INFECTION AND IMMUNITY, Nov. 1976, p. 1221-1227Copyright © 1976 American Society for Microbiology

Vol. 14, No. 5Printed in U.S.A.

Isolation of a Temperature-Sensitive Dengue-2 Virus UnderConditions Suitable for Vaccine Development

KENNETH H. ECKELS,* WALTER E. BRANDT, VENTON R. HARRISON, JACK M. McCOWN, ANDPHILIP K. RUSSELL

Division of Communicable Diseases and Immunology, Walter Reed Army Institute ofResearch,Washington, D.C. 20012

Received for publication 21 May 1976

Dengue virus, type 2, in viremic human sera and after passage in cell culturesproduces mixtures of small and large plaques when assayed in LLC-MK2 cells.Clones of dengue virus type 2 obtained by plaque selection in primary greenmonkey kidney cell cultures were tested for temperature sensitivity in vitro andfor virulence by intracerebral inoculation of suckling mice. Sublines of a small-plaque clone were found to have lower nonpermissive temperatures than theparent virus by both plaque formation and release of infectious virus into theculture media. Small-plaque sublines were significantly less virulent in suck-ling mice than was the parent virus. Sublines from a large-plaque clone were nottemperature sensitive and closely resembled parent virus mixed-plaque mor-phology. When small-plaque sublines were serially passaged using undilutedinocula, reversion occurred as evidenced by the appearance of large plaques andreturn of mouse virulence. Small-plaque virus could be maintained throughseveral serial passages without reversion by using low-input inocula. Desirablepassage history as well as temperature-sensitive and attenuation characteristicsof the S-1 small-plaque subline make it appear suitable as a vaccine candidatevirus.

Passage in cell culture has resulted in re-duced in vivo virulence for numerous flavivi-ruses. These include yellow fever (11), WestNile, and Russian spring-summer encephalitis(8), Central European tick-borne encephalitis(4), Japanese encephalitis (3), and Kyasanurforest (7) viruses. Plaque selection has beenused as a final step in the purification of manyof these viruses; however, plaque size and viru-lence have been related only rarely. Mayer (4)described a small-plaque variant of Central Eu-ropean tick-borne encephalitis virus obtainedfrom a persistently infected human amnion cellculture. The small-plaque variant had reducedvirulence for mice by the peripheral route whenit was compared with the parent virus. Paul (7)obtained a spontaneous variant of Kyasanurforest virus by repeated passage in monkeykidney epithelial cell cultures. In addition tosmall-plaque and lesser in vivo virulencemarkers, replication of the variant was re-stricted at 40°C, a permissive temperature forthe parent virus.Mixed populations of large and small plaques

have been frequently found in wild dengue vi-rus type 2 (DEN-2) isolates from humans andmosquitoes in both Asia and the Caribbeanregion. Previous studies with DEN-2 demon-

strated that large- and small-plaque clonescould be readily derived from isolates in firstcell culture passage; the clones were identicalby neutralization tests, but the small-plaqueclone had a markedly reduced virulence forsuckling mice (P. K. Russell and P. Sukhava-chana, unpublished data).Many naturally occurring and induced tem-

perature-sensitive mutants of other types of vi-ruses have been associated with lower animalvirulence (2). Indeed, a lower permissive tem-perature of replication than that of the parentvirus has been associated with some virusescurrently in use (1) and which have been pro-posed for use (6, 12, 13) as live-virus vaccines.This paper describes the isolation and purifica-tion of small- and large-plaque clones from ahuman serum isolate of DEN-2 virus. Plaquesize is correlated with temperature sensitivityand with suckling mouse virulence for theseclones.

MATERIALS AND METHODS

Virus. DEN-2 virus, PR-159 strain, was recoveredfrom serum obtained in Puerto Rico on 19 August1969 from a 31-year-old male patient with uncompli-cated dengue fever. The serum was free of bacteriaon culture, and radioimmunoassay for hepatitis B

1221

1222 ECKELS ET AL.

surface antigen was negative. The virus was identi-fied as DEN-2 by plaque reduction neutralizationtests using reference antisera and was found to beantigenically identical to the PR-109 strain from thesame epidemic (9).

