Neutralization of adenovirus infectivity and cytotoxin in various cell cultures

Journal of Virological Methods, @ Elsevier/North-Holland

2 (1981)

Biomedical

NEUTRALIZATION

321-330

321

Press

OF ADENOVIRUS

INFECTIVITY

AND CYTOTOXIN

IN

VARIOUS CELL CULTURES*

E. SCHRADER

and R. WIGAND**

Institut fiir Hygiene

und Mikrobiologie

der

Universit&t des Saarlandes,

D-6650

Homburg

(Saar),

F.R.G. (Accepted

5 February

1981)

The neutralization was

determined

cells

from

cercopithecus,

The results showed

adenovirus

sensitivity

neutralization.

this method

5 and 11 by homologous

in various mouse),

rabbit,

concerning

similar

results;

of human

by CPE inhibition

and

cell cultures

or in HeLa

specificity

is suitable

cells made

were

Immunofluorescence

and heterologous

(HeLa,

similar

subtle

Vero,

impermissive in HeLa

immunological

rabbit

antisera

secondary

kidney

by IUdR

in all cases. Crude

neutralization

for demonstrating

HEL,

inhibition.

and purified

cell cultures

relations

virus

gave similar

between

adenovirus

types. The neutralization different

from

concluded

from

the replicative Hence,

either

of the early

the virion;

cytopathic

the cytotoxin

the results

that

cycle in infection

factor

was found

(‘cytotoxin’)

showed

to be active in part

the virus function(s)

blocked

and for the initiation

of the abortive

kind of cells may be used for neutralization

a pattern

of cross-reactivity

of the cell cultures

by antibody

appear

infection

only. It is

to be identical

in non-permissive

for cells.

tests.

INTRODUCTION

Neutralization munological

is not only the procedure

to identify

virus types and to measure the im-

host response, it is also the basis for classification

of virus species in many

virus families. Adenoviruses multiply and spread slowly in cell cultures. Hence, for neutralization tests either a high virus concentration or a long observation period is required. The procedures used vary widely with respect to the kind of cell cultures, the virus concentration,

time of reading, criteria of neutralization,

macro- or micro-procedure

(Sohier et al., 1965; Stevens et al., 1967; Tan and Wigand, 1979). American investigators often prefer monkey kidney cell cultures (Stevens et al., 1967) which show a cytopathic

*

Aided by a grant

from the ‘Bundesministerium

** All correspondence Abteilung D-6650

des Homburg

Institutes (Saar),

and

requests fur

F.R.G.

Hygiene

for reprints und

fur Jugend, should

Mikrobiologie,

Familie

und Gesundheit’,

be addressed

to Dr. R. Wigand,

Haus

Universitatskliniken

47,

F.R.G. Virologische im LKH,

322

effect with human holzer

adenoviruses,

and Bingham

using microtitre

capsomeres,

multiplication.

but are not or only partially

developed

as the neutralization

method

in Vero cells

their results both in monkey kidney and

of the cytotoxic

in view of the limited

Those virus components

permissive for them. Hier-

an elegant neutralization

plates. These authors interpret

in Vero cell cultures vertex

(1978)

permissiveness

are known

activity

of pentons

and/or

of these cells for adenovirus

to produce

an early cytopathic

or

cell-detaching effect (Everett and Ginsberg, 1958; Pereira, 1958; Rowe et al., 1958b). However, Hierholzer and Bingham (1978) determined similar homologous neutralization titres of adenovirus

