Comparative study of rabies virus persistence in human and hamster cell lines.

0022-538X/81/010001-06$02.00/0 Vol. 37, No. 1

Comparative Study of Rabies

Virus Persistence

in Human

and

Hamster Cell

Lines

0. G. ANDZHAPARIDZE, N.N. BOGOMOLOVA, Y. S. BORISKIN,* M. S. BEKTEMIROVA, AND

I.D. DRYNOV

ResearchInstitute forViral Preparations,Moscow, USSR

Persistent infections by rabies virus in BHK-21/13S and HEp-2 cells were

studied comparatively. No evidence of interferon production, selectionof

virus-resistantcells,orintegration of the viralgenomecould be found. Persistingviruses

replicated efficiently at 34, 36, and 40°C. Both persistently infected cultures

released defective interfering virus particles.Acyclicalpatternof infection,which

wasnotcharacteristic of the persistently infectedHEp-2system,wasobserved in persistently infected BHK cultures. The virus from persistently infected BHK

cultures lost its virulence for mice, whereas the virus frompersistently infected

HEp-2 cultures retained mouse-killing capacity formorethan3years.

Persistent infectionby rabies virus has been studied extensively in cell cultures of hamster

origin (6, 8, 16); in general, the mechanism of rabies virus persistence is currently attributed

todefective interferingparticle (DIp)-mediated

cell sparing, as evidenced from several studies

with BHK-21 cells (6, 8, 17). However, a host

effect with respect to DIp generation (7) or

acquisition of virulence (3) has been observed

whencells distinctfromconventionalBHKcells were used. This communication is an examina-tion of the roleofseveral factorsthat may con-tribute to rabies viruspersistence intwo differ-ent cell cultures. The results indicate that the hostcell type maystronglyinfluencethe

estab-lishment and course of infection and the

viru-lence of the persistingvirus.

MATERIALS AND METHODS

Cells and viruses.Primarycultures ofJapanese

quail embryo(QE)cellswerepropagatedasdescribed

previously (2). BHK-21/13S and HEp-2 cells were

grown inEagle and 199media, respectively,

supple-mented with 10% bovine serum and antibiotics. A

Pasteur strain of rabies virus that was adapted to

growth inQEcells (2) wasused for the initiation of

persistent infection andas a control standard virus.

Bothpersistentlyinfected cell linesweremaintained

at360C.

Assayof viralinfectivity.The infectious titer of

rabies viruses was determined by the intracerebral

inoculation of mice orbya plaque assaywith CER

cells (kindly provided by J. J. Holland and T. J.

Wiktor) withaSephadex overlay (10).

Directfluorescent-antibody technique.The

di-rectfluorescent-antibodytechniquewasdescribed pre-viously (2).

Cellcloningexperiments. Carriercellswere

dis-persed,counted,andplatedatdilutionsthatprovided

veryfewcells per flask.Single cellsweremarked3 to

4days after seeding and observed until a colony had

formed. These coloniesweretransferredtoindividual

tubes, and thedeveloped subcultures were examined.

Immunogenicity of persistingviruses. The

im-munogenic activity of the viruseswasdetermined by

the standard National Institutes of Health potency

testinmice(11).

Concentration andpurification of rabies virus.

Rabies virus was pelleted from the culture fluid at

20,000 rpmfor2 hina 6x250mlrotorofanMSE SS

65 centrifuge, suspended in NTE buffer (13), and

clarified througha30%sucrosecushionat61,000xg

for 40 min. The visible band at the interface was

aspirated, diluted with NTE buffer, and pelleted at

45,000 rpm for2hin anSW50rotorofaBeckman L5

65centrifuge. Virus suspended in 0.5 to 0.8 ml was

layeredon a 5 to30% linearsucrosegradientin NTE

buffer made with the aid ofanLKBUltrograd gradient

mixer. CentrifugationwasperformedinanSW40rotor

at 34,000 rpm for60min. Gradients wererecovered

from the bottomthroughaflowcell ofaGilford

2400-2spectrophotometer, withacontinuousmonitoringof

theoptical density(OD)at260nm.

