document

C U L T I V A T I O N OF R A B I E S VIRUS IN H U M A N D I P L O I D CELL S T R A I N WI-381

T. J. WIKTOR, M. V. FERNANDES AND H. KOPROWSKI

From The Wistar Instilule of Anatomy and Biology, Philadelphia, Pennsylvania

Received for publication February 12, 1964

Although growth of rabies virus in tissue cul- ture has been reported by several investigators in the past (1-3), none of the systems described could be utilized for a detailed study of the kinetics of rabies virus infection, primarily be- cause of the absence of reproducible cytopathic effects. The adaptation of rabies virus to human diploid cell strain WI-38 in tissue culture re- ported in this presentation makes available a tool for the quantitative study of rabies infection. In addition, because of the apathogenicity and high antigenicity of these tissue culture adapted strains, it is now possible to undertake a study of their use as a safe and effective vaccine for man.

MATERIALS AND METHODS

Virus strains. Three rabies strains were used in this study: a) standard challenge virus (CVS), a fixed rabies virus propagated in mouse brain2; b) Pitman-Moore virus (PM), a fixed rabies virus propagated in rabbit brain2; and c) Flury high egg passage (HEP) (4), a chicken embryo adapted rabies virus at the 225th egg passage.

In addition, an attenuated poliovirus type 1 strain (CHAT) grown in a human diploid cell strain (HDCS) and Eastern equine encephalo- myelitis (EEE) virus grown in primary chicken fibroblasts (PCF) were used in the interference experiments.

Rabies virus pools, consisting of a 20% suspen- sion of the infected tissue in 50% normal calf serum in distilled H~O, were kept at -70°C.

Tissue culture. The six tissue culture systems used were: a) human diploid cell strain WI-38, derived from embryonic lung (HDCS) (5), b) pri- mary chicken fibroblasts (PCF), c) primary green monkey kidney cells (PGMK), d) embryonic mouse fibroblasts (EMF) at the fourth subcuI-

This work was supported, in part, by United States Public Health Service Grant AI-02954 from the National Institute of Allergy and Infectious Diseases and by the World Health Organization.

Supplied by the National Institutes of Health, Bethesda, Maryland.

tare, e) a dog kidney epithelial cell line (DK) ~ and f) C-13 line of hamster kidney fibroblasts (C-13) (6).

The standard methods for propagation in vitro of the six tissue culture systems have been de- scribed elsewhere (5-7). Eagle's basal medium (8) in Earle's balanced salt solution supplemented with 10% inactivated calf serum and containing 25 ml of 5.6% sodium bicarbonate, 105 units/L of penicillin and 105 /~g/L of streptomycin or 5 × 104 ~g/L of Aureomycin were used through- out. Cultures of WI-38, DK, C-13 and EMF were transferred twice weekly, using a 0.25 solution of trypsin (Difco 1/250) in phosphate buffered saline (PBS) supplemented with the antibiotics described above.

Test for the presence of yirus

I. Intracerebral inoculation of animals. The presence of HEP Flury virus was determined by intracerebral inoculation (0.03 ml) of 4-day-old Swiss stock mice and the presence of CVS and PM strains by intracerebral inoculation of 4- to 5-week-old mice of the same stock. For titration purposes, 10-fold dilutions of the virus were inoculated into 5 to 10 animals/dilution. The mice were observed for 21 days and the endpoint was calculated according to the method of Reed and Muench (9). Young adult Syrian hamsters and guinea pigs weighing approximately 350 gm were also inoculated with 0.1 ml of virus at differ- ent dilutions and observed for 21 days. Rhesus monkeys weighing 6 to 8 lb were injected intra- thalamically with 0.25 ml of the virus and ob- served for 60 days.

2. Immunofluorescent antibody staining (FA) of tissue culture preparations. Tissue culture prepa- rations were stained directly by the technique of Coons and Kaplan (10) as modified by Gold- wasser et al. (ll). The antirabies-globulin con- jugate was supplied by the National Rabies Laboratory, Atlanta, Georgia.

Supplied by Norden Laboratories Inc., Lin- coln, Nebraska.

353

354 T . J . WIKT()I~. M. V. FEIINANI)ES AND H. KOPR()WSKI [VOL. 93

Tenfold dilutions of the virus to be t i t rated were prepared in tissue culture medium, and 1 ml of each dilution was mixed with 5 ml of the cell suspension to be tested (about 5 × 10 ~) cells). The cells were plated on Petri dishes containing eoverstips, and the cultures were incubated at 37°C. After 4, 7 and 10 days of incubation the eoverslips were removed, fixed and stained with the globulin conjugate. The highest dilution of virus causing fluorescence of cells was considered as the end point of the titration.

Cytopathic effect. Virus-infected tissue culture preparations were examined daily for the presence of eytopathie effects. Coverslip preparations were also stained with May-Greenwald Giemsa, after fixation for 20 rain in Bouin's fixative, in order to determine the presence of inclusion bodies and confirm the destructive effect of the virus on the cell.

Serum neutralization test. The test was per- formed in young adult mice injected intraeere- brally according to the standard method de- scribed elsewhere (12). Anti-rabies sera were obtained from three sources: a) dried anti-rabies 3'-globulin prepared in horses at the Metchnikoff Research Institute, Moscow; b) anti-rabies serum prepared in horses at the Pasteur Institute, Paris; and e) human anti-rabies serum fi'om a man who had been immunized by the classical Pasteur treatment and had, in addition, received three booster inoculations of Flury HEP virus.

