Seven complementation groups of respiratory syncytial virus temperature-sensitive mutants.

D022-538X/78/0027-0459$02.00/0

Copyrighti 1978 AmericanSocietyforMicrobiology Printed in U.S.A.

Seven

Complementation

Groups

of

Respiratory Syncytial

Virus

Temperature-Sensitive

Mutants

H. B. GIMENEZt AND C. R. PRINGLE*

Medical Research CouncilVirologyUnit, InstituteofVirology, Glasgow Gll 5JR, Scotland

Receivedforpublication13April1978

Fifteen temperature-sensitive mutants of the RSN-2 strain of respiratory syn-cytial virus have beenclassified into sixcomplementation groups, two of which appeared tobehomologous withtwoof the threecomplementationgroups of the A2strain describedbyWrightetal. (P.F.Wright,M. A.Gharpure,D.S.Hodes,

and R. M. Chanock,Arch. Gesamte Virusforsch.41:238-247). Thus seven

com-plementationgroups ofrespiratorysyncytial virus, designated A, B,

C,

D, E, F,

and G, have been defined. The frequency and type ofmutant isolated varied

accordingtostrain;groupCwasuniquetothe A2strain,and groupsD, E, F,and

Gwereuniquetothe RSN-2strain. Thehighest

complementation

indexeswere

obtained

by

preincubatio

fo

ation atres ye mperaturefor40to 50h inthecaseof A2 strain mutants o fr 1-2 strainmutants.Genetic recombinationwas notdetected.

Temperature-sensitive (ts) mutants have been isolated fromtwostrains ofhuman

respi-ratorysyncytial(RS)virus. Seven mutantswere

isolated from the A2 strain by Gharpure et al. (5) and classifiedintothree groups (designated

A, B, and C) by genetic complementationtests

(11). Five of these seven mutants belonged to

complementation group A. Another36 mutants

were isolatedfromthe RSN-2 strain of RS virus

byFaulkneretal. (4), andaminimumof three

complementation groupswasrecognizedonthe

basis ofgenetic and physiological studies. We now reportfurther genetic characterization of the tsmutantsof theRSN-2 strain and identi-fication of thehomologiesofthe

complementa-tion groupsof the A2 andRSN-2strains.

Opti-mal conditions forcomplementationwerequite

distinct in the two strains. Definition of these conditionswas anessentialpreliminaryto dem-onstration of interstrain complementation. We suggest thatasimilar approachmay be

appro-priatewith other viruses which do notgrowto

hightiter and wherecomplementationhas been

difficulttodemonstrate.

Fifteen of the RSN-2 ts mutants have been

classifiedinto six

complementation

groups, two

of whichappearedto behomologouswith

com-plementation groupsA and B of the A2strain.

Thus sevencomplementation groupsare

recog-nized now in RS virus: groups A, B, and C, representedbyA2strainmutants,andgroups A, B,D, E, F, andG,byRSN-2strainmutants.

Genetic recombinationbetween

complement-t Presencomplement-t address: Faculcomplement-tad de CienciasExactasy Natu-rales,Universidad de BuenosAires, Buenos Aires,Argentina.

ing ts mutants was not detected in agreement with the previousfindingsofWright et al. (11) and Faulkner et al. (4). It is likely, therefore, that the RS virus genome is an unsegmented molecule andthat there may be at least seven

primarygeneproducts.Theidentificationof

be-tweensixandeightvirus-specified polypeptides

inpartiallypurifiedRS virus and in extracts of

RS virus-infected cells reinforces this conclusion (3, 7, 13). However, because five of the seven groups are represented by single mutants, the

occurrence of intracistronic complementation cannotbe excluded.

MATERIALS AND METHODS

Cells. The BS-C-1 and HeLa cell lines were ob-tainedfrom J. F. Williams,Institute of Virology, and

propagatedinrollerbottle culturesin Eagleminimum essentialmedium with twice the normal concentration of amino acids (Glasgow modification) and supple-mented with 5% fetal calf serum (virus and myco-plasma screened,Gibco-Biocult, Glasgow).

Virus. The A2 strain wild type and mutants tsl (A2), ts2 (A2), andts7 (A2) wereprovided by Linda

Richardson, National Institutes ofHealth,Bethesda, Md.The origin and characteristics of the RSN-2 wild type andts mutants have beendescribed previously (4). In this paper, to avoid confusion, the strain of originofindividual mutantswill be indicatedin paren-thesesfollowingthe mutant number.

