isolate 4 strains of virusfrom 272 ticks nollected there (Table 1). In autumn the

Savea was again searched for ticks as it is shown inTable 1. From a total of 524 specimens of Ixodes ricinus.not a single virus strain could be isolated. After col-lecting 430 nymphs and 43 adults of Ixodes ricinus inMay 1972 we sprayed again te aroa bordering the road to•the fortress with Gardens(R . Virus isolation experimentswith thusi ticks were not successful, indicating thatthe virus cycle may indeed have been interrupted by ouprevious lowering of the density of ticks with Gardona .

Small mammals: In Taggenbrunn (Carinthia) wecontinued the small mammal control program with the aimof eradicating the virus in this focus. In 1971, in April,when the first excursion was made after the winter not a

- • single mouse could be trapped. It is appuront from Table 6that by summer the small mammal populations had somewhatbuilt up and probably as a result of our control efforts -as a rule the highest number of free-living mice are found

"¶ ,in October and November - had decreased again by autumn.In May of 1972 we again found a vary low population den-sity of mice. It is of special interest that in this year,

0Y> 1972, no virus could be isolated from ticks although264 nymphs and 19 adults were collected and tested for thepresence of virus (see also Table 7).

62

Although it is too early to draw a dafinite con-clusion this could possibly mean that we are on the uayof reaching our goal a? interrupting the virus cycle inthe focus.

S+ (1,4) Discussion and Conclusions.---

The high number of new foci of TBE virus enablesus to conduct now further studies on methods to eradi-cats the virus in nature. Nou we will try to interruptthe virus cycle on the one hand by spraying tle focifromarthe.9goynd or from the air with Gardona R or with

* ~rdalathion. H• and on the other hand by reducing the popu-lation density of the small mammal species in certainfoci; In connection with the results of this program ourfirst tests are very encouraging and we hope, that at theend of this yoar the effectiveness of our methods willfuri)62-be substantiated.

S(i,5) summary.

'in search of endemic areas of TBE in differentprovinces of Austria 6 nbw foci in Lower Austria, Burgen-l land, Carinthia and Styria wora found by virus isolationsfrom ticks. The effectiveness of Malathion(H) againstticks (Ixodes ricinus) was proved in tda field trials. A

S°ruduction of between 85 ý. and 95 b of the tick populationswas achieved.

'In order to eradicate the virus in well known

permanent focus in Hochostorwitz Gardona M van sprayedin autumn 1971 ard in spring 1972. After spraying no viruscauld be isolatoc 'rom ticks collected in this area.

The control program drsmai1_-mammrPals in Tagqenbrunnwas continued. The population densities of these animalswere kept low and subsequently also in this permanentfocus we failed to isolate virus from ticks, collectedthore in 1972.

4

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(2)_ SurveX in Lower Austria with sara of forest workers.

In the hemagglutination-inhibition (HI) test a sur-vey was conducted with sera of forest workers from differentdistricts in Lower Austria. The purpose of thie "study wasto improve our knowledge on the distribution of TOE virus inK . thtis,,province but also to find a group of persons who are

° 4>'. at ,high risk 'f infection. It is intended to perform afield trial in volunteers as soon as the first batch of aninactivated vaccine will be available, hopefully, sometime during 1972. This batch is being made for us by a la-boratory in Bosat Britain whure we have sent 3 strains ofTBE virus,(tipk suspensions).Thie laboratory has had someexperieno* idtr'he production of a vaccine against Loupingill. This disease is caused by a virus that is very closolyrelated to TBE virus.

The results of the survey are presented in Table 8.It can be seen that of the 830 sera tested lU1 (12.2;6)had antibodies to the virus. A field trial is consideredin those districts wheru the percentage of workers withantibodies exceeds lD•.

(2,1) summary.A survey was conducted with sore of forest war-

kers from diffurent districts in Lower Austria. Thus, ourknowlvdge on the distribution of TeE virus in this provincewas improved and groups of persons, who are at high risk ofinfection, were found. It is intended to perform a fieldtrial as soon os an inactivated vaccine will be available.

S3 _Surtsey .with sera of Carnivora from Switzerland.

In July 1970, the Institute of Veterinary Bacterio-logy and Parasitology, Univeisity of Brn, received thebrain of a dog from Hallau (Kenton Schsffhausen) for diagno-sis of rabies. An agunt could be isolated from the dogwhich proved to be T8E virus. This was the first isolationof this virus in Switzorland, where in 1965 we had diagnoseda human case of TBE and, therefore, already then had claimedthat the virus must be endemic also in this country (14).Since then v iumber of human cases were reported by otherworkers.

",• "

- .6-

"' .o. .We yore contacted by Drs.-Stock and Wandeler from"" "' the Institute mantionod" above and asked to conduct a sur-

'•vey with see~q of Carnivore that had been shot during the

ra•bies "oradicatiori.program in Switzerland.

• "': " ,h totql-sOf• 519 sera of foxes, "U of badgers,2ofbse'ehf-*martena and".onea of a-polecat was raceived

o and -test~id in.the H'I-teet a gain3t TBE vi'rus, Uf 560 sera,•,• ,91 waer positive.

'o./ The lat'%W were then also examined for antibodiesI'•against this virus in the tissue -.ulture neutralizationi'•••• 2 • .,Btest.'EoileOf .these,, eut15 were',toxic for the cells and 76 gave

S•. It is a well known fact that sera fron animals" that are shot may become contaminated with nonspecific

,• ~inhibitors for arboviruaes.,such ale bile. Since all ani-•_.•"mela had been k'lled~by shooting Wb were not convinced""'• about th6 Validity of our'resultas. In: order to pick up

" " nrinepecifi6""inihibitors all 61 positive sera were tested. , , I.•n the Hl-te~st against bemliki and Sindbis entigens of

. "• , gzoup A and Yellow Fever, D~hgue 2 and West Nile antigens%- of grdup 'B.'

, .... •48 sera only reacted with TQ.E~eat~agO5'2 iua er;'•' also positive with oqJhsr--9roV-P'6*ý1usee and 22 sera

S o gAV9 .0_-:Pcs-ti• Test with group 8 and groupA viruses.• • Un6ur these circumstanees we ?eel that only'the HI-tast•'•-with- thos'e-48 sera TeaCting exclus'ively with TeE virus,, • .... hould'bb-bonsidered as a valid result due to specific:,\"•: ~antibodi'es...',

-,""In Fig.3 the geogrdphic distribution of the•,•samples is shown. From it it is striking that positive: as well as'negative sera are distributed more or less

,o ovsr the whole arQ&.under investigation. However, Lon-e• idering tfi`6 home range of foxee which, according to the

i~o"experiences of our Swiss colleagues, can be 50 km oro more, many of these animals were shot a long distancu

• away from the fucus whore they had acquired the infec-.. • tion.

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"A lees random distribution is seen when the results"are listed according to the age of the Carnivora (Table 9).

o •Of the 31 foxes aged between 1 1/2 and 4 months none wasfound tn have antibodies against TBE virus. Then a numbeaof positive sera increasing with ege wqre observed. As.oxes are very good indicators for the presence Qf TBEvirus in larger regions it can be stated with certaintyths't TBE virus must be more widely distributed in Switzer-land than hitherto known.

(3,1) Summary.

Of 560 sera of Carnivora shot in Switzerland48 specimens were found to contain antibodies against

. TBE virus in both the HI and the neutralization test. Thegeographic distribution of the positive sera shows thatTBE virua must be more widely distributed in Switzerlandthan hitherto known.

Experimental Laboratory Investigations

(1) Studies on the enceptor substance for TBF virus andother arboviruses of groug B.'

(1,1) Introduction and earlier results.

From brain-lipids, the receptor substance forgrg~up 8 arboviruses could be isolated (8,9,22). Thissubstance" Wed tuo--s-tCphosphoinositide .TPI). Itwas a very strongq'ihibitor-of the hemagglutination (HA)

~ as wall as or the infectivity of group 8 arbaviruses (9,15).It reacted with TBE virus in a two step reaction. An ini-tial reversible step, caLused mainly by electrostatic attrac-tion, is followed by a eccond one, causLed by strongerforces which are not any m,.,e susceptible to the interac-tion of electrically cherOed molecules (16).

(1,2) Preparation of receptor substance.

TPI is now prepared in our laboratory as a routineprocedure. We use a method of extraction, precipitation andchromatography which has been extensively deswribed in thereport of 197U (15). Some refinements were introduced (16)and now we are able to obtain from 1U00 g monkey brain 300to 4UU mg TPI in form of its Ca-salt.

vt

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(1,3) Chemicel composition of TPI-Ca preparation.

,The composition of 9uq preparations was now esti-meted more exactly by quantitative photometry of thinlayer chrdmatograms. Theee chromatograms were obtained byapplying'the m6thod of GUNZALEZ-SASTRE and FULCH-PI (10),using chloroformLmethanol-4N ammonia 9:7,:2 as solvent and"Kieselgel HR" (Merck) with 0.1b exalats as adsorbent (15).The intensity of tho spots, stained with DITTMER's rea-gent (6), was now measured quantitatively with a JOYCE"Chromoscan". The areas beneath the extinction graphswere measured with a pWccimeter (ýieometrical intogration).By dividing the integrated graphs by the number of phos-phorus atoms in the formula of the substance forming themeasured spot, values corresponding to the molar amount ofeach substance were obtained; due to the big difference ofthe relative concentrations of the main product (TPI) and

Sthe imputi1-'(phaphatidylserine=PS), it was necessary tomeasure two series of dilutions. From one (Table 10) the

e ... ratio of TPI to DPI could be calculated to be I00 t 9.88;the other (Table 11) yielded the ratio ofDPI to PS as100 : 7.32. From these figures the percentage of TPI, DPIand PS in our product was calculated to be. 90.40ý, 8.957 _and 0. 65, 2_eApecjt_.lel¥..

°0, (1,4) Chemical synthesis of intermediate products on theilk way for. a total- synthesis of TPl.