Inactivation of virus for vaccine production. In- fected tissue culture preparations were inacti- vated either by the addition of phenol at a final concentration of 0.5% and incubation at 37°C for 48 hr (12) or by treatment with t3-propiolactone (BPL) at a final concentration of 1/6000 and incubation at 4°C for 24 hr (13, 14). The inacti- vated vaccine was stored at -20°C until used.

Twenty 5-week-old mice were inoculated intra- cerebrally with undiluted vaccine t~ test for the absence of infective virus.

Immunization of animals. The potency of in- activated virus vaccine was determined by the standard Habel test (12). In order to check the potency of live virus vaccine (HEP strain), young adult mice were inoculated twice intraperitone- ally with 0.5 ml of virus at 7-day intervals and challenged intraeerebrally with CVS virus 3 weeks after the first inoculation.

Rhesus monkeys were injected intramuscularly twice at 4-day intervals with 2 ml of undiluted

live virus vaccine and serum collected for the neutralization test before inoculation and on the I4th day after the first iniection.

RESULTS

Propagation of virus in IVI-38. As shown in Tables I and II, rabies virus could infect WI-38 either as monolayers ol' dispersed cell cultures, but serial transfer of either the medium or the cell extracts in a homologous tissue culture system resulted in a gradual decrease of infectivity which was finally lost after 4 to 7 passages, de- pending on the virus strain used (15).

The results obtained through FA staining of the infected tissue culture system indicated that very few cells produced rabies virus antigen at an)" given time after infection with medium from the preceding culture. Since, in addition, the small amounts of infective virus (Table I) ap- parently made it impossible to propagate the virus either by transfen'ing the medium or the cell-free extract from infected cultures, a different method of cultivation was used in an a t tempt to adapt rabies virus to growth in WI-38.

l)ispersed WI-38 cells in suspension were ex- posed to rabies virus and plated into culture bottles or Petri dishes. When the cell sheet be- came confluent after 3 to 4 days of incubation, the cells were transferred, either trypsin or Ver- sene being used to detach the cells from the glass. After six consecutive passages, the number of fluorescing cells in the culture series treated with trypsin had greatly increased, but in the series treated with Versene had remained stationary or even decreased. Virus in the latter series was finally completely lost, whereas infectivity of the trypsin series could be maintained through about 20 subcultures before there was total cell lysis.

This stage was preceded by a gradual increase in the number of cells showing the presence of large eosinophitic and 1)olymorphic intracyto- plasmic inclusions (Fig. 1, B and C) (15). By the 19th subculture passage virtually all cells showed the presence of these large inclusion bodies and apparently were unable to divide.

Staining of the cultures of this series with con- jugated antiserum initially revealed the presence of small fluorescing gramfles dispersed through- out the cytoplasm of most of the cells. In the course of subsequent p~sages large cytoplasmic masses gradually began to accumulate and finally, at the level of the 18th to 19th passage,

1964] CULTIVATION OF RABIES VIRUS IN HDCS WI-38

TABLE I Ada 9tation of Flury high egg passage rabies virus to growth in human diploid cell strain WI-$8

Procedures f o r V i r u s Transfers a

Passage No. Subculture Cell-mixing Cell-free medium

11 13 15

17 19 21 23 25

45 47

49

59 61

FA CPE BMI b

+ -- 1.5 +

+ + - - 3.6 + + + +

+ + + + + 4.5 } + + + + + + + + + + + + + + + + + + 4.6 I + + + + + + + +

completelysis

+ + + + + + + + completelysis

+ + + + + + + + comple t e l~ i s

FA CPE

+ + + + + + + + + e ~ +

+ + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + c + + + +

f + + + + + + + +

t + + + + + + + +

BMI FA

+ + +

4.5

% + +

4.0

5.5

+ + + +

+ + + + current passage

CPE

a

noninfectious

noninfectious

+ + + + $

+ + + +

355

BMI

1.5

a Key: Percentage of cells destroyed (CPE) or fluorescing (FA): + + + + , 90 to 100; + T + , 50 to 90; + + , 10 to 50; + , 1 to t0; - , 0.

b BMI = Baby mouse LDa0/0.03 ml of medium (log,0). c = used as living vaccine.

most of the cells of the culture contained these masses, which corresponded in shape, size and location to the intracytoplasmic inclusion bodies described above (Fig. 1D).

Following lysis of the infected cultures at the 20th passage level, at tempts were again made to propagate the virus by transfer of cell-free media. As shown in Tables 1 and II, these attempts were unsuccessful, and portions of the infected culture had to be mixed with noninfected homologous cells at each transfer passage in order to main- tain the vitals in the tissue culture system. With this technique, fluorescence of all cells was ob- served on tile 3rd day after cell transfer, and HEP, CVS and PM strains of rabies virus could be maintained for an unlimited number of pas- sages (Tables I and II).

The virus could be preserved for a long time in tissue culture cells frozen at -70°C (5). Further- more, there was no loss of infectivity when these cells were recovered from the frozen state.

