EVALUATION OF TOXORHYNCHITES SPLENDENS AS A BIOASSAY HOST FOR O--ETC(U)JAN 81 0 M WATTS. B A HARRISON, A NISALAK

UNC LASSSIFEOi NL

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Fvruluation of Toxorhynchites .~s arbthissay Host for Dengue ViruSe Interim

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Watts, D.M., Harrison, B.A., Nisalak, A.,Scott, R.M., and Burke, D. S.

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bioassay host for the detection and propagation of dengue viruses. Alldengue virus serotypes and strains attained titers in T. splendens comparableto those observed for 2 strains of Aedes aegypti. Peak virus titers occurredin Tx. r;,Uendens approximately 6 days postinoculation; however, speciJficfluorescence for all viruses was not observed in 100% of mosquito heads until12 days postinoculation. A 100% correlation was noted between specificfluorescence in Tx. spendcns heads and the recovery of virus from correspondin

WO ~ 43 IIF NOVPT IS OB1SOLETEI J A ,, 1 7 3 lo s O F I N o 6 5U N C L A S S I F IE D

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thorax-abdomens. The volume of inoculum tolerated by Tx. splendens wasapproximately 5 times greater than that injected into Ae. aegypti. Thus,for a given volume of inoculum, the number of Tx. splendens required forvirus assays was appreciably less than that needed for Ae. aegypti. Theoverall survival rate for Tx. splendens following lntra|thoracic inoculationwith dengue viruses was 92%, compared to 41 and 42% for 2 strains of maleAe. aegypti. These findings imply that Tx. splendens would be more efficientthan Ae. aegypti as a laboratory assay host for detecting dengue viruses inblood of infected patients and for use in experimental investigations\

SECURITY CLASSIFICATION OF THIS PAGE(Ien Dees Entered)

. . .. - II

V ON OF TP)XORIIYNCHITES SPLENDENIS AS A BIOASSAY

HOST FOR DENGUE VIRUSES

By D)ouglas M. /Watts,- Bruce A. Harrison, 2 Ananda Nisalak,

"obcrt M. Scott and Donald S./Burke1

The views oi iw autiors do not purport to reflect the positions of

!,cplrtMeTL0 toh re .\rmv or the Department of Defense.

tDo i::irt:-:ent , ci 'oio:y, U.S. Army >ledical Component-AFRIMS, APO SanF-..1C1sC0, . ,6)4h (international mail, Rajvithi Road, Bangkok 4,

Mhail,1d) .

2-Department 2"dical Entomology, U.S. Army Medical Component-AFRIMS.

3Department ,.,iru:- Dsieas.es, Walter Reed Army Institute of Research,

'ash ingto:r, 10. 2U 12.

Pri,,nt ;hd,;: U.S. Army Medica[ Research Institute of Infectious

Di ,,asos, Fr Iccick, >arvland 21701, to whom correspondence should be

add resSa..

2

.\b ll-t at o:.:orhvn chites splendens, a non-hiematophagous mosquito

was evaluated ,Is a bioassav host for the detection and propagation of

dengue viruses. All dengue virus serotypes and strains attained titers

in T. splendens comparable to those observed for 2 strains of Aedes

aegvnti. Peak virus titers occurred in Tx. splendens approximately 6 days

postinoculation; however, specific fluorescence for all viruses was not

observed in lO0"' ,of mosquito heads until 12 days postinoculation. A

100% correlation was noted between specific fluorescence in Tx. splendens

heads and the recovery of virus from corresponding thorax-abdomens. The

voluo:l of inocuium tolerated by Tx. splendens was approximately 5 times

grc,!:!,r tha Lut injected into Ae. aegypti. Thus, for a given volume

01 ulc:n, L:ie number of Tx. splendens required for virus assays was

apoJ--iaiv' icss than that needed for Ae. aegvpti. The overall survival

cat 4, r TK. s p-.ndens following intrathoracic inoculation with dengue

viruses was Ij, coiipared to 41 and f2g" for 2 strains of male Ae. aegyti.

These fizidiii, imply that Tx. splendens would be more efficient than

Ao. aeoypti ... :i laboratory assay host for detecting dengue viruses in

blood of ia: uLd patients and for use in experimental investigations.