Polyacrylamide gelelectrophoresis.Samplesof

purified virusweredissociated andseparatedon7.5%

cylihdrical gelsinasodiumdodecyl sulfate-phosphate

systemasdescribedbyWeberetal.(15).

Electropho-resiswasconducted in 14-cmtubesat3mApertube

for 20 to 24 h. Gels were fixed and stained in a

methanol-glacialacetic acid-Coomassiebrilliant blue

R-250mixture and after destainingwere scannedin

theGilfordspectrophotometerat550nm.The

molec-ular weight standardswere bovinealbumin,

ovalbu-min, and myoglobin (Schwarz/Mann, Orangeburg,

N.Y.; 68,000, 45,000,and18,600,respectively).

Preparation of rabies virus ['H1RNA and

[tHl-cDNA. For thepreparation of[3H]RNA, RNA was

extractedfrompurifiedrabiesvirus(a totalof5 x10'0

50% lethaldoses[LD50])that had beenpropagatedin

QE cellsby treatmentwith sodium dodecyl

sulfate-phenol-chloroform (9).Aportionof this RNA

prepa-ration was used forlabelingbythe direct introduction

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2 ANDZHAPARIDZE ET AL.

of tritium into the polynucleotide in the course of RNA treatment with 3H atoms (12). [3H]RNA

ob-tained in thismanner wasdialyzed against25volumes

ofO.lxSSC buffer(lx SSC is0.15MNaClplus0.015

M sodium citrate) containing 0.5% sodium dodecyl

sulfate inanMMC Amiconsystemthrougha10PM

membrane and stored under ethanol at-25°C. The

specificactivity of thepreparationwas0.9x106cpm/

Ag-[3H]complementary DNA ([3H]cDNA) of rabies

vi-rusRNAwasobtainedessentiallyasdescribed

previ-ously (1) with50U of Escherichia coli DNA

polym-erase (Sigma Chemical Co.,St. Louis, Mo.) and

3H-labeled deoxytriphosphates ([3H]dGTP and

[3H]-dCTP[AmershamCorp., Arlington Heights,Ill.],

spe-cificactivities,10.3and25Ci/mmol,respectively;and

[3H]dATP [Isotop, Leningrad, USSR],specific

activ-ity, 7.2 Ci/mmol). The composition ofthe reaction

mixture, the isolation ofcDNA, and thepreparation

of thehybridwerethesame aspreviously described

(1).Thespecific activity of cDNAwas6.7 x107cpm/

ug as determined from the specific activities ofthe

input3H-labeleddeoxytriphosphates. After synthesis,

cDNA sedimented inan alkalinesucrosegradientat

apositioncorresponding to 8to 9S and annealedto

excessvirion RNAto an extentof about 90%.

Hybridization of carrier cell DNA to rabies

virus [3H]RNA orcDNA. The DNA was isolated

from carrier cultures at a level of 104 passages for

persistently infected HEp-2 cultures(HEp-2p.j.)andat

alevel of117passagesforpersistently infected BHK

cultures(BHKp.i.)byachloroform-isoamyl alcohol (24:

1)technique,including thestepsof RNase andpronase

digestions between subsequent deproteinizations, as

described by Cooper and Temin (4). The DNA

ob-tained in thismannerhadaratio ofabsorbanceat 260

nm toabsorbance at280nmof1.8 to1.9,whichwas

comparabletothat ofcalfthymus DNA(Serva,

Hei-delberg, Germany). The composition of the annealing

reactionmixtures and theprotocol for the

hybridiza-tionwereidenticaltothose describedpreviously(1).