After maintaining the CVS and HEP strains for 47 tissue culture passages with the cell-mixing transfer technique, at tempts were made to propa- gate the virus by transfer of tissue culture medium. This time it was possible to maintain virus in serial passages, and this is the method now employed for propagating rabies virus in the HDCS WI-38 (Tables I and II).

Figure 2 graphically illustrates events following exposure of a culture of WI-38 to infected culture medium representing the 49th passage of HEP in WL38. At a multiplicity of two, as used in this experiment, there seems to be a lag period of 24

356 T. J. WIKT()ll. M. V. FEI(NANDES AND H. KOPIIOWSKI [VOL. 93

TABLE II Adaptation of standard challenge rabies virus to growth in human diploid cell strain WI-38

Procedures for Virus Transfers a

Passage No. Subculture Cell mixing Cell-free medium

7 9

11 13 15

17 19 21 23 25

45 47

49

59 61

FA CPE AMI b

+ - 3.3 4-

+ + - 3.0

+ + + - 3.4 4-4-4-+ + 4.0 4-4-+4- +

+ + + + a ~ + 4.1 + + + + a ~ + + 4.0

4 - + + + 4 - + + 4-4-4-+ + + + 4 -

completelysis

¢ + + + + + +

+ + + + + + +

FA CPE AMI

+ + + + + + + + + b ~ + 4.5

+ 4 - + + + + + + + +

4 - + + + + + + + + +

+ + + + 4-

+ + + 4 - + + + + + + b + +

I + + + + + +

+ + + + + + current p a s s a g e s

2,

3.5 4.4

4.3

FA CPE AMI

+ -- 3.7 + -- 1.4

noninfectious

+ + + + + + + +

n o n i n f e c t i o u s

+ + 4 - + + +

+4-4 -+ ÷ +

Key: Percentage of cells destroyed (CPE) or fluorescing (FA): + + + + , 90 to 100; ---~-+, 50 to 90; + + , 10 to 50; + , 1 to 10; - , 0 .

b AMI = Adult mice LD~0/0.03 ml of medium (log,0). Used for preparation: (a) Phenol" (b) BPL inactivated vaccine.

hr before new virus is either produced inside the cells or released into the culture medium. Once this occurs there is a rapid increase in the produc- tion of infectious virus which reaches its peak between the 2nd and 3rd day after exposure, when 80 to 100% of the cells show innnunofluo- rcscence. All cells eontimm to 1)e fluorescent during the culture's remaining lifetime. On the 4th and 5th days after infection there was a de- crease in concentrations of intracellular and free virus, and loci of degenerated cells were noted for the first time. The cytopathic effect became more pronounced, leading to lysis of the culture 3 to 4 days later.

Interference and interferon. The difficulties en- countered in propagating rabies virus during its initial stage of adaptation to WI-38 could have been explained by the presence of an inhibitor sub-

stance produced by WI-38 as a result of exposure to the virus. The interferon-like nature of this substance is suggested by the fact that it was easier to maintain the virus in cultures treated with tl\vpsin, which inactivates interferon (16- 18), than with Versene, which apparently has no effect.

Before attempting to demonstrate the presence of interferon in rabies-infected WI-38, resistance of such cultures to reinfection with homologous and heterologous viruses was investigated. Be- cause HEP virus is not pathogenic for adult mice injected intracerebratty, it was possible to study interference in WL38 infected with this strain of rabies virus and re-exposed to another strain, such as PM, which retained its pathogenicity for adult mice.

Cultures of WL38 infected with the HEP

t964] CULTIVATION OF RABIES VIRUS IN HDCS WI-38 357

© Figure 1. HDCS WI-38. A, control culture incubated for 7 days. Staini~lg May-Greenwald Giemsa

(X 500); B, WI-38 infected with HEP virus at the 47th passage after incubation for 7 days. Note the presence of CPE and intracytoplasmic inclusions. Staining May-Greenwald Giemsa (X 500); C, WI-aS infected with HEP virus at the 47th passage after 3 days of incubation. Inclusions are present. Staining May-Greenwald Giemsa (X 1500) ; D, WI-38 infected with CVS strain of virus at the 10th passage level after 5 days of incubation. Note the presence of large fluorescing inclusions. FA staining (X 1500).

358 T. J, WIKT()It, M. V. FEIINANDt'S AND tt. KOI'II()WSKI [VOL, 93

g >

'zo

~o

~o

,?, 3.0 z 2D

2 .,J

DAYS AFT~'R , EXPOSURE

FA GPE

i - io

o

= , .

1 t f I 2 3 4 5 6 7 8

+ +++ ÷+÷+ "~"*'*+ -+++ **~t ,+,+ **+* 4 .+ +++ .+¢ ++.~

Figure 2. Growth curve of HEP virus in HDCS at 49th passage level as determined by FA in homologous tissue culture systein.

strain of virus at the 14th t)assage level and show- ing 100% fluorescing cells were trypsinized, counted and exposed, in suspension, to the PM strain of virus. After adsorption for 2 hr at 37°C with constant agitation, the cells were centri- fuged, washed three times with new medium and planted in Petri dishes containing coverslips. After 5 and 10 days of incubation, respectively, the eoverslips were stained by FA and the super- natant medium was titrated in adult mice by intraeerebral inoculation.