Complementationtests. Complementation tests werecarried outby the method described byFaulkner etal. (4),with theadditional modificationsdescribed in thetext.Thecomplementation index was calculated asthe ratio:yieldfrom themixed infection (tsx+tsy) at39°C/sumoftheyields of the single infections (tsx) +(tsy)at39°C,where theyieldsareassayedat31°C, 459

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460 GIMENEZ AND PRINGLE

and tsx andtsy are anypair ofmutants. The multi-plicity of each mutant was the same in mixed infection andself-infection; i.e.,the totalmultiplicitieswere not

adjusted. Previousexperiments showed that this did not bias theresults.

Preparationof mutant stocks. RS virus does not grow tohigh titer,therebylimitingthemultiplicityof infection which can be achieved. The following

method of stock preparation wasfound to give the highestvirus titers. Each mutant was inoculated into a 30-ml (1-ounce) bottlecontaining 10' BS-C-1 cells

and incubated until cytopathiceffect was extensive. The monolayerwasscrapedinto suspension, and the

whole culture wasinoculated immediately into a 2-liter (80-ounce) roller bottle containing 108 BS-C-1 cells. The culture wasincubated for 1 h at 31°C to allow absorption of the inoculum. Then 40 ml of incubation medium wasadded, and the culture was

incubatedat310Cfor 5days.The incubation medium was discarded andreplaced by30ml offresh incuba-tionmedium. Incubationwascontinued at310Cfor a further48 to 72huntilcytopathiceffect wasalmost

complete.The attached cells wererapidly scrapedinto the medium, which was immediately dispensedinto vials and frozen inliquidnitrogen. Approximately10' PFU wereobtained from oneroller bottle culture.

Infectivityassay. Infectivities were measured on

BS-C-1cellmonolayers.Dilutions ofvirus-containing fluidswereinoculatedin0.2-mlvolumesandincubated for 30 min at 310C forabsorption. Then the

mono-layerswereoverlaid with 5 to7mlofEaglemedium

containing 0.9% agar and 2% fetal calf serum. The

infectedmonolayerswereincubated for 5 to 7 days at

39°C (therestrictive temperature) or7to 10daysat 31°C(thepermissivetemperature) inC02-gassedand humidified incubators. The monolayers were fixed with Formolsaline and stained with Giemsa. At both temperatures, RS virus plaques appeared as darkly staining heapsofcells,asdescribedpreviously(4, 12). The nonsyncytial plaque morphology of mutant ts2

(A2), which according to Gharpure et al. (5)

distin-guishesthismutantfrom the other A2 strainmutants, was notobserved in BS-C-1cellsbecauseallstrains of human and bovine RS virus so far examinedproduce

the sameuniform focalplaqueonBS-C-1 monolayers

under agaroverlay (3).

Growthcurvedetermination.Replicatecultures of2 x10 HeLa cells in 30-ml(1-ounce) screw-capped

bottleswereinfected withapproximately1PFU of the wild type of either the A2 or the RSN-2 strain per cell. The infected cultures were incubated at390Cfor 30 min toabsorb the inoculum. The inoculumwasthen removed by two washes of coldmedium, and finally 5 ml ofwarm incubation mediumwas added and the bottles were placed in a water bath at 390C. Two bottles were removed at intervals up to 44 h after infection and frozenat-70°C. The infectivities were measured onBS-C-1monolayersat310Cin thenormal

manner.

RESULTS

Growth curve of wild-type virus at

re-strictivetemperature.Figure1showsthatthe

growth of wild-typevirusof the A2 and RSN-2 Y

I

I

-J

x 04

-LI

z

0 0

-J

I-0

C-)

P-4

--4

-u m

0

I

I-I

HOURS

FIG. 1. Growth of wild-typevirus (ts+) ofthe A2 andRSN-2strains in HeLa cellsat39°C. Monolayers ofHeLa cells in 1-ounce (ca. 30-ml) bottles were

infectedwith 1 PFUofts+(A2)orts+(RSN-2)percell asdescribed in the text and incubated in a water bath at39°C. Replicatecultures were harvestedby freeze-thawingatvarious timesafter infection, and totalinfectivity wasassayed on BS-C-1 cell

mono-layers at 31°C. (a- ) ts+ (RSN-2); (---) ts+

(A2).

strains in HeLa cells at 390C was essentially similar. The yieldof theRSN-2 strain virus in

HeLacellswaslower, althoughthe cultures had

been infectedatthesamenominalmultiplicity.