Cells. Primary green monkey kidney (PGMK)cells were obtained from Lederle Laboratories, PearlRiver, N.Y., as frozen suspensions. Lots of cellswere tested by the supplier for the presence of sim-ian adventitious agents by inoculation of growthfluids in PGMK, Lederle 130 human diploid cells,and BSC-1 cells. Hemadsorption tests using guineapig erythrocytes were also performed on monolayersof the pretested PGMK cells prior to their certifica-tion. Certified PGMK cells were grown in Eagleminimal essential medium containing 25 mMHEPES (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) buffer (Grand Island Biological Co.,Grand Island, N.Y.) supplemented with 10% fetalbovine serum (Microbiological Associates, Be-thesda, Md.), 2 mM L-glutamine, and 100 jg ofstreptomycin sulfate and 50 ug of neomycin sulfateper ml. All sera used in cell culture media werethoroughly screened for adventitious agents andbacteriophage prior to use. Upon formation of amonolayer, the PGMK cells were used immediately,or a 1:2 subpassage was made and secondary cellmonolayers were used for virus passage and plaqu-ing. Growth medium with 2 or 5% fetal bovine se-rum was used to maintain confluent cell monolayersfor virus passage.A continuous line of rhesus monkey kidney (LLC-

MK2) cells was grown in medium 199 containing 20%fetal bovine serum. The LLC-MK2 cell line was sub-passaged weekly and used for plaque assay ofDEN-2virus.

Plaque assays. Monolayers of LLC-MK2 cells in25-cm2 plastic flasks were used for plaquing andcharacterizing DEN-2 virus clones. After virus inoc-ulation and adsorption for 1 to 2 h, flasks received 7ml of medium 199 containing 10% fetal bovine se-rum, 36 mM NaHCO3, 0.02% diethylaminoethyl-dextran, 0.5% 100x concentrated Eagle basal me-dium vitamins and amino acids, 1% purified or No-ble agar (Difco), and antibiotics.

After incubation at a designated temperature for6 days, flasks received 4 ml of a second overlaycontaining 1% Noble agar and 1:6,000 neutral red inHanks balanced salt solution. Plaque flasks wereincubated at 35°C for 4 h and overnight at roomtemperature before plaques were counted.

For plaquing in PGMK cell monolayers, the firstoverlay medium contained Eagle minimal essentialmedium supplemented with 10% fetal bovine serum,36 mM NaHCO3, 0.02% diethylaminoethyl-dextran,1% Noble agar, and antibiotics. After virus inocula-tion and adsorption for 1 to 2 h, flasks received 7 mlof the first overlay. After 6 days of incubation at35°C, a second overlay containing 1% Noble agarand 1:6,;000 neutral red in Hanks balanced salt solu-tion was added. Plaques were counted the followingday.

Viral replication. Adsorption was carried out for1.5 h at 35°C, using undiluted virus suspensions in25-cm2 plastic flasks (Costar, Cooke Engineering,

Alexandria, Va.) containing 2 x 106 cells. The inocu-lum was removed by washing the monolayers threetimes with Hanks balanced salt solution, and incu-bation was started at the indicated temperatures 2 hafter addition of the virus. Samples of cell culturefluids were removed during the experiments, di-luted 1:10 in growth medium, and frozen at -70°C.Animal inoculation. One- to two-day-old suckling

mice were inoculated intracerebrally with 0.02-mlvolumes of diluted virus. The mice were observed forsickness and death for 21 days.

RESULTS

Plaque purification of DEN-2 virus. Six pas-sages of the DEN-2 isolate were made in PGMKcells using undiluted cell culture fluid for theinoculum at each passage. A large stock of seedvirus was prepared at the 6th passage. Thisseed virus is referred to below as the parentvirus. The original human serum isolate andthe parent seed virus contained similar mixedpopulations of large (3 to 5 mm)- and small (1 to2 mm)-plaque-forming virus (Fig. 1). Virus ti-ters gradually increased from 1.7 x 104 to 3.0 x106 plaque-forming units/ml during the first sixpassages in PGMK cells.

Plaque assays in LLC-MK2 cells were usedthroughout this study to characterize theplaque morphology of all DEN-2 isolates andclones. All clone purification was performed incertified PGMK cells, where plaque morphol-ogy was variable and not distinctive. Plaquepicking in PGMK cells was therefore necessar-ily followed by characterization of the selectedclones in the LLC-MK2 plaquing system. Pas-sage history of the clones is summarized inTable 1.

