An immunological method to determine antigenic variation in three strains of myxoma virus

Journal of Virological Methods, S (1982) Elsevier Biomedical

21- 26

21

Press

AN IMMUNOLOGICAL

METHOD TO DETERMINE ANTIGENIC VARIATION

IN

THREE STRAINS OF MYXOMA VIRUS

