Isolation of Temperature-Sensitive Conditional Lethal Mutants of “Fixed” Rabies Virus

(1)

Isolation

of

Temperature-Sensitive Conditional Lethal

Mutants

of

"Fixed" Rabies Virus

H FRED CLARK AND HILARYKOPROWSKI

The Wistar Instituteof Anatomy andBiology, Philadelphia, Pennsylvania 19104

Received for publication 13 November 1970

In an attempt to induce temperature-sensitive (is) conditional lethal mutants

ofrabies virus, stocks ofaplaque-purified substrain of strain CVS fixed rabies virus were subjected tomutagenesis by HNO2, 5-fluorouracil, or 5-azacytidine. It was

necessary to prepare virus stocks from clones of mutagenized virus selected at

randomandtotestsubsequently each stock for possibletscharacteristics by

measur-ing its relative capacity for growthatpermissive (33 C) and nonpermissive (40.5 C)

temperatures. Five tsmutants were detected intestsof 161 clones of mutagenized virus. Each of themutants exhibited aremarkably low incidence of reversion and

little demonstrable "leakiness." One of the five ts mutants (ts2), which formed formedvery smallplaques, andanother (tsl), which formed plaques of only slightly reducedsize, werefurthercharacterized. Virus tslwasmorethermostableat40.5 C than the parental virus, but the ts2 mutantwasunchanged in this respect.The tsl

virus exhibited normal pathogenicity for mice, butts2virus causedaveryirregular death pattern. Both deaths and survivors immune to rabies virus challenge were

noted inallgroupsofmiceinoculated with ts2virus, regardless of the virus dose.

Although the viraletiologyofrabies has been

recognized for nearly a century (13), application

ofmodernvirologicaltechniques to the

study

of

rabies virus has been possible only since its

adaptation to growth in cell culture in 1958 by

Kissling

(6).

Rabies virus does not

consistently

produce acytopathic effect or plaques in

mono-layer cell culture. The

practical

study

of

geneti-callyhomogeneousclones of rabies virus became

possibleonlyafter the

development

ofan

agarose-suspended cell

plaquing

technique (15).

Unfor-tunately, thisplaquing system tolerates onlyvery

narrowranges of temperature and thusis not

ap-plicableto direct assayfor

temperature-sensitive

(ts) conditional lethal mutants of rabies virus.

Furthermore,

plaque

sizes vary for artifactual

reasonsinthis system,soit hasnotbeenpossible

to selectplaque-sizevariants from "wild" rabies

populations.

TIhe CVS "fixed" strain of rabies virus

repli-catesinBHK

monolayer

cellculturesat tempera-tures

ranging

from 25 C to over 40 C.

Plaque

assay is performed at 35 C. Maximum yield of released virus isobtainedat33C,buta tempera-tureof35 C is usedforclone outgrowth, asthat is the lowest temperature supporting cell multi-plication. In an attempt to isolate tsmutants of rabies virus, we have selected atrandom clones of virus populations treatedwith chemical

muta-gens and determined their relative capacity for growth at 33 C (permissive temperature) and 40.5 C (nonpermissive temperature). The isola-tion by thesetechniques offive tsmutants and the

preliminary characterization oftwo mutants are

described in this report.

MATERLALS AND METHODS

Cel cultures. BHK subclone 21 (BHK-21) cells

werepropagated inEagle's basal mediumwith double concentrations of vitamins and amino acids (BME)

with0.225% HCO3-and 10%fetalcalfserum (FCS)

added (BME FCS 10). BHKsubclone13S(BHK-13S) cells were propagated in BHK growth medium (10) with0.17%HCO3-and 10% FCS.

Virus. CVS strain virus that had been passed 112

timesin primary hamsterkidneycell culture (7) was

kindly suppliedto usby P. E. Halonen.Thevirus was passed twice in BHK-21 cells, clone-purified three times in BHK-13Scells, and passed two or three more times on BHK-21 cellspriortouse.Virus stocks were

routinelyprepared in BHK-21 cell cultures infected at acellmultiplicityof 0.1 to 10.0 andincubatedfor 72 to 96 hr at 33 C in a medium consisting of BME plus 0.1% bovine serumalbumin (BNME.1BA).Cell super-natantfluidsclarified by centrifugation at 800 X g for 30minat4 Ccomprisedthevirus stock.

