Archives of Virology 67, 203--215 (1981) Archives of Virology © by Springer-Verlag 1981

Association of Human Cytomefalovirus (HCMV) with Mink and Rabbit Lunfl Cells

By

HELENA HART and MAI~Y NORVAn Department of Bacteriology, University of Edinburgh Medical School,

Teviot Place, Edinburgh, Scotland

With 6 Figures

Accepted October 15, 1980

Summary

The association of human cytomegalovirus with mink and rabbit lung cells was studied. Strain AD-169 was used which was free of Mycoplasma and other contaminating agents. I t was found to be incapable of productively infecting mink lung cells. Infection appeared to be initiated but aborted at an early stage. This was indicated by indirect immunofluorescence, assays of culture supernatants and cell lysates for infectious virus, electron microscopy of ultra-thin sections of in- fected cells, labelling of virus and viral DNA with aH-thymidine and assay of vitally-induced DNA polymerase at various times after infection. On the other hand, using these methods, AD-169 was found to infect rabbit lung cells, the virus being produced in low amounts over a period of up to one month after infection. At this time, focal areas of infection were still apparent and 15 per cent of the cells expressed nuclear viral antigens as shown by immunofluorescence. The viral genomc was assumed to have become latent in some rabbit cells with a few being capable of producing infectious virus.

Introduction

Human cytomegalovirns (tICMV) normally replicates in the nuclei of human fibroblasts, although there have been reports of other cells being susceptible to infection. Two human systems have been described; firstly, lung epithelial cells being productively infected with slow release of virus over several weeks and ac- companying cytopathic effect typical of cytomegalovirus (12); and secondly, amnion epithelial cells being productively infected, the virus yield being lower than from equivalent fibroblasts and the replication cycle being slower (4). In addition, human embryonic kidney epithelial cells which are normally non- permissive for cy~omegalovirus replication can be converted into a permissive state by treatment with iododeoxyuridine before infection (15).

0304-8608/81/0067/0203/$ 02.60

204 HELENA HART and MARY NORVAL:

Various non -human sys tems have also been descr ibed in a t t e m p t s to f ind a permiss ive cell for HCMV repl icat ion. Guinea-pig embryo cells were abo r t i ve ly in- fected, wi th no new virus par t ic les being produced (5). E a r l y vi rus ant igens were formed and cellular D N A synthes is s t imula ted . However , the D N A po lymerase a c t i v i t y in the infected cells d id no t have the proper t ies of the enzyme induced by HCMV in permiss ive cells (8). Mouse f ibroblas ts can also be abo r t i ve ly infected, the block in rep l ica t ion being a t the level of v i ra l D N A synthes is (1).

I n addi t ion , DARAI and FLUGEL (2) found t h a t epi thel ia l - l ike mink lung cells were h ighly suscept ible for the repl ica t ion of var iee l la-zos ter and suggested t h a t th is migh t be a su i tab le ceil line for the growth of o ther Herpes viruses, l ike Eps te in -Bur r v i rus and cytomegalovi rus .

F ina l l y FAgBER et al. (3) discussed the rep l ica t ion of HCMV in r abb i t lung f ibroblasts . They no ted a ey topa th i e effect in th is sys tem af ter 5 to 7 days which increased for 2 weeks and then remained unchanged over a per iod of up to 28 days . CMV ant igens could be de tec ted in the nucleus in 40 per cent of the cells b y im- munofluorescencc a t this t ime. Al though no infectious vi rus was de tec ted in t he cul ture supe rna t an t 3 days af ter infection, v i rus could be recovered b y co-cul t iva- t ion of the r a b b i t lung cells wi th h u m a n embryo lung cells even 28 days post- infection.

We decided to inves t iga te fur ther the associa t ion of HCMV wi th r a b b i t lung cells and to ascer ta in whether mink lung cells could be infected. This inc luded an examina t ion of infected cells b y electron microscopy, g rowth curves over per iods of up to 20 days , label l ing with 3 t I - thymid ine and subsequent, ex t r ac t ion of v i ra l DNA, immunof luorescenee over per iods of 32 days and fina.lly measu remen t of D N A polymerase a c t i v i t y inside infected nuclei.