Initial plaque purification was performed bylocating a single, well-isolated plaque afterneutral red staining, removing a plug of agarover the plaque, and growing the isolate inPGMK cells under fluid culture media. Aftertwo of these cycles, clones designated 5 and 6were selected for further purification. Sublinesof these two clones at the 10th passage levelwere carried to the 14th passage level by threecycles ofplaque purification, the progeny ofoneplaque being directly plaqued again under agarfor each cycle. Table 1 lists the results of the S-1subline, one of eight sublines of clone 6, and theL-1 subline, one of five sublines derived fromclone 5. Although all of the small-plaque sub-lines retained their small-plaque morphology,this was not true for the progeny of the large-plaque clone. All five sublines of clone 5 afterthree cycles of plaque purification containedmixed viral populations similar to the parent,passage 6 virus. Whereas small-plaque popula-tions could not be eliminated from these prepa-

INFECT. IMMUN.

TEMPERATURE-SENSITIVE DENGUE-2 VIRUS 1223

Fe,

HUMAN PARENTISOLATE

FIG. 1. Original DEN-2 human isolate and passage 6, parent virus, plaqued in LLC-MK2 cells.

rations, the percentage of large plaques rangedfrom 73 to 85, whereas the parent virus poolcontained approximately 70o large-plaque vi-rus. In Fig. 2 the homogeneous character of theS-1 subline at the 14th passage level is com-pared to the plaque mixture found in the parentpreparation and in the b-1 subline preparation.The S-1 subline at the 14th passage level was

further purified by direct plaque picking to the18th passage level. Virus pools prepared at the18th and 19th passage levels, as listed in Table1, still retained homogeneous small-plaque pop-ulations, with plaques not exceeding a diame-ter of 2 mm.

In vivo virulence of the purified clonal sub-lines. Small-plaque sublines had reduced intra-cerebral virulence for suckling mice when theywere compared with the parent virus. Viru-lence ratios listed in Table 2 measure the infec-tivity in cell culture compared to the infectivityin suckling mice for the small- and large-plaquesublines and parent virus. Although significantdifferences in virulence were not seen betweensmall- and large-plaque sublines, the small-plaque sublines were clearly less virulent forsuckling mice than the parent virus. Also, the

mean survival time for mice inoculated withthe small-plaque viruses was at least 2 daysgreater than for mice inoculated with the par-ent virus. A higher plaquing efficiency for thesmall-plaque sublines may account for thelower virulence ratios. However, a longer meansurvival time in suckling mice would indicate areal difference in lethality for these viruses ascompared with the parent virus.

Effects of temperature on plaque forma-tion. The first four small-plaque sublines (S-1to S-4) at the 14th passage level had plaque-forming titers in LLC-MK2 cells similar tothose of a large-plaque subline (L-1) and theparent virus (6th passage level) when assayedat 35°C; however, only the parent virus and thelarge-plaque subline formed plaques at 39°C(Table 3). The S-1 subline was selected for theremaining experiments. Plaque formation bythis virus was compared with the parent virusat increasing temperatures from 35 to 39°C (Ta-ble 4). The nonpermissive temperature for thesmall-plaque subline was between 38 and 39°Cin LLC-MK2 cells. An effect of increasing tem-perature on plaque formation by the parentvirus was to gradually decrease the size of both

VOL. 14, 1976

1224 ECKELS ET AL.

the small and large plaques until (by assump-tion) only the large plaques were visible at3900. In a separate test, 4000 was found to benonpermissive for plaque formation by the par-ent virus.

Effects of temperature on viral replicationin cells in fluid culture. The S-1 subline wascompared to the parent virus at 35 and 3900 in

TABLE 1. Plaque size and titer ofDEN-2, PR-159virus after passage and clone selection

in PGMK cell culture

PlaqueVirus Pas- size in Titer (PFU/sage LLC-MK2 ml) at 350Ca

cells (mm)Human serum 0 1-2, 3-5 1 x 103Parent, passage 6b 6 1-2, 3-5 3 x 106Clone 5c 10 1-2, 3-5 1.1 X 104Clone 6c 10 1-2 5 x 104L-1 (from clone 5)d 14 1-2, 3-5 9 x 104S-1 (from clone 6)d 14 1-2 9 x 105S-le 18 1-2 2 x 105S-i 19 1-2 1 x 105

a PFU, Plaque-forming units.b First six passages made using undiluted cell culture

fluid as the inoculum for each successive passage.c Clones derived by plaque selection at passages 7 and 9.dClonal sublines derived by plaque selection at passages

11, 12, and 13; five large-plaque sublines derived from clone5 and eight small-plaque sublines derived from clone 6 inidentical fashion.

e S-1 subline further plaque purified at passages 15, 16,and 17.

