HAL Id: hal-00901391

HAL Id: hal-00901391

https://hal.archives-ouvertes.fr/hal-00901391

Submitted on 1 Jan 1982

HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ANTIGENIC VARIATIONS AMONG CALF DIARRHEA CORONAVIRUSES BY

IMMUNODIFFUSION AND

COUNTERIMMUNOELECTROPHORESIS

S. Dea, R.S. Roy, M.A.S.Y. Elazhary

To cite this version:

S. Dea, R.S. Roy, M.A.S.Y. Elazhary. ANTIGENIC VARIATIONS AMONG CALF DIARRHEA CORONAVIRUSES BY IMMUNODIFFUSION AND COUNTERIMMUNOELECTROPHORESIS.

Annales de Recherches Vétérinaires, INRA Editions, 1982, 13 (4), pp.351-356. �hal-00901391�

ANTIGENIC VARIATIONS AMONG CALF DIARRHEA CORONAVIRUSES BY IMMUNODIFFUSION AND COUNTERIMMUNOELECTROPHORESIS

S.

DEA,

R.S. ROY M.A.S.Y. ELAZHARY

Département

^de Patho%gie ^et Microbio%gie, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, Québec, Canada, J2S 7C6

Résumé

VARIATIONS

ANTIGÉNIQUES

CHEZ LES CORONAVIRUS DE LA

DIARRHÉE

DU VEAU

DÉMONTRÉES

PAR IMMUNODIFFUSION ET

CONTRE-IMMUNO-ÉLECTROPHORÈSE. -

Les

techniques

^{de contre-}

immuno-électrophorèse (CIE)

et d’immunodiffusion

(ID)

ont été utilisées _pour étudier les relations

antigéniques

^{existant entre}

cinq

isolats de coronavirus bovins. Par la

technique

^{de CIE deux}

antigènes précipitants

^ont été observés pour chacun des isolats. Un des

antigènes

^{identifié comme}

l’antigène

^{« M » s’avéra} commun aux

cinq

isolats tandis que le second

antigène,

^{identifié comme}

l’antigène

^{« m} » variait selon les isolats. Considérant ce deuxième type

d’antigène,

les isolats étudiés ont pu être

placés

^{dans deux} groupes

antigéniques,

les membres appartenant ^{au même} groupe étant indifférenciables pour les deux

antigènes.

^Des réactions d’identité

partielle

^ont été notées entre les virus des deux différents groupes par la

technique

d’immunodiffusion.

Members of the coronavirus group

naturally

infect domestic animals

causing

^{a wide}

variety

^of disorders

involving

^a number of different _organ systems

(Kapikian, 1974 ; McIntosh, 1974 ;

^Pen-

saert and

Callebaut, 1978).

In neonatal calves, their

multiplication

^{in the} intestinal tract induces

a severe diarrhea

syndrome

^{which may lead to}

death

(Stair

et al., 1972 ; Gouet et al.,

1978 ;

Dea et al.,

1981). ).

Studies on the

antigenic relationships

among various coronaviruses have demonstrated the

complex antigenic variability

which exists in this group. The transmissible

gastroenteritis

^virus

(TGEV)

^of

pigs

^{shows little or no}

antigenic

variation between isolates and is

antigenically

related to human coronavirus

229E,

canine

diarrheal coronaviruses and the feline infections

peritonitis

^virus

(Bradburne,

1970 ; Mcintosh,

1974 ; Reynolds

^et al.,

1977 ;

Pedersen et al.,

1978).

Different

serologic

types ^are

recognized

for infectious bronchitis virus

(IBV)

of chickens and for mouse

hepatitis

^virus

(MHV);

^the

hemagglutinating encephalomyelitis

^virus

(HEV-67N)

^of

pigs

^is

probably

^{related to both}

viruses and to calf and human OC-43 coronaviru-

ses

(Bury

^and

Stokes, 1968 ;

^{Tevethia and}

Cunningham, ^{1968 ;}

Bradburne,

1970 ; Cunning-

ham,

1970 ; Kaye

^et al.,

1977 ;

Pedersen et al.,

1978). Antigenic

variations have also been demonstrated among coronaviruses isolated in humans

(McIntosh

et al.,

1969 ; Bradburne, 1970 ;

Kapikian, 1974).

This

manuscript

reports ^the

comparison by counterimmunoelectrophoresis (CIE)

and immu- nodiffusion

(ID) techniques

^{of five calf coronavi-}

rus isolates

using homologous

^and

heterologous

sera.