, -J

3

At least 2 species of the Aedes subgenus Ste.omvia hlve been tested

as bioassav hosts for dengue viruses. Aides a lhop ictus (Skuse) has been

shown to be susceptible to infection with all 4 den),ue virus serotypes

following intrathoraic inoculation (Rosen & Cubler 1974, Kuherski L ::osen

1977). Infectivity titers attained in this species were hiigher than those

observed in the LLC-MK 2 cell plaque assay of Yuill et al. (1968), the

technique most commonly used to isolate these viruses. 1: addition,

dengue viruses were isolated more frequently from sera of dengue patients

by using Ae. albopictus as compared with LLC-MK, cells (Rosen & Gubler

1974). Subsequently, Woodall et al. (1979) and Gubler et :1. (1979)

found that Ae. aegypti (Linnaeus), was also a sensitive and reliable host

for isolating dengue viruses from patients. Virus isolation rates from

dengue patients in Southeast Asia obtained by using Ac. acyoti and Ae.

albopictus (Gubler et al. 1978, 1979, Kuberski et al. 1977) w re

consistently higher than rates previously reported using other assay

techniques (Russell et al. 1968, Winter et al. 1969, Hlalstead et al.

1969a, b, Nimmannitya et al. 1969).

In addition to the above studies demonstrating the suitability of Ae.

aegypti and Ae. albopictus as assay hosts for dengue viruses, Kuberski &

Rosen (1977) suggested that Toxorhynchites amboinensis (Doleschall) was

also a suitable host. Furthermore, Burton & Rudnick (1979) reported that

Toxorhynchites splendens (Wiedemann) was susceptible to dengue virus

infection following intrathoracic innoculation. Although these 4 species

appear to be suitable assay hosts, Rosen & Gubler (1974) reported that

a Culex species and an Ainigeres species were resistant to dengue virus

infection following parenteral inoculation. These differences in

susceptibility indicate that mosquito species vary in their efficiency as

t _ ,4 - '-- , 4. f . ' "- < -. :9.J

4Iassv iio.'{i Lsor 1 c.lontte viruses; however, comparative studies documenting

thI have not )tcn pu bLished.

This -stud\ was co0d'UCtd to eval nate Tx. sp endens, a commion

Sout'Aeast A'-itn mosquito, as a hMoassav host for dengue viruses, and to

comp:ire IL,, stasceptibilitv to dengue virus with that of different Ac.

ae-'pvtA strains. Additional. evaluations were based on the size of the

2 soecies, the post inocitlation survival and the time required to detect

virus specific fluorescence in the 2 species.

MLTI!d'RIALS AN) METHODS

A, e_,_,:Li mosquitoes were obtained from colonies maintained in

the 1)_Iart.nrt of ,.edical ntomology, Armed Forces Research Institute

of .'cJical >'ic es (AFRI.IS), Bangkok, Thailand. Colony 1,,'1, of unknown

:

5

identity was determined by plaque reduction neutralization tests (PRNT)

in LLC-MK, cells employing dengue virus types 1, 2, 3 and 4 monospecific

antisera (Rlussell & Nisalak 1967). Antisera were produced by injecting

rhesus monkeys intramuscularly and subcutaneously with 103.5 plaque

forming units PFU/mil of each dengue virus serotype. After approximately

one month, blood was obtained from monkeys; sera were obtained by

centrifu ;ition at 270 x g for 30 min.

Mosquitoes were immobilized for inoculation in 50-ml test tubes in

an ice water bath. Inoculation of mosquitoes with virus was performed

with a needle and apparatus similar to that described by Rosen & Gubler

(1974). Five or more Tx. splendens and 20 to 40 Ae. aegypti were

inoculated with each virus dilution, 0.85 ,l per Tx. splendens and

0.17 .,l per .. aeslypti. Viruses were diluted in medium as described

above and su ple::ented with 500 units/ml of penicillin and 500 g/ml of

streptomycin. 'Iosquitoes were incubated at 320 for various time intervals,

sacrificed and stored at -70' for virus assay.