The incremental yields, however, were

compa-rable. This result isprobablyareflection of the

pasthistoryof thetwostrains andthe factthat

the inoculum virus titers were determined on

BS-C-1 cell

monolayers.

The RSN-2 strain had

beengrownexclusivelyonBS-C-1 cells and

ex-hibited some adaptation to these cells (4),

whereas the A2 strain had beengrown predom-inantlyin HeLa cells.

Effect of theperiodof incubationat31°C

oncomplementationyields.Factorsaffecting

theefficiency ofthe standardcomplementation test wereexaminedtoimprove consistencyand reducevariability between experiments. Itwas

confirmed thatcomplementation indexes were

higher andmorereproducible when HeLacells

wereusedashost for the mixed infection rather thanBS-C-1 cells (4). Anotherimportantfactor

was the length of the period of incubation at

permissive temperature

(310C)

before removal

of the inoculum and incubation at restrictive

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SEVEN RS VIRUS COMPLEMENTATION GROUPS

temperature (390C). Table 1illustratesthat the

complementation index in a mixedinfectionof

two A2strainmutants wasincreased from 100

to 813 by increasing the incubation period at 31°C from 1 to7 h.Other experiments showed

that increasing the310C incubation period

be-yond 7 h did not further enhance the comple-mentation index.

Effect ofthe period of incubation at

re-srictivetemperature oncomplementation.

Theeffectofincreasingperiodsofincubationat

390C on the complementation index is

illus-tratedinTable2.HeLa cells wereinfectedwith

two complementing mutants from the RSN-2

strain, usingtwodifferent periods ofpreliminary

incubation at310C, namely, 1.5h and the

opti-mal7h. Thecomplementationindexes increased up to 88 h after absorption and subsequently

declined.Atall times thelongerincubation

pe-riod at

310C

gave higher complementation

in-dexeswiththese RSN-2strain mutants, as

ob-servedpreviouslywith A2 strain mutants (Table

1).

Optimum incubation periodsat390C.

Ini-tiaily,

theoptimumperiod of incubationat390C

had been determined for mutants of the A2

strainandfoundtobe between 40 and 50 h. This

isillustratedinFig.2aformixed infection by tsl

(A2)and ts7(A2). However,severalexperiments

showed thatcomplementationbetween mutants of the RSN-2 strainwas poorunder these

con-ditions. The experiment with mutants ts17

(RSN-2)and ts19 (RSN-2)illustrated inFig. 2b

shows that muchlongerincubationwasrequired

to obtain satisfactory complementation with

RSN-2 strain mutants. The optimal

period

of

incubationat

390C

wasbetween 80and90h, i.e.,

TABLE

1. Increase

in

compkmentation

index(C1)

with timeof absorptionat31°C

Absorp- Incuba-Mutants Host cell h?n pe. to_{niodat at390C}teCI

31°C (h) (h)

tsl (A2) + HeLa 1 47 100

ts7 (A2) 2.5 45.5 160

4 44 240

7 41 813

TABLE 2. Complementationindexesinmixed

infectionofHeLa cells with mutantstsl 7(RSN-2)

andtsl9(RSN-2)

Absorption Complementationindexat time of incubation timneat310C (h)at390C:

(h) 63 72 88 96 112 135

1.5 19 75 78 76 6 10

7 64 90 210 145 13 38

2000

x

21600

61200

z 800

e 400

I

0

C-40 60 80 100

HOURS

FIG. 2. Complementation index after various

pe-riods of incubation at restrictive temperature. (a)

Complementationindexes in amixed infection with two A2 strain mutants, tsl (A2) and ts7 (A2). (b)

Complementationindexes in a mixed infection with two RSN-2 strain mutants, tsl7 (RSN-2) and tsl9

(RSN-2). (c) Complementation indexes in an

inter-strain mixedinfection,tsl(A2)andtsl9 (RSN-2).

twice as long as in the case of the A2 strain

mutants.Underthese conditionsof infection the observedcomplementationindexeswerehigher

thanpreviously reportedfor anypairof RS virus

ts mutants. The data illustrated in Fig. 2a, b,

andc wereobtained inasingleexperiment,and therefore the indexes are directly comparable.

Figure 2c illustrates the complementation

in-dexes obtained in a mixed infection with one

mutant from each strain, tsl (A2) and

tsl9

(RSN-2).

It isapparentthat interstrain

comple-mentation experiments between the A2 strain

and theRSN-2 strainrequire careful design and evaluation.