Materials and Methods 1. Viruses

The FBK cell

culture-adapted

^{Nebraska calf}

diarrheal coronavirus

(NCDC) (Mebus

et al.,

1973)

was

kindly supplied by

Dr. Bass of Norden Laboratories, Lincoln, Nebraska. Four other bov- ine coronavirus isolates recovered from diarrheic calves in Quebec and

designed

^{as bovine}

coronaviruses

BCQ.1,

2,

3,

and 4

(Dea

et al.,

1980a, b)

^were also used in this

study.

^Infectious

bovine rhinotracheitis

(IBR)

virus strain Colorado, and bovine enterovirus type ^II

(BE-11),

^both

adapted

^{on Vero} cells, ^were used as controls.

2. Cell cultivation and virus

propaqation

Procedures for the

growth

and maintenance of Vero continuous cell cultures and for the

propagation

of the bovine coronavirus isolates in these cells ^were described in a

previous

report

(Dea

et al.,

1980b).

Infected cell cultures were

harvested when maximal

cytopathic

^effects

occurred ;

cells and culture medium ^were freez- ed-thawed three times, ^clarified

by centrifuga-

tion at 8 000 ^x _g for 20 min at 4 °C and stored at

- 70 °C or used

immediately

for cell inoculation.

In the present

study,

^the NCDC, ^BCQ.2 and BCQ.3 viruses were used at their 24th to 28th passages ; BCQ.1 and BCQ.4 were used at their 10th to 15th passages.

3.

Preparation

of coronavirus

antigens

Approximately

200 ml of Coronaviruses - infected Vero cell culture fluids were freezed- thawed three times and

centrifuged

^{at 5 000} x g for 30 min at 4 °C. The clarified

suspensions

were then concentrated to a volume of 10 ml

by

ultrafiltration

through

^a XM100A Diaflo mem-

brane

(Amicon

Co.,

Lexington, ^Mass.).

^The

concentrates were

ultracentrifuged (Model L5-65,

Rotor

T65,

Beckman Instruments Inc., Palo Alto,

Calif.)

^{at 100 000 x} g for three hours on the top ^{of a} cushion of 2 ml sucrose 30 %

(w/v).

Each viral

pellet

^was

suspended

^{in 1 ml of 0.05 M}

Tris-hydrochloride (pH ^8.0).

^{The presence of}

virus in the

pellet

^was then checked

by

^electron

microscopy.

^{For this} purpose, the

suspensions

were

negatively

^{stained as}

previously

^described

(Dea

et al.,

1979)

with 2 %

phosphotungstate

acid

pH

^6.5.

4. Antisera

production

Antiserum to each coronavirus isolate was

prepared

ⁱⁿ New-Zealand albino rabbits follow-

ing

the method used in a

previous study (Dea

et al.,

1979).

Before use, the antisera were inactiva- ted at 56 °C for 30 min and absorbed

against

bovine liver

powder (Difco

Lab,

Detroit, Mich.),

and Vero cells to eliminate the unwanted non-

specific reactivity.

Titration of

neutralizing

^anti-

bodies was conducted as described

previously (Dea

et al.,

1979)

in 96-well Microtest culture trays

(Falcon

Plastics,

Oxnard, Calif.) plated

^with Vero cells. The titers were

expressed

^{as the}

reciprocal

^{of the}

highest

^serum dilution neutraliz-

ing

¹⁰⁰

TCID 50 ⁽⁵⁰

^% tissue culture infective

doses)

of viruses.

5.

Counterimmunoelectrophoresis (CIE)

The test was

performed,

^{as described in a}

previous

report,

using

^Kodak

projector

^slides

coated with 1 % _agarose

prepared

^{in 0.025 M} Trisbarbital

buffer, pH

^8.6

(Dea

et al.,

1979).

Cathodic wells were filled with viral

antigens

diluted 1:5 in the buffer and immune sera were

placed

ⁱⁿ the anodic wells.

Electrophoresis

^was

conducted at a constant

potential

^{of 150 volts}

for 90 min at 4 °C. The slides were examined for the presence of a

precipitin

reaction after an

overnight

^{washed in 0.85 % NaCl and}

staining

for 15 min in 1 % tannic acid.

6. Immunodiffusion test

tID)

The test was carried out in 0.75 % Noble agar (Difco Lab., Detroit,

Mich.) prepared

^{in 0.15 M}

phosphate

^buffered

saline (PBSI, pH

^7.5.

Eight

^ml

of warm agar ^was

poured

^{into 60 x 15 mm}

petri-

dishes and allowed to

solidify.

^Patterns

usually

consisted of six

peripheral

^wells

(4-mm diameter) punched

^{at 4.5 mm from a} central well

(7-mm diameter).