Mosquito heads were severed from the thorax and head-squashes were

prepared and assayed for dengue virus antigen by the direct fluorescent

antibody technique as described by Kuberski & Rosen (1977). Anti-

dengut, virus sera employed were obtained from humans, 14 days or more

after acute deng:ue virus infections. Sera with hemagglutination-inhibition

(HI) titers ot i:040 or greater to all 4 dengue virus antigens were

pooled and the .;amma globulin fraction was obtained bv ammonium sulfate

precipit;tion. Estimation of protein concentration was determined by the

Biirec :,iothod (Cornall et at. 1949). Antisera were conjugated with

fluorescLin i;oLhiocvanate (riTC) as described by Goldman (1968). The

titer of the c,nti late was; capible of detectin.z dengue virus antigen

in mosquito heads at a 1:16 dilution; however, a 1:4 dilution was used

6

in all experiments. 1ead-squZIS1.hes 1-11m viru.S-inoctLaIItcd ,nd III in>cul;ted

mosquitOes were examined with the 1():-: and 25X oh cc ti ,t-s (d- '(,We'r) of J

Leitz Orthoplan microscope.

The thorax-abdomens (TIh-Abd ) of t Cst nIIsqu it 0Cs k. r L .i dI

pools and/or as individul specimens bv tile direct pli(itIL- Lecim i que

in LLC-MK2 cells. Pooled specimens were pla(ced in 1 .5 ::,1 ,nd individual

specimens, in 1.0 ml of RPM[ 1640 mcdiuim, suppuieniented ;I,-- duscribed above,

and disrupted by sonification. Suspe, nsions were centri;"u .Cd for 30 min

at 12,000 x g at 4'. Replicate cultures of LLC-!K.) el.< Iwre inoculated

with each suspension, 0.3 ml per culture.

Survival rates for male Ae. aegypti and both sexes of l.:. sph':-iens

were determined by recording the number of dead mosquitoes Ltch ca

postinoculation. Mosquito size was based on the mein we i'iKS 01

individual mosquitoes of each species.

RESULTS

Comparative results of the propagation of low and hi ,h riouse brain-

passaged dengue viruses in Ae. aegvti are presented in Table 2. iihe

dilution of virus inoculum at which fluorescence was detuctcd in

Ae. aegvpti was comparable for the different virus strains, regardless

of the passage level of the virus and/or the generation of the mosquitoes.

Virus-specific fluorescence was usually detected in 2 or more Ae. acgvpti

heads up through 10-4 dilutions of dengue virus types 1, 2 and 3, while

the dilutions of dengue virus type 4 that produced fluorescence were

considerably lower.

All 4 dengue viruses replicated to high titers in Tx. splendens

(Table 3). Except for strain 050 of dengue virus type, 4, virus-specific

7

tlf cscIc aS e t ed in ont ()it r tiore T:-: -;;)I endciy , heauds nii t IIr-I i

tLhe 10) d i Iit t.i on I o r c~ic :1 v i ms

GvTLI L~ tv Li t ers i o r ie~c.rc ii .I- I I, and ini

AL.! e''t n I'*%- I udn are summa rized i n Tab tc 4 . All viruscs,

exceptL the Pro' ot ''pc st ri i ns- of dengue vi rus types 3 ind 4 , atta ined

appreciab 1v hi slier titers in both mosquito species than in LLC- [K, cells.

Table 5 shoWs the infect ivity titers attained by dengue viruses in

individual Ae. ziecIvpti and in Tx. splendens af ter 14 days incubation

at 320. On the his is of mean titers, the amount of each virus type

recovered was hi ;Ier for Tx. splendens than for Ae. aeigypti.

FIG. 1 shows the perinuclear pattern of fluorescence most commonly

observed in brain 4:in,;lia cells of Tx. splendens infected with denguu

viruses. Fluore-scence Was uIsually more intense and more prevalcnt in

heads of Tx. sp lenidens inoculated with deigucik virus types 1 aind 2 thanI

those inoculatcd wIth denguei ty1 )es- 3 and -4.

Dengue viruses were recovered in LLG-M K.) clls- fromt Poole"' Ti-A!hd

suspensions that corresponded to one or more fluorescence-positive heads

of Ac. -avp i ad Tx- spleIns for each Virus dilution. Virus was

not recovered Crom 'Fh-AbLd Suspensions for dilutions of inoculum that

did not produce Fluorescence in mosquito heaids. No evidence of virus-

speci fic fluore.scenIc wa's observed in hecads of control mosquitoes, nor

was viruis recovered from their Thi-Abd.