ThedatainTable3 show that itwasnecessary to measure complementation indexes in inter-strain

experiments

over a range of incubation

timesspanningtheoptimaofthe two strains. In

this

experiment,

twots mutants oftheA2strain, representingcomplementation groupsAandB,

werecrossed with fourmutually complementing

461

VOL. 27, 1978

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TABLE 3. Interstraincomplementation; effect of varying the period of incubation at 39°C

Complementation indexes in mixed infection at time of incubation (h) at39°C":

Mutant 37 43 50 61 75

t.s ts2 ts1 ts2 tsl ts2 tsl ts2 tsd ts2

(A2) (A2) (A2) (A2) (A2) (A2) (A2) (A2) (A2) (A2)

tsl(RSN-2) 0.8 <1 96 <1 4 50 2.7 1 5.5 <1

tsl7(RSN-2) <1 0.4 73 <1 2 1.3 <1 1.1 1.1 <1

tsl9(RSN-2) 5.4 0.2 535 <1 7 78 ND <1 ND <1

ts20(RSN-2) <1 <1 67 <1 2.6 86 0.5 <1 3.7 <1

a

Optimum

values are indicated in italics. ND, Not done.

mutants[tsl(RSN-2),ts17(RSN-2),ts19 (RSN-2), andts2O (RSN-2)]oftheRSN-2strain.

Mu-tant tsl (A2) complemented all fourRSN-2 ts mutantsoptimallyat43h,whereasmutant ts2

(A2) complemented mutants tsl (RSN-2), tsl9

(RSN-2),andts2O(RSN-2)at50 honly. Mutant ts2 (A2)didnotcomplementmutantts17

(RSN-2) at any time, and both mutants have been

assigned to the same complementation group

(group B) onthe basis of these and additional

data. Mutants tsl (RSN-2), tsl9 (RSN-2), and

ts2O (RSN-2) complemented both tsl (A2) and ts2(A2) ateither 43or50h,and therefore did not belong to complementation group A or B.

Since these three RSN-2 tsmutants werealso

complemented by ts7 (A2), representing group

C, they are assigned to new complementation

groups, whichhave been designated D, E, and

F,respectively (seeTable4).

The seven complementation groups. Of the remaining 32 RSN-2 strain mutants, only

one [mutant ts6 (RSN-2)] complemented each

ofthe six mutantsrepresentinggroupsA, B, C,

D, E, and F. Mutant ts6 (RSN-2), therefore, representedtheseventhcomplementationgroup

(Table 5). Table 5 also shows thatmutantts26

tecombinants was very low, they could have (A2),tsl (RSN-2),tsl7 (RSN-2), tsl9 (RSN-2), ts2O(RSN-2),andts6(RSN-2),but not tsl (A2).

Mutantts26

(RSN-2), therefore,

wasconsidered

tobelongtocomplementationgroupA.Another nineRSN-2 tsmutants complementedthe

mu-tantsrepresentinggroupsA, C, D, E, F,andG,

but not tsl7 (RSN-2) (Table 6) oreach other

(datanotshown). Therefore,thesemutantshave beenplacedincomplementationgroup B. The remaining21RSN-2 straintsmutantsfailed to complement,orcomplemented sporadically,and

couldnotbe classifiedintoanycomplementation

group.

Absence of genetic recombination. No wild-typerecombinants have been found previ-ously in the progeny ofmixed infections of ts mutantsofeithertheA2strain(11)orthe

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RSN-2 strain (4). Nevertheless, ifthe frequency of RSN-2) complemented mutants ts2 (A2), ts7

TABLE 4. Interstraincomplementation,ts7(A2) versus tsl(RSN-2), tsl7(RSN-2),tsl9(RSN-2), and

ts2O(RSN-2)

Complementation indexes in

Incuba- mixed infection' withRSN-2mutants:

Astuntion

time

mutant

ati

_{at 39 C (h}time _(RSN-tsl _(RSN-tsl7 _(RSN-ts19 _(RSN.ts2O

2) 2) 2) 2)

ts7(A2) 43 76 26 17 3.3

49 ND 25 13 5.8

60 17 5.3 8.3 2.3

67 4 4 2.3 1.4

'Meanof threeexperiments.Theoptimum values are indicated initalics.ND, Notdone.

escapeddetection in the earlier work.Therefore,

theyieldfromthecomplementationexperiment

illustrated in Fig. 2b was examined to

determine whether thegeneration ofwild-type

virusbyrecombination had contributed tothe

highyields obtainedinthisexperiment.Aseries

ofcloneswasisolated from thecomplementation

yieldsat69, 75,88, and 100h,and theseclones

were screened for ability to form plaques at

39°C. All317clones examinedweretemperature

sensitive like the parental virus; therefore, the

wild-type recombinant frequency, even under

conditions favoring the recovery of wild-type

virus, couldnothaveexceeded 0.03%.