In.cOopevation with the-Institute'for PharmaceuticalroAK Chemistry, thete are also 6xperiments under way to obtaina synthetical receptor substance.' The main difficulty isthe synthesis. of the sterically right triphosphoinositol.Direct phoapharylation of inositol proved unsuccessfuland the,synthesis had to be started from 'aceto-bromoglu-'oose..j.ra series, of 21 steps, the final product, TPI,should be raaphod. Prosontly, the hydrolysis of step 9:2.3 .,.

di-o-alyl.,.2&w(deoxy) 8-nitro-0-benzylgldcooide to 2,3di-o-allyl-6-(duoxy) 6-nitroglucose is investigated. Thelast mentioned compound is expected to be transferred into

•.!!•; -deuxy-l-nitro-4.5-di-o-allyl-inositoI by treatment with

Ba-hydroxido. From this first inositol derivative 2,3,6-tri-benzyl-4.5-di-o-allyl-myoinositol shall be obtained in five"furthe, steps; This compound should be able to combine withdiacyl-glyceraol and POCI and, aftur phosphorylation and

2removal of the shielding groups, should yield finally TPI.

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(2) 'Concentration and purification of. arboviruses.

(2,1) roncentration of TBE virus by an aqueous two phaseSystem.

It was tried to concentratq and to purify TBE virususing a two phase system, contaiiffili polyethylene glycol(PEG) and dextran (1). A similar system has been usedalready for the concentration of Japaness encephalitisvirus (19).

Brains of infected baby mice were extracted witha berate saline buffer of pH 9 and treated with protamin

__÷ _ sulfate (4). To such a virus suspension PEG (M'iW 6,_.U)and dextran (1'1W ll0,UuU) was added to a final concentra-tion of 10• and 0.5b respectively. From 3U ml total solu-tion about 0.5 ml dextran rich phano separated which con-tained practically all of the added virus. This resultedin an approximately 5U-fold concentration of the originalvirus preparation (Table 12). UsinU tissue culture fluid,>° the virus apparently disappeared. I;t seemed to be adsorbed

onto a precipitatu from which it could not be eluted again.

(2,2) Purification of arbovirusos by chromatography onporous glass.

It was considered to treat arboviruses with enzymesto increase the HA-titre of badly hemagglutinating prepa-rations, especially of viruses from the Bunyamwera super-group. To obtain a good action of the enzymes, the virusbatches have to be purified prior to the enzyme truatmentin order to removo material which could possibly competeas substrate for the enzyme with the adsorbed impuritioeon the surface of the virus particles. It was also advisable

* to develop a purification procedure in order to separatethe virus from the enzyme after the treatment.

A very promising technique seemed to be chromato-graphy on a recently developed medium; controlled porousglass (12). The principle is the same as in gel-chromato-graphy. wowever, tho poares of the glass can be made ofnearly equal diamcter and all interconnecting. This causeethat a separation which would need 20 to 30 hours using an

roganic gel like Sephadex can be performed in as many mi-nutes, using CPG. Furthermore, columns filled with glass canbe sterilized uither chemically or by autoclaving beforu andafter use. The technique has been us9d already to purifyplont viruses (12) and bacteoriophages (11).

¢10

Preliminary experiments were performed with a

column filled with glass granules with a pore diameter of332 A. The column haA. 11mm diameter and was 100 cm long.For these tests, West wIa rs `ýs used as a model. The

I virus was extracted from infected &A-by 06uea brains with10 vol. of borate saline 6f pH 9, centrifuged at'20,OQO gfor 30 minu~tes, then ultracentrifuged at 105,000 g for "3 hours. The lediment was homogenized in about 1/10 of theoriginal volumie of the same buffer and the undissolvedresidue was sedimanted at 3,800 g for 30 minutes and dis-carded. The supe.natant had an HAwtitre of about 20,000.2 ml of this partly purified preparation were applied tothe column and eluted with borate saline, pH 9, at a 7lowrate of 2 ml/min. Monitoring with UV at 280 A revealed avery sharp peak at 30 ml which contained all the HA-acti-vity. A broad puak with a shoulder at 66 ml and a maximumat 80 ml represented smaller particles and molecules. Aboutequal amounts of viral and impurity protein were containedin this preparation ap could be judged from the integra-tion of the curves. An excellent separation of virus fromprotein moleculoes was also obtained when an equal volumeof 4• al~buidn wa6 admixed to tho virus before it was appliedto the qolumn. Again a very sharp peak with HA-activityoccurred at 38 ml.. The blbUmin appeared as a very broad peakwith a maximum at 62 ml. The original impurities could beobserved as a shoulder, at 80 ml (Fig.4). In this case, theratio of viral to nonviial protein was 1:7. Therefore, this

umethod supmed to be very appropriate for a separation ofarbovirusep from added enzymes. In thso experimunts how-ever, the yield of the viral HA was only IU to 2u per cent.oecause of the obvious advantages of thie method, we con-tinued our experiments; After trying different elution,media and a glass with a smaller pore diameter (240 A), itwas finally possible to obtain yield as largo as 75 to 85

,' o0 per-cent of HA. Now we use a 0.U5 MI Tris buffer, pH 8.2 +U.1 M NaCl (T-ris saline) and a coating of the poroua glasswith polyethylene glycol (MW=2U,UO0) (13).

Uur model virus (T8E) was extracted from infectedbaby mouse brains with borate saline, pH 8.5, and thentreated with protamin sulfate (4). This resulted in preci-pitation of acidic impuritics. By CPG-chromatography furtherimpuirities and also thu excaes of thu added protamin couldbe separated from the virus (Fig.5). This procedure couldbe -repeated in a way that a now batch of virus was applied

,when the protein-impurities of the previous sample were just'leavinr, the column. With 4 ml portions of virus applied tothe column und 9 flow rate of 2.5 ml/min it'was possible Lo

K.

Kp

0'

•A purify 20 ml of TBE virus in little moro than three hoursI ~ (Fig.6).

Further expeýriments with coated CPG were performed:Superhatants of low-dpeed centrifugatione of homogenatesfrom mouse brains infected with Sindbis virLu and withWest Nile virus were applied to the column without previousultracentrifugation or precipitation. Remaricbly. the 2U to60 fold amount of viral HA uas obtained by CPG c.romatogra-phy alone (Table 13). However, with the very poorly hem-agglutinating Tahyna virua (Bunyamwera supergroup) no in-crease of the HA-titre could be'obeekvad. It is hoped

<Vl_ i' that the intended enzymatic treatment of such preparationsprior•to chromatography will yield reasonably good hemag-glutininsi '

(2,3) Summary.

The receptdr substance for arboviruses of group Bcan be preparid from monkey brain in good yield. Thin layer.chromatograms'of such prepoiations were evaluated quantita-"tively by photometry.The product consisted of 90.4; TPI,8.9% DPI, and 0.7, PS. But also chemical synth.;sis of TPIwas undertaken. Starting from glucose, step lu of a plannedchain of 22 steps'could already be prepared.

TBE virus could be purified and concentrated 40-100 fold by partition in an qquious two phaso system con-taining PEG and dextran. In order to separate arbovikusesfrom enzymes to be added for purification, exclusion chro-matography on porous glass was investigated. Model axperi-ments showed that added albumin and protamin could be ra-moved from arboviruses by this technique. Coating the glasswith FPG'resulted in a dramatic increase'in the yield ofviral HA. When low''speed supernotants of mouse brains, in-fected with group A or B arboviruses were applied to porousglass columns, the purified virus showed 20 to 60 timesmor6 HA than the aupernatant. It was not possible to ob-tain hemoaglutinati6g prepa2ations of Bunyamwera super-group viruses by chromatography alone and enzymatic truat-ment had to be considored.

jL) Chronic Infection of Mice with Langat Virus.

DENK and KOVAC (5) studied the hietopathology ofLangat virusinfection in mice. They observed seven monthsp.i. residual encephalitis in animals, which had survived

A 4,

infection without any clinical signs of disease. Thischronic encephalitis caused by a member of the TBE com-plex of grdup B arbuvirtses was investigated by us indetail by vrological and serological methods.

(3,1) Perqitenc'e of Langat virus in the brain of miceafter s.c.infection.

Eighty mice (strain GP, NIH), weighing 15 g, wereinfected subc:utaneously with 1000 LD (for baby mice i.c.)

" C of Langat virus (strain TP 21). 47 mice survived infectionand uere considered as being chronically infected. Begin-ning 3 weeks p.i., at weekly intervals, 4 mice ware killed

°• r and their se8ra and brains wore pooled.

The brai6 suspensions (1lU dilutions in PBS) weretested, for the.,presence of virus, CF-antigen and interfuron.The sera were agerched Iur antibodies, virus and interferon.

"0 " As can be read in Table 14, traces of virus were

found in some of the brain suspensions until the end ofthe oxperiment (1 3 th week p.i.). Brains also contained"CF-antigen of Langat virus in titers ranging from 1:8 to1:32. Interferon was neither dutectod in breain nor inserum. Also no virus was found in the serum, obviously dueto ito content of antibodies, which wure demonstrated intiters from l.8Ukt_9 1020. Those antibodies were of the

"r ,IgG-fypu as zovealed'by-thq 2-morcapto-ethanol-tust (18).This indicat estHtt the infeldtion.utspnao longer in theacute stage.

S(3,2) Behaviour of Langat virus after i.c.inf&ction"'dnd

of TBE virus after s.c.infuctidn.

Ten mice each were infected i.c. with lUUO0 I00,10 and 1 LD5 . kfor baby mice i.c.) of Langat virus. Allanimals but 4, which had been infected with I LDSO, died.Those survivors neither showed virus nor complement fixingLangat antigen in the brina. Thus application uf the virusby the intracerobral instead of thL subcutaneous routo doesnot causo chronic infection. Besides, much smallur quanti-ties of virus are needed for lethal mncophalitis as com-pared with those necessary by the po:ipheral routo.