Transfectionexperiments with DNA from

car-riercells. Confluentmonolayersof 2x107BHKcells

were pretreated for 5 min with 300

iLg

of

DEAE-dextranperml in 0.15 MNaCl. Afterthe aspiration of

DEAE-dextran,cellswereexposedtoDNAdissolved

in 0.15 M NaCl. The DNA exposure levels ranged

from0.5 to 7 mg perflask. Aftera15-min adsorption

at roomtemperature,growth mediumwasaddedand

cells were incubated at 36°C. Transfected cultures

weresplittwiceweekly,with periodic assays of cultural

fluid forinfectiousvirus and of cells for viral antigen

byimmunofluorescence.

RESULTS

Establishment and time course of per-sistent infection. Confluent monolayer

cul-turesofHEp-2 or BHK cells were infected with

rabies virus at a multiplicity of 1 LD50per cell.

Aftera1.5-hadsorption period, growth medium

was added and the cultures were incubated at

36°C.Nosigns ofcelldestruction were observed in

HEp-2p.i.

at 7 to 10 days postinfection. In contrast, in

BHKp.i.,

manycells diedby

postin-fection day 4, and the addition of fresh BHK

cellswasrequired during eight subsequent

pas-sages to preventcompletecelldestruction.After

the persistent infection had stabilized,

BHKp.i.

werepassed every5 to 7days,withnosigns of

visible cytopathology.

Differentpatterns of infection werefound in the two persistently infected cultures (Fig. 1). For

BHKp.i.,

cyclical fluctuations of antigen-pos-itive cellsranging from 100toless than1%were

observed, although the antigen never

disap-peared completely. The persisting virus

gradu-ally lost its virulence for mice; soon after 50 passages of

BHKp.i.,

mouse-lethalviruswas no

longer detectable.

In contrast to

BHKp.i.,

a significantly more

constantproductionof infectious virus and viral

antigenwasfound in

HEp-2p.i.

(Fig. 1).

Properties ofpersistingviruses. Starting

from theearly stages ofinfection,thepersisting viruses fromboth cultures

characteristically

dis-playedincubationperiodsthatwereabout twice

aslongin miceasthose withstandardvirus(12

to 14 versus 6 to 8days).The virus from

HEp-2p.;.

retained its virulence for mice formorethan 3years, whereas the virusfrom

BHKp.i.

lostits

pathogenicity after 50 passages ofthe culture. Thisloss of

mouse-killing

abilityremained after three intracerebral passages of the virus in

suck-ling-mousebrains.

Persisting viruses could induce plaques on

CER cellmonolayers.Thespecificityofplaques

wasverifiedinaplaque reduction neutralization

testwith antiserumtooriginalviruspropagated

in QE cells.

BHKp.i.

virus plaques were 0.1 to

0.15 cm in

diameter,

whereas

HEp-2p.i.

virus

formedtiny pinpointplaques.

The immunogenic ability of unconcentrated

persisting viruses was poor. However,

concen-FIG. 1. Timecourseofpersistent infections by

ra-bies virus in

HEp-2p.,.

(A) andBHKP,.. (B). Symbols:

0,infectivity;0,percentage ofantigen-positivecells.

(Abscissa) Number of cell transfers; (left ordinate)

percentageoffluorescingcells;(right ordinate) virus titerinlogLD50permilliliter.

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[image:2.496.264.455.470.606.2]

trated viruses displayed their immunogenicity

evenatlatestagesof persistence(Table 1).

To determine the effectoftemperatureonthe multiplication of persisting viruses, BHK cells wereinfectedwith

HEp-2p.i.

virus frompassage

134,

BHKp.i.

virus frompassage163,orstandard virus and incubatedat34, 36,and400C.Samples

ofculture fluidwere taken on 3, 4, and 5days

postinfection and titratedin miceorbyplaquing. As shown in Table 2, the virus from

HEp-2p.j.

replicated almost equally well atdifferent

tem-peratures. The virus from

BHKp.i.

replicated

somewhat better at 340C than it did at 400C. However, there were nosignificant differences in the numbers of antigen-positive cells when BHK cultures infected with

BHKp.i.

virus and

incubated at various temperatures were com-pared (datanotshown). The original virus

dis-played pronouncedtemperaturesensitivity (Ta-ble2).