A.r;A

t/o;f*!.v eSx 4

LLC-MK2 cells and at 37, 39, and 400C inPGMK cells (Fig. 3A, B). The parent virusreplicated well in the primary cells at 390C,whereas in the continuous cells infected at thesame time its replication was substantially de-pressed at the same temperature. Replication ofthe S-1 subline in primary cells was signifi-cantly depressed at 390C, but in continuouscells replication was totally nonpermissive at390C. When infected primary cells were incu-bated at 400C (Fig. 3B), replication of the par-ent virus was further depressed, whereas repli-cation of the small-plaque subline was totallyrestricted at this temperature, 10C higher thanin the continuous cells. The effect of tempera-ture on the S-1 subline at 10C intervals wastested in LLC-MK2 cells (Fig. 4). Whereas thenonpermissive temperature was again 390C,there was also a substantial depression of repli-cation from 37 to 38°C.

Stability of small-plaque sublines on pas-sage. It was observed that genetic stability ofsmall-plaque sublines on serial passage inPGMK cells was influenced by the multiplicityof infection (MOI). Parallel lines of S-1 and S-6viruses were passed three times using eitherdiluted inocula (MOI, approximately 10-4) frompassage 14 to 17 or undiluted inocula (MOI,approximately 10-1) from passage 14 to 17 (Ta-ble 5). With both S-1 and S-6 sublines, serialpassage with a relatively high MOI resulted in

PARENTFIG. 2. Parent DEN-2 virus and clonal sublines S-1 and L-1 plaqued in LLC-MK2 cells.

INFECT. IMMUN.

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TEMPERATURE-SENSITIVE DENGUE-2 VIRUS 1225

TABLE 2. Infectivity of selected small- and large-plaque sublines ofDEN-2 virus in cell cultureand suckling micea

Virus Suckling mice log LLC-MK2 cell culture PFU/ Ratio: LD5/PFULD,dml ml at 35TCS-1 (passage 14) 3.4 9 x 105 0.003S-2 3.6 1.2 x 106 0.003S-3 4.4 9 X 105 0.03S-4 3.5 1 X 106 0.003S-5 4.3 6 x 105 0.03S-6 3.3 1.2 x 106 0.002S-7 4.6 1 x 106 0.04S-8 4.6 1.1 X 106 0.04

X = 0.019 ± 0.018L-1 (passage 14) 4.3 4 X 105 0.05L-2 4.6 4 x 105 0.10L-3 4.8 3 x 105 0.21L-4 4.8 4 X 105 0.16L-5 3.8 3 x 105 0.02

X = 0.108 ± 0.078Parent (passage 6) 5.6 3 x 106 0.13

x = 0.168 0.06ba LD54), 50% lethal dose; PFU, plaque-forming units.b Mean + a- LD5,/PFU ratio for five determinations.

TABLE 3. Plaque formation by small- and large-plaque sublines and the parent DEN-2 virus

in LLC-MK2 cells at 35 and 39°CPlaque-forming units/ml

Virus350C 39°C

S-i 9.0 X 104 <5 x 10S-2 7.0 x 104 <5 x 10'S-3 2.9 x 10; <5 x 10'S-4 1.7 x 10; <5 x 101L-1 9.0 X 104 2.8 x 104Parent (passage 6) 1.6 x 10;1 1.7 x 104

TABLE 4. Plaque formation by the S-1 and parentDEN-2 viruses in LLC-MK2 cells at increasing

temperatures

Plaque-forming units/mlTemp (°C)

S-i Parent

35 1.7 x 104 1.5 x 10537 4.5 x 103 1.4 x 10538 5.0 x 103 7.5 x 10439 <1.0 x 10' 2.9 x 104

reversion of the homogeneous small-plaque vi-rus to a population containing large plaques.The appearance of large plaques was accompa-

nied by loss of temperature sensitivity and in-creased virulence for suckling mice. The small-plaque characteristic as well as reduced mouse

virulence and temperature sensitivity could bemaintained by passage of diluted virus at a lowMOI for at least six passages. Yields of infec-tious virus produced at 7 days were approxi-mately the same for cells inoculated with eithera high or low MOI.