In the next oxperiment, 2U mice were e.c.infectedwith 10 LD05 (for baby mica i.c.). Lf TBE virus (strain

-S0

1 -13-

Hypr). Eight mice survived; howeve', -neither the virus norI, TOE antigen could be detected in the brains. Tfits, TOE

virus; an agent closely related to Langat vir(ih ,'does notcause chronic. Infection. in mice.

(3,3) Susceptibility of chranically inrected animalsfor a challenge infection witti Seinliki Forest

SI Virus.

A group of 46 mice infected chronically with Lan-get virus as well as a control.group of 29 noninfectedmice with the same weight and age were challenged with3 LDs1 of Semliki Forest (SF) virus four weeks after Langatvirueinfection. In Table 15 it can be seen thae 78•6 ofthe chronically infected and 62• of the control mice suc-

Fcumbed SF infection. Average survival bime was •.9 and° 4.8 'days rpopectively. From that we conclude, that chronic"

oLsngit virus infsctiQn neither prove t8infection with Psecond virus nor does it make the mice more suiceptibleLn to it.

(3,4) Influence of the interferon inducer Poly I:C onthe chronic Langat virus infection.

interferon was neither found in sere nor in thebrains of chronically infected mice: therefore we triedto eliminate the virus from the brain by the interferon

9 inducer Poly I:C, which is very affective against TeE inmic;e (17) and which is known to induce interferon not onlyin the serum but also in the brain (3). The experiment issummarized in Table 16, Out of 31 chronically infectedmice 10 were sacrificed aod thair brains testid individuallyfor virus and antigen: 8 mice showed antigen and 5 there-from also tho virus. Eleven of the remaining 21 mice were10• treated each i.p. with 20U gg Poly I:C, lU mice retimined

nontreated and served as contruls. Four days after Poly I:C?° application antigen was demon3trable in the brains of all

S• •5 mice tested, virus was four d 4 times, u40 the 5 carre-F spunding cuntrol mice 4 showud the antigen and 2 also thevirus. Aftestd days antigen tsds found in the brains of five

treated as well as of five n(ntreated mice. Virus could bedetected in 2 treated and in I nontreated mouse. Thus bymeans of Poly I:C neither virus nor the antigen could beeliminated frum the brains uf chronically Langat virus in-

So fected mice.

" °"o• , •-14-r o

"•36) 'biscussion.

K• Our experiments clearly indicate,.that Langat virusK may poesist after peripheral infection in the brain for a-1n•ng time..Similar results have been presented by PRICE(20), whc.infocted mice with Kyasanur rarest Disease virus,which is also a virus of the TBE complex. Hoauuvr, viryo.persistence suems not to be a general feature of theseviruses,.beoause after peripheral infection with TBE, virusdid not persist in'the brains of mice. But also after i.c.Langat .virus infection of mice virue'persistence could notbe"•asperved.

; UsWe assume that after peripheral infection due toextraneural virus replication nonspecific defense of theorganism is well stimulated. Thua the brain is protectedand virus cannot multiply to the extent, that is necoes-sary for the development of encephalitis. After i.c.infac-

° •tion nonapecific defense is not stimulated or too late andths anidial therefore always dies after infection.

: g nd.• impc~rtant mechanism uf nonspecific defense" againdt Irus infection is the interferon response of the

host, I hf'h thaoretically could be responsible for the-"above'-mntioned resistanceaof s.c.infected mice against

Langat virus. Howover, this does not seem tO be the casebecause intorfuron never was found in chronically infucted.mice,'uhich also were uqually susceptible for.a challengeinfection as noninfectec animals.-Besides, Poly I:C hadnu influence on the content of virus and antigen in thebrairli

I ecently, .attention has bean focusod on the per.-sistenca uf measles virus in human brain and its relationto Subacute Sklearosing Panenoephalitis. The results ofour study provide some evidence that, at least in thebrains ofmico, else arbuviru.sea.may reach a stat.; of chro-nic infuction..n view of these findings one wuonars whetharthis couid not happen in the brain of man tuu. This themore, because we were frequently told by the clinicians thatoccasionally TBE can take a rather protracted course withlong lasting disorders in the EEG.

(3,6) Summary.

The chronic Langat virus infection of mice wasstudied virolioically in detail. The virus ,ae5 recuvuzud"from thL brains until the ,nd of the study (13th week), Also

9

0

CF antigen was. fqund in the brains in titers.ranging from18*to 1:32. In sera no virus but HI aniibodiep of the-

Ig(-typo were found against thi8.virus. Interferon wasnuithe: detected in the brains nor in the surum. The chro-nically infected mica never did show any sign of diseaseand were eq!;nlly susceptible for a second infection withSFV as normal mice. Their brains cannot be freed from thevirus by applination of the interferon inducer Poly TsCo

(4) Study on the possible immunosuperessive effectof the TBE virus'**-

It has been published that some viruses - prado-minantly oncogenice viruses.- have an immunosuppressiveeffect.

was investigated by us on the antibody-producing systems

in mice. This was cione by injecting a suspension of gooseu•ythrucytec intrapseritoneally into infected and nonin-,fected animals an conparing t-, titer of goose cell agglu-tinating antlbodles that had beL~n formod in both groupsof mice by thu 1'ourth day thereafter.

SU Initially the concentration of goose erythvocytesin 0.2 ml saline was determined in noninfected micegiv'.iig rise to sufficiently high titers. The resulty maybe soen in Table 17. Due to these results a concentva-tion of 2% was chosen fur all the experimbnts.

Exoerimunt 1: Threw groups of 10 mice each wers infacteewith about 10 LU of TBE virus, Atrain "Hypp". One, 2 and3 days thermafter mice of une group were immunized, and4 days aftor immunizatiun the animals were bled. Nonin-fected controls were immunized and bled io the same man-ner. Antibody titers against goose orythrucytos in allgroups of enimals (infucted and noninfuctod) were 1:256.

Experiment 2: In this experiment mice weru infected witha very high dose of virus (1U 5 LD5 0 )O Four groups were im-munized, 192,3 and 4 eays p.i. roapuctively. Althoughthosu mice which had receivud rud blood cells 4 days p.i.weor bled in e moribund state thuy exhibited the samelevels of antibodies against the goosu cells as the con-

/'

0j

.= ---

-:".

.ý-o*-i ýj~e on immuo-

trols. Thus, TBE virus 0 _ -" mIsuppressive effect, although it multiplies.a... a.t......producing tissues of infected mice.

(4,1) Sr"""'a"y... ran'umsafyt-. - -. , ion, T-

By immunizing mice with goose-erythrocytes atuodifforon.. s after infection, TBE virus was not foundto have.an imrmunbsuppresive effect.

•+ Studiess'on bEatien te • -

° + ~~(1) Diagnostic studles,;: ",, .:

0 Since the beginning of-1971 w. usually diagnoseTBE by.means of the 2-mercaptoethanoi-test, which was

°o ,.,, described in last year's Final Technical Rbpqrt"(15).This test gives excellent resulta and isuwell acceptedby the physicians at the hospitals, because it makes anearly diagnosis possible. unly in a few cases a second"serum sample is required and the CF-tost must be per-formed#'-' ... ...

The 288 cas,' Uf; TBE, diagnuse by thoso methods

in the .year 1971 in ",he 1ifferunt .provinc4i of Austriaare lidtod in detail in Table 18. Fu comparison, Table 19.,.gives the results of the' first three months' of the sea;-"bour"l49-7-2. *S-o far, there seems to be about the samt inci-denc6 of.TBE.in 19?'2 ad in 157l.; Howover, it will be ofgreat iriipurtaic," if the simwrore will be hot and dr*-or__.cool and rainy.

.(,iy' Summary.

0 In the yoar 1971, 288 cases of TBE had been re-

corded. From the first cases recorded in 1972 it is like-ly that during this year 1972 the disease will have about

the same inoidence as last year.0-+

V~ TRIBEC VIRUS

(1) Survey for-antibodie3 acainat Tribee virus withsera of cattle.

Soo

arbovirusea and was first isolated from ticks in Czecho-Aý slovakia. Some Czech workers claim that this agent may be

voth --Viii.-f 9-

Because of the possible clinical importance ofthis virus and its transmission by thd'same'tick, ixodesricinus,. which. is the etodr qfn.TTE virus, we attemptedto get some~iqtormat~on~on.its i'ncJ~dence in Austria bymeafsof -serýa oic survey wictsera of cattle. These

"- doarbostic animils are kniown.to devesla p •ntik6dies again6t

thi•' virus..

St trs, a total of 1825 s~era was tested in the... tiaeizatueon f t es pousin blekU-3U. TcIDc ofthe virus andchith vmbryo celals. .tAntbuadias ware .det cted in 14 soerairldicating he,.oat.incJ4erce of Tr ibc virus in Austria,

From Table 2U~ it is strlki~ng that all positive"sera but 2 came from cattle frum Carinthia. Unly ane posi-tive serum each was in tahe samples from Salzburg and Our-. .nland. Titers of.posi5ve sra eranged between t:h andl:4U. -(2C,) Summary!,

, Antibodies against Tribec virus were found in sarasf. cattle frPm Carintthia. Sara of cattle fpor, other di-

' s-,ctasteteum e Styrian Thu Tyroles Upper Austrig, !4anlz-r- % Jenl andand.) were negative with' only b e zc:I.aaons.

°% " :4u. . , .

K -~

'2 " 0

SURVEYS ON THE It6CIOE•?CE OF ARBUIRUSES

£°)A Investigations on the role of birds as hostsI IS .• of arboviiusea in Austrla;

K ,'; (Introductiot -

.-Lo s we-irknown that-birdi cay represent impor-tF_.t-'ti8ts of arboviruses. Due td the fact that m•ny

- -. species migrate over large distances every year, theymay also introduce viruses which do not normally circu-late in certain regions, paRticularly in moderate zones.

So for, in Austria no inveaeigat.ons on the roleof birds as hosts of arbovirueap have been carried out.In order to elucidate their Importance from the arboviro-logical aspect, we started'a field pcogram in the Nau-siedlersee area during fail 1970 which uase continued in1971 and 1972.