Virus cloning methods. All virus assays were per-formed by the agarose-suspended BHK-13S cell plaque technique (15). For virus cloning experiments, selected plaqueswereaspirated with a Pasteur pipette 295

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CLARK AND KOPROWSKI

and suspended in 1.0 ml of BME.1BA. To prepare

clonedvirus stocks, 0.5 ml ofplaquesuspension was added to 1.5 X 106 BHK-21 cellssuspendedin 0.3 ml

of growthmedium.Themixturewasincubatedin

30-ml plastic tissueculture flasks (Falcon Plastics Co.)

standingonendfor30min at 33 C.After this

adsorp-tionperiod, 5.0 ml ofgrowth medium was added to each flask, and the flasks wereplaced flat and incu-bated at 35 C for 4 to 6 days, after which the super-natantfluidswere harvested.ClonedCVSvirus stocks prepared in this manner contained 107-5 to 108.7

plaque-forming units (PFU)/ml.

Chemical mutagenesis: nitrous acid. The method of

Granoff (4) wasemployed for chemical mutagenesis. Two partsofvirus suspension (undiluted virusstock) weremixed withone partof4.0 M NaNO2 in water and onepartofacetatebuffer, varyinginpHfrom4.0to 4.8, at roomtemperature. Atvarious intervals, samples ofthereaction mixturewereremovedand thereaction

was stopped byimmediate 10- to 25-fold dilution in BME.ABA. Eachsamplewastitrated immediatelyto

yield plaquesfor selection aspotentialmutants. 5-AzaC and5-FU.In experiments employing these

mutagens, 5-azacytidine (5-AzaC; Calbiochem, B

grade; reference 5) or 5-fluorouracil (5-FU; Calbio-chem) was incorporated in themaintenance medium

of BHK-21 cells infected with rabies as described

above.

Assayofclonesfor ts mutants. BHK-21 cellcultures

were inoculated with virus of each clone tested, at

multiplicities of infection (MOI) of 10 to 100, and

incubatedat40.5C.Viralyieldsafter 24 or 48 hr were measured. Virus clones giving yields at 40.5 C that

were10-fold(or more) lessthannormalcontrolclones

weresubsequentlyretestedbycomparingtheirgrowth

atboth40.5and33C.

Mouse infectivity titrations. Groups of six 4- to

5-week-old femaleICR micewereinoculatedintothe

left cerebral hemisphere with 0.03 ml each of serial 10-fold dilutions of virus in BME.1BA. Inoculated animalswereobserved foratleast 28daysafter

inocu-lation forthe onsetofparalysisor death.

RESULTS

Inactivation of rabies virus by HNO2. Rabies

virus was

rapidly

inactivated

by

HNO2

at rates

that are

markedly

pH-dependent.

For

example,

infectious virus titers were consistently reduced

from 100-fold to

1,000-fold

after incubation for

1 min in 1.0 M nitrous acid under conditions

varying from

pH

4.4to4.9. At

pH

5.8 and

6.1,

less than 10-fold reductions in virus titers were

observed after incubation

periods

as

long

as 10

min. In control reaction mixtures of

pH

4.4 in

which

NaNO2

was

replaced

with

NaCl,

no

reduc-tionin virus titerwas

detected,

thus

establishing

the

specific

role ofthe nitrous acid in the virus

inactivation.The

high

degree

of

stability

of rabies

virus under acidic conditions of

pH

54.5 has been

previously

reported

by

Turner and

Kaplan

(18)

and

by

Kuwertand Liebenow

(8).