For each

plate,

^the

peripheral

^wells

were filled with the

prepared

^antisera

(25 !1)

^and

the central well was filled with one of the coronavirus

antigens.

^The

petri-dishes

^were incubated at room-temperature in a humidified chamber and were examined for

precipitin

^arcs

at 24,

48,

72 and 96 hours over a narrow

oblique

beam of

light.

Results

Typical

coronavirus

particles

^{with an} average diameter of 120 nm and surrounded

by petal-

shaped projections

^{13 to 17 nm}

long

^were

visualized

by

EM in the

antigen suspension prepared

from NCDC-infected Vero cell culture fluid

(fig. ^1).

^{Similar results were obtained with}

the four other isolates. No other viruses were

observed in all cases.

The antisera

produced

^{in this}

study

^{had a}

neutralizing

^{titer of 160 to 320}

against

^the

homologous

viruses. In

preliminary experiments,

soluble

antigens

extracted from Vero cells infected with the different bovine coronavirus isolates ^were detected

by

the CIE and ID

techniques using

^their

homologous

^antisera.

Non-infected Vero cells

similarly

^{treated did not}

react

against

^{the same antisera nor did cells}

infected with IBR or BE-11 viruses. No

precipitin

reaction was noted with normal rabbit serum.

With the ID test, the

precipitin

reactions could be

readily interpreted

after 72-96 hour’s incuba- tion. The results obtained

by

^{CIE were}

usually

difficult to read

directly

^after

electrophoresis.

The immersion of reacted _agarose slides in 1 % tannic acid solution intensified

antigen-antibody complexes

within the agarose.

In order to

study

^their

antigenic relationships,

each of the 5 coronavirus isolates ^was reacted

by

^CIE

against

^{the different}

prepared

^antisera.

One or two

precipitin

lines could be observed between the

antigen

^and

antibody

^wells

depend- ing

^on the antiserum used. As illustrated in

figure

2, two

precipitin

^{lines were} detected for NCDC virus when tested

against anti-NCDC,

and anti- BCQ.1 sera, whereas

only

^one

precipitin

^line

appeared

^with anti-BCQ.3 and anti-BCQ.4 sera.

Two

precipitin

^{lines were} noted with anti-BCQ.2

serum

(not illustrated).

The results obtained

by

^CIE

using

^all

possi-

ble combinations of

antigens

and antisera

are

reported

ⁱⁿ table 1. Each virus when tested

against

^it

homologous

^antiserum

produced

^the

two

precipitin

^lines

designated

^as coronaviral

antigens

^{« M » and « m} »,

according

^to

migration

distance.

Antigen

^{« M » which}

migrated

^nearest

from the

origin

^{seems to be common to all}

isolates since it was

recognized by

^{each antise-}

rum when tested _{with any} of the coronaviruses studied. However, the « m

» antigen

^{was shown}

to differ _among the isolates as revealed

by

^their antisera. The

anti-NCDC,

anti-BCQ.1 and BCQ.2

serums

recognized

^{the « m}

» antigen

^of

^NCDC,

BCQ.1 and BCQ.2 viruses while not

reacting

^with

BCQ.3 virus. The « m

» antigen

of NCDC-virus

was not detected

by

the anti-BCQ.4 serum.

Only

anti-NCDC serum did not detect the ^{« m »}

antigen

of BCQ.4 virus. Furthermore, the anti- BCQ.3 serum revealed the two

precipitating antigens only

when reacted

against

^{BCQ.3 and}

BCQ.4 viruses.

The results obtained with the ID test demon- strated also that the 5 isolates studied have at least one common

precipitating antigen

^detect-

able

by

each antiserum. However,

despite

^the

fact that the _presence of other

precipitating

^anti-

gens ^was unclear,

antigenic

dissimilarities whprp noted among the isolates

by interchanging

^the

antisera

position

in the lateral wells. As illustrated in

figures

^{3 and}

4,

the results obtained with the antisera

suggested

^a

partial identity

^{between the} NCDC and the BCQ.3 viruses and between the BCQ.1 and BCQ.4 viruses; ^a reaction of total

identity

^was noted between the BCQ.3 and BCQ.4 viruses. From results which are not illustrated in this paper, the

NCDC,

BCQ.1 and BCQ.2 viruses were shown to be

indistinguisha-

ble.

Discussion

In this

study,

^two

precipitating antigens

^were

demonstrated

by

^{CIE in}

semi-purified

prepa- rations of bovine coronaviruses cultivated in Vero cell cultures. These results differ from those

published previously by Hajer

^{and Storz}

(1978)

who have

reported

^four

precipitating antigens

ⁱⁿ

the alkaline intestinal extract of bovine coronavi-

rus LY-138 infected-calves.