First in fall 1970, we directed our attention tostarlings .Sturnua vulparis), a opeciee which occurs Invery high population densities in the Neusiedlersee area.M'iany attempts were made to capture a nepresentative num-o = bar of this species usidg aesaral methods at day and at

night. It proved, however, to be extremely difficult toobtain larger numbers of blood samples of starlings;despite of a high expenso only.32 sera could be collec-

S' ted. We therefore tried'to get parmission for capturingall bird species o1urring in the reed zone of the Neu-siedlersee'area. which we obtained in 1971. Thus, numerousbird species'could be netted in reasonably high numbersduring ýerious seasons in 1971 and 1972.

In total, so far tra following investigationswere carried out: -

(a) V~rologJcal and seroldgical survey with 32 segaof starlings.

(b) Virological and serological investiget.ions with488 sera ofr 21 bird species (migratin6 and non-migrating)collected during autumn 1971.

W() Serological survey with 125 saera of 6 bird speciescollected during late winter 1972.

(d) Virological and aerological investigations with149 onra of 16 migrating species during spring 1972.

I7

(1,2) Methods.

Birds were captured with Japanese mist nets.<24 In the case of the starlings the nets were placed

in open areas as well as in the reed zone in the vicinityof the sleeping sites of these birds in the Western partof the Neusiedlersee area. As soon as one starling hadbeen netted the others shunned this locality, so that wewere forced continuously to change the capture places.Ue also tried to force the swarms into the nets - at day/ 0 by driving a car over the meadows behind the swarms, atniqht )y.stortl.ng them by boat or by foot, This latter

z;;,t"jd was also unsatisfactory as the sleaping sites arein most cases situated in places whnch are inaccessive

4 to a boat (too much reed), although the water level sums-times reaches a height of 1 m. At any rate, the success

K was very poor.

As we got the permission also to capture birdsother than starlings in 1971, all further collections ofbirds wore carried out in the read zone near the villageNeusiedl in the Northeastern part of the lake. The netswete put up early after sunrise and removed in the ,eve-ning.

With the exception of the starlings which werebled from the wing vein, blood was taken from all otherbirds from the jugular vein. Birds were marked and ro-leased immediately after puncture. Some birds were recap-tuped some days later;,these specimens were however ru-leased without taking a second blood sample.

A small part of the blood was immediately frozen"in dry ice and then kept at -80°C until virus isolation

6 experiments weru done. The main part of the blood (about0,05 to 0.2 ml) was immediately diluted in 0.5 ml of PBS,kept in ice for some hours and then frozun at -2D C untilserological examination.

Virus isolation trials wmre carried out by i.c.in-oculation of the blood into baby mice. The mice were ob-Served over a period of two weeks.

Some of the stearling sera were tested four neutra-lizing antibudiee against Tohyna virus in a tissue culture

of the cell line GMK-AH-1 with methods paeviously describedo+ (2).

All other aera were tested for hemagglutination in-hibiting antibadies according to the reference method de-

. scribed by CLARKE and CASALS (4)..The sera were treatedwith acetone and tasted. in a dilution of 1!10 z-a--st4 tu 8 units of coitain antigens (sue below). They woreregarded as positive when they reacted in this d1lution;due to the fact that already the blood samples had beendiluted immediately ater puncture, thck real dilutionswuro higher, ranging from 1:20 to 1:50, or even more.

(1,3) Virological and serological survey with sara of•, o , •starlinge..

During the peiod from September 23 until Ucto-bar 15, 1970, blood samples of 32 starlings •(Sturnu VUl-Uia_• wore collected. From those 23 sampl3s (Nr.1-23)were tested for noutralizing antibodies against Tahynavirus, 29 .ora (Nr.4-32) were tested for humagglutinationinhibiting antibodies against the following antigens: Yel-low Fever.(YF), Dengue II, West Nile (Wiq), TBE, Sindbisand Sumlikl.

In three samples antibodies against arbovirusascould be detected. Serum Nr.5 reacted positively in thuhemagglutinatiun inhibition (Hl)-tust against YF andDengue II; norum N-.9 ruactud positively in the HI-testagainst DOenuu II and in the neutralizat'ion test (HT)

q•ainst Tahyna virus; serum Nr.12 hao neutralizing anti-bodies against Tahyna virus.

Frum nune uf the blood samples virus coulo beisolated.

(1,4) Vir.ologicsl and serological survey with sera of21 migrating and non-migrating bird speciescaptured in autumn.

Un September 3,1971, we started collections ofblood samples of bizds netted in the reod zone of theNeusiedlersou and continued these captures until October22,1971.

r 2During this period a total of 488 blood samplesf frin 21 bird species w:-s obtaired.

.21-

No virus could be iaolatad from any of the samplestested.

A serological survey was done with all sera bytesting them in the HI-test agaq'nst the following anti-goqns:.TeE, WN, Uukuniei*, Chikungunya, Samlikit Sindbis,Calovo and Tahyna. .Tho results are shown in Table 21.

(1,5) Seralogical survey with 6 bird apecies during

In order to investigate the question whether birds

, ,which normally do not leave Central EurDpe may act ashosts of.arboviruses, that are endemic in the Neusiedler-see area, blood samples were collected of birds nettedin the reed zone of the Neusiedlersee area during theperiod from Fpbruary 16 to April 8,1972.

Altogether 125 individuals belonging to 6 specieswere capturec The oera. were tested In the HI-test againstthe antigens mentioned under (2,4). The reau:lts of thesestudios are shown in Table 22.

9 4, * (1,6) Virological and serological survey with 15 migra-ting birds just at arrival is. Austria in ipring.

•:In ordertu_ts&,d._ths question. of a possible intro-S...•t ""duc'ti~n"•ij?-iiba-bu-i-uses by-.•da, fJ~m•t~opical and subtropi-

S"-2 6eb91'56i a 'ieidstudy was carried aut in thereed zonef o"of the Neusiedlersee during the period from March 15 toM ay 17,1972. In this investigation only those birde werej included which had newly arrived from abroad, so that thedetection of a possible viremia could be expected. Alto-gather, 149 birds belonging to 16 species were captured.Thu blood samples swere te~ost for virus an6 for hemmagglu-tination inhibiting antibodies againat the.antigens men-tioned under (2,4).

The results of this invwstigation are shown in

Table 23.

(1,7) Discuesion.

In the investigations described above, altogether"794 birds belonging to 28 species (of 12 familios) wereincluded from which so faor 639 individuals were tasted forvirus and 786 for antibodies.

A'

-0,

S"Uuf-of-thoeblood .f a Rybin (Erithicus rubeculi)S60a strain of a virud was .isolated. The agent could serially

be pzopagated in baby mice.. Rcisoletion .from the bloodSapcz:men was successful. A crude antigen was pzepaiod from

V? the brains of mice infected with the 3rd passage of'thisvirus and tested in the cumplemontfixatiun test with soreageinat tie folldwing viruses: TBE, 41N, Uukuniemi, Tribec,Caluvu ard Tahyna. None of the sera reacted with th& agent.Thus the virus dwes not appear tu be identical with anr

Saof the arbovirusoa which aru knouwn to ba endemic in Austria.Table 24 summarizes the results obtained in the

HI-tests. All positive sera will be tested for neutrali-zing antibodies in near future. From. Table 24 it will beseen that most positive reactions occurred with Uukuriimivirus. ERNEK et al. (7) also found in 5 of 25 sera of anumber of bird species captured in Slovakia hemagglutina-tion irihibiting antibodies against Uukuniemi. Our resultsindicate that this virus probably also occurs in Austria.

viruses might bu traced back to infections acqur.ed in

Central Europe while most other positive findings (Chikun-0gunya, Semliki, Sindbis, WN, YF, Dengue II) found in mi-

grating birds (Locustella luscinioides, Acrocehalus mela-_Snpogan, Acrocophalus aciroaceus, Apropop'alus erundina-caut.. Sturnus vulQarisa) may reflict prior infections withgroup A and B viruses in Africa or in the Neditorranean

08 reogion.

o Uf particular intorqst is, however, thu findingof hemagglutination inhibiting antibodies against Semlikivirus in a Boardbd Tit (Panurus Vsrmicus).and in BlueTits (Parus caeruluus) thus indicating that th3se birdshad boen infectecd with a group A arbuvirus. The popula-tions found in Austria normalyv du not leave CentralEuruopo during winter, vury raroly do they migrate, huw-ever, to thu Ijurthourn parts uf the N'uditurranean rugion.lit any rato, no arbovirus of gruup A has so far beun isu-latud in Europe, and the rcsults obtained give, therefore,

K a further nint to the (purhaps occasional) occurrence ofa Uroup A arbuvirus un thts cuntin.nt.

Furthermore, 'he finding of antibudles againstSi virus in a 8lue Tit cannot be clearly intorpretud, Per-

0 haps this particular bird had already once bcen in the Iqu-

I -,

o •-23-

diterranean region, where UN virus io endemic; on the otherhand it cannot be excluded that I,. virus may occasionallyoccur in the Neusiedlersee area, where its vector Culexmodestus is found.

4 Finally it should be stressed that in none o? the754 sera teated in the HI-test antobodies agai4nst Tahynavirus could be detected while among 23 starlings examinedin the Neutralization test antibodies were found in twospecimens. By contrast, in 3xperimental studios SIMKOUA (24)

S0 , found that young starlings (1-6 days old) are not ascep-tible for Tahyna virus and neither develop viremie nor

°- - neutralizing antibudies. Thus, the question arises whetherelder ineividuals are suwceptible to the virus or whetherthe two positive tests ay .be due to nonspecific virus in-hibitorc in the sera. Furthermore, the question, whetherbirds are itvolved in the circulation of the Tehyna virusremains open.

(1,8) Summary.

During the period from September 1970 until May 1972° • blood was. taken 'from ?74 birds telonging to 28 species which

- uwere captured in the Neusiedlarseo area in the east ofAustria.