DISCUSSION

Table 7 summarizes the classification ofRS virusts mutants intocomplementation groups.

Thereisamarked difference in the distributinn

sandstrain

ofogin

For

instance o the7

stami

iunts into

compleme

n

group

A.whereasonly1

Of-the

5 tr mut-antsappearsinthis

groul.

Similarly,

10 ot tne 15 RSN-2

strainmutan`ts

wereclassifiedingroupB,butonly1of the7A2

strain mutants. Group C is unique to the A2

strain,andgroupsD,E, F,andGareuniqueto the RSN-2 strain. These differencesmay indi-cate inherentdifferencesin themutabilityof the twostrains.Ontheotherhand,theA2 mutants 462

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TABLE 5. The sevencompiementationgroupsdefined by inter-andintrastrain mixedinfections

Complementationindexes ingroup:

Group Mutant A B D E F G

ts26 tUl7 tsl tsl9 ts2O ts6

(RSN-2) (RSN-2) (RSN-2) (RSN-2) (RSN-2) (RSN-2)

A tsl(A2) 1.5 72 96 555 67 3

B ts2(A2) 14 1.6 50 78 86 5

C ts7(A2) 11 26 76 21 6 28

A ts26(RSN-2) - 11 6 10 4 23

B tsl7(RSN-2) - - 550 536 100 7

D tsl (RSN-2) - - - 150 9 23

E tsl9(RSN-2) - - - - 68 114

F ts20(RSN-2) - - - 60

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aHighest valuein three tofiveexperiments.

TABLE 6. Assiwnmentof RSN-2mutants togroupB ComplementationindeX

Mutant

ts17

tsl9

ts3 ts4 | 5 |sl2MU t16 tsN8 ts23 ts25 ts37 (RSN-2) (RSN-2) (RSN-2) (RSN-2) (RSN-2) (RSN-2)

(R)N-

(RSN- (RSN-2) (RSN-2) (RSN-2)

______________________________________________ 2) 2)

tsU7(RSN-2) (2) 650 0.4 0.2 0.7 1 1.5 0.5 1.2 0.6 0.3

(3) (160) (2) (120) (0) (1) (0) (2,000) (300) (89) tResult ofsingle

experiment;

optimum

timeofincubation for RNS-2mutants.Figuresinparentheses are

numberof PFU in self-infection.

TABLE 7. Distributionofmutantsinto complementation groupsaccordigto strain

Comple- No.isolated

mentaon_mentation Prototype_{mutant A2 RSN-2}

grOUp strain1 strainl Total

A tsl(A2) 5

ts26(RSN-2) 1 6

B ts2(A2) 1

tsl7(RSN-2) 10

C ts7(A2) 1 0 1

D tsl (RSN-2) 0 1 1

E tsl9(RNS-2) 0 1 1

F ts2O(RSN-2) 0 1 1

G ts6(RSN-2) 0 1 1

Total 7 15 22

Unclassi- 0 21 21

fied

were isolatedfrom HeLa cells and the RSN-2

mutants from BS-C-1 cells, and it is possible that the host cell may influence the type and frequency ofmutants. Similardifferenceshave

been observed with ts mutants of the human

rhabdovirus Chandipura virus after

5-fluorour-acilmutagenesisindifferent hostcells(Gadkari

and Pringle, unpublished data). However, the

differentfrequenciesmaymerely reflect the

dif-ferentprocedures employed ineachlaboratory.

A

high_proE2rtion

(21/36)