In vivo stability of the S-1 subline was testedafter one passage in suckling mice. Brain ho-mogenates from mice dying after intracerebralinoculation of the S-1 and parent viruses wereassayed for the presence of small and largeplaques in LLC-MK2 cells. Only virus capableof producing small plaques was recovered frommice inoculated with S-1, whereas the parentvirus-inoculated mice yielded progeny virusthat produced a typical plaque mixture.

Tests for adventitious agents in the S-1 vi-rus pool. The S-1 virus pool prepared at the19th passage level was tested for adventitiousbacterial and viral agents. Tests for bacterialand mycoplasmal contaminants were negativeafter inoculation of the S-1 pool in Trypticasesoy, thioglycolate, and mycoplasma broths.Tests for adventitious viral agents were done inadult and suckling mice and in four types of cellcultures. Young adult mice remained healthyafter receiving a combination intracerebral andintraperitoneal inoculation of the S-1 subline.Suckling mice inoculated by the same routeusing neutralized S-1 virus also remainedhealthy over a period of 3 weeks. Tests aftertwo passages of the S-1 subline at 14-day inter-vals in WI-38, primary rabbit kidney, primaryrhesus monkey kidney, and PGMK cell cul-tures were also negative for cytopathic or mor-phological changes in the cell monolayer.

DISCUSSION

The plaque size variants observed in the un-passaged DEN-2, PR-159 isolate and in the pas-saged parent virus appear to be due to viral

VOL. 14, 1976

1226 ECKELS ET AL.

6

5

.4CL

2

3

2

6

5

4

3

2

PARENT_35C

S-l 35C

/ a<+__-- --* ~PARENT

/10 0/S- -1 39C

.a ............... PARENT,. 40C

.....------- ...........---....- D........ EloO -1 40C

2 3 4 5 2 3 4 5DAY POST INFECTION DAY POST INFECTION

FIG. 3. Growth ofDEN-2 parent and S-i viruses at 35 and 39°C in LLC-MK2 cells (A) and at 35, 39, and400C in PGMK cells (B).

6

5

2a-

0~

4

3

2

2 3 4

DAY POST INFECTION

FIG. 4. Effect of increasing temperatreplication of the S-i subline in LLC-MK

subpopulations with differing virutemperature sensitivity charactersmall-plaque clone was derived fromvirus and multiple sublines were is(purified. Homogeneity of the originalsubstantiated by the similar chardemonstrated by the sublines. Theisolate pure large-plaque clones in s

peated attempts strongly suggests ahigh-frequency mutation from largea mixture of small plaques and largThere appears to be a much-lower.mutation of small-plaque to large-plphology.There is a strong positive corre

tween small-plaque morphology, teisensitivity, and reduced suckling mlence. The reason for reduced in vivcof the small-plaque clones is probal

36 C suppression of viral replication processes at thebody temperature of the host. The degree ofsuppression at a given temperature is deter-

37 C mined in part by the virus and in part by thehost cell. We have shown in this study thatviral replication is suppressed at different re-strictive temperatures in continuous and pri-mary monkey kidney cells. Virulence or atten-uation might be a reflection of which cells in acertain host are effectively replicating the vi-

0 38 C rus. Thus, measurement of temperature sensi-tivity in vitro cannot generally be used to pre-dict attenuation for a given host, but it is a

O 39 C practical screening method for mutants or var-iants in many virus-cell systems. In the case ofa DEN-2 small-plaque clone, temperature sen-

5 6 sitivity appears to be a very useful laboratorymarker, which is associated thus far with at-ure on the tenuation.

L2 cells. The small-plaque clonal subline S-1, at the19th passage level, which has been tested more

rlence and extensively than other DEN-2 clones, has sev-istics. A eral characteristics that indicate its potentialthe parent as a vaccine candidate. These include tempera-[oated and ture sensitivity, reduced neurovirulence forIaclone. was suckling mice, and, in other studies to be re-*acteristics ported (Harrison et al., in preparation), re-failure to duced virulence for primates and low neurovir-pite of re- ulence for rhesus monkeys. In addition, the S-1ponsistento subline is free of detectable adventitious agentsplaques to and has only been passaged in cell culturese plaques. acceptable for human vaccine development.-frequency Passage experiments indicate that the geneticLaque mor- purity of the S-1 subline can be maintained

during serial passage by using a low MOI.dlation be-mperature ACKNOWLEDGMENTSLouse viru- We thank C. Hampton for technical assistance and J.) virulence Lowenthal, S. Berman, and D. Dubois for helpful discus-bly partial sions.