The results of this exl)eriment, as shown in Table III, indicate that the PM challenge virus apparently could not multiply in the HEP- infected cells, although it did infe('t and multil)ly in WI-38 controls.

Since, ms shown by Walker (19), cultures chronically infected with mmnl)S virus were found to be resistant to challenge with homol- ogous but not with heterologous virus, it be- came of interest to check the interfering capacity of rabies virus strains grown in w i - 3 8 , not only against another strain of rabies but also against re-exposure to heterologous virus.

One day after monolayers had formed, rabies- infected cultures containing I00Q fluorescing cells and control WI-38 cultures were exposed to challenge with serial I0-fold dilutions of either polio or EEE virus. After 1 hr of adsorption the inoeulum was removed and, when the cultures had been washed three times with PBS, agar overlay was added. The cultures were incubated for 3 days at 37°C and then stained with neutral red solution at a 1 : 10,000 concentration in order to determine the number of plaques of either

polio or t';EE. Three Petri dishes were used for each dilution of challenge virus.

The results of challenge, summarized in Table IV, show eoml)lete resistance of rabies-infected WI-38 cultures to infection with at least 5 X 104 1)laque forming units (PFU) of poliovirus and at least 5 × 103 PFU of EEE virus.

In order to analyze the interference phe- nomenon in greater detail, WI-38 cultures in- fected with rabies virus were trypsinized and nfixed in different proportions with fresh cells. One day later, when only a part of the culture showed fluorescing (!ells, the cultures were ex-

TABLE III

Resistance of Flury high egg passage- (HEP) in- fected human diploid cell strain W1-38 to rein- fection with Pitman-Moore riras .~train of rabies

(2ells Showing Immunof luorescent LD~0 T i t e r of

WI-38 Cul tures An t ibody S ta in ing Medium in Adu l t Infec ted wi th M i c e

5" 10 ('

i % HEP b . . . . . . . . . . 100 100 0 Nothing . . . . . . 1 20 103.6

" Days after exposure to Pitman-Moore virus. At 14th passage level in WI-38.

TABLE IV

Resistance of rabies-infected human diploid cell strain WI-38 to polioviras challenge

Infec t ion of WI-38 wi th Rabies

Virus s t ra in

Standard challenge virus

Flury high egg pas- sage

Average No. of P laques

Observed a f t e r Chal lenge wi th

Po l iov i rus

Cells , showing immuno- pRabies- [ Con-

f luoresc ingl infec ted I t rois an t ibody

_ _ _ _ s t a l l i n g

30 ~ 11.3 22.0 60 ~ 9.0 20.0 80 b 6.3 55.0

100 b 0 57.0 100 b 0 55.0

Reduc- t ion In P laque

No.

%

49 55 82

100 100

(' Experiment 1. b Experiment 2.

1964] CULTIVATION OF RABIES VIRUS IN HDCS Wt-38 359

posed to challenge with a dilution of poliovirus, as described above.

The results of these experiments, shown in Table V, indicate that the degree of resistance to poliovirus depended largely on the apparent number of cells containing rabies viral antigen, since the reduction in plaque number in a culture of WI-38 showing only 30 to 60% fluorescing cells was much less than that observed in cultures showing either 80 or 100% fluorescing cells.

Repeated attempts to isolate an interferon- like substance from WI-38 infected with rabies virus at different stages of infection, using methods of interferon-isolation found successful for other virus-host systems (20), were unsuccess- ful.

Identity of the WI-38-adapted virus. Although repeated tests have shown that HDCS cultures were free of contaminating viruses, it seemed

prudent to check the identity of the virus after serial passages in HDCS. Therefore, serial 10- fold dilutions of HEP before adaptation to HDCS (Table VI) were mixed with normal and anti-rabies sera, respectively, incubated for 1 hr a t 37°C and then injected intracerebrally into baby mice. The results, presented in Table VI, indicate clearly that the HDCS-adapted virus was neutralized by anti-rabies serum. Similar results were obtained when CVS and PM virus strains were tested.

Modification of properties of HEP strain of rabies virus after continuous passage in a human diploid cell strain. Although no cytopathic effect was observed in HDCS infected with HEP at early passage levels, degeneration and cell lysis were noticed at the 13th passage (Table VII), and the cytopathic effect became more pro- nounced during subsequent tissue culture pas-

TABLE V Resistance of rabies-infected human diploid cell strain (HDCS) WI-38 to challenge

with poliovirus and eastern equine encephalomyelitis (EEE)

Challenge Virus

Polio

EEE

HDCS Infected with Rabies Virus Strains a

Standard challenge virus Pitman-Moore Flury high egg passage Standard challenge virus Flury high egg passage

Average of Plaques Observed af ter Infection with Challenge Virus Dilut ions

10 -3 10 5 10--6 I0-7

R b R N e

m

C ~

C

10--4

R N K

- - i f - 0 0 0 0 0 0 0 }C 0 0 0

N • ___N

0 153 0 0 0 ~4 0

R N 0} 0 3 0

Passage level in WI-38: CVS at 49th, PM at 10th and HEP at 14th. b R = Rabies-infected. c N = Noninfected controls. d C = Complete cell lysis.