Since tlie in itIil experim-,ent found T\. spIendens as susceptible to

infkct ion with dnicviruses as Ac. aecvp Li, further experimients were

not. conducted on Ac. aog,-,vpt i, except. for survival suis

Ta 1)1e 6 shows thie timie req u ired for deng ue virus-s peel Fic fluorescence

to aIppear in heaids (f Tx. s-o lenldcns- and tlieC recoveryv rate of these v iruse s

from e r respond in , iInd LVi dnI Tli-Abd fo 1 1owil g oe ii Iation of mosquitoes

b

to 10 3.0 '/ of each viriio. E::cept fr strii:

001 of dcnIAuLL vir ,.i type' 1, sjp cific luor,-;ccricn was nOt ob. rved -:i T .

soli teasl hla l 00 ,SI\' 6 xs inctih lat lion1. On+: 0i s- p'c il La I firu-; ',flce

was preaclt in :::~st I tuitoc.s inuculated with all viruses exccpt strain2 5 77 o 1 den Vue vi1 s t .pe 3. Spefiic f uwre:ae, e was Dsu ryed in -ill

Tx. spIelndenlS inoculated with the latter viruIs on day 12 . Dengue viruses

were recovered from 100, of Th-Ahd suspensions prepared from individual

mosquitOeS that were positive by head-squash. Most viruses attained peak

titers on day 6. Thereafter, titers tended to decrease, especially for

denueL viru.s t.pe, 1. In oeneral, den-ue viruses type 2 and 4 exhibited

the idibest titers.

!iL2 survi val rates Cor male ,\c . aegypt i and male and female T:, .

sp j1-ndis, during the 14-day period after inoculation with dengue viruses,

are presented in T,ble 7. Survival rates rana ed from 25 to 63 for

As. ae,!,-)ti, aid fromn 75 to 100,-" for Tx. spl.endens.

The MeAn wci'ht of 10 individual Tx. splendens was 6.76 mg for :aies

and 7.65 mg for females, in comparison to 1.38 mg for male Ae. aegvpti.

DISCUSSION

Den}gue virus-specific fluorescence was detected only in heads of

mosquitoes whose corresponding Th-Abd contained virus. In a previous

report, specific fluorescence was apparently observed in heads of Ae.

a lbop!iat,.s, but virus was not recovered from corresponding Th-Abd

susp)ensions (Rosen & Gubler 1974). However, the frequency of these

ohservations was exceptionally low and associated with dengue virus

tvpe., 3 and 4. On the contrarv, the recovery of virus from Th-Abd in

the absence of detectable fluorescence in mosquito heads was not uncomlmon

(Rosen & Gub icr L974). Since Th-Ahd from virus susceptibility experiments

-AFt

0 1o -io: (jl " LI it e ol n Idi'v 3 ind i o r 11 v rs LIS S r, L V 1 1-C

on dv9 'or d-ueviruis tvpe 3 :1I!-4 Appairent ly, this is rt1 itecI

to theC t ire euire'd for denu'ueC virusesS to sprea!,'d to theL htead u

mCosq it teS , ~IS i ud iI cd by t hie re COV''r v of v i rLus f rorn cuor re. c s d in

TihI-A !d an ld t 1 e si 11seouCI Ieut deL2t0c t io n fi s1~ it c c 1t 1-uo rcs c uicc in :l;iuse: its

hieads, enl li.1V, J, 12 1 _ Linid 16 postL lnoc U l' i iln

D" 1I _, s Lit v a1t t LIa li ed peaik v irus t itLr s i n Th- Ahd cof T x . sl pde v ns

an: r- 1:-1:1 LL! Iv l IVS p)ost i !IOc t it a un L 1 ) a 'el r :iat th11i S t i 1),e s le2c i

flue rCe 12 eI I' is dtCt 2Li ill thc he Cud 01 on i v k)1 ( .1Oe C m qi to0 A cor-pa-ranie

p er io d o C ti :is cs r2qire f7llIio r dn l-i IItI 'Vir uSeIS to0 reaut 1 i 7inM.11 :L t l: - -

i n sei T u-Ai'd ; o e a Ibpi lt tuis llowetv e r s specc i f i c f Iu s- r e s ceun c u55 : a r osent