Materials and Methods

Cells

Rabbi t lung cells were prepared by trypsinizat ion of the lungs of a 1 year old New Zealand White rabbi t and grown in Earles based Eagles medium (MEM) containing 100 i .u. /ml penicillin and 100 ~xg/ml s treptomycin and supplemented with 2 to 5 per cent rabbi t serum. After the cells had formed monolayers they could be split I :3 every week and were fibroblastic in appearance. They were used between pass 8 and 20.

Mink lung cells (Mv-l-Lu) were obtained from the American Type Culture Collec- tion, number CCL 64, and were cultured in MEM containing 2 to 5 per cent new bern calf serum and antibiotics as above. They were epitheliM-like in appearance and exten- sive microvilli on the eetl surface could be seen on electron microscopy of ul t ra- thin sections.

Human embryo lung cells (HEL) were prepared as described for the rabbi t lung cells except tha t the medium was supplemented with 5 to 10 per cent new born calf serum instead of rabbi t serum.

Incubat ion of all cells was at 37 ° C in an atmosphere of air containing 5 per cent COz. All cells were free of contamination with Mycoplasma.

Virus

Human cytomegalovirus (HCMV), strain AD-169, which had been plaque purified by Dr. J . M. DeMarchi and was free of other contaminat ing viruses as tested by Dr. G. Dural, was obtained from Dr. J . Cameron, Ins t i tu te of Virology, Glasgow. I t was also free of contamination with Mycoplasma species.

Assoc ia t ion of Y[CMV w i t h M i n k a n d R a b b i t L u n g Cells 205

In/eetion o/Cells Cells were in fec ted as mono laye r s , a l lowing i h o u r for a d s o r p t i o n a t 37 ° C, or in

suspens ion w i t h gen t le s h a k i n g for 2 h o u r s a t r oom t e m p e r a t u r e . T h e cells were w a s h e d before m a i n t e n a n c e m e d i u m c o n t a i n i n g 2 pe r cen t of t h e a p p r o p r i a t e s e rum was added.

Ultrastructure Cells were h a r v e s t e d us ing glass b e a d s a n d p r e p a r e d for e lec t ron microscopy . T h e

b locks were sec t ioned on a n L K B U l t r a t o m e 11 a n d t h e sect ions v iewed on a I~ i tach i I-IU 1 i A e lec t ron microscope .

Immunofluorescence A n ind i r ec t immunof iuo re scence t e s t was pe r fo rmed on ace tone- f ixed (10 m i n u t e s

a t 4 ° C) cells g rown on cover-s l ips us ing a v a r i e t y of h u m a n conva le scen t sere which h a d a h igh c o m p l e m e n t f ix ing t i t r e to cy tomega lov i rus . Sheep a n t i - h u m a n i m m u n o - g lobu l in c o n j u g a t e d w i t h f luorescein i so th iocya n a t e (Wel leome Reagen t s ) was added , d i lu t ed 1/10 in p h o s p h a t e buf fe red sa l ine (0.01 ~ pI-I 7.2) c o n t a i n i n g 2 pe r cen t foeta l calf s e r u m to reduce non-specif ic s ta in ing .

Growth Curve o/AD.169 in Rabbit Lung, Mink I~ung and HEL Cells F o r th i s expe r imen t , t he r a b b i t l ung cells were f i rs t a d a p t e d to grow in t h e p resence

of new b o r n calf s e rum i n s t e a d of r a b b i t se rum, as t h e fo rmer was f o u n d to be toxic for t t E L cells on s u b s e q u e n t assay.