TABLE VI Se~oneutralization of Flury high egg passage strain of virus before and after adaptation to

human diploid cell strain WI-38 in baby mice

WI-38 Pass- age Level

49

Serum Used for Neutra l iza t ion

Normal Anti-rabies Normal Anti-rabies

Mortal i ty Rat io of Mice Inoculated with Mixtures of Serum and Dilut ions of Virus a

Undiluted 10 lb 10 2 l0 s 10 4 I0 s 10 6

7/7 I 9/9 9 / 9 1 7 / 7 1 3 / 7 / 0 / 6 / ° / 7 1 0 / 7 / ° / 8 1 ° / 6 1 /

8/8 8/8 8/8 8/8 8/8 4/8 0/8 i io /8 t l 1o/8 I

a Nominator = no. of mice who died after inoculation; denominator = no. of mice inoculated. b 10' to 106 represent serial dilutions.

360 T . J . WIKT()H, M. V. Ft.;I~NANI)t-:S AND H. K()PI~()WSK[ [VOL. 93

sages (see above). At the 47th passage hwel the virus at, a 10 -6 dilution caused lysis of a portion of the eukure and the endpoint coincided with that obtained by staining the tissue culture preparations with fluorescent antiserum. Results of titration of virus preparations in baby mice, summarized in the last. eotumn of Table VII, indicate that there was no direct correlation between infectivity of the virus for baby mice and the development of eytopathic effect.

Since these data seem to suggest that in the course of continuous propagation in the hmnan

diploid cell strain, WI-38, viru~ particles were seh'cted which had gl'e'~ter affinity for the tissue culture system in which the5: were propagated, it t)ecame of interest to study the infectivity of HEP virus at different passage levels in WI-38 for tissue culture cells derived from various animal species.

Figure 3 shows the results of titrations based on imlnunofluorcscenee (see '%Iaterials and Methods") of HEI ' virus in six different tissue culture systems: WI-38, in which it was propa- gated; PGMK, EMF and DK cultures; the C-13

TABLE VII Cytopathic effect and immunofl,orescence ~4f Flur# high egg passage strain of rabies virus

at different passa(te terels in human diploid cell strain WI-38

Passage L e v e l in T e s t

WI-38

0 CPE FA

13 CPE FA

47 CPE FA

D i l u t i o n of Virus ~ LD~0/ml in B a b y

Mice (log,0) 10 -1 io '-' 1(}-~ I0 -~ Iff ~ I0-~

+ + - _ + + + + + + + + -- + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + +

6.00

6.00

7.00

Key: Percentage of cells destroyed (CPE) or fluorescing (FA) as recorded on the 12th day after infection: + + + - t - , 90 to 100; + + ÷ , 50 to 90; + + , l0 to ,50; ÷ , 1 to 10; - , 0.

HEP STRAIN FOR BABY MICE & MONKEY INOCULATION, 8~ FOR TISSUE CULTURE SYSTEMS

BEFORE 8, AFTER ADOPTATION TO GROWTH IN HDCS

Human Diploid CelIs S~roin

INFECTIVITY OF BY INTRACEREBRAL

0 Passage Level 13 Passage Level 47 F~ssage Level

Primary Green Monkey Kidney Ce~ls

Embryonic Moose Fibroblasts

Dog Kidney Epifheliel Cell Line

Neonatal ltomster Kidney Fibroblosts Line BHK- 21 CLone 15

Primary Chicken Fibroblasts

Baby Mice Inoculalion

i + 0 ' NT Monkey , . . . . ~ Inoculohon

Figure 3. Infectivity of HEP strain for tmby mice and monkeys by intracerebr~d inoculation, and for tissue eulture system, before, and after "~daptati(m to growth in HDCS.

1964] CULTIVATION OF RABIES VIRUS IN HDCS WI-38 361

line of neonatal hamster kidney fibroblast; and cultures of PCF.

The results show that, whereas the nonadapted HEP virus was equally infectious for PCF and baby mice, it was of very low infectivity for WI- 38, EMF and DK and was noninfectious for Passage

PGMK tissue. The susceptibility of the C-13 line Level in to infection seemed to lie somewhere between wi-38 that of WI-38 and PCF. At the 13th passage level in WI-38, the infectivity of the virus for C-13 0 and chicken fibroblast remained the same, but 49 the virus became much more infectious for WI- 38, PGMK, EMF and DK.

Finally, at the 47th passage level, the infectiv- ity of the virus for the cell strain in which it was propagated increased greatly, almost reaching the level of infectivity for chicken fibroblast. There was a simultaneous increase in infectivity for the four other tissue culture systems.

Since it was known that monkeys become sick and die when inoculated intracerebrally with HEP virus of chick embryo origin, the mortality endpoint being only slightly lower than that obtained in baby mice (4), it was of interest to virus determine the pathogenicity of the tissue culture- Dilutions adapted virus for the same species.

Two monkeys injected with two dilutions of egg-adapted HEP virus before its adaptation to WI-38 became sick (on the 12th day after 10-1 inoculation) and were sacrificed when moribund 2 days later (Table VIII). At the same time two 10- 2 other monkeys injected intracerebrally with higher concentrations of HEP at the 47th passage level in WI-38 showed no signs of illness during a 10-8

TABLE VIII Results of intraeerebral inoculation of monkeys 10-4

with Flury high egg passage virus before and after adaptation to human diploid cell strain WI-S8 10-5

Passage Level in WI-38

0

47

Experi- ment No.