of the RSN-2 strain

_______not-e

cassifed

intoo

men

~~~asuggesf~L

etp

mutationalevents or alesion

affecting

fusible

gene

Droduct.

Two of thethreeunnamedcomplementation groupsof theRSN-2 straindescribed by

Faulk-ner et al. are equivalent to group B (mutant

ts23) andgroup E(mutanttsl9).Theidentityof

thegrouprepresented bymutanttsl5 is obscure

because themutanttsl5 usedby Faulkneretal.

(4) couldnotberecovered fromstorage.

The absenceof recombination and the

appre-ciable number of complementation groups (7

from22 mutants) arecharacteristic features of

otherparamyxoviruses.Forexample,tsmutants

ofSendai virushave been classified intoseven

groups (8), fiveand apossible sixth complemen-tation group have been described for measles virus(2), and five have been claimed for New-castlediseasevirus(10). Therefore the size and

structure.ofthe RS virusgenome may be similar

tothatof other

paramyxoviruses.

The.datecion

ofcomplementAtinn

betLeen

mutantsofRSvirusdependedon

severalfa

whichfincluded

thenos1

cee

type,multiplicity of 27,

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infection, durationofpreliminaryincub at

permissive-temperature.

andperiod Of incuba-tionatrestrctve temper

Attempts

to

im-prove

complemen

by creation ofviral

ag-gregates using

poly-L-ornithine,

nucleohistone,

orcoprecipitation byPEG-6000wereineffective.

It wasconfirmedthat HeLa cels wereabetter IMt tha

_{M-U-_}

-

_ceffs

e ad_a that this was a

conseauenceof the greatertemperature

sensitiv-it-y

(lowerleak) ofmutantsinthesecell

(dt

not shown). The multipcy ofi onused

was thehcouldbeachieved

(about

1

PFU/cell

at the best), but the low intectivity --

Sed

-w'i

titers obtaine w Ith S viru were

probal

resposbefor teproiDiemencoutrdn

~mplementtionanalysis.Aperiodof

absorp-tionat3100of 7 h increased the

complementa-tionidex-witho_ut

increaigtes-yielda

otmutantsof

bothstrainsequally.

However,

the

optimal

time

of incubationat39°C differed (Fig. 2). The

rea-son for this difference is not clear since the growthcyclesof the wild types of thetwostrains

in HeLa cells at

390C

did not show obvious

differences (Fig. 1).

Possibly

amplification

of

gene productswas required inthe case of the

RSN-2 mutants, whichhad lowereffective

mul-tiplicitiesofinfectioninHeLa cellsthenthe A2

strainmutants.Althouh22tsmutantshave been

grouped

satisfactorily

into seven groups, it is

possible that these conditionswere not

appro-priate for complementation between mutants

withlesionsinothergeneproducts. The

example

of RS virus may be instructive in the

genetic

analysisof other animal viruseswhere

difficul-ties have been encountered in

demonstrating

complementation.

Some phenotypicproperties of the A2 strain

mutants in groups A,

B,

and C have been

de-scribed (1, 6, 9), the most

striking being

the

nonsyncytial

cytopathology

associated with

mu-tant ts2

(group

B).

It may be

significant

that

noneof theRSN-2mutants

presently

classified

ingroupBinduced the

nonsyncytial

cytopathic

effect in

HEp-2

cells

reported

for ts2

(A2).

Therefore the homologyof thesemutants may

bespurious,and absence of

complementation

in

thiscase mustbe treated with caution. Thesame

reservationmayapplytogroupA,sincetsl(A2)

is thermosensitive and ts26 (RSN-2) is ther-mostable(unpublished data).Therefore seven is the minimum number of complementation groups resolved in the present study, if

intracis-troniccomplementationisdiscounted. ACKNOWLEDGEMENTS

This workwasasistedby financial supporttoH. B. Gi-menezfromthe British Council, the Medical Rearch Coun-cil, and the World Health Organization.

LTERATURE CTD

1.Belshe, R. B., L S.Richardson, T. J.Schnitzer,D.A. Prevar, E. Camargo, and R. M. Chanock. 1977. Furthercharacterization of the complementation group Btemperature-sensitive mutant ofrespiratory syncytial virus. J.Virol. 24:8-12.

2. Breswhkin, A. M., F. Rapp, and F. E. Payne. 1977. Complementationanalysis of measles viru ts mutants. J.Virol. 21:439-441.

3. Cash,P., W. H.Wunner,andC. R.Pringle.1977.A companson of thepolypeptides of human and bovine respiratory syncytial virume and murine pneumonia virus.Virology 82:369-379.

4.Faulkner, G. P., P. V. Shirodarla,E. A. C.Follett, and C. R.Pringle.1976.Respiratorysyncytial virusts mutants and nuclear immunofluorescence. J. Virol. 20:487-500.

5. Gharpure,ALA.,P. F.Wright,and R. LCho 1969. Temperature-sensitive mutants of respiratory syncytial virus. J. Virol. 8:414 421.

6.Kalica, A. R., P. F. Wright, F. MLHetrick, and R. IL Chanock.1973.Electronmicroscopicstudies of respi-ratorysyncytial virus temperature-sensitive mutants. Arch.GesamteVimrforach.41:248-258.

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