2 × 106 ceils were p u t i n to 5 m m P e t r i d ishes (Steri l in) in 5 ml m e d i u m a n d i n c u b a t e d ove rn igh t . T h e y were in fec ted a t a m u l t i p l i c i t y of 0.3, a n d t h e i n c u b a t i o n c o n t i n u e d ove r a pe r iod of 20 days w i t h a c h a n g e in m a i n t e n a n c e m e d i u m e v e r y 4 days . A t intei~vals, p l a t e s were r emoved , t h e cu l tu re s u p e r n a t a n t was col lected a n d t h e t r y p s i n - ised cells f rozen a n d t h a w e d twice fol lowed b y u l t r a s o n i e a t i o n for 1 m i n u t e s to release cei l -associa ted v i rus . B o t h were t h e n a s sayed acco rd ing to t h e m e t h o d of I-IAMELI~ a n d L~rSSIEa (7) us ing t t E L cells in Mie ro t i t r e p la tes (Nune) .

Labelling o/ Virus with aH-Thymidine A t var ious t imes a f t e r in fec t ion of cells w i t h AD-169, the m e d i u m was rep laced

w i t h M E M c o n t a i n i n g 1 pe r cen t s e r u m a n d 2.5 ~Ci/ml (methyl-SH) t h y m i d i n e (The R a d i o c h e m i c a l Centre , A m e r s h a m ) , a n d i n c u b a t i o n c o n t i n u e d for a f u r t h e r 48 hours . The cu l tu re s u p e r n a t a n t was h a r v e s t e d , clar if ied a t 10,000 X g for 10 m i n u t e s a n d t h e n e i t he r p r e c i p i t a t e d w i t h a n equa l v o l u m e of cold s a t u r a t e d (NHd) ,S04 or cen t r i fuged a t 30,000 r p m for 1 h o u r in a n S W 4 0 ro to r The depos i t was s u s p e n d e d in a smal l v o l u m e of buffer , 0.01 ~ Tris-HC1, 1 m ~ - E D T A , pI-I 7.1 (6). I t was l aye red on to sucrose dens i t y g r a d i e n t s (20 to 70 pe r cen t ) a n d cen t r i fuged a t 25,000 r p m for 18 hou r s in a n S W d 0 ro tor . F r a c t i o n s were co l lec ted f rom t h e b a s e of t h e tube , p r e c i p i t a t e d w i t h cold 10 pe r cen t TCA a n d c o u n t e d a f t e r f i l t r a t ion t h r o u g h glass f ibre discs (14). U n d e r t he se cond i t ions H C M V has a d e n s i t y of 1.219 g /ml (10).

Labelling o/ Viral DAL4 with all-Thymidine Cells were g r o w n in 4 rol ler bo t t l e s (2 51) un t i l a l m o s t con f luen t ( abou t 2 × 107 cells

pe r bot t le ) , t h e n 2 in fec ted w i t h AD-169 a t a m u l t i p l i c i t y of 1, a n d 2 lef t as controls . 5 days pos t - infec t ion , (methyl-~H) t h y r n i d i n e was a d d e d as descr ibed above . 48 hours la ter , t h e cu l tu re s u p e r n a t a n t was col lec ted a n d cells r e m o v e d w i t h glass beads . Af te r c la r i f ica t ion of t he cu l tu re s u p c r n a t a n t t h e v i rus par t ic les were depos i t ed b y een t r i f uga t i on a t 30,000 r p m in a n S W 4 0 rotor . T h e y were r e suspended in a sma l l v o l u m e of buf fer a n d pur i f ied on a sucrose g r a d i e n t as above . The cells were f rozen a n d t h a w e d twice fol lowed b y u l t r a son i ca t i on a n d t h e cel l -associated v i rus par t ic les s imi la r ly pur i f ied on sucrose dens i ty g rad ien t s . The f rac t ions f rom t h e g r ad i en t s w i t h d e n s i t y 1.20 to 1.23 g /ml were pooled, d i l u t ed a n d t h e v i rus par t ic les depos i t ed b y cen t r i f uga t i on a t 40,000 r p m for 1 h o u r in a n S W b 0 rotor . The pel le t was t r e a t e d wi th pronase , 2 m g / m l (Boehr inger) , a n d SDS, f inal c o n c e n t r a t i o n 1 pe r cent , a t 37°C for 8 hou r s a n d t h e v i ra l D N A b a n d e d on a 10 to 40 pe r cen t sucrose d e n s i t y g r a d i e n t (10). F r a c t i o n s were col lec ted f rom t h e ba se of t h e t ube , p r e c i p i t a t e d w i t h cold 10 pe r c en t TCA a n d c o u n t e d a f t e r f i l t r a t i on t h r o u g h glass f ibre discs.