Infectivity Ratio of Monkeys Injected Intracerebrally with Baby Mouse

LDso a (logto)

0 . _ _ _ A _ _ _

1 1//1

1 0/1 0/1 2 [ o/8 i o/2 I

3.5

1/1

" Nominator = no. of monkeys who died after inoculation showing symptoms of rabies; denom- inator = no. of inoculated monkeys.

TABLE IX Influence of age on susceptibility of baby mice to

infection with Flury high egg passage before and after adaptation to human diploid cell strain WI-38

Mortality Ratio of Mice Injected Intracerebrally a

3 days 7 days 8 days 9 days 14 days

7 / 7 b 81/8 8//8 0 / / I0

8/8 8/8 8/8 o/8 0/10

- Inoculated with 104 baby mouse LD60 of virus. b Nominator = no. of mice who died after

infection with HEP virus; denominator -- no. of mice inoculated.

TABLE X Results of inoculation of adult mice with brain

tissue obtained from baby mice injected with egg- adapted and human diploid cell strain WI-~8- adapted Flury high egg passage (HEP) virus, respectively

Dilutions of Baby Mouse Brain

Inocula

10-1 10-2 10-3 10- t 10- 2 10-a 10-1 10- 2 10- 8 10- t 10- 2 10-s 10- ~ 10-~ 10- 3

Mortality Ratio of Adult Mice Inoculated with Baby Mouse Brain

Infected with HEP a

Egg-adapted

Nsb RS b

0/6 0/6 0/6 2/6 3/6 1/6 6/6 6/6 5/6 1/6 3/5 1/6

WI-38-adapted

S RS L

4 / 5 1 0 / 5 0/51

0/6 0/5 ! 0/6 0/5~ 0/6 0/5 1/6 0/5 o/6 0/5 o/6 o/5 o/6 o/5 o/6 o/5 0/6 0/5

0/5 0/5 O/4

-Nominator = no. of mice who died after inoculation of baby mice brain tissue infected with HEP; denominator = no. of adult mice inoculated.

b NS = normal serum; RS = serum with rabies antibodies.

6-month observation period. In the second experi- ment (Table VIII), groups of eight and two monkeys were injected intracerebrally with 106.5 and 10 ".5 baby mouse LDs0 (0.25 ml) of the

362

HAMSTERS

I00 ~_ _ n . . . . . . . . , Iii+:/+ +:?],~ , I I , + l~ I I I, i~+ ~ 0 PASSAGE

I~ ; t'i'l ' I " II I++ +I'++\ I t i l t I,I I I t ]i+]r'~ "

I :+ + t~ +;I;+;i+~), II+;, )i!+ =i :hhhlx" ~"

:+ +oi++, ++++++ , ' ,

m,1,+++, ' ' I ' ]++ i [ ; [ l~ t+ ;~ \ if+ It,,, I , , I) I', ~,~+r+~I~/l\ ,, I ' ''I I i) , \

0.7 1.7 27 3.7 4:7 5.7 67

LOGIo DILUTION OF VIRUS

T. J. WlKTOR, M. V. FERNANDES AND H. KOPROWSKI

GUINEA-PIGS

I00 . . . . . . . . . . ,

+'~ 0 PASSAGE

s o

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[VOL. 93

. . . . . . I; mortality of baby mice

~ % mortality of test animo!s

~ immune challenge

F - ] ~ act immune challenge

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Figure 4. Pathogenicity of HEP strain for hamsters and guinea pigs before and after adaptation to HDCS and resistance of surviving animals to challenge with CVS virus.

HEP virus at the 47th passage in WI-38. None of the animals showed signs of sickness during a 60-day observation period.

Thus, adaptation to WI-38 not only did not increase pathogenicity of the virus for primates, but the adapted virus apparently lost its lethal properties for monkeys when injected directly into the central nervous system.

Since the egg-adapted HEP virus is only pathogenic for baby mice less than 10 days old (21), the susceptibility of baby mice of different ages to the HEP virus after adaptation to WI-38 was investigated. The results of the experiment in which concentrations of 1(0 baby mouse LD+o of egg-adapted and WL38-adapted HEP virus were injected intracerebrally into baby mice (Table IX) show that mice nine days old or younger are equally susceptible to the two virus strains.

In order to investigate the behavior of the virus after passage through baby mouse brain, baby mice were injected intracerebratly with

serial 10-fold dilutions of the HEP virus before and after adaptation to WI-38 and sacrificed as soon as they showed signs of illness. Serial 10- fold dilutions of a suspension made from the brain tissue of these animals were then injected intracerebrally into young adult mice. As can be seen in Table X, only in one instance did passage of WI-38-adapted HEP produce a rabies virus population which seemed to become patho- genic for adult mice, whereas rabies virus patho- genic for adult imce has been recovered from the brain tissue of baby mice injected with all dilu- tions (except 10 -z) of egg-adapted HEP, con- firming previous observations (4, 21). However, when the remaining adult mice were challenged with a lethal dose of CVS injected intracerebrally, there was solid resistance to reinfection through- out the infectivity endpoint of the preparations (as measured either in baby mice or in tissue culture).