Effect of 5-FU and 5-AzaConthe

replication

of

rabies virus. Incorporationof5-FU incell main-tenancemedium at aconcentrationof 2mm led

to a depression in rabies virus yield (after 48 hr

incubation at 33 C) of from 65 to 85%. Under

similar conditions, 5-AzaC at a concentration of

25

yg/ml

led to a 97 to

>99%

reduction in rabies

virus yield.

Selection of rabies virus mutants. Initially, an

attempt was made to select a heat-resistant CVS

virus variant for production of a standard pool

for chemical mutagenesis,toobviate the problem

of the lability of rabiesvirus at highnonpermissive

temperatures. Virus suspended in BME.1BA was

heatedat 60 C, and samples removed at various

time intervals were titrated for residual

infectiv-ity. Several plaques were harvested from the

titration of the last sample yielding surviving virus

(5, 10, or20 min) andused to prepare new virus

stocks whichwereagain heated at 60 C. After five

such cycles of heating and regrowth of clones of

surviving virus, no thermoresistantvariants were

obtained. The

half-life

at 60 C of the original

CVS stock (suspended in BME.1BA) and of

all

the tested progeny clones (suspended in BME

FCS 10) wasapproximately 36 sec. A clone

des-ignated CVS-HPP (CVS-heated and

plaque-purified) was selected from thesixth heating

ex-periment foruse as abasic stock formutagenesis

because, although it was notdemonstrably more

heat-stable than the parental virus, it was

as-sumedto begeneticallyhomogeneous as a result

of

six

consecutive

clonings.

Five tsmutant clones weredetected in studies

of 161 clonesof

mutagen-treated

CVS-HPPvirus.

The

incidence

of

mutant clones was 1 (tsl) of 44

treated

with

HNO2,

2

(ts2

and

3) of

91 grown inthepresence of 5-AzaC, and2 (ts4 and 5) of

26 grownin thepresenceof5-FU.

The relativecapacity of each mutantvirus (in

the 2ndor3rd BHK-21 passageafter clone

selec-tion)

for

growth

at 40.5 and 33 C is shown in

Table 1. Although CVS stock virus gave yields

at

40.5

Cequalto 0.5 to 5.0% of thoseobtained

atthe

permissive

temperature,themutant

viruses,

withasingleexception, revealednodemonstrable

replicationatthe

nonpermissive

temperature. The

small amount of tsl progeny virus detected at

40.5 C wasdeterminedtoberevertantnon-tsvirus.

The results suggest that there is no measurable

"leakiness" ofthesemutants at40.5 C. Anexact

determination ofreversionrateswas not

possible

becauseofourtechnical

inability

to

plaque

rabies

virusatthe

nonpermissive

temperature.

However,

the total absence of detectable virus in 40.5-C

cultures infected with four of the five mutants

indicatesthattheincidence ofrevertantsin those

mutant stocks was less thanone in

108 particles,

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TS MUTANTS OF "FIXED" RABIES VIRUS

TABLE 1. Temperature sensitivityof CVS rabies virus and mutantsa

Virus yield (releasedvirus, PFU/ml) Permissivetemp(33 C)

24hr

9.0X 105 9.0 X105

2.2 X 104 2.3 X 104

104

2.2 X106

1.6 X105 5.5 X105

48hr

2.0 X 108

8.5 X 107

1.2 X 106 107 1.4 X 107

1.4 X 106

1.2 X 107 7.5 X 106

Nonpermissivetemp (40.5 C)

6hr

101 2.0 X 102

24hr

1.6 X 104

<5.0 X 10'

102

<5.0 X 10'

<5.0 X 10' <5.0 X 10' <5.0 X10'

<5.0 X 10'

48hr

Fractional yield (40.5C/33 C)

24hr 48hr

2.2X 106 0.018 0.011

<

<5.0 X10' <0.0000056 <0.000000059 6.0 X 104

2.0 X 102

<5.0 X 10' <5.0 X 10'

<5.0 X 10'

<5.0X 10'

0.0045 <0. 00023 <0.00050 <0.0000023 <0.000031 <0.0000091 0.050 0.00002 <0.00000036 <0.0000036 <0.00000042 <0.00000067

aBHK-21monolayersin 30-miplasticflaskswereinfected atamultiplicityofapproximately10.Afteradsorptionfor 30minat

33 C,monolayerswerewashed twicewithphosphate-buffered saline,treated with 1.0 ml ofpolyvalentrabiesantiserum 1:1,000 for10minat33C,andagainwashedtwice withphosphate-bufferedsaline priortoaddition of cellmaintenancemedium.Times arecalculated fromthe initiation ofadsorption.

the minimum amount of virus in the inocula

tested.