Incontrasttothe originalvirusgrown inQE

cells, persisting viruses from

HEp-2p.i.

and

BHKp.i.

couldreadilyestablishacarrierstatein

BHK and Vero cell cultures, with a similarly fluctuating pattern of infection in BHK cells

(datanotshown).

Detection of rabies virus DIpin

persist-ently infected cultures. Several lines of evi-dence suggested the presenceof interfering ac-tivity in ourpersistently infected cultures: (i)a consistently observed autointerference

phenom-enonin initial virus dilutions when titratedby

plaquing; (ii) alonger incubation period of per-sisting viruses in mice whencompared with that

ofstandardvirus; and (iii) acyclical patternof

infection in

BHKp.i.

thatwasreportedly dueto

[image:3.496.48.241.475.579.2]

DIp (8). Totestdirectly for thepresenceof DIp,

TABLE 1. Immunogenicproperties ofrabies viruses

releasedbypersistently infectedculturesa

Passagelevel Infectiousvi-

Immuno-Source ofvirus of persist- rustiter(per genicity

entlyinfected mll) index

cellsb

HEp-2.i. 29-31 3.1x10"PFU 2.4

123-126 3.7x107PFU 0.94

BHKpi. 36-39 2.1x108PFU 2.2

163-164 2.5x107PFU 0.57

QEcellsacutely 5.5logLD5o 2.5

infected

aPersistingviruseswereconcentrated109-foldandinjected intraperitoneallyinto mice. For each dilutionof thevirus,

groupsof 14animalswereused. Theimmunogenicityindex is

expressedasthe ratio of thereciprocalof thehighestdilution

of the virusprotecting50%ofthe mice tothat of thestandard

reference vaccine supplied byTarasevichState Institute of

BiologicalStandardizationandControl,Moscow.

'Asimultaneouscomparisonbetweenviusesof different

cell passages waspossible due to the storage ofthe early

passagecarrier cellculturesinliquidnitrogen.

TABLE 2. Replicationofpersistingviruses in BHK

cells atdifferenttemperatures

Timepjt- Titer atfollowingtemp Source of virus inoculation _ _ C_ _

(h) 34 36 40

HEp-2p.L 72 3.0 2.5 3.0

96 4.25 3.5 2.75

120 3.75 3.0 4.0

BHKp.5. 72 4.5 4.0 3.0

96 NTb NT NT

120 NT NT NT

QEcells acutely 72 4.5 3.5 2.0

infected 96 6.0 3.5 2.0

120 6.0 3.75 2.0

a

All

of the titers are expressed in log

LD50

per

milliliter,

except for

BHKp.L,

wherethe titers are expressed in log PFU permilliliter.

b

NT,

Not tested.

persistingviruses were

sedimented

from 800- to

1,500-mlvolumes and inoculatedinto mice in a mixturewith 10-fold dilutions of standard virus.

Nodifferenceswereobservedbetween the titers

of the virus mixture and the standard virusonly.

However, after a

preliminary

one-step amplifi-cation on BHK cells (6), the titer ofamixture

was0.75 to 1.5log

LDio

perml lowerthan that

of similarly amplified standard virus. When BHKcellswereinfectedwithundiluted and

100-fold-diluted culture fluidfrom

HEp-2.i.,

the final virustiterwas 1

log

LD5o

perml

higher

in the

lattercase.Releasedvirusesharvested from

per-sistently

infected cellsatdifferentpassagelevels

wereconcentrated and

analyzed

insucrose

gra-dients. Viruses from

HEp-2p.i.

or

BHKp.i.

formed ODbands that couldnotbedetectedin

concen-trated

materials

frommock-infectedorstandard

virus-infected

QE

cells

(Fig. 2).