However, antigens

from

purified

LY-138 coronaviral

particles

^in-

duced

only

^three

precipitin

^lines.

Other

investigators

have described

multiple precipitating antigens ⁽²

^or

³⁾

^{in crude or}

purified preparations

^{of human}

(Bradburne, 1970 ;

Hier- holzer et al.,

1972 ; Hierholzer, 1976 ;

^Yaseen et al.,

1981 ;

Schmidt and

Kenny, 1981),

^avian

(Tevethia

and

Cunningham, 1968 ; Kaye

^et al.,

1970)

and

porcine (Mengeling, 1972 ;

Bohac et

al., 1975 ; ^{Bohac and}

Derbyshire, 1975)

coronavi-

ruses. In all these studies, the number of coronaviral

antigens

detectable was related to the extractive

procedures

^{or the viral concentra-} tion or the

sensitivity

of the ID and CIE

techniques employed.

The

antigen

identified in this

study

^{as the « M »}

coronaviral

antigen appeared by

^{CIE common}

to the five bovine coronaviruses tested. The second

antigen

^{named the « m}

» antigen

^was

shown to differ _among these isolates. Conside-

ring

^this

antigen,

the 5 isolates could be

placed

ⁱⁿ

two

antigenically distinguishable

groups. The first _group of viruses

(BCQ.1

and

BCQ.2)

were

related for both

antigens

^to the NCDC virus while the other _group

(BCQ.3

^and

BCQ.4)

differed from the NCDC virus

by

^{the « m}

» antigen.

In the ID studies, the presence of the two

precipitating antigens

^was uncleared. This differ-

ence in the number of

precipitin

^{lines observed}

by

^{ID and} CIE have also been

reported

^with

the TGE virus of

pigs ^(Bohac

^{et al.,} 1975 ; ^Bohac and

Derbyshire, 1975).

Reactions of

partial identity

^were however noted between the two groups of viruses identified

by

^{CIE. The}

viruses from the same group ^were shown to be

indistinguishable.

^This type of reaction suggest- ed the _presence of

multiple antigens

^{in which at}

least one is common to all the isolates.

Considering

^{that in a}

previous study

^{it was}

demonstrated that the

growth

in cell cultures of the five isolates could be neutralized

by

^{each of}

the antisera used in the present

study (unpublish-

ed

data),

^we cannot confirm yet ^the presence of different serotypes of bovine coronaviruses.

Further studies will be done to

identify correctly

the coronaviral

antigens

described in this

study

and to understand the

antigenic

differences observed.

Accepted

^for

publication,

^{26 October 1982.}

Summary

The

antigenic relationship

among five bovine coronavirus isolates ^was studied

using

^the

counterimmunoelectrophoresis (CIE)

^and immunodiffusion

(ID) techniques. By

^{CIE, two}

precipitat- ing antigens

^were observed for each coronavirus isolate. One

antigen

identified as the « M »

coronaviral

antigen

^{was found to be common to the} five isolates while the second

antigen

^identified

as the « m » coronaviral

antigen

differed between the isolates.

Considering

^{the later}

antigen,

^the

isolates studied were

placed

^{in two}

antigenic

groups, members of the ^same group

being indistinguishable

^{for the two}

antigens. By ID,

reactions of

partial identity

^were noted between viruses of the different _groups.

References

BOHAC J., DERBYSHIRE J.B., THORSEN J., 1975. The detection of transmissible gastro-enteritis ^viral antigens by immunodiffusion. Can. J. Comp. Med., 39, 67-75.

BOHAC J., DERBYSHIRE J.B., 1975. The demonstration of transmissible gastroenteritis ^viral antigens by immunoelectrophoresis ^and counterimmunoelectrophoresis. Can. J. Microbiol., 21, 750-753.

BRADBURNE A.F., 1970. Antigenic relationships among coronaviruses. Arch. Ges. Virus Forsch., 31, 352-364.

BURY D.M., STOKES K.J., 1968. Antigenic variations in isolate of infectious bronchitis virus. Vet. Rec., 82, 157-160.

CUNNINGHAM C.H., 1970. Avian infectious bronchitis. Adv. Vet. Sci., 14, 105-148.

DEA S., ^ROY R.S., BEGIN M.E., 1979. Counterimmunoelectroosmophoresis for detection of neonatal calf diarrhea coronavirus : methodology ^and comparison with electron microscopy. J. Clin. Microbiol., 10, 240-244.