Of the ts mutant clones, one, ts2, produced

unusually small plaques, whereas the size of plaques producedby the other fourwasnormalor near-normal (Fig. 1). The small-plaque mutant

ts2 and the near-normal plaque-forming ts

mutant, tsl, wereselected for further study. Preliminarycharacterization ofmutants tsland

ts2. Theeffect ofthe cell MOI ontheexpression of the ts state ofmutants tsl and ts2 was tested

in several experiments in which postinfection

treatmentofcells with antiserumwasomitted, so that minuteamountsofrevertantvirusmightnot

be neutralized by antibody. Insuch experiments the ts state of mutant stocks was slightly

ob-scuredat6 and24hrpostinfection because of the

release of unadsorbed inoculum virus observed

both at permissive and nonpermissive

tempera-tures. However,data obtained at 48 hr postinfec-tionclearly revealed thatno newinfectious virus

wasproducedat40.5C in culture inoculatedwith tsl orts2 virusstocks atMOIranging from0.13

to 8.0, whereas virus replication was readily demonstrated in cultures inoculated with wild-type virus at similar MOI.

As a furthertestofthestability of thetsstate

of these mutants, serial undiluted passages of

second BHK-21 passage mutantstocks were per-formedin BHK-21 cellsatthepermissive tempera-ture until the 10th passage level was attained.

The results oftemperature-sensitivity determina-tions of virus tested at each passage level are givenin Table 2. Both tsl and ts2retained their

ts mutant characteristics unaltered through nine successivepassagesat33 Candthen exhibited

re-version atthe 10th passagelevel. The conditions

leading to thisabrupt conversion are not under-stood, but it is evident that both mutants are

normally very stable when propagated at the permissivetemperature.

The possibility that clones tsl and ts2 may

represent thermolabile mutants was tested by thermoinactivation studies at 40.5 C (Fig. 2). The results indicate that clone ts2 virus is

inacti-vatedatarateverysimilartothat of the parental virus (half-life, ca. 4.1 hr). Clone tsl virus is

ac-tually more thermoresistant than the parental stock (half-life, 7.5 hr).

Pathogenicity for mice. Theresponse of

wean-ling mice to intracerebral inoculation with serial dilutions of parental type CVS and ts mutant

viruses is depicted in Fig. 3. Typicaldose-response effects were observed with CVS, CVS-HPP, and

mutant tsl viruses. The plaque-purified CVS virus appeared to be slightly less virulent for mice than theoriginal CVS stock, causinggreater

than50% mortality onlyatdosesabove 10 PFU, whereas the wild CVS stockwashighlylethaleven

in doses of less than 1 PFU. Mutant tsl virus appeared slightlymorepathogenicthan the CVS-HPP stock from which it was derived, giving a

dose-response curve similar to that of the CVS stock.

Mutant ts2 virus exhibited an extremely

aber-rant dose-response relationship. Both rabies-specific deaths and survivors were observed in groupsof micereceiving virus doses varying from

ca. 1 togreaterthan 10' PFU.

The incubation period was prolonged in mice

infected withtsmutants. Deaths ofmice infected

with tsl mutant virus occurred about2 days later

than inmiceinfected with comparable doses (in

PFU) of CVSorCVS-HPP virus. Deaths of mice

VOL.7, 1971

297

6hr

<5.0 X 10'

1.6 X 102

Virus ExptA CVs ts2 ExptB Cvs tsl ts2 ts3 ts4 ts5

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CLARK AND KOPROWSKI

I

CVS"..i...,.