Pathogenicity of WI-38-adapted HEP for hamsters and guinea pigs inoculated intracere-

1964] CULTIVATION OF RABIES VIRUS IN HDCS WI-38 363

brally was investigated in experiments in which they were inoculated with serial 10-fold dilutions of egg-adapted and WL38-adapted HEP. The results, shown in Figure 1D confirm the fact (4, 21) that a fraction of the virus population of the egg-adapted HEP is pathogenic for ham- sters and guinea pigs. In contrast, not a single animal injected with WI-38-adapted HEP showed signs of sickness; most of the animals, however, became resistant, as did adult mice, to challenge with a lethal dose of CVS (Fig. 4).

Immunizing properties of HE P virus after adaptation to the human diploid cell strain WI-38. Adult mice were injected intraperitoneally twice at 7-day intervals with tissue culture fluids, representing the 13th and 47th passages of HEP in WI-38, respectively. Twenty-one days after the first injection, the CVS mouse-adapted strain was tit, rated intracerebrally in the two groups of immunized animals and in nonvaccinated con- trols. The results of the challenge inoculation (Table XI) indicate that both preparations of the virus immunized mice against challenge. The greater degree of resistance shown by mice in- jected with the virus at the 47th passage level may reflect either a greater dose of virus used for immunization or greater antigenicity of the virus particles.

Although HEP virus grown in chick embryo has been used for immunization of human sub- jects, poor antibody response following primary vaccination suggested that it was incapable of multiplying in tissue of primates and man after parental inoculation (21). It was thus of interest to investigate whether the virus, after adaptation to WI-38, would show increased immunizing capacity for primates injected parentally. Three rhesus monkeys were injected intramuscularly twice at 4-day intervals with undiluted tissue culture fluids, representing the 47th passage of the virus in WI-38 and containing 4 X 10 ~ baby mouse LDs0 (Table XII). For comparison, another group of three rhesus monkeys was injected similarly with a chick embryo prepara- tion of HEP virus prior to its adaptation to WI- 38. Sera obtained from these animals 14 days after the first vires inoculation were submitted to a neutralization test. Results of the test indicate the presence of a large concentration of neutraliz- ing antibodies in sera obtained from the three monkeys vaccinated with the WI-38-adapted virus and the apparent absence of detectable

antibodies in sera of animals injected with the egg-adapted virus.

Immunizing properties of inactivated rabies vaccines prepared with strains of rabies virus adapted to grow in the human diploid cell strain WI-38. Four preparations of tile CVS strain at different passage levels in WI-38 and one prepara- tion of the PM strain at the 12th passage in WI-38 were inactivated, either with phenol or with BPL (see "Materials and Methods") and tested for the absence of live virus.

Since intracerebral inoculation of adult mice with undiluted inactivated vaccines failed to reveal the presence of infectious virus, these five lots of vaccine were submitted to a standard

TABLE XI Results of protection test s in adult mice with live rabies vaccine prepared from human diploid cell

strain WI-38 adapted Flury high egg passage

Passage LeveI m

WI-38 Vaccine

Baby Mouse Vaccine LD6Q

Inoculated (log10)

Titratlon of Challenge Virus (CVS)

Protection Index

LDso in LDso in vaccinated control mice (loglo) mice (log10)

4.15 6.50 1.63 6.50

13 6.00 224 47 7.00 74,000

a Two iutraperitoneal injections of vaccine given at 7-day intervals. Intracerebral challenge 21 days after the first injection.

TABLE XII Development of neutralizing antibodies in monkeys

immunized with live Flury high egg passage strain of rabies at different passage levels in hu- man di ~loid cell strain WI-S8

No. of Animals Tested

Virus Passage Level m WLSS

Vaccine

47

Baby Mouse Vaccine

LD6o Used for Immuni-

zation b

4 X 108 < 1 : 2 <1:2 <1:2

4 )< l0 T <1:2 <1:2 <1:2

Antibody Titer in Monkey Sera Obtained a

14 days Before ira- after first munization inoculation

of vaccine

<1:2 <1:2 <1:2 >1:250 >1:250 >1:250

Standard Challenge Virus =f= 1000 LDso for mouse inoculum.

b Vaccine inoculated on 1st and 5th day.

364 T . J . WIKTOR, M. V. FERNANDES AND H. KOPROWSKI [vor.. 93

TABLE XIII Effect of two inactivating agents on potency of rabies virus prepared from standard challenge

virus and Pitman-Moore strains grown in human dioloid cell strain WI-38 (Habel test)

Titration of Challenge Vaccine Virus (CVS)

LD~o titer in Protection Index Virus strain Passage level mice before in-

in WI-38 activation Inactivating agent (log 1~)

CVS

PM

13 15

9 47 12

4.12 4.00 4.50 4.30 3.80

Phenol Phenol BPL~ BPL BPL

LDso in vac- LD~o in con- cinated mice trol mice

(iog~o) (logx0)

4.90 6.26 4.98 6.26 1.11 5.63 0.74 5.63 1.57 5.63

23 18

30,380 70,750 11,500

° BPL--Beta Propiolactone.

ttabel test (see "Materials and Methods"). This test showed that, whereas the potency of the two CVS preparations inactivated with phenol was very low, the immunizing capacity of the three vaccines prepared from CVS and PM strains and inactivated by BPL was extremely high (Table XIII). I t should be of interest to note that a high degree of protection was obtained even though the infectious titer of the virus preparation, prior to inactivation, was relatively low.