206 HELENA HART and MARY NORVAL;

DNA .Polymerc~e Assays The infected cell lysates were prepared and the assay carried out according to the

method described by HI•AI, FUR.VKA'WA and PLOTKIN (8), except that activated calf thymus DNA was used in place of salmon sperm DNA as a template, and each assay contained 10 ~Ci (methyl-aI-I) thymidine 5'-triphosphate (30 Ci/m mole; The Radio- chemical Centre, Amersham). The nuclear fractions (5--30 ~l) were used to assay the

Fig. 1. Thin section of AD-169 infected rabbit lung cell showing an enlarged and involuted nucleus ( × 4800)

Association of HCMV with Mink and Rabb i t Lung Cells 207

DNA polymerase act ivi ty. At 15 minutes intervals during incubation, 50 ~1 samples were removed, spot ted on to W'hatman filter paper discs which were then fixed in cold 10 per cent TCA washed five t imes in cold 10 per cent TCA then twice in cold ethanol, before being air-dried and counted in PPO-toluene scintil lator in a Packard Tri-Carb liquid scintillation instrument. Protein content of the samples was es t imated by the method of LowJ~Y et al. (11).

To distinguish viral DNA polymerase from cellular DNA polymerase, (NHa)eSOa at a final concentration of 54 mM was added to the assay mix (8).

Results

Cellular Changes After In]ection

W h e n AD-169 was used to infect r a b b i t and mink tung cells a t a mu l t i p l i c i t y of infec t ion of 0.1 to 3, there was an effect on the cells easi ly seen b y l ight micro- scopy and appear ing f i rs t 2 to 3 days pos tdnfec t ion . The r a b b i t cells became rounded and pi led up in cer ta in areas. This effect pers i s ted for up to 2 months . The mink cells also became rounded a l though, in th is case, the re were no focal areas of infect ion. The cells con t inued to d iv ide and the effect g r adua l l y dis- a p p e a r e d over a 3 week period.

U l t r a - t h in sections of bo th cell t ypes were examined in the e lec t ron microscope a t 5 and 15 days post- infect ion. No abnormal i t i e s or v i ra l s t ruc tures could be seen in the mink lung cells a t these t imes. However , in the r a b b i t lung cells, a l though no whole v i rus par t ic les or dense bodies could be seen, the nucleus of ten looked abbe r r an t , being enlarged and involu ted . This is i l lus t ra ted in Fig. 1.

70

6O

5O

% cells with 40 positive nuclear immtmofluorescence 30

20

i0

2 4 6 8 10 12 14 16 18 20

Days after infection

Fig. 2. Percentage of rabbi t lung ( . .) and mink lung (c~ ..... o) cells showing positive nuclear immunofluorescence at various times after infection with AD-169

Immuno/luorescence

Ind i r ec t immunof luorescence was carr ied ou t on A I ) - t 6 9 infec ted r a b b i t a n d m i n k lung cells a t var ious t imes up to 20 days post - infect ion, using 3 se lected h u m a n sera which gave nuclear immunof luorescence wi th AD-169 infec ted h u m a n embryo lung cells. The infec t ion was a t a mu l t ip l i c i t y of 1. I n the mink lung cells, there was posi t ive immunof luorescence showing in i t i a l ly as uni form nuclear s ta in-