antisera

in monkey

kidney,

Vero, and the highly permissive

HEK

tube cultures. Furthermore, the pattern of cross-reactivity for adenovirus antisera found by Stevens et al. (1967) in monkey kidney cells was similar to that obtained in HeLa ceil cultures (Wigand et al., 1965). On the other hand, neutralization of cytotoxin displays a pattern of specificity different from that in neutralization of virus infectivity (Daring et al., 1972). The question is of practical importance, namely whether sensitivity and specificity of adenovirus neutralization are influenced by the type of cell culture, whether they are different in systems with abortive virus multiplication, or in permissive cells, when virus multiplication is inhibited by a DNA inhibitor. Is this indeed a ‘cytotoxin neutralization’? These questions were studied by comparative neutralization of adenoviruses and their cytotoxins by homologous and selected heterologous adenovirus antisera in various cell cultures. Adenovirus 5 (Ad5) and adenovirus 11 (Ad1 1) were chosen as members of subgroup C (III) or B (I) respectively. They differ in a characteristic manner in the specificity of neutralization of virus and toxin respectively (Doring et al., 1972): Ad5 toxin, but not the virus, is neutralized by all three heterologous antisera of the same subgroup (Adl, 2,6). Ad1 1 toxin is neutralized by Ad3,7, and 14 antisera, while Ad1 1 virus shows some neutralization by Ad14 and Ad21 antisera of the same subgroup (Wigand et al., 1965). Although the virus components producing early cytopathic changes and cell detachment do not destroy the cells (Pereira, 1958) we refer to them in the present paper under the term ‘cytotoxin’. METHODS

Cell cultures HeLa and Vero cell cultures were prepared in Eagle’s MEM, enriched with 4% lactalbumin hydrolysate and 5% calf serum. Human diploid fibroblast cultures (Flow 2002 and MRCS) were obtained from Flow Laboratories; they were processed to secondary tube cultures. Primary rabbit kidney and mouse kidney cultures were prepared by standard procedures from kidneys of 10 day old rabbits or Swiss mice respectively. For cell lines and tibroblasts, tube cultures were seeded with cells sufficient to grow to a confluent monolayer within 3-4 days. Primary or secondary kidney cultures grew to monolayer in about 7 days. The maintenance medium (1 ml/tube) contained 2% calf serum or, for fibroblast cultures, 2% fetal bovine serum.

323

Viruses Prototype

strains of human

in HeLa cell cultures.

adenovirus

Concentrated

5 (Ad75) and 11 (Slobitski)

virus stocks were obtained

were propagated

from packed cells by

repeated freezing and thawing. Viruses were stored in aliquots at -20°C. Purification procedures For the preparation of purified virus particles concentrated material was centrifuged to remove cellular debris; it was then centrifuged onto a cushion of cesium chloride with a density of 1.4 g/ml in a Spinco LI16.5 centrifuge (at 19,000 r.p.m. for 1 h in a rotor SW40). The virus bands at the interface were resuspended in phosphate-buffered saline (PBS) and treated with an equal amount of Frigen 113. After a second centrifugation onto a CsCl cushion, the virus bands were centrifuged in an isopycnic CsCl density gradient (31,000 r.p.m. for 20 h, rotor SWSO). The virus band was harvested and dialyzed against PBS before use. The supernatant of the first cushion centrifugation was recentrifuged under the same conditions. The second supematant, which contained only minimal infectivity, was used as crude cytotoxin. For purification, this material was adsorbed to and eluted from DE1 1 (Whatman) columns at pH 7.2 as described earlier (Gelderblom et al., 1968). The material was free of cellular proteins, as detectable by complement futation with anticellular antiserum. Titration of infectious virus and cytotoxin Relative infectivity titres of crude and purified

virus were determined

by inoculating

each of two HeLa or other tube cultures with material diluted in 4-fold steps and observing the cytopathic effect (CPE) for 4 days. Cytotoxic activity of crude or purified toxins was determined and titrated in tube cultures after dilution in 2-fold steps and read 1 and 2 days post inoculation (p.i.). Test for virus multiplication To determine

whether

or not adenovirus

was replicated

in non-human

cell cultures,

culture tubes showing maximal CPE were drained, washed twice with 2 ml of Hanks’ solution, and supplied with fresh medium, in order to remove the inoculated virus. The cultures were then frozen and thawed twice, inoculated into HeLa cell cultures, and observed for 6 days for the appearance of CPE. Antisera Standard adenovirus rabbit antisera, prepared by repeated intravenous inoculation of unpurified virus without adjuvants, were used. One Ad5 antiserum was prepared by immunization with purified virus particles. Neutralization After titration of crude or purified virus in the respective cell cultures, a modified version of the Rowe procedure (Rowe et al., 1958a) was applied for neutralization. A

324

virus concentration

was chosen in which the CPE started

reached a level between cultures

2+ and 4t (complete)

used. Virus and antiserum

min at 37°C; examined

daily

thereafter,

to appear l-2

4 days p.i., depending

were mixed in equal amounts

two cell culture

tubes were inoculated.

for CPE, and the final reading was performed

days p.i. and

on the kind of cell

and incubated Culture

for 60

tubes were

2 days after the virus

controls had shown a definite, albeit slight CPE. This was mostly between 3 and 5 days p.i. Antisera were diluted in 4-fold steps, and lack of CPE or minimal (+) CPE was judged to signify the neutralization endpoint. For the neutralization of cytotoxin, the antigen was used with 4 toxin units, the sera were diluted in 2-fold steps, and tests were read 1 and 2 days p.i. Because of the importance al., 1972) a concomitant

titration

of the antigen

of the cytotoxin

concentration

used (Doring et

was always carried out.

To demonstrate cell detachment (Rowe et al., 1958b) by cytotoxin, the respective cell culture tubes were shaken vigorously, in comparison with uninoculated control cultures. A gross detachment of the cell sheet from the glass was readily discernible. Neutralization with IUdR-inhibited virus IUdR at a concentration of 20 pg/ml inhibits the formation of infectious virus, while capsid proteins are formed and the CPE proceeds normally (Wigand and Schmieder, 1973). HeLa tube cultures were inoculated with virus/serum mixtures; at 4 h p.i. the medium was removed and the tubes were washed twice with 2 ml of Hanks’ solution. Thereafter, 1 ml maintenance medium per tube was added, with or without 20 pg/ml IUdR. Neutralization was observed as usually. Irnmunofluorescence neutralization Neutralization was performed with crude or purified virus diluted to contain about 104-10’ f.f.u. per 0.2 ml. Virus/antiserum mixtures were incubated as stated above, inoculated into two Leighton HeLa culture tubes and incubated for 3 days. Indirect immunofluorescence was carried out according to established procedures (Kron et al., 1974). F.f.u. were calculated from 100-200 microscopic fields. A lo-fold or higher reduction of f.f.u. was considered as evidence for neutralization. RESULTS Virus susceptibility of various cell cultures (Table 1) A cytopathic effect was seen with both Ad5 and Ad11 in all cell cultures studied. A similar CPE was produced by crude and purified virus. Short-term titrations showed no major difference in susceptibility between the cell cultures, although the progression of CPE was different from one cell culture to the other. Hence, similar virus dilutions could be used for neutralization tests in various cell cultures. While both viruses multiplied readily in all human and in Vero cell cultures (Hasler and Wigand, 1978) no multiplication was found in cercopithecus and mouse kidney cells. Only Ad5 showed a multiplica-

325 TABLE

1

Susceptibility

of various

cell cultures

to virion and toxin

of adenoviruses

Cell

virus

Effect

culture

type

CPE

multiplication

CPE

cell

HeLa

5 11 5 11 5 11 5 11 5 11 5 11

+ + + + + + + + + + f +

+ + + + + +

+ +

+ + f +

+

+

Diploid fibroblasts Vero Cercopithecus kidney Rabbit kidney

Mouse kidney

tion in rabbit

kidney

of virion

5 and 11

Effect

detachment

+

cells. This was found in accordance

Pereira (1957) who observed multiplication group as Ad 11, in rabbit kidney cells.

of toxin

with the results of Kelly and

of Ad5, but not of Ad7 of the same sub-

Susceptibility of cell cultures to cytotoxin (Table 1) Cytotoxin-containing preparations of both viruses showed a cytopathic effect as well as cell detaching in HeLa cells, but only detaching activity in fibroblast cultures. Surprisingly, only Ad11 toxin was found to be active in Vero cells, while in the other three cell cultures neither of the cytotoxins produced any visible change or detachment. The negative effect in these cultures was confirmed by inoculating them with 50 or 10 toxin units for Ad5 or Ad1 1 respectively as determined in HeLa cells. Crude and purified cytotoxins showed the same behavior. Neutralization of adenovirus 5 (Table 2) Both homologous antisera showed similar neutralization titres with crude and purified virus in all cell cultures studied; also the IUdR-inhibited Ad5 showed the same neutralization pattern in HeLa cells. The heterologous antisera from the same subgroup showed some neutralization in rabbit kidney and occasionally a delay of CPE in the other systems, when an 1 : 5 dilution was applied. Ad5 toxin was neutralized in high titre by all three heterologous antisera of the same subgroup, in accordance with earlier observations (Doring et al., 1972).