Jus oSLLV2 L ; L (I VSpu0sic no u I :.i t i onii, whtich approximatLes our findings

for fc.spi, 11''71 A slijit lv 1on~er period of time wal; required for

fit11reseenCtle t' o ppl:ar in T:x. spy~ lndens; inoeul ited with denguek virus

t Vjs I - ; o id 'I ill t- i ison t o the Other seroLepeS . Similar f indinl-s

we rt- ohse rvt~i I or()I- S rtvp- inl A\L'. a ho pi t us (Rosen & Guib 1r 1974-

TC.0 e-!- I-s Ijj'jid to heI~irx n'tl 5 tim-es heavler itca

Wei 'It) t i111 ; '1 1 'S. I) vt i . Acceerd i n I y the la rge r Tx . si' I endells

a ill r- L Ill r t I 1!e 0) 1- C Vo Tms iri o nw iii i 11 pi 11' puC r xilti l v 5 r imos tie

amo inilt ill j e - I it' i L 111 el I e .- iA" . As :I rest, kI the number v f

nosq(i i toe; rec~ji i ri~ Ior v i riicu 1.issv 1,:I:: rek-dIi-ed susti Ii Ily. The

lair er vo [tius o C i Il(i Ii tli- tO I C. t 011 C~ -'X. sjl L'1111S i 11 CO1Ip): i A 501

T~~~~ 1 0 ,

C~t to lic L' 1:'i I1 he IIvi , I I.- L For cl blo en I I I

IAvo.o 11 t. tL 1md0.' T:'0V10:;;aIIL>K L s c.'r

.\. anId I1x. s) I eilici ( we rz re;ad ilIyN in11fec(:t d Wi tl 111

S c ro0t V Pe0S Il S L as d i I'_2c it st ra ins of don',, vi ' ruses. V irris t i t or s

wore u suia L I, i ia::r i n T':: . s I Ion dn ; at Iit.,r f i d i n ts i d i cato (d thit Lii is

IO ILrL U.1 1_1 000 Otc font :1550': ho t . Since fc!!ales of Tx.:-: zjk-iLoacns

do noL ta.se 1)lood moaf s(ts 1949)) , both s;exes can he used in the

laimraitorv i itOlt risk 01nua inlfct 10n by mos quito bitc.Telre

c o o : ::.s a ! ns. in c omparison withi Aed es s pece s fac ilit ates

inoculatioin, a11 1 -'.:s ioUse of a larcr %'0im o l.k!0f inoenil n_1o, and resuILs

in tloO roria~~ao quantities of virus. Also, tne nunmber 01

T:,. st lenduns reoliircd %,as lower due to a Iii-hur survival rate. Al thou-1i

Aefo''t1 caln "c reared micli faster than Tx.. spIindens , the findings

im;)L mcd that th2 Ilatter spec ies would he a more efficient- host for

dia.cn'S in,, Ci,. enguc virus infect ion and for use in experimental stud ies.

r, . i ii 1, Nt iu I i kh ml Iit I I s WiiSo Wl o Blit 1 )mhbt i lilt] !

P~lu US L Or I00!. 1nL t , lill 1c:1 1 s statlic(' , and M'r. SLI':tl 1-11C11:1 laV

Chi1. ' i ripa i t:t i to or pliotto,:,raiit it cs is t ance

I- I iiA I '! I C IlF

B.\Mi,. )1. 1 ) . "ic 91 tuv.t3 1 I t,-,r ,! it ; it '- . 3.i:J r .

I ' .N, ... ' XI(,K. ]97). L'n 3I d i ,,:', rr i'v l ft .dt i -r

GOLDMAN M. L908. !%,2re seent antibody mL LI is Acad e Mic Press , New

York, 303 )., pp. 104-8, 178-81

GORN.\LL, A. G., C. I. BARDAEI3I, & N. N. DAVl). 1949. Determination of

serum proteins by men:s of the Bi Urc reaction. J. Biol.

(Cihiii. 177:751-no.

G ,? L3R, 0b. 1, D., HED,)., ROSEN J .J. C. HITCHICOCK, JR. 1978.

Epiemiologie . clinical and virologic observations on denLiie

in Lhu Kin ,doI of Tonga. Am. J. Trop. Ned. Hvg,. 27:581-89.

CUBLER, D. SUIIARYONO, 1. LUBIS, s: ERAN & .. 3. SARCYSO. 1979.

Epidet:ie dengue hemorrhagic fever in rural Indonesia. I.

Vir,Ioical and epidemiological studies. Am. J. Trop. Mted.

Hv:. 28:701-10.

3iAL STFA), S. B., S. NI\MANNITYA & 'M. R. MARGIOTTA. 1969a. Dengue and

ch,iillgunya virus infection in man in Thailand, 1962-1964.

Ii. Observations on disease in out-patients. Am. J. Trop.

Med. IMg. 18:972-83.

HAIA:SD, S. 5., S. IUDOMIS.\KDT, J. E. SCANLON & S. ROIIITAYODHIN. 1969b.

Dcn.,tic and chiktiingunva virus inFection in man in Thailand,

1962-1964. V. Epidemiologic observations outside Bangkok.

A\m. J. Trop. Med. HyM. 18:1022-33.

K B SK , T. T. & L. ROSEN. 1977. A simple technique for the

dut ection of dengue ;ntingen in nlOS(litoes by

immunof ioresccnce\. Am. .1. Tirop. MOd. I. 26:533-37.

AA

_____~~~~~~ __ ix!~ I). hi.: I~i ' !,)d.Ju .

l , rit )I-% b.-; I' s i a!1 i t :i L I I1r: nId sc iu a

1 i Aii A "ea' 7. 7aI I'I .. .a ii .u

:i>VNIx S. 1, . iL\LSYKNP COIPc>' V~ R. >I (I I A 1909-~.

Ikuuc ind c h iktins-unvai Vi rU-, in foct i on i n man i n Tha iIand

1902'-1904. 1 . Obse rvat ions on hosp itall zud pit icn ts w~ith6

hemlorrhni-ic Cever. AnT. Tro p . Med . Hyg . 18 :9 5-+- 71 .

I .~, . ~p dL: 1 97 'I'. ,h iuse oft mosc ui toes to detect and

p rL):alsaAte dengu4Le viruses. Am. J. Trop. Med . Hvc . 23:1153-60.

IU',I.K. I A\. NIS.',I..\K. 1967 . Dcnsuet virus identific:ation by the

I Ii,,ue redIUet 011 flk2ut raI iZatiOn test . J. Inniunol . 99: 291-9b)

RUSSIIK . , g. M . YU I' I. I.,.A\. >:SALAK .S . UDOMSA\KI)1, D. J . GO'LD &

... .. .. .. .UNT!~ .19 o,- . Aui insu lir outbreaik of deudue

:rhgefever. II . Virolos:ic and serologic studie~s.

A:; . Trop . 'Ied . Iivp. 17:600-8.

KiTI.P. i. S. NANTAPANITCH, A. NISAL.\K, S. UDC)MSAKDJ , R. INT. DEWLY

. .._RUSSNLL. 1969. Recurrence of epidemic dengue

heor rhagic FVV'er inl an inlsLt r setting. A\m. J . Trop. Med.

lia.18:573-79.

JOA.L.P., C. G. MIOORL-,, G. E. SATHF3R &- G. KUNO. 1979. The

laboratory diagnos is of denguie by the mosquito inoculation

t oin iqic , pp . 1 51)-0!;, in Den s-ue in tile Cairibbein , 1977.

(SCI' lut if iC Puhi . No. 375). Pain American Health

Organ i 2a:Lionl, DihLgo ).C., 16 p).

T6 . 'I., P. SUKHAVACIIAN.\, A. NISALAK &. P. K. RU!SSELLI. 1968.

Ikengu-viriis recovery hY d irC~'t and de'.1 lavd pliqaucs in

LL-K)ctlts. Am. . Ttr''. Nod. fiyvg-. 17:441-48.

S ___NU..,MMO.im

TAIBI.L 1. tlisl-rv o d n.tUie viruses u. l,,''ud in t!c :,:.quit., bi..i iv

Loil I 11'a i ons

110ST/No. M.,

VIRUS S K ['Y P F. t',SSAG I I DA I

DEN-I 77-OU Mouse! 3 Human 197-

Hawxi i Mhuse/l 6 tluman 1 944

DEN-2 74-3379 Mouse/S 5Human 1974

78-189-18A Tx. splondens/l Ae. aegyptl 1978" Aw -~'n~ CX L MouaC ,!29 tHumaln 1944

DEN-3 77-2797 Mouse/5 Human 1977

77-21S77 Mouse/3 Human 1977

- Mouse/ 25 Human 1956

DEN-4 77-050 Mouse/3 Human 1977

H---, L Mouse/32 Human 1936

Suckling mouse brain passage.

Prototype viruses.

4L ..... ....

-.

7 71

77

C - - I

7£

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To L:L 42 (579/ 1360) 41 (535/1295) 92 (294/320)

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