HP (VS

FIG. 1. Plaque morphology ofmutanit tsl and ts2 rabies viruses, oftheparental CVS-HPP rabies

substraini,

[image:4.491.106.395.69.366.2]

midof wild CVSvirus.

TABLE 2. Temperature sentsitivity of tsi and ts2 virus after unidiluted serial passage in BHK-21 cells atpermissive temperature5

tsl Virusyield(PFU/ml) 1s2 Virusyield

Passage level

33C 40.5C 33C 40.5C

3 1.6 X 107 <5.0 X10t 5.5 X 107 <5.0 X 10° 4 1.8 X 108 2.0 X10' 2.0 X 107 <5.0 X 100 5 1.2 X 108 5.5 X 101 1.4 X 107 <5.0 X 100

6 2.0X 108 <5.0 X 100 1.5 X 108 <5.0 X100

7 1.6 X 108 10' 1.5 X 108 .<5.0 X 10°

8 1.9 X 10' 5.0X 10° 1.2 X 108 <5.0 X 10°

9 1.3 X108 1.5 X101 2.9X10 <5.0 X 100 10 6.0 X106 1.5 X 106 5.0 X 105 1.2 X 103

aTechniques for theperformanceoftemperaturesensitivity

determinationsaredescribed in Table 1. In the above

experi-ments,only48-hr virusyieldswereconsidered.

infected with ts2 viruswere delayedfrom4 to 10

days. When mice surviving ts2 infection were

challenged with CVS virus (110

LD5o

inoculated

intracerebrally,28daysafter thets2inoculation),

all animals receiving doses of ts2 virus of 160 PFU orgreater wereprotected.The 50% protec-tive doseofts2viruswas23 PFU.

To determine whether mouse deaths were the

48 TIME IN HOURS

FIG.2.

Tlhermal

inactivatiomi

ofCVSrabiesvirus

amid

ofts mutantstsland ts2at40.5C.Allvirusessuspended in BME. IBA. Datafrom several experiments are

in-cluded.

result ofmutant or ofrevertant virus, virus was

recovered from thebrains ofrepresentative mice

dying after infection with tsl or ts2 virus.

(Be-cause of very low virus titers in mouse brains, brainisolate stocks hadtobepreparedinBHK-21

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VOL.7,1971TSMUTANT'S OF "FIXED" RABIES VIRUS29

.:-HPc .-Vs

80j

2

40-2.0 -10 0 0 10 2.0 3.0 40 5.0 6 0 VIRUSTITER/ LOGI0 (PFU /0.03ML)

FIG. 3. Pathogenicity ofCVS rabies virus and ts mutantsforweanlingmice.Groupsof6to12 micewere

inoculated intracerebrally with 10-fold dilutions of

virusandobservedfor28daysafterinoculation.

cellsbythesamemethods usedforpreparationof

stocks from plaques, prior to performance of temperature sensitivity determinations.) Virus from three ofthe sixmice that died of tsl virus infection retained the ts mutant characteristic,

whereasvirus from three others was ofrevertant nature. Virus from three of the four mice that died ofts2 virus infectionwas revertant with

re-gardto both temperaturesensitivity and

plaque-sizecharacteristics; virus from the fourth mouse

retained both the ts and small-plaque charac-teristicsoftheinoculum.

DISCUSSION

We have described the selection of five ts mutantsfromatotal of 161 clones ofpopulations

of CVS strainrabies subjectedtomutagenesis by

nitrousacid, 5-FU,or5AzaC. Theimportanceof chemical mutagenesis for induction of the ts statewasnotdetermined.

Each of the five rabiestsmutantsdetected has exhibited no measurable "leakiness" atthe

non-permissive temperature. The incidence of

re-versiontothenon-tsstatehasbeenlow,andlarge

stocks ofts mutant virus were prepared without difficulties. This experience with the rabies

mutants differs from the general experiencewith

ts mutants of other viruses (1) but parallels ob-servations of ts mutants of the morphologically

similar vesicular stomatitis virus described by

Flamand (2, 3) andby Pringle (InThebiology of

large RNA viruses, Academic Press Inc., in

press).