DEA S., ROY R.S., BEGIN M.E., 1980a. Physicochemical ^and biological properties of neonatal calf diarrheal coronaviruses isolated in Quebec and comparison with Nebraska calf coronavirus. Am. J. Vet. Res., 41, 23-29.

DEA S., ROY R.S., BEGIN M.E., 1980b. Bovine coronavirus isolation and cultivation in continuous cell lines. Am. J.

Vet. Res., 41, 30-38.

DEA S., ROY

R.s :

ELAZHARY M.A.S.Y., 1981. La diarrh6e n6onatale due au coronavirus du veau. Une revue de la litt6rature. Can. Vet. J., 22, 51-58.

GOUET Ph., CONTREPOIS M., DUBOURGUIER H.C., RIOU Y., SCHERRER R., LAPORTE J., VAUTHEROT J.F., COHEN J., L’HARIDON R., ^{1978. The} experimental production of diarrhea in axenic and gnotobiotic calves with enteropathogenic ^E. coli, rotavirus, coronavirus and in a combined infection of rotavirus and E. coli. Ann. Rech.

V

et., 9, 433-440.

HAJER L, STORZ J., 1978. Antigens of bovine coronavirus strain LY-138 and their diagnostic properties. ^{Am. J.}

Vet. Res., 39, 441-444.

HIERHOLZER J.C., PALMER E.L., WHITFIELD S.G.. KAYE H.S., DOWDLE W.R., 1972. Protein composition ^of coronavirus OC-43.

Viro%gy,

^{48, 516-527.}

HIERHOLZER J.C., 1976. Purification and biophysical properties of human coronavirus 229 E.

Viro%gy,

75, 155-165.

KAPIKIAN A.Z., 1974. The coronaviruses-International Symposium ^on immunity ^to infections of the respiratory system in ^man and animals. Develop. Biol. Standard., 28, 42-64.

KAYE H.S., HIERHOLZER J.C., ^DOWDLE W.R., 1970. Purification and further characterization of an « IBV-like ^» virus (coronavirus). Proc. Exp. ^Biol. Med., 135, 457-463.

KAYE H.S., YARBROUGH W.B., ^REED C.J., HARRISON A.K., 1977. Antigenic relationship ^{between human} coronavirus strain OC-43 and hemagglutinating encephalomyelitis ^virus strain 67N of swine : antibody

reponses in human and animal sera. J. lnfect Dis., 135, ^201-209.

McINTOSH K., KAPIKIAN A.Z., HARDISON K.A., HARTLEY J.W.. CHANOCK R.M., 1969. Antigenic relationship among coronaviruses of ^man and between human and animal coronaviruses. J. /mmuno/., 102, 1109-1118.

McINTOSH K., 1974. Coronaviruses, a comparative review. Curr. Top. Microbiol. Immunol., 63, 85-129.

MEBUS C.A., STAIR E.L., RHODES M.B., TWIEHAUS M.J., 1973. Neonatal calf diarrhea : propagation, attenuation and characteristics of a coronavirus-like agent. Am. J. Vet Res., 34, 145-150.

MENGELING W.L., 1972. Precipitating antigens ^of hemagglutinating encephalitis virus demonstrated by agar gel immunodiffusion. Am. J. Vet Res., 33, 1527-1535.

PEDERSEN N.C., WARD J., MENGELING W.L., 1978. Antigenic relationship of the feline infectious peritonitis ^virus

to coronaviruses of other species. Arch. Virol., 58, 45-53.

PENSAERT M., CALLEBAUT P., 1978. The coronaviruses: clinical and structural aspect with ^some practical implications. Ann. Med. Vet, 122, 301-322.

REYNOLDS D.J., GARWES D.J., GASKELL G.L., 1977. Detection of transmissible gastroenteritis ^virus neutralizing antibody ^{in cats.} Arch. Virol., 55, 77-86.

SCHMIDT O.W., KENNY G.H., 1981. Immunogenicity ^and antigenicity ^{of human} coronavirus 229E and OC43. Infect.

lmmun., 32, 1000-1006.

STAIR E.L., RHODES M.B., WHITE R.G., ^MEBUS C.A., 1972. Neonatal calf diarrhea ^- Purification and electronmicroscopy ^{of a} coronavirus-like agent. Am. J. Vet. Res.. 33, 1147-1156.

TEVETHIA S.S., CUNNINGHAM C.H., ^1968.

Antigenic

characterization of infectious bronchitis virus. J. lmmunol., 100, 793-798.

YASEEN S.A., JOHNSON-LUSSENBURG M., 1981. Antigenic ^{studies on} coronavirus. I. Identification of the structural antigens ^{of human} coronavirus, strain 229E. Can. J. Microbiol., 27, 334-342.