15 Arch. ¥irol, 67/3

208 HELENA HART and MARY NORVAL:

ing of over half of the cells, bu t th is number decreased r ap id ly and all were nega t ive b y 6 days post- infect ion. I n contras t , in the r abb i t lung cells, pos i t ive immunof luorescence was seen in 50 per cent of the cells 1 day = af te r infect ion and, a l though this number diminished, 15 per cent were st i l l posi t ive 20 days af te r in- fection. These resul ts are shown in Fig. 2. The ceils were not subeu l tu red dur ing this t ime. In i t ia l ly , the posi t ive immunofluoreseenee was associated wi th in-

t

Fig. 3. Indirect immunofluorescence on AD-169 infected rabbi t lung cells a 2 days post-infection and b 10 days post infection

Association of t tCMV with Mink and Rabbi t Lung Cells 209

d i v i d u a l cells (Fig. 3 a) bu t severa l days pos t - infec t ion the posi t ive cells were in a form of mul t inuc lea r g i an t cells (Fig. 3b) .

I n add i t i on to indi rec t immunofluorescence, an t i - complemen t immunof luores- cence was carr ied out on the infected r abb i t lung cells and gave the same p ic ture , the cells being cu l tu red for 32 days in this case.

Log 10

p f u / m I

7 .0

6 .0

5 .0

4 .0

3 .0

2 .0

1.0

m

0 1 2 3 4 5 6 7 8 9 10 11 I2 13 14 15 16 17 18 19 20

DAYS AFTER INFECTION

Fig. 4. Assay of infectious AD-169 in culture supernatant and cell-associated at various t imes after infection. (Human embryo lung cells; supernatant • . . . . . . . . . . , , cell-associated o - - - - - o; mink lung cells, supernatant A A, eelI-associated a- - a; rabbi t

hmgeel ls , supernatant x -- l, ee l lassoeiated a - - D)

Growth Curves Culture supe rna t an t s and cell lysa tes of m ink and r a b b i t lung cells were assayed

a t var ious t imes pos t - infec t ion wi th AD-169 wi th the resul t shown in Fig. 4. In- fec ted H E L cells were used as a posi t ive control. I t m a y be seen tha t , in the case of the mink lung cells, the t i t res of virus in the cul ture s u p e r n a t a n t and cell- assoc ia ted d ropped r ap id ly unt i l no infect ious v i rus was a p p a r e n t 6 days a f te r in- fection. The g rowth curve of AD-169 in r a b b i t lung cells showed a di f ferent p ic ture . There was a fa i r ly s t e ady and low level of infectious vi rus bo th in the cul ture s u p e r n a t a n t and cel l -associated which pers is ted as such over the 20 d a y per iod of the exper iment .

Labelling o/ Virus and Viral DNA with 3H-Thymidine 3H- thymid ine was a d d e d to infected rabb i t , m ink and h u m a n embryo lung

cells be tween 5 and 7 days post- infect ion, wi th subsequent a s say of the cul ture

15"

210 HELENA HAIler and MARY N0t~VAL:

supernatant by sucrose density gradient centrifugation for labelled acid-insoluble material of the density of ChfV, i. e. 1.22 g/ml. While a large peak of radioactivity was obtained at this density in the ease of the culture supernatant from infected H E L cells, no labelling was found in the culture medium of infected mink lung cells. With the rabbit lung cells, again there was no peak of labelling at this density. However, the picture in this case was not entirely clear due, it is thought, to the substantial degree of cell 1 ysis which occurred in these cells when infected. This resulted in some labelling at a slightly lighter density than CMV and which may be sufficient to mask a small amount, of viral-labelled material.

Acid- insoluble c.p.m.

2,000

1,000

Acid- insoluble c.p.m.

6, 000

4,000

2,000

5 I0 15 20

Fraction number

Fig. 5 a

5 10 15 20

Fract ion number

Fig. 5 b

1.14

1.12

1.10

1.08

1.06

1.14

1.12

1.10

1.08

1.06

1.04

Density g/ml

Density g /ml

To clarify this situation, the material with density 1.20 to 1.23 g/ml was taken from these gradients and the viral nucleic acid, if any, extracted and subsequently banded on a 10 to 40 per cent sucrose gradient. In addition to the culture super- natant, cell lysates were also examined in this way. The result may be seen in Fig. 5.