So far, 639 individuals weme tested for virus andK 786 for antibodies; from these, 754 samples were teoted

for hemagglutination inhibiting antibodies against Uuku-[o , niemi, Calovo, Tahyna, Chikungunya, Semliki, S.ndbis, T8ES°•and UN only. Of the remaining 32 sara (all from starlings)r 29 samples were tested for henegglutination inhibiting

antibodies against YF, Dengue II, UN, TBE, Sindbis andSemliki, and 23 samples for neutralizing antibodies againstTahyna virus.

Out of the blood of a robin a strain of a virus(probably an arbovirus) was isolated which could, howevez,not yet be identified.

Hemagglutination inhibiting antibodies were foundin the following birds (in brackets: antigens against whichantibodies were dotected/number of positiva samples): Spot-ted Irake (TBE/l), Green Sandpiper (Calovo/l), Savi's Warb-rlo (Sindbic/b, TBE/l, WN/2), 3edge Warbler (TBE/l), Peed

Warbler (Uukuniemi/4, Chikungunya/2, Semliki/5, Sindbis/1,WN/3), Great Reed Warbler (Chinkungunya/l), Bearded Tit

_V

"_ " -- r- . .. .. oT. ...

"(Semliki/l), Robin '(Uukinlemi/l), Blue Tit (Uukunimi/5,I

.Senliki/4, WN/1), Roed Bunting'(Uukuniami/2, Chikungunya/1)"and Starling (YF/1, O6 gue 11/2), Moustached Warbler(Chinkungunya/1, Sindbis/1,. TBE/l, WN/2). In addition,nutu•alizing intibod3ed'against Tahyna virus were found

. .the iebult bf the study appears to indicate thepresence of Uukuniemi virus in Austria, although most ofthe other positive findings can be explained by prior in-fections acquired in Central Europe or (in the case of ;ni-grating birds) in the Wiediterranean region or in Africa.

Uf particular interest are, however, reactions withantigens of group A arboviruses uith the blood of birdswhich do not migrate or, at least, not farther than theMediterranean region. This is a hint for the occurrenceof a group A arbovirus in Europe.

iiL.(21' -Studies on the activity and ecology o? arbovirusesin Turkey.

In 1965, Db.Radda took part in an expedition toTurkey in order to collect zoological material for theMuseum of Natural History in Vienna. Thun he was also ableto collect mors than 200 blood samples of domestic animals.

...ý The results of a serulogical survey with these sera showedthat in the surroundings of Ankara West Nile (W4) virus oran agent very closely related is active. In the south-oastern part of Anatolia, in the Vilayet Hatay, he foundanimals with antibodies against one virus of group A andone of group B. From the results of the neutralizationtest the latter seemed to be Tick-burno encephalitis (TBE)virus (2.1).

Very simil' results weoe obtained by SERTER (23)who also made investigations on the *activity of arbovirusesin Turkey.ý He diagnosed three human cases of TBE with sero-"logic methods and could domonstrate the presence of anti-bodies in human beings against TBE, West Nile, Dongue II(D II), Tahyna and Sindbio viruses in the surroundings ofIzmir.

Because uf these findings we felt that further stu-- dies on the incidence of arbovirusas in Turkey wore indi-

cated. In 1971, Dr.Radda was awarded a medical fellowshipby the Council of Eurupa in urder to conduct studies on the

41

activity an .eculogy dtiboviruses.inAhdtotiia. He workedfrom-Siptember 20 until November 20,1971, and again from

4P. March 17 until April 15,1972t at the Department of Micro-biology and Infectious Diseases of the Madical faculty,Ege University in Bornova/Izmir.

h At first, collections of seraefrom human beings• Z (healthy people as well as outdoor patients) were made.

A total of 270 serum specimens were obtained and shippedto our laboratory for testing (see Table 25). IP addition,263 sera from sheep deriving from 8 different places

K (Isparta,. Konya, Tire., Canakkale, Aliaga, I!laso, Menemen,Edremit) which had been slaughtered in Izmir w,:; c_..•tested (see Table 26). The results summarized in Table 25pruvide further evidence for the occurrence of at leastone virus-of group A and two viruses of group B in theIzmir area. The group A agent appears to be related toSemliki virus while one of the group 9 agents must be.very ciose to or identical with WN* virae.'Surprisinglynone of the human-seri e6acted with TaE virus in the hem-agglutination inhibition (HI)-test. However, among these from sheep 5 wore positive when tested against thisvirus.

of In order to catch small mammals in the surroundingof gemalpese, a small town about 30 km east of Izmir, whereone humei caso of TOE had been observed (22), small mammal

eV• traps wore set up in different habitats. In 14 trappingnights (491 trap-units) 82 individuals of different speciesof small mammals wore trapped (see Table 27). These animalsobvihuely were nut caught in a focus of TOE viruc a3 it isshown by the lack of antibodies against this antigen(Table 25). Yet 4 sera gave positive results (3 Plus musacu-lug, 1 A.udemus spec.) in the HI-test with the Semlikior two group B antigens. Dr.Radda also collected 90 humansera from residents of Istanbul and 95 sora From Ankara indifferent hospitals. The eare of both groups of humansyielded gimilar results and providud some evidence fur theactivity uf unu ur, perhaps, two viruses of group B (seeTable 25).

Sera that had bean positive in the HI-test againstTOE or uther group 8 viruses were also tested against TOEvirus in the neutralization test using L cells for the

"antibody assay. This was dune in order tu prove with thismore specific test that at least some of the antibodiesagainst group B viruses actually were due to infections

"with this virus, Thus in 9 sera nuutralizing antibodiesagainst TBE virus were f-und (ae Table 28).

During fall of 1971 it was hardly pussible to c,1-lect any ticks because of their inactivity at that time ofthe year. However, in spring 1972, n total of 782 ticks,mostly larvao, nymphs.and adults of Ixodes ricinus, but alsoscoe specimens of Haemaphysalis punctata, Rhipicephalusbursa and Hyalomma aeqyptium could be collectod in pineforests and shrubs slung fields on places whero also smallmammals had been trappod during the fall of 1971. Virusisolation experiments from these ticks were not success-ful (see Table 29). Finally the plant associations wereanalysed in the plac3s where field studies on ticks andtheir small mammal hosts.weru done (see Table 30).

(291) Summary.

Ilk° Sara from humans, sheep and small mammals were in-vostigatod for adtibodies against A and B arbovirusee.About 5, w•re positive. It seems that cne of the threeprobable causative agents is relatud to Semliki Forestvirus, the other two appear to be West Nile and Tick-borneencephalitis virus. Attempts at isolation of TBE virusfrom ticks collucted in Turkey were not successful.

(3) Survuy with human sera from West Camuroon.

•During a zoological collecting trip from 1anuaryuntil February, 1971, Dr.Radda was able to do some medicalstudies at thu Presbytoriar General Hospital in 'anyomen,

•o0 West Cameroun. These studies wusr granted by a fellowshipof the "Nutring Wissonschaftlicher Verb~nde (jsteerrichs".In order tu purfurm a serological eurvey on the activity

Q oof arboviruses in West Cameroun 173 serum specimens ?romoutdour patiunts of the local population wero culloctedand shipped to our loburatury in Austria.

Jýo> Sora wero treated with acetone to reniuve nunspeci-fic inhibitors in the hemagglutination i,; :.bition (HI)-test.The test was done follouing the claasical procoduruo du-scribed by CLARKP and CASALS (4). The following antigens,prepared from sucrose-acetune treatud baby-mouse brains,wore used: Semliki Forest (SF), Sindbis (Sind.), YallowFever (YF), Iurray Ualley Encephalitis (iiVE), West Nile (WN),Denguce II (D 2), Tick-burne Encephalitis (TBE7).

IV

-,27--

"The results of the HI-test can be sesn in Table 31.,Lt•y-aeven oser from a total qf 173 'sera tested gave posL-1t1vo reactions. Many gera showed cross reactions with aurethan-one antigen.

All the 40 sera of the first group which wore poai-tive in the HI-tast were tested agalh in the neutralizationtest (NT) in green aunkey cells (strain GPMK-AHI),2 againstthe following virusest: O'nyongnyong, Chikungunya, Zika.,Uganda S. These viruses Pre known to occur in Africa, Theresults can be seen in Table 32. Titers of hemagglutinationinhibiting and neutralizing an'ibodies of all positive saraere listed in Yable 33. Upon reading the results of theHI-tebt it aill be noted that a few saga reacted to e lowtiter with Semliki and Sindbis antigens which are gaouparbovirusos. These ars probably cruse reeztione which warspossibly caused by infections, with agents closely relaetd

tu or identical with J'nyongnyqng and Chikungunya viruses,as it can be seen from the results with the more specificNT. The pattern of hemagglutination inhibiting antibodies

V I against the group B viruses (YF, NVJ, WN, D 2v TBEE tested3Ls somewhat puzzling. However, from our experience andthat uf other workers it is a reaeonable assumption thata number of group B viruses are actzve in the area wherethe sera were ccllertod. This would also explain the hightiters against OiVE virus, althuugh this agent dues not cc-cur in Africa. un the other hand, some of antibodiesagainst YF virus (seo sera Nr.22,42 and 57) could be causedby infections with this virus. Nunu of the persons, whoseblood wcs included in the survey, had prcviuusly been vac-cinated against Yellou Fever. Besides, at least one typeof Dengue virus appears tu be endemic in the area under-investigation (see sera 21,33,80 and 92). In addition,Uganda S and Zika viruses are probably present in WestCameroon, as it can be concluded from the result uf the dT.

(3,1) Summary.

A serological survey on 173 sora of thu residentsof West Camerouon ruveale• the activitiy of at least twoarbuviruses of group A, probably Ulnyongnyong and Chikun-gunya as well as fuur virusoo of group 8 including Uganda S,Zika, Yellow Fever ont, a member of the De--uu viruses.