DISCUSSION

The process of adapting rabies virus to WI-38 was beset by difficulties familiar to other investi- gators (3) who have attempted to grow rabies virus in other tissue culture systems. Initially less than 10% of the WI-38 cells became infected with rabies virus and the low yield of infectious virus made it impossible to maintain the virus by the passage of infected medium or cell extract into fresh cultures. The virus could be maintained, however, in a series of subcultures made by direct transfer of infected cells.

Although the difficulties in adapting rabies virus to WL38 may be caused by relative hetero- geneity of the cells in relation to their susceptibil- ity to rabies infection, it is more probable that an inhibitory mechanism engendered by the virus-infected cells was operative. Whether the inhibitory effect was mediated by the production of an antiviral substance is impossible to say, since up to now no such substance has been isolated from the rabies-WI-38 tissue culture system even though interference by rabies- infected WI-38 cultures against homologous and

heterologous virus challenge was quite pro- nounced. Although attempts to isolate an inter- feron-like substance remain futile, trypsin, which is supposed to destroy interferon (16-18), did favor virus propagation, whereas the use of Versene led to the loss of infectious virus in the course of several tissue culture passages. It is possible, of course, that methods currently used to extract interferon do not apply to the rabies- WI-38 system.

Rabies virus could not be propagated in- definitely in subcultures of WI-38 because of the development during several cell generations of cytoplasmic inclusions which were so large that they interfered indirectly with the mechanism of cell mitosis (15). In order to circumvent loss of the cultures, a mixture of fresh cells had to be added at each subculture passage level. This finally led to the adaptation of the virus to growth in WI-38 where it could be maintained in serial passage by subinoculation of cell-free media on fresh WL38 cells.

It is interesting to note that rabies virus, once adapted to WI-38, could be propagated in other, unrelated tissue culture systems which were, in general, resistant to infection with normdapted virus. Thus, while the virus was adapting to WL 38 there may have been a selection of virus par- ticles with greater affinity for attachment to the receptors of WL38 cells. These receptors may be identical for many tissue culture systems, re- gardless of their origin.

A different character of the WI-38-adapted virus population was suggested by the study of the properties of HEP Flury virus grown in WL

1964] CULTIVATION OF RABIES VIRUS IN HDCS WI-38 365

38. While the nonadapted virus was still patho- genic for hamsters, guinea pigs and monkeys injected intracerebrally, the WI-38-adapted strain completely lost these properties. Also, in contrast to the high pathogenicity for adult mice acquired by the nonadapted strain after one passage through baby mouse brain, the increase in pathogenic properties of the WI-38-adapted strain for adult mice was of very low order indeed.

The greater antigenicity of WI-38-adapted HEP Flury for intramuscularly injected monkeys, as compared with the egg-adapted virus, may be due to its having grown in the fibroblast-like WI-38 and, therefore, being able to replicate in the muscle tissue. Conversely, however, the antigenieity of WI-38 preparations may be explained by the presence of large amounts of dead (noninfectious) virus, and this may also have accounted for the excellent immunizing properties of BPL-inaetivated CVS and PM virus vaccines. In this case, the infectivity titer of the viruses before inactivation was much lower than that observed with the brain tissue-propa- gated homologous virus (12), and yet the anti- genie power of the WI-38 vaccines was com- parable to the corresponding Semple4ype vac- cine (12).

Because of the infectivity of rabies virus for all warm-blooded animals and because of its bizarre and ubiquitous behavior in the infected host, study of the host cell-virus relationship in the tissue culture systems of various animal origins is highly indicated. Apart from the fact that WI-38-adapted virus seems to grow in at least six other tissue culture systems, methods used for the adaptation of rabies virus to WI-38 may be employed for cells of other species which hitherto resisted supporting growth of the virus.

Acknowledgment. The authors are deeply indebted to Jacqueline Kessel and Jean McClure for their excellent technical assistance.

SL-MMARY

In spite of the initial infectivity of three strains of rabies virus for the human diploid cell strain WI-38, the virus could not be serially propagated in this cell system with conventional methods of passaging infectious material. However, by following the mixing-cell transfer technique,

three strains could finally be adapted so that they could propagate indefinitely. The adapted HEP virus caused a eytopathic effect in WL38 and its infectivity, for four other tissue culture systems of different animal origins increased during the course of its propagation in WL38.

After adaptation to WI-38, the HEP strain seemed to lose its lethal properties for monkeys injected intraeerebraUy, while acquiring a high degree of immunizing capacity for the same species.

Inactivated vaccines prepared from two other strains of fixed virus (CVS and PM) were found to be highly antigenic after adaptation to WI-38. This fact and the availability of the WI-38- adapted live HEP virus seem to indicate that production of effective and safe anti-rabies vaccine for man is now feasible.

Finally, because of good antigenic properties demonstrated either by the live attenuated HEP virus or by inactivated CVS and PM viruses grown in WL38, an ideal medium for the produc- tion of human vaccines, effective and safe im- munization of man against rabies may not be too far away.

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366 T . J . WIKTOR, M. V. FERNANDES AND H. KOPROWSKI [VOL. 93

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