Using HEL cells infected with AD-169, a peak of labelled acid-insoluble mate- rim with density around 1.10 g/ml was found both in the culture supernatant

Association of HCMV with Mink and Rabbit Lung Cells 211

(Fig. 5a) and cell-associated (Fig. 5b), which was not present in uninfected control cells. The same experiment using mink lung cells showed no such peak, indicating tha t viral DNA was not being replicated in these cells (Figure not shown). Wi th the rabbi t lung cells, no labelling was apparent a t this densi ty in the cell lysatcs (Fig. 5 d) but a small peak was seen in the material from the culture supernatant

(Fig. 5 c). This may represent small amounts of I)NA in virions released from in-

fected cells.

150

100

50

i i I I

5 10 15 20

Frac t ion number

Fig. 5 c

Acid- insoluble c.p.m.

1.12

1.10

1.08

1.06

1.04

Density g/ml

6, 000 1. t2

1.10

Acid- Density insoluble 4,000 i. 08 g/ml c. p. m.

1.06

2, 000 ~ 1.04

5 I0 15 20

Fraction number

Fig. 5d

Fig. 5. Cells infected with AI)-169 and uninfected controls were labelled with aI-I- thymidine, the virus was purified from the culture supernatants and cell lysates, its DNA extracted and run on a sucrose density gradient. The graphs show the acid- insoluble counts per minute (cpm) of the fractions from the gradient a culture super- natants of human embryo lung cells (o o) and human embryo lung cells infected with AD-169 (® .), b lysates of human embryo lung ceils (o o) and human embryo lung cells infected with AD-t69 (o o), c culture superna~ants of rabbit lung cells (o o) and rabbit lung cells infected with AD-169 (.---~ .), and d lysates of rabbit lung cells (o o) and rabbit lung cells infected with AD-169

( . -- o)

40,000

DNA Polymerase Activity

I t has been reported tha t DNA polymerase induced by HCMV is different from the normal cellular DNA polymcrase (8, 9). I n particular, it was found to be s t imulated by concentrat ions of 50 to 100 mM (NH4)2SO4, which inhibit cellular D N A polymerase act ivi ty.

The infected cells were harvested at various times after irffeetion and the nuclear fractions prepared. These were assayed with and without 54 m ~ (NH4)2S04 and the counts per minute (cpm) of 3H-TTP incorporated into acid-soluble material in 45 minutes per ~tg protein was calculated. A typical assay curve for H E L and mink lung cells is shown in Fig. 6 (a) and (b) and the tota l results are

10,000

30,000 Acid- insoluble c . p . m .

20,000

Acid- insoluble c . p . m .

given in Table 1.

I I I

15 30 45

MINS,

Fig. 6 a

60, 000

40,000

20, 000

15 30 45

MINS.

Fig. 6 b

212 HELENA HART and MA~Y NORVAL:

Fig. 6. a DNA polymerase activity of nuclear fraction (90 gg protein) from AD-169 infected human embryo lung cells with (o o) and without (. -) 54 rn~ (NH4)2SO4. b DNA polymerase activity of nuclear fraction (200 }zg protein) from AD-169 infected mink lung cells with (o o) and without (. .) 54 m~a

(NH4)2SO4

Association of HCMV with Mink and Rabbit. Lung Cells 213

Considering HEL-infected cells, there was an increase in DNA polymerase ac t iv i ty 4 days post-infection by about 15-fold compared to the uninfected cells. This enzymatic ac t iv i ty was st imulated over 2-fold by the presence of 54 mM (NH4)2S04 in the assay mix while the control ac t iv i ty was inhibited. In infected mink lung cells DNA polymerase ac t iv i ty was st imulated, a peak of 12-fold 2 days after infection and gradually decreasing thereafter. However, this enzyme seemed to be inhibited by (NH4)2S0a at all times. A very similar picture emerged for the rabbi t lung cells with a 2-fold increase of DNA polymerase act ivi ty 5 days post- infection, and there was no st imulation by (NH4)2S04.