BHKp.i.

virus

usually

formed one broad

band,

whereas two

bandscould oftenbeobservedin

HEp-2p.i.

virus gradients

(Fig.

20).

The

infectivity

peak

sedi-mented ahead of the OD band

(Fig.

2B),

sug-gesting

that OD structuresweresmallerin size thanrabiesvirusvirions.The

polypeptide

com-position of

purified

ODstructureswassimilarto

that of standard virus

propagated

in

QE

cells (Fig. 3).Themolecular

weights

ofthe

polypep-tides were

78,000,

62,000,

43,000,

and

26,000,

whichare in agreementwith

reported

data on

rabies

virus proteins (14). Thus,

we concluded thatthese ODstructures weretruerabies virus

defective

particles.

Purified defective

particles

were not infectious when

injected

intracere-brally into mice and did not protect animals against

challenge

with virulent rabies virus. However, when these

particles

weremixed with theinfectiousvirus and

amplified

onBHK

cells,

a

reproducible

reductioninthevirus

titer,

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4 ANDZHAPARIDZE ET AL.

'x

IsoK

°

o . I 1/:.6

6

A

4

2

3

2

I

[image:4.496.57.458.54.570.2]

ib I5

FIG. 2. Sucrosegradient profiles ofrabiesviruses

releasedfrom acutelyinfected QEcells(A)and

HEp-2p.,.

(B). (C) ODprofilesoftwoparallel gradients (I,

HEp-2p..;II, BHKp.i)recordedonthesame sheetof

paper. ( ) OD at 260nm; (....) infectivity.

(Ab-scissa) Fraction number; (left ordinate) OD at260

nm;(right ordinate) virus titerinlogLDroper

milli-liter.Centrifugationwasfrom lefttoright.

ing from 0.5 to 1.25 log LD50 per ml, was ob-served.

Examination of cell clones isolated from

persistentlyinfectedcultures. Todetermine

whether the resistance of persistently infected

culturestohomologous viruschallenge was as-sociated with the selection of rabies virus-resist-antcells,wecloned

BHKp.i.

and

HEp-2p.i.

atthe levels of 100 and about 70% ofantigen-positive

cells, respectively. A total of 32 clones were

0.5

B

C

FIG. 3. Polypeptide composition of QE

cell-prop-agated standard rabies virus (A) and DIp from

BHKp.L.

(B) and

HEp-2p.1.

(C). (Ordinate) ODat 550 nm.

obtained from

HEp-2p.i.,

17of which were anti-gen positive and virusproducing. The

antigen-free clones were susceptible to rabies virus to

thesame extent as werecontrol uninfected

HEp-2 cells. Of26

BHKp.i. clones,

24 were antigen

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negative and rabies virus susceptible. This result is intriguing because 100% of the cells in pre-cloned

BHKp.i.

contained viral antigen. More-over, two antigen-positive clones from

BHKp.i.

were spontaneously destroyed after four pas-sages, whereasall antigen-negative cloneswere splitatleast another five times without notice-ablechanges.

Attemptstodetect interferon production in

BHKp.i.

and

HEp-2p.i.

BHKp.i.,

HEp-2p.i.,

and control uninfected cellswere infected with the Indiana serotype of vesicular stomatitis virus andaninterferon-susceptible measles virus var-iant. At 72 h postinfection, challenge viruses weretitrated by cytopathogenicityonBHK (ve-sicular stomatitis virus) or HEp-2 (measles vi-rus) cells. Challenge viruses consistently multi-plied equally well both inpersistently infected cultures and in uninfected control cultures, sug-gesting the lack of detectable interferon produc-tioninpersistently infected cultures.