0 ? ' /'

0

if

-REFERENJCES CITED

()Albortson,P.A. (1958): Particle fractionation inlipid two-phase systems. The compositionof some phase systems- and the behaviourOf someniodel particles in them.

- Bioc~hm.Biophys.Rcta 27.L 3378-395.*

(2) Aspdck,H. and Ch.Kunz (1972): Llntersuch~ingen Oberdio (iborpinterung von Tahyna- und Calovo-Virus ina Arnphib.ion und Reptilian.Zbl.Bakt., I.Rbt.Orig., 216, 1-8.

(3) Catalana,LoW. and S.Saran (1970).: Protection against:Vj~ herpes virus and oncephaloanyucarditis vixus

with a double-stranded RNA inducer ofinterferon.Proc.SocExptl.Siol.Muid., 133 68-687.

(4) ClarkeD.H. an~d J.C8s01s (1958): Tochniquos forhu.magglutinating and harnogglutinationinhibition with arthropud-burnte virusus.

H Amur.Trup.Mud.Hyg., :7j 561-575.

(5) Donk,H. und 6,,Kovac (1969): Luie Neuropathologio derdurch Langat-Virue bed~ington Encephalu-rryelitis der wuil~en I'laus.Acta nourapath., 12, 1-172..

(6) Uittmer,ZJ.C,..nd R..L.Lustor (1964): A simplo

* 0ciecAific- spray fur the dotoctiun of phosphci-1l.pids on thin~layiir chromatograms.

K ~aJlipid Ras.j 5 1 2 6 .(7) Ernek,E:.,, (.Kozuch, M'.Sokeyova, K.Huduc and

C.Fulk (1971): Antibodies tu arbovirusuain birds in Czechoslovakia.Acta viral., 15,_ 335.

()Friech-NiggumayurW. (1967): Diu chumisctse Strukturdur bei d'or Adsorption von TBE-ViJrus anErythruzytun wirksamen Iiozeaptorsubstanz.Arch.Hyq.3akt., 151., 585-598.

b~epso'albe c

K,>"

A

(9) Frisch-aigge rteyerL. (1971): Polyphosphoinositidesas raecptor substances for certain groupsof arboviruses.-Acta virol., .15, 119-125.

(10) GcnzaleZ-Sastre,F. land 3.Folch-Pi (1968): Thin-lay.eor-chrom3tography of the phosphoino-

J.oiipld Res., " 532-533.

"(11) Gschwondnor,H.H, W.Haller and P.H.Hofschnaider (1969):Large-scale preparations of viruses bysteric chromatography on columnz of con-trolled pore glass._Bichem.Biophys.Acta 19 46U-469#

(12) Haller,W. (1965): Chromatography on glass ofcontrolled pore size.Nature, 206. 693-396.

(13) HiattC.W., A.Shelokov, E.J.Rossnthal and

"J.M.Galimoro (1971): Treatment of con-trulled pore glass with puly (ethyleneoxyde) to prevent adsorptiun of rabiesvirus.3.Chrometogr., 56, 362-364.

o .. (14) Kunz,Ch., W.Frisch-Niggemeyer, 3.Loew and A.Hadda(1966): Epidomiologicalp woological andbiochemical Studies on tick-borrio ance-phalitis (TBE) virus.Annual Report.Contract Number 91-591-EUC-3649.

(15) Kunz,Ch,, H.Aspdck, W.Frisch-Niggumeyer, H.Hofmannand A.fHoddo (1970); Studios on Eick-bo ieencephalitis and kthor arthrupod-burnevirus diseasus-.Annual Report.Contract Number UAJA 37-69-C1451.

ReSproduied: fIro~me f ' a

°O

(16) Kunz,Ch.9 H.Pspdck, LhFrisch-Niggomayer, H.Hofmann,A.Radda and G.Wiedermann (1971 ): Studieson tick-borne encephalitis and other ar-r thropod-borno virus diseases.Annual Roport,

* Centract.Number BAJA 37-70-C2462.

(17) Kunz,CA~. undl H.Hofrnann (1969): Die BeeinflussungI ~der oxperimentellon, Fr~Jhsomrner-Meningo-anzophalitis (FSME)-llirusinfektion durchI dio Interfeorn induzierande Substanz

k Poly I:C.Zbl.Bakt., I.Abt.Orig., 211, 270-273o

(18) Kunz,Ch. unci H.Hofmsnn (1971): Diu Fr~hdiagnon'oder Fr~hsummor-Ileningoonzaplielitis imHgmaggluti'notionshammungetest durchBehandlung des Serums mit 2-MuIrcapto-fithano1.Zbl.8akt., I.Abt.Oriq., A. 218, 273-279.

(19) NskaiH. (1965): concentration of Japanese once-phalitis virus by aqueous polymer twophase systu.a.

(20) Price,1W.H. (1966): Chronic disuasu and viruspersistunce in mico inoculated withKyasanur Purust disoasu vi-rus.Virology 29, 679-681.

(21) Radda,A.: Antibodies against group A and B arbo-viruses In dumcsstic animals from Turkey.Ege Universitesi, Tip Fak.Mocmuasi(Izmir).In presse.

K0 '.22) Schlosingur,.U~. and P.M.Gsterrieth (1972):0ý0 Arboviruses.

1ý Int.Virol.2, 2nd Int.Congr.Virol.9Budapest 1971, 158-169.

I Reproduced frem (4best availablo =COPY.

(23) Sort&,'sF. '(1968): Ego 8519aainde Karioler1e BulaaanMc.k-borne) VizrOs loienengo-Ansofeaitleri.

* Ego Univorsiteso± Tip Fek.-12ecmuaei (Izmir~),

(24) SiakovaqR. (1962): Tahyna virus in birds.Repr Vioduce 6,.19U

43

00

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n- NO.CO NO n- --31 07 '.a n

Z - ?

K • Table 4: Number of ticks (Ixodes ricinus) collectod 1971 and1972 in different atreas and virus strains isolated

t herefrom

Virus strain No. Pool size Location Date

34253 5 females £nzesfeld 5bpt.l1,1971

% 34260 5 Males Hirtanberg Septo19,19?

34262 5 females Hirtenberg Sept.19,1971

34352 28 nymphs Micheldorf ct. 3,1971-

S33540 20 nymphs Hochosterwitz July 7,1971

33547 20 nymphs Hochosterwitz July 7,1971

33576 20 nymphs Hochosterwitz July 7)1971

33578 12 nymphs Hochosteruitz July 7,0971

33586 20 nymphs Taggenbruin July 8,9101

34324 20 nymphs Taggenbrunr act. 1,19-I'1"V.

34326 4 females +2 males Taggenbrunn Oct. 1,1971

364t#3 .20 nymphs Allhau II April 4,1972

36445 20 nymphs Allhau II April 4,1972

36449 20 nymphs Allhau II April 4,1972

36455 7 famales -

I male Allhau II April 4,1972

rt° 36505 28 nymphs Hohenegg II April 30,1972

"" 36593 20 nymphs Jauling/Enzes-fold May 10,1972

36765 20 nymphs hirtenberg May 24,25,1972

36793 20 nymphs Hirtenbor6 dlay 24,25,1972

36801 5 females +"5 males Hirtenb9rg May 24,25,1972

36854 54 nymphs + Graz/Autobahn June 3,19725 adults

0~

0e

4 I

04 in I -q CHC

41 EC -q NiI I' H*rl

E1 =1 H l n Lo L )v

_P z ~ ~~c 0 )HC4 0 HCC3p-%'

i-l-I N4 H ('

41. co 0

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Q CD

omeq0 4.) m'4 c- N.~ ' CS in. C-r!r q I 1 - _1

00 0

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(a -1 0

H~ mNHH 0

ti teH -f _CI U 0H t)U 1 t a

in 0411-1a ll 11 1IA.

al

co Ol u 6 411 H*

rill k m 4)10 Cll k 443I 4H oH- (41l 41.- r4

ZllC 0 Ak0 MiDl Ca .0.0 Fu - 4

, 4

Table 6: Results of small mammal trapping in Taggenbrunn(July 1971 - June 1972).

Excursion No. Date Trapped animals

1 July 7-8 1971 16 Apodemus Spec.5 Clathrionamys cilareolue

!• 1 Piicrotus Spec.

2 August 9-11 1971 ..... 14 Apademug spec.

2 Clethrionomys glareolus3 Iicrotus spec.

3 Sept. 1u-12 1971 8 Apodemus spec.0o 1 Microtus Spec.

4 uct. 1-3 1971 8 Apodemus Spec.S1I Clethrionamys clareolus

5 Nov. 5-8 1971 11 Apodemus spec.5 Clethzionamys qlareolus3 Microtus spec.2 Sonex araneus

6 May 13-15 1972 4 Apodmus spec.I Microtus Spec.

0

"o "

0

0

S0

-4L-

Table 7.- WJi.b•r of ticke (Ixodes ribiius) collected inTaggenbrunn in three subsequent yeare and numberof virus strains isolated therefrom.

:i u mbe r ao

K Exour•siol . nymphs strains adults strainsI.date collected isolated collected isolated

"S _ept-, 1-5,

-- "69 . 204 - 64 5

I -May 27,1970 668 17 938

:. July 8, -1S••°1971 • 205 1 39

Sept 1-12

1971 194 lU

'•, ct. 1-3p"" 1971 288 11

day 13-15,1972 264 19

F- •

4--1

- 4

Table 8: Hesults of HI-tests uith ssea of forest uorke-rsand TOE virua.