Table 1. DNA polymerase activity o/nuclear ]factions o] human embryo lung (HEL), mink lung and rabbit lung cells, and niter injection with AD-169

Increase activity Increase in acid- with

Increase in acid- insoluble (NH4)2SO4 in acid- Pro- insoluble cpm in %- insoluble tein cpm in presence of activity cpm in ~zg/ 45 rnins/~g (NH4)2SO4 without 45 mins/izl ml protein in 45 mins/p.1 (NHa)~SOa

HEL cells 460 7.8 59 336 73 4 days after infection 16,200 17.8 910 33,690 208

Mink lung cells 120 2.9 41 108 90 1 day after infection 1,750 6.3 278 1,950 111 2 days after infection 3,350 6.6 508 3,240 97 5 days after infection 3,000 8.9 337 1,500 50

10 days after infection 600 5.0 120 600 100

Rabbit lung cells 250 2.6 96 175 70 2 days after infection 533 3.8 140 500 93 5 days after infection 1,000 4.8 208 1,225 109

10 days after infection 290 2.2 132 272 94

Diseussion

We were interested to understand the association of AD-169 with mink and rabbi t lung cells. All experiments detailed here indicated tha t mink lung cells were not capable of supporting replication or persistence of HCMV. There was a~ initial toxic effect, followed by recovery of the cells within a few days. Immuno- fluorescence results indicated tha t infection was ini t iated but aborted at an early stage as no positive cells remained 6 days after infection. Growth curves of virus in the medium and the cells corroberated this. No virus or viral-induced structures were seen on electron microscopy of u l t ra- th in sections and there was no induction of viral DNA polymerase act ivi ty detectable in infected cells. In addition, no labelling of virus or viral DNA with aH-thymidine in infected cells was obtained.

I t has been reported (3) tha t rabbi t lung cells may be infected with AD-t69, the virus being produced for the first 3 days after infection, and not thereafter, although it could be recovered by co-cultivation with human embryo cells even after 28 days in culture. Immunofluorescence showed 40 per cent of the cells with nuclear fluorescence at this t ime and in 10 per cent it was thought tha t some early

214 HELENA HART and MARY NORVAI3 :

proteins had accumulated to give large fluorescent granules, perhaps indicating" a block in replication. Our experiments would support the idea of a long-term persistent infection of rabbit lung cells with IICMV, but in our system small amounts of infectious virus were released from the cells at a fairly constant rate over 4 weeks after infection. This could be measured by assay of the culture supernatant for infectious virus, and also by assay of viral DNA in the culture supernatant after labelling with 3II-thymidine. Measurement of DNA polymerase activity did not indicate induction of an enzyme with the properties of I-ICMV DNA polymerase. However, this is probably not a sufficiently sensitve method if only a small number of rabbit lung cells from the culture were capable of produc- ing virus at any time and the block in the remainder is at or before this point.

The mechanism of the low grade persistent infection is not clear but it seems evident that there is a restriction of viral replication in rabbit cells, as has already been described in mouse cells (1). I n addition, recent work b y MOOARSKI and STI~S~:I (13) indicated tha t t ICMV genome can persist over long periods if the virus is used to infect human fibroblasts at high multiplicities. Non-product ive cells continued to grow and some of these spontaneously converted to product ive viral infection. I t was assumed tha t all sequences of the viral genome remained in the cells after infection and tha t viral gene expression was restricted in some way in the non-product ive cells. The same si tuation m a y apply to the rabbi t lung cells. Certainly, focal areas of infection could be seen even 28 days after infection and infectious virus was eontinously being produced at low level over this time. I t m a y be tha t some host funct ion is required to complete the viral replieative cycle which is seldom present, perhaps only being found in the rabbi t lung cells at certain stages of the growth cycle. At other times the viral genome would become latent. I t is still expressed, however, in 15 per cent of the cells as early antigens as shown by nuclear immunofluoreseence up to one month after infection.