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TEMPERATURE-SENSITIVE DENGUE-2 VIRUS 1227

TABLE 5. Characteristics ofthe S-1 and S-6 sublines passaged at relatively high and low MOI inPGMK cells

PFU/mlaVirus, passage MOI LD5,,I/PFU Plaque morphology at 35°C(P) 35°C 39°C

S-1, p-14 9 x 10) ND" 0.003 SmallS-1, p-17 10-4 5 x 10) <5 x 102 0.008 SmallS-1, p-19 10-4 1 x 10 <5 x 10' 0.040 SmallS-1, p-17 10-' 3 x 10` 5 x 103 0.1 Large and smallS-6, p-14 2 x 106 ND 0.0008 SmallS-6, p-17 10-4 1 x 10" ND 0.006 SmallS-6, p-20 10-4 1 x 10 ND ND SmallS-6, p-17 10-1 1 x 10) ND 0.2 Large and small

a PFU, Plaque-forming units.b Suckling mouse intracerebral 50% lethal doses (LD5,,)." ND, Not determined.

LITERATURE CITED

1. Food and Drug Administration. 1974. Tests for in vitromarkers. Code of Federal Regulations 21:630.16 (b)(3). U.S. Government Printing Office, Washington,D.C.

2. Ghendon, Y. Z., A. T. Marchenko, S. G. Markushin, D.B. Ghenkina, A. V. Mikhajeva, and E. E. Rozina.1973. Correlation between ts phenotype and patho-genicity of some animal viruses. Arch. Gesamte Vi-rusforsch. 42:154-159.

3. Hammon, W. McD., S. Rohitaydhin, and J. S. Rhim.1963. Studies on Japanese B encephalitis virus vac-cines from tissue culture. IV. Preparation and char-acterization of a pool of attenuated Oct-541 line forhuman vaccine trial. J. Immunol. 91:295-305.

4. Mayer, V. 1963. Two variants of tick-borne encephalitisshowing different plaque morphology. Virology20:372-390.

5. Mills, J. V., and R. M. Chanock. 1971. Temperature-sensitive mutants of influenza virus. I. Behavior intissue culture and in experimental animals. J. Infect.Dis. 123:145-159.

6. Mills, J. V., I. van Kirk, D. A. Hill, and R. M. Chanock.1969. Evaluation of influenza virus mutants for possi-ble use in a live virus vaccine. Bull. W.H.O. 41:599-

606.7. Paul, S. D. 1966. Some biological properties of two

variants of Kyasanur Forest disease virus. Indian J.Med. Res. 54:419-424.

8. Price, W. H., R. W. Lee, W. F. Gunkel, and W.O'Leary. 1963. The virulence of West Nile virus andTP 21 virus and their application to a group B arbovi-rus vaccine. Am. J. Trop. Med. Hyg. 10:403-422.

9. Russell, P. K., and J. M. McCown. 1972. Comparison ofdengue-2 and dengue-3 virus strains by neutraliza-tion tests and identification of a subtype of dengue-3.Am. J. Trop. Med. Hyg. 21:97-99.

10. Schluter, B., B. Bellomy, and A. Brown. 1974. Patho-genesis of temperature sensitive mutants of Sindbisvirus in the embryonated egg. I. Characterizationand kinetics of viral multiplication. Infect. Immun.9:68-75.

11. Theiler, M. 1951. The virus, p. 39-136. In G. K. Strode(ed.), Yellow fever. McGraw-Hill, New York.

12. Wagner, R. R. 1974. Pathogenicity and immunogenic-ity for mice of temperature-sensitive mutants of ve-sicular stomatitis virus. Infect. Immun. 10:309-315.

13. Wright, P. F., W. G. Woodend, and R. M. Chanock.1970. Temperature-sensitive mutants of respiratorysyncytial virus: in-vivo studies in hamsters. J. Infect.Dis. 122:501-512.

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