District of Niumber of uorkers tNumber of workeitsLauer Austria tested with antibodies

I 8: Baden 164 36 (22,

Wr.fleustadt 64 11 (1?hJ)

?Neunkirchen 174 11 (6N)Pxrei~baum 46 1

St.P~lten 154 25 (166)

Lilienfleld 165 9 (5•)

Scneibbs 63 8 (13%)

T c t a 1 83U 101

0

U"

C

K >o

IVIa 30

C34

00 ILS0 InV4F

VV

oC 0.n4-0 -C4 to c t e0 -P ( 0i

0 ~ J4.)0 CL I- Ho0 I'

0 4) 5.4 Q

430 CD 3IC1

0 co

co C C 1) 3 -

2! cH D N

ca , * rfI 1 4 3N

CD 0

o 4co t)~

0 0 (

.0 4 H3 N- 4K> IC zI k 0

CD H DN33 4

Nq 0 0 -1

Table 10:1 W~antil~ta*vc cvalustion of thin layer chr-c;2atograme a"' polyphioopnoinaouAAidar3 R atioTPI :DPI.

af S rom

AP 'ADP A AS AP/3 .D!,/2-

35 1-4.0j 0.8 * 105.3 7.(! 10 6.65

161. 10.0 n.d, 53.7 5.0 100 :1~.32

359 22.8 1.2 119. 6 Al. 4 100 ; 9.54

184 17.3 n.d. 5-1.3 8.6 100 1-34.03.

M.-~an L'atlo TPY UPY:'J 10LLI: 9.38

ATP?: Arann brtionan bf:se lion)~ a'nd axtinct;;ion gz.;3

of TPI F1pot

ADPI : Area betw-en bets. .line nand o.-tinc{;i~on grca;.-h

of DPI spotjA

PS5 Arcai botbwecn basoe linr. and oxtinztiori nr',rih

of PS spot obmaurdr

n.de Not dete~rmined quantitativoly.,

A2.2. values arbitrary i'nit~o.

.944-.

Al

~LD Table 11: Qiuantitative evaluation of thin-layer chrcmato-

graeO of polyphosphoinoaitides II. g Ratio

Vto

ATPI ADPI APS ADPI/2 DPI : PS

662 153 6.5 76.5 100 : 8.50

685 264 6.5 132.0 100 493

582 176 7.5 88.0 100 :8.52_ _ *675 220 8.5 110.0 100 : 7.27

M'lean ratio UPI : PS = IOU : 7.j2

A ATPI ;Area between base line and extinction graph of

o ATPI : Area between base line and extinction graph ofDPI spot

APS : Area between base line and extinction graph ofPS spot

* : Value too large to be measured exactly

All values given in arbitrary units

2

__ _ •-45-

Table 12: Concentration of TBE virus by an aqubus twoS... phase system.

* ~Expeririant No.

1 2 3 4

*IVolume (ml) 25.5 25.5 25.5 25.5

Original MA-titre : 2,560 5,120 2,560 12,800

Units : 65,000 130,000 65,000 326,000

Volume (ml) : 29.5 29.5 29.5 29.5

PEG-phase HA-titre :32 32 16 128

Units 940 94U 470 3,770

Yield : L5. 7- 0.? 1.2%

Volume (ml) : 0.4 0.5 0.5 0.5

o Dextran-phase HA-titre : 256,010 256,000 204,600 512,000

ov, Units : 102,000 128,00U 1029000 256,000

- •Yield )l00> 98• • 100 .

Concentration-factor lOOx 5My 40x 20x

The original was already purified once by the two phasesystem

Arbitrary units resulting from multiplication of titreby volume

II

Tb -13: Ic 2eas of eAmmg 82%,192a-virus prsp

ralawt by CP,:-c, rzatogrerhy.

Sindbis virus:Applied to co19u 2 r-1 512 1024

16201 64 128

20 2 '4. 256 5-1221 2 oi 128 25622 2 nl 64 128

"27,648

Uest NW1e virus

Applied to column 2 ml 8 16

Eluate,Fraction No.:

--15 2rl ....

1s 2 ml 54 12817 2 ml 256 51218 2 ml 128 25619 2 mn 32 6420 2 .a1 16 3221 2 ml 8 1622 2 ml 4 823 2 ol 2 4

"•-• • 1,020

9. ,

-47-

Table 14: Per;16tenme of Langat virus in the brains of mice.

Week Brain .. Serum

4- Virus Interfe.-on CF antigen Vfirus Interi"cron HI antibodies

3 + -- 1:8 - - 1:160

4 + 1:32 1 - :132U

5:15 -1 1: 80

6 + - 1:16 6 - - U1160

7 + - 1:8 - 1:320

8 + - 1:16 - - .1-320

9 + " 1 8--1320

10 1:16

11 1:6 -1:32U

12 - - 1:16 - - 1:320- .

J1 + - 1:32 - - 1:323

0 0

0 I

74,, .

I.N

Table 15: Susceptibility of chrcnically infected animalafor a challenge infection with Semliki 'oreatvi.rus.

oup Numbe qi.f ice Numbe o ib -Average---- ___--infected with succumbed to survival

•o~~~F FV ....... 9t-ne ... ime.

N'"ice chroni-dally infec- 4"6 36 (78%) 4.9 daysted with

0 7 Langat virus

Control 29 18 (626) 4.8 days

7>

.2

43

CD

43

14 0 0

s43 -

43 0 D0

.D kt) U) U0G

M M 43o E24343Z i

IDD

(44 >

43r

44

C- 4-c1 0 -0 0 0.c i C

CD 0 0ý-

.. i 6r 'D k CD c

641

.3 0.

F- 0 0

rc.n

Table 17: Immunization of mice with goo0e erythrocytes- (0.2 mi/mouse).

Concentration of Titer of antibodies against4 goose erythrocytes in goose erythrocytes 4 days

K'; after injection

S0.M005 neg.

o.01 neg.

a*0.05 neg.

0.5 1.4

1.01:

5.0 1.128

- 10.0 1:256

06

V 3K

K

VI CN 13 0 a 0 nI V~)

U) riI CN NNN wa N. M N: Cte .CZ C') C) t 4 uI Ill

If >

I Ua

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E UN.

r-I 4

O 0 TU #- H 3 t 004 C4

m6 - 0 C") 0 C3 0E N%% N N% IN.

C- N 0i0 V 14

LID 0 C4 Z30 0 co 11 C4V-1 0 cmN r-1 0 N1

H NN I N 94

03 %0 r4 ri 0D it -

<C 1 in tr o If1 U

.D 0- 0i rl 00 C) E -

L- : 34 ) N N 04 H N t. U N 1 C

q C14 * Ni- Uf %0 0

C. U);>0 .-3 L: If Z)

0 0 II I.II

H It CMc

OD G.a C . . . . . . .13

r-4 0 0 0 0 X: k I it

P-0 ý (D 0 3 "1 ' 4 0 D -4 0 N It CZ -1mI 0) Q') FAi- M IICL0 A 1--1.

to $4 0ýl 0: o 4 L3 : 3i0. 0 c 00 301 0 cc m InI

41 N ' N. U N1

Table 19: Caoe of TBE in 1972 until June in Austria.

Pro~ince A.ril May ýJune

SVienna 3/0/0 14/o/oLowe1 /ustria 10/0* 10/2/1 16/4/1

Carýnthia 3/2/0 17/1/1

Styria

Upper Auatria 1/1/0 4/0/0 15/0/1

Burgenland I/O/U 1/0/0 1/0/0

Salkburg

Tho Tyraol 1/0/0

T o. t a 1 3/1/0 21/4/1 64/5/3

TBE confirmed by 2-ME/CF/uncertain (HI positive,2;.MC and CF-tests uncertain)

r'0

K. ,/,

Table 20: Antibodies agai.nst Tribec virus in srea of cattlefrom~ different provinces o? Austria.

Pr:ovince . Number of' sera Number oftested positive sera

Styria 68U 0

The Tyrol 225 0g•, Upper Austria* 180 0

Salzburg 254 1

Carinthia -A7.3.-- 12

. .. 8urgenrarii 13 1

T o t a 1 1825 14

00I• • •I

II

Table 21: Virological and serological survey uith sera ofmigrating and non-migrating bird species.

Species Number of blood Number of sarasamples tested tested fo. anti-for virus/virus badies/niimberaisolation positive sera+

Fam. AROEIDAE1. Little B.ittern

(Ixcbrychusmýnutua) 1/- 1/-

fFam. RALLIDAE2. Water Rail

(Rallus aquaticus) 1/- 1/-

3. Spotted Crake(Porzana porzana) 1/- 1/1 TBE

Fam. SCOLOPACIDAE4. ,Snipe

(Gallinago gallinago) 2/- 21-

Fam. ALCEDINIDAE5. Kingfisher

(Alcedo atthis) 2/- 2/-

Fam. HIRUNDINIDAE+• •6. Swallow

6, (Hi.undo rustica) I/- 1/-

Fam. NIUTACILLIUAE7. White Wagtail

(O•ttcilla alba) 2/- 2/-8. Blue-headed bagtail

(rotacilla flava) I/- I/-

Famin, MUSCICAPIDAE"9. Savi's Warblai.

(Locustella '.uscini-oides) 6/- 6/-

10. Mioustached Wart:1er(Acraocephalusmlelanopogon) 4u/- 40/ 1U, 1CH, JI'

11. Sedge Warbler(Acrocephalusschoenobaenus) 17/- 17/-

Table 21: (Flokw sheet, cant.)

12. Marsh Uarbler(Acracephalus palu-stris) 3/- 3/-

13. Rteed Uarbler(Acrocephalus arun-dinaceus) 187/- 187/ 2UJ, 2CH9

SSFDq IWN14. Great Reed Liarb1er

(Acrocephalus arun-dinacaus) 15/- 15/ICIh

15. Bearded Tit(Panurus biarmicus) 37/- 37/ISFLi

16, I Chiff'chaff cly(Phyllascopus cly

* bita) I/- /'47. Robinj ~(Erithacus rubiacula) 1/- -

/ 1 18. PEMIZduliE T1ai. RenuiZDTit*f(Reiniz penrdulimus) 36/- 36/-

F am. PARIDAE0/19. Blue Tit

I ~~~(Parus caaruleus) 52/-5/SD N20. Great Tit

(Parus major) I/- I/-

10, rao EMBERIZIDAE

21, Reed Buntingrj;(Emberiza scoheniciusAl11 81ls ICHT a t a 1 488/- 488 4U,5BCH,

8SFDp ITBEp3WN

+)For abbr'eviations see Table 21 A.