The question of la tency of cytomegalovirus is an impor tan t one as this virus persists in man over m a n y years, even in the presence of the host immune response. The cell type(s) and site(s) in the body of this persistence arc uncertain bu t there m a y be a mechanism similar to t ha t demonst ra ted here for rabbi t lung cells, whereby cells are infected Intently, with a small proport ion of the populat ion being capable of product ive infection at any one time. The virus so produced could in- fect new cells and perpetuate the chronic state.

Aeknowledfments We wish to thank the Cancer Research Campaign for funding this work.

Referenees 1. BOLDOGtt, I., GONCZOL, E., GARTNER, L., VACZI, L., MmnELSON, S. : Latent infec-

tion of mouse cells with human cytomegalovirus. Bull. Cancer (Paris) 63, 4i 1--419 (t976).

2. DAtgAI, G., FLUGEL, R. M. : Mink lung cells : a non-primate ceil line highly suscep- tible for varicetla-zoster virus. J. gen, Virol. 41, 179--I81 (1978).

3. FAIRER, I., WUTZLE~, P., SO~WEIZER, It., SeROSSIO, M. : Human cytomegalovirus induced changes in rabbit cells. Arch. Virol. 59, 257--261 (1979).

4. FIGUEROA, M. E., GEDER, L., ~)A~P, F. : Infection of human amnion eells with cytomegalovirus. J. mad. Virol. 2, 369--375 (1978).

Association of HCMV with Mink and Rabbit Lung Cells 215

5. •IORETTI, A., FURUKAV/A, T., SANTIOLI, D., PLOTKIN, S. : Non-productive infec- tion of guinea, pig cells with human cytomegalovirus J. Virol. 11,998--1003 (1973).

6. GI~AYZEL, A. I. : Uridine incorporation into the media and RNA of cultured rheu- matoid synovial cells. Arthritis Rheum. 16, 419--421 (1973).

7. HAMEJ, IN, C., LUSSlEt~, G.: Concentration of human cytomegalovirus from large columns of tissue culture fluids. J. gen. Virol. 42, 193--197 (1979).

8. HIRAI, K., FU~:AWA, T., PLOTKI~, S. : Induction of DNA polymerase in WI-38 and guinea pig cells infected with human cytomegalovirus. Virology 70, 251--255 (1976).

9, HUA~-G, E.-S.: Human cytomegalovirus. I I I . Virus-induced DNA polymerase. J. Virol. 16, 298--310 (1975).

10. ttUA~G, E.-S., CHE~, S.-T., PAGA~O, J. S.: Human eytomegalovirus. I. Purifica- tion and characterization of viral DNA. J. Virol. 12, 1473--148t (1973).

11. Low~Y, O. H., ROSEBROUCH, N. J., FARR, A. L., RANDALL, R. J. : Protein measure- merit with the Folin phenol reagent. J. biol. Chem. 193, 265--275 (197t).

12. MICHELSO~-FISKE, S., ARNOULT, J., FEBVttE, I-I.: Cytomegalovirus infection of human lung epithelial cells in vitro. Intervirology 2, 354--363 (1975).

13. MOCARSKI, E. S., STINSKI, M. F. : Persistence of the cytomegalovirus genome in human cells. J. Virol. 31, 76t--775 (1979).

14. NO,VAt, M., 1V[ARlVIION, B. P. : Attempts to identify viruses in rheumatoid synovial cells. Ann. Rheum. Dis. 35, 106--t13 (1976).

15. ST JEOl{, S., RAPP, R.: Cytomegalovirus: conversion of nonpermissive cells to a permissive state for virus rep]ication. Science 181, 1060--i061 (1973).

Authors' address: Dr. MA~Y NORVAL, Department of Bacteriology, University of Edinburgh Medical School, Teviot Place, Edinburgh, E t t 8 9AG, Scotla~ld.

Received August 4, 1980