evaluation of toxorhynchites splendens as a ...toxorhynchites splendens (wiedemann) was susceptible...
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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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SECURITY C LASSIFICATION OF THIS PAGE (iflien [)at* E'n a.eJ
REOTDCMNTTO ALSL V READ INSTRUCTIONSREPOT DOUMENATIO PAGBEF.ORE COMPLETING FORMIREPORT NUMBER 2. GOVT ACCESSION NO. 3. RECIP.I N'S CATALOG NUMBER
______________ 7 y _ . __7/__-4. TITLE (find Subtitle) S. TYPE OF REPORT & PERIOD COVERED
Fvruluation of Toxorhynchites .~s arbthissay Host for Dengue ViruSe Interim
6. PERFORMING ORG. REPORT NUMBER
1. AUTHOR(a) 8. CONTRACT OR GRANT NUMBER(&)
Watts, D.M., Harrison, B.A., Nisalak, A.,Scott, R.M., and Burke, D. S.
~~9.ENFRMIG OGANZATON AMEANDADDESS10. PROGRAM ELEMENT. PROjECT, TASKAREA & WORK UNIT NUMBERS
US Army Medical Research Institute of Infectious 62A770A3. Diseases, SCRD-UIP-S 3MI62770A870/BA-070
1I CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATEUS Army Medical Research and Development Command An R1
SOffice of the Surgeon General, Dept of the Army 13. NUMBER OF PAGESWashington, DC 20314 23 pages
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UnclassifiedI5a. DECLASSIFICA*ION! DOWNGRADING
SCHEDULE
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Approved for public release -distribution unlimited
17. DISTRIBUTION STATEMENT (of the abs tract entered In lflack 20, If different from, Report)
18. SUPPLEMENTARY NOTES
Information regarding reprints not available at this time.
k 9. KEY WORDS (Continue on reverse side if necessary and identify by block number)
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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SECURITY CLASSIFICATION OF THIS PAG(WlI/m Da Ei'urs )
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)
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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..
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.\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
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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
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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
:
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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
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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
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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
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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
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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
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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 .
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(Cihiii. 177:751-no.
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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
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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 &
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S ___NU..,MMO.im
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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 ..... ....
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D IN-! 3379 5 NT NT 4/4 4/4 4/4 2/4 0/4
Ne, iuiacx (C 29 NT NT 4/4 4/4 4/4 3/4 0/4
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23 4', 5/. 4/4 3/4 3/4 NT NT
D E-. 034 3 4/. 4/4 3/4 4/4 0/4 0/4 NT
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smb- 16 7.7 8.0 6.1 6.7
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DEN-3 smb- 7 . 7.7 7. h 4.9
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DLN-1 00[ 1 48 (105/ 217) 47 (101/ 216) 75 36/ 48)
i 4.,,,L 1 -o3 (loo/ 2,0) ,2 ( 97/ 230) 95 59/ 62)
PE;-2 337) 25 -,S / 175) 43 ( 76/ 175) 90 38/ 42
,.'..' :uin .: U: _9 3 77/ 18(o) 36 ( 64/ 180) 94 51/ 54)
I",,-3 2707 4 4 91/ 209) 38 ( 53/ 139) 100 48/ 48)
(i-LU7 23 63 ( IlI/ I0) 26 ( 47/ 180) 93 28/ 30)
DEN-- 0_o 3 27 ( 47/ 175) 55 ( 97/ 175) 94 34/ 36)
ti--.I 32 31 ( h7/ 219) 35 ( 78/ 220) 94 45/ 48)
To L:L 42 (579/ 1360) 41 (535/1295) 92 (294/320)
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