Search for virus-specific sequences in

DNA of the carrier cell cultures. At a Cot ranging from 137 to 6,567 mol.s/liter and a carrier cell DNA concentration of5mg/ml, the percentageofhybridization wasintherangeof

from 5to7% with eithera[3H]cDNAora

[3H]-RNAprobe. Control calfthymusDNA did not

differsignificantly from carrier cell DNAin its annealingrate.However, the cDNA probe

pres-entinthe reaction mixture wasstillcapable of specific annealing to excessvirion RNA atthe end of thehybridization period (59to62%ata

Cot

of3,283 mol.s/liter). Repeatedtransfection

attemptswith carriercellDNA toinduce

infec-tious virus or viral antigen in BHK cells were also unsuccessful.

DISCUSSION

Persistent infectionsby rabies virus have been established in rabbit (5) and reptilian (16) cell cultures, but have been studied extensively in

BHK-21 cells (6, 8, 16). However, an apparent hostcelleffect has been observedonrhabdovirus DIp generation in human cells (7) and on the virulence of rabies virus in neuroblastoma cells (3). We have studied thehost cell effecton (i) the course ofpersistent rabies virus infection, (ii) properties ofpersisting virus, and (iii) the

contribution of several factors currently re-garded as the mechanisms of viralpersistence into the establishment and maintenance of a carrierstate.To thisend, wehaveuseda well-characterized

BHKp.i.

system and

HEp-2p.i.

of

humanorigin.Incontrast to

BHKp.i.,

inwhicha cyclicalpatternof infection has beenobserved, the course of infection in

HEp-2p.i.

was more

constant. The persisting virus from

BHKp.i.

at

the level of50cellpassageslost itsvirulence for

mice; this is in accord with the finding of

Fer-nandes et al. (5). In contrast, the virus from

HEp-2p.;.

retained itsvirulence formiceformore than 3 years despite the production of DIp. Recently, Wunner and Clark have presented

evidence that the virulenceof rabies virus is not

mediatedbyDIp (18).

An examination of persistently infected cul-tureswith respect to a mechanismofvirus

per-sistencerevealedthatneither interferonnor

in-tegration ofvirus and cell genomes seemed to

playarole.Temperature sensitivity ofpersisting viruses was poor and never reached the level of that of the original virus. A selection of

virus-resistant cells is alsounlikelydue to the observed

susceptibility

of virus-negative clonesto

reinfec-tion. Both carrier cell cultures were found to

produce DIp, which is apparently inherent to

rabies virus persistence (6, 8, 17). The viruses

readily establishedpersistent infections in Vero

and BHK

cells,

incontrast totheQE cell-prop-agated non-DIp-containing virus, whichcaused

regular cytopathic effectinthesamecells.This

finding

suggeststhat in

BHKp.i.

the generation

andprotective effectof DIp are prerequisites for

the initiationandmaintenanceof persistent

in-fection.In

HEp-2p.i.,

however, the role ofDIp in

viruspersistence is questionable.In contrast to

BHKp.i.,

persistent rabies virus infection could bereadily andconsistently institutedinHEp-2

cells,

without anysigns of initial cytopathic ef-fect. Wealso did notfind anyevidence for the

rapid generation of DIp within the firstpassage

of rabies virus-infected HEp-2 cells (data not

shown). These observations leadus to suggest

that, in

HEp-2p.i.,

DIp are

produced

as a

by-product of rabies virus

replication

which is not necessaryforHEp-2cellsparing.

Thus, host cellsmay exhibit pronounced

ef-fects on the course of persistent rabies virus

infectionand thevirulence ofthevirus,whereas

inherent viralproperties, e.g.,thegenerationof

DIp, could neverthelessbepreserved in various cellsystems.

ACKNOWLEDGMENTS

WeareindebtedtoJ. J.Holland,B.Janis, and T. J. Wiktor foranintroduction into rabies virusplaqueassaysandtoA. V.Shishkov for in vitrolabelingof RNA. The expert technical assistanceofI.Snyatkova,T.Luckjanova,and I.Kasatochkina isgratefullyacknowledged.

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