Kt

-56-

Table 21 A: Abbreviations.

TBE = Tick borne-encephalitis

U = Uukuniemi

CH = Chikungunya

"04 SFD Semliki Forest Diseaee

WN = Weot Nile

I •C = Calovo

Sind = Sindbia --... -

YF = Yellow Fever

rI IVE = Murray Valley Encephalitis

D2 Dengue II2Onyong= U'nyongnyong

UgS Uganda S

00

"4 I

0

'S,€ - -

Table 22: Serological survey with birds captured during

Species Number of sere tested forantibodies/number of positiv"aera +

Fam. TRUGLUDYTIDAE1. Wren

(Troglodytestrogiodytee) 1/-

Fam. MUSCICAPIDAE2. Bearded Tit

(Panurue biarmicus) 3/-

Fam,.....Rf!ZIDAE3. Penduline Tit

(Remiz pendulimus) 23/-

Fam. PARIDAE4; Blue Tit

C" (Parus caeruleus) 69/5U, 2SFDS. Great Tit

(Paerus major) 1/-

Fam. EMBERIZIDAE6. Reed Bunting ++)

.(Emberiza schoeniclus) 28/lU

+) Abbreviations see Table 21 A

++) Reed Buntings found in Austria during winteur haveusually spent the summer in other parts of Europesituated north of Austria.

4- -

•.,• -58-

Table 23: Virological and serological aurvey,.wit1 ±"i*r•--returning from tropical and subtropical hiber-nation areas.

r .. Spepiep Number of birds Number of seracaptured so far/ tested for antibo-Number of blood dies/Number of po-

........................-. smpfes tested sitive sera +)for vr 8ir r . .isolation

."" Fam. SCULUPACIDAE1. Snipe

(Gallinago galli- - . ......nago) .. 1/- 1/-

2. hadshank(Tringa totanus) 3/3/- 3/-.

3. Green Sandpiper(Tringa ochropus) 1/1/- 1/TSE, C

Fam. MUTACILLIDAE4. Blue-headed Wagtail

(Motacilla'flava) 1/1/- : l-5. White Wagtail

(Motacilla elba) 3/3/- 2/-Fam. MUSCICAPIDAE

0,Savils Warbler '. -

- (Locustalla lusci-

".9/ITBE, lSind,

7. Moustached Warbler"("Acrocophaluemelanopogon) 9/8/- 9/1TBE, lind,

o ;" IWN"•8. Sedge Warbler

(Acrocephalusschodndbaonus) 8/8/- 8/1TBE

9."Reed Warbler(Acrocophalus"scirpaceus) 40/24/- 37/2Ut, Sind,

2WNlb. Great Reed Warbler

(Acrocephalus arun-dineceus) 35/24/- 32/-

0

y -9= " -" U'- Table 23: (Flo 8heet;. cont.) " . i:;

Species Number of 'bi£•ds iyjmber of oe-a0 captured do far/ testaed for antibo-

Number of blood diea/flumber of pa-samples tested sitive sere ÷)

4- for virus/virus

11. Garden Warbler(Sylvia borin) 1/1/- 1/-

12. Blackcap.XSylvli ~attrica-i;* •~ ~~ pila), // /

Sw:13. ChiffohWr

,'• ;collybita) i/I/- i/-S14. Willow abe

(Phylloscopustrochilus) 2/2/- 2/-

1.Robin(Lrithacus rubecula) 18/18/uimus not 18/IU

Reed Bntingyet idantifiiadFam. EMBERIZIDAEI,6,16. Reed Bunting

(Emberizaschoeniclue) 16/15/- 15/-

,..%•":T o a 1 150/!18 141/3U, ICV

"3Sind 4TBE,bb atn I virus not yet identi-

.•% ,.•.•fled

-. ;.' " +)Abbreviations see Table 21 A

a C

Atz.0

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6 ~ ~ ~ ~ .G-'4CF L M 0 a0 Z 9

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to 0+)

$14 "4 Il4)3 z 2e%0 -i a -

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C3 (0 .3 4-) 64 : 14c0 3 r-i H ,-4 W a)r-4 oC 00 1 0 40) 0

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cC~LO LA-4 CL +o

-64-

Table 25: Results of the hemagglutination inhibition testsS• I with. eer• from Turkey.

, - N~mber of p6sitiver sera from

Izmir Izmir Kemalpasa Istanbul Ankara.Antigen (human) (sheep) (small mcamm.) (human) -(human)

SemlikiForestDisease 6 - 1 - -

-• ~~Sindbis...West Nile 17 4 - 1 4

Tick-borne 5

encephalitis

Dengue II 1 2 2 - -

Murray Valley 7 4 2 7 3encephalitis

Yellow Fever 4 2 - 1 3

T o t a 1 23*/270 12*/263 4*/82 8*/90 5*/95

Number !f pnsitive/investigated sera

* Some of the sera showed cross reactzins with more thanone antigen

K

-65-

Table 26: Results of the survey with 263 sheep sera frombestern Anatolia.

Geographic: [Numbar of Number of Titers of the positiveregion sera inve- positive sera in the HI

stigated sera

Kenya 17 1 WN 1:2U

Isparta 92 4 2 iN 1:20, 3 TSE 1:20,6 .2 YF 1:20 "

Tire 30 1 WN 1:10

""r Canakkale 21 -

Edremit 23 - ..

Aliaga 24 1 MuE 1:10

(,henemen 33 3 2 RUE 1:10, D2 1:10

""lilas 23 2 2-TIE 1:10, 1 MUE 1:10,

4,o

" "T t a1 263 12 4 i;, 5 TBE, 2 YF, 4 NrE,2 D2

""+ (a-l

+ Abbreviations see Table 21 A

4,4 '

4<

%I

-65-

Table 27: Results of small mammal1 trappings in-Kremalp~asa77k and results of HI-tests.

4-Spbcies Number of i~umber:o? +~ ~sera tested positive sera

rius muaculus spicilesus 37 3 (SFD, liVE, D2)J?0

Apademus epee. 28 1 (FiVE)

Cricetulus miqratorius 3

Cracidura spac. 12-

Crocidura suaveolens 1

Suncus etruscus I -

To0tal1 82 4

+ Abbreviations see Tabla 21 A

&,

i;, ,

Table 28: Results of the neutralization t~ist with

TBE virus.

Serum Ne. Species beri'"I£ng Antibody titer

254 Homo Izmir 1:80

"73/71 Ovis Isparta 1:80

98/72 (ivis Milas 1:40

281 Homo Istanbul 1:20

284 Homo Istanbul 1: 5

286 . Homo Istanbul 1:10

292 Homo Istanbul .

62 homo Ankara T;i -

70 Homo Ankara 1:80

A,

•5

K -68-

Table 29: Results of tick collections near Kernalpasaand Belkav&

-- Sparmies--and-dwu- f~~ia nwtt- otalmental stage (Kemalpasa) Pineturn

(F-oroiat, elkave)

Ixodes ricinusLawvat6 70 271 341Nymphs 51 3U41 355Adults 6 18 24

Hyalomma av.pyetiurýAdults 5 5

Rhiipicephalus bursaAdults 2 2

Haemaphysalis punctataLarvae 52 52

KAdults -3 3

T Total1 179 603 782

-69-

Table 3U0: Plant communities in habitats of ticks and their*small mammal hosts in possible foci of 7hE virus

in Kemalpassa and Balkave.

Spocies Frequoncy of species in thehabitat macchia habitat pinatum(Kemalpose) (Bolkave)

(Juarcus chocif era ++++

Phillyrea media +Zicsrninum fruticans ++ +Cistus creticus ++Laurus nabilis +Paliurus spi4ns-

christi+Platanus orientalis +Ruscus acutifolius +Pyrus a~nygdnlifolius +Cyclamen neapolitanum ++Vitex agnus-castus +Crataegus monogyna ++ +"Companula- l-yrr~t-- ----Pistacia terobinthus +(4uercus ilex +(Juercus infectoriaAsparaigus acutifolia +Cirsium Spec.

"I ~Rubus Spec.Taraxcicum officinela"Origanum virens-PMarrubium Spec.Rumax Spec.Rainunculus arvensisPinus halupunsisUrchis analotica +

Frequency very high ...frequency high ++

frequency low +rare

Table 31: Results of hemagglutination inhibition testswith 173 human sera from West Cameroon.

Number of positive sera

S: Antigen Group 1 Gypup, 2- ,,.'Total

Semliki Forest

Disease 2 1 3

Sindbis 5 1 6

Yellow Fever 16 16 32

Murray VoelcyEncephalitis 31 17

West Nile - 2U 4 24

Dengue II 17 2 19

Tick-borneEncephalitis 5 0 5

I T o t a 1 40*/98 27*/75 67*/173

" Numbe r of sera positive/investigated

* Many of the sera showed cross ieactions with more thanone antigen

0

o0

K

-71-"C"

*Table 32: Results of the neutralization tests(N)wt- thu 40 positive sera of gruup 1.

Virus Positive reactions

Olnyongnyong 7

S•. °Chikungunya 12

Zika 2

UgEandh S 14

I T o t a 1 22/40

•2" -

- Number 'of sera

- positive/investigated

.0

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M CD c .0 00 0 3 0""C4 " N"H " -i

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4 000000000 300 00 00

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43c ca w o 0 0 0 0 0 o : o : c

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added albumina by CPG-chromatogr~p7

t;,4 -I.'AAiL

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pretamin by GPG-chroaatogrepbj

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0

KEY WUJRDS:

Arboviruses in Austria; eradication of TOE fzci; TOE ecology;

0 ~TOE in Swaitzerland; receptor for TBE (Synthesis); concentra-tion of arboviruses; purification of arboviruses; persistent

#i~i arbovirus infection; Langat virus; birds as hosts of arbo-

viruses; arboviruses in Turkoy; arboviruses in Cameroon.