326

TABLE

2

Homologous cultures

and

heterologous

neutralization

of adenovirus

5 (virus

and cytotoxin)

in various

cell

or conditions

Cell culture

Antigena

HeLa

w

Neutralization

with antiserum

Adl(160)

640

1280

_d

2560

cru

640

HeLa (IUdR)

CN

640

2560

MRC-5

pur

640

5 120+

Vero

prepared

Ad5(2)b

Ad5(1)

CIX

160

640

pur

640

5 120+

CIU

640

2560

pur

160

2560

pur

640

2560

against Ad2(80)

Ad6(1 280)c

10

10

10

Cercopithecus kidney Rabbit kidney Mouse kidney

5120+

pur

2560

HeLa

pur

640

2560

5

5

5

Immunoflu.

CN

640

2560

5

5

5

Hela

toxin pur

640

640

160

160

160

CN

640

640

160

160

160

a

Purified

b

Antiserum

’

In parentheses

d

-islessthan1:5.

-

or crude

respectively.

Ad5(2)

was prepared

homologous

against

purified

neutralization

titre.

virus.

Neutralization of adenovirus 11 (Table 3) Homologous

neutralization

titres were in the same range in all cell cultures,

except

for Vero and rabbit kidney cells with crude virus. Furthermore, a weak cross-reactivity was seen with both Ad14 antisera and with one of two Ad21 antisera in almost all systems including immunofluorescence, while Ad3 and Ad7 antisera of the same subgroup failed to show any neutralization. For Ad1 1 toxin, which could be tested in both HeLa and Vero cells, fairly low homologous neutralization titres were observed. Type 3, 7, and 14, but not Ad21 antisera showed cross-reactions, which again was found similar in previous studies (Doring et al., 1972). Immunofluorescence neutralization The immunofluorescence neutralization

procedure

is more laborious than the conven-

327

TABLE

3

Homologous cultures

and heterologous

neutralization

of adenovirus

11 (virus

and cytotoxin)

in various

cell

or conditions

Cell

Antigen

culture

Neutralization

with antiserum

prepared

Ad11

Ad11

Ad3

Ad7

(1)

(2)

(640)

(320)

against Ad14 (1) (160)

Ad21 (2) (160)

(1) (320)

(2) ( 160)a

pur

640

160

-

-

(5)b

-

-

(5)

CIU

320

80

-

-

(5)

(5)

-

(5)

HeLa (IUdR) Flow 2002

CN

pur

320 640

80 160

-

-

(5) (5)

(5) -

-

(5) 10

CN

640

160

-

-

(5)

(5)

-

(5)

Vero

pur

320+

160

-

-

(5)

(5)

-

(5)

CN

40

20

-

-

-

(5)

-

(5)

5

-

5

(5) 20

5

5

5

20

HeLa

Rabbit kidney HeLa

pur

1280

20 320

-

-

Immunoflu.

CN

320

80

-

-

20

5

10

10

10

10

-

40

10

10

10

10

10

-

-

30

5

(5)

5

5

10

-

-

40

10

10

10

10

40

-

-

CN

w CN

Vero

toxin

pur CN

a

In parentheses

b

(5) = significant

homologous

neutralization

delay ofCPE

titres.

by 1 : 5 diluted

serum.

tional tube method. Hence, it does not lend itself to routine use. The method appears to be suitable, however, to investigate subtle antigenic relationships between virus types. For example, the relation between

subgroup

C viruses (Ad 1,2,5,6),

only irregularly

seen by

the CPE inhibition, is clearly shown by immunofluorescence (Fig. l), even if the stringent criterium of a lo-fold reduction of f.f.u. is applied. For subgroup B, the antigenic relation between Ad1 1, 14, and 2 1, and the lack of relation of Ad1 1 to Ad3 and 7, is also clearly indicated

by immunofluorescence

(Fig. 2).

DISCUSSION

The neutralization of virus infectivity is essentially proceeding during the incubation period of virus and antibody, and the cell culture indicates what has happened during that step. The dilution by inoculation into the tissue culture tube may have some influence, but is identical for all kinds of cell cultures. Secondary effects after the inoculation of

328

5rn

Antiserum 50) ’

Type L-

Antiserum Type 32 14(l) 21(21 I(1 1

1

-c

r4

d

3-

I? LL 2CF P

Fig. 1. Immunofluorescence Results

with purified

neutralization

virus. Nos. in columns:

Fig. 2. Immunofluorescence

neutralization

of adenovirus reciprocal

1 -

5 by homologous

and heterologous

antisera.

serum dilution.

of adenovirus

11. Figures

in parentheses

correspond

to

those in Tables 2 and 3.

virus-antibody complexes onto the cell cultures are probably of minor importance. Although it is conceivable that a virus-antibody complex is still infectious for one kind of cells and not for another (Kjellen and Schlesinger, 1959), our findings militate against such a possibility in adenoviruses. In fact, homologous neutralization titres as well as the specificity of neutralization were identical within the experimental error in almost all cell cultures tested for Ad5 and mostly so for Ad1 1, irrespective of whether or not the cells permit virus multiplication or whether the virus multiplication was inhibited by a DNA inhibitor. We conclude that the virus function(s) which is blocked by antibody appears to be the same for the replicative

cycle in infection

and for the initiation

of the

abortive infection in non-permissive cells. This function appears to be similar for monkey and rodent cells, although the mechanism of the abortive infection may be different in these systems (Philipson et al., 1975). It appears that in several systems the homologous neutralization titres are somewhat higher in purified This may be explained by the presence of soluble viral amount to at least 80% of total virus proteins synthesized expected to compete with virus particles for antibody

as compared with crude virus. proteins in crude virus, which (White et al., 1969). These are molecules during the neutrali-

zation period. The similarity of neutralization titres and specificity in various cell cultures is advantageous for practical purposes. It would probably be possible to standardize neutralization titres obtained in different laboratories by providing reference antisera, even if different kinds of cell cultures are used. Thus, the sensitivity and specificity of neutralization in conventional tube culture tests appear to be sufficiently reliable to be used as the basic for classification of virus species. HeLa cell cultures serve as well for neutralization as the more expensive monkey kidney

329

cell cultures, with

and instead of the tube procedure

comparable

proposed

results

(Tan

and Wigand,

by Rowe et al. (1958a)

than systems which require these conditions

microneutralization

may be carried out

1979). A short-term

and similarly

procedure

like that

used in this study is more expedient

7 or more days of incubation

(Wadell, 1972). Even under

a virus dose of 100 TCIDse cannot be provided.

It is of interest that the cytotoxin

showed no effect whatsoever in monkey

and rodent

kidney cells, which may be due to a lack of receptors on these cells. The immunological specificity of cytotoxins in Ad5 and 11 was found earlier to be different from that of infectious virus (Doring et al., 1972); this has been confirmed in the present study. While the ‘early CPE’ produced by the cytotoxin is maximal at 24 h p.i. and then decreasing, the CPE due to virus particles is progressive even in non-permissive cultures. Hence, the adenovirus neutralization in monkey cells and in other non-permissive cells cannot be due to toxin neutralization, as proposed by Hierholzer and Bingham (1978) but rather to neutralization of the virion, which is no longer able to produce a CPE. This has unequivocally been demonstrated by a) the pattern of cross-reactivity in neutralization, b) the progression of CPE, and c) the failure of cytotoxin to produce a CPE in these cells. Difficulties in virus identiticaton in human cell lines sometimes arise in virus stocks producing or containing great amounts of cytotoxin, particularly in subgroup C viruses because of the subgroup-specific cross-neutralization of their toxins (Doring et al., 1972). This difficulty would probably be non-existent in cell cultures not susceptible either to early CPE, like human tibroblasts, or to cytotoxins in general (monkey kidney), or to the cytotoxin of subgroup C (Vero cells). A certain degree of cross-neutralization of infectious virus is found in some subgroup C antisera (Wigand et al., 1965). This is corroborated by the definite, albeit low-titred fluorescence of Ad5 (Fig. 1).

heterologous

neutralization

found in immuno-

ACKNOWLEDGEMENT

The diligent technical acknowledged.

assistance provided by Mrs. I. Maurer and D. Keller is gratefully

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