In addition to temperature sensitivity, mutant

tsl exhibited increasedthermostability at 40.5 C, whereas mutant ts2 induced abnormally small

plaques in BHK-13S cells and asharplyreduced incidence of lethal infections in mice. Mutants with known multiple markers should provide

valuable

flew

tools for

biological

studies of the

rabies virus. The

only

invitro marker

previously

reported

forafixed rabiesstrain is the RCT 40-character found in a rabies strain

adapted

to

growth

at 26 to 28 C

by

Selimov and Nikitina

(16).

Whether the

multiple

markers of thetsl and

ts2 mutants

represent

multiple

mutations or

multiple

manifestations of

single-point

mutations

remainsto bedetermined.

The behavior of

small-plaque

ts mutant

ts2

in mice is of

particular

interest.

Although

virus

virulence invivo has not

usually

been a

primary

concern of those

studying

ts

mutants,

reduced

virulence has been described for

chemically

in-ducedts mutantsofSemliki Forest disease virus

(1)

and influenza virus

(9, 12).

Reduced

patho-genicity

in vivo has also been described in the case of

small-plaque

mutants offoot-and-mouth

disease virus induced

by hydroxylamine

(11),

small-plaque

mutants of Newcastle disease virus

induced

by

nitrous acid

(4),

and a

small-plaque

mutant of vesicular stomatitis virus

(VSV)

in-duced

by

ultraviolet

irradiation

(17). However,

to

our

knowledge,

a

pattern

of

pathogenicity

suchas

that caused

by

ts

virus,

in which

virus-specific

deaths and survivors both occur in animals

inoculated with doses

varying

from 1 to 106

PFU,

has not

previously

been described. The

explana-tionfor this unusual death

pattern

is not

clear;

it

is

tempting

to

postulate

asituation in whichdeath occurs

only

if revertant virus appears in lethal

amounts before

multiplication

of mutant virus

induces

protective immunity. Preliminary

studies

haverevealed that the brains of three of thefour

mice

dying

afterts2 infection containedrevertant

virus,

whereas a fourth contained ts virus. The

three mice

yielding

non-trs

virus died aftera

near-normal incubation

period

of10

daiys;

the mouse

yielding

tsvirusdied

only

after avery

prolonged

incubation

period

of28

days.

The

possibility

that

ts virusmaybemutantinacharacter criticalfor

rabies

pathogenesis

will

befurther

explored

in the

hope

thatinformation may beobtained

concern-ing

the unresolved

question

of how rabies virus

causesdeath.

Asnoted

above,

rabies virusis thesecond virus

oftherhabdovirus groupfrom which tsmutants

havebeen described.tsMutantsofVSV havebeen

obtained in

large

numbers from virus stocks

sub-jected

to chemical

mutagenesis

and an incidence

of 0.9to

5.0%

ts mutants in wild VSV prepara-tions has been estimated

(3,

4, 14).

Comple-mentation between

appropriate

ts mutants of

VSV has been demonstrated to be

unusually

ef-ficient,

and recombination has been

demon-strated. It hasbeen

possible

to

place

the

majority

of VSV tsmutants withinoneofeitherfour

(14)

orfive

(3) complementation

groups.An

adequate

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CLARK AND KOPROWSKI

comparison of the genetic properties of rabies virus with those of VSV will require: (i) deter-mination that the known rabies mutants

repre-sent single-point mutations, (ii) performance of

tests for complementation and recombination, and (iii) the isolation of many more ts rabies

mutants,anaccomplishment which will

undoubt-edly require the development of new and more

efficient means for screening rabies virus clones

for thepresenceofthe tscondition.

ACKNOWLEDG MENTS

We gratefully acknowledge the excellent technical assistance

ofMaria-Elvira Soriano.

This investigation was supported by Public Health Service Research Grants ROI-AI 02954 and R22-AI 07988 from the National Institute of Allergy and Infectious Diseases and by funds from the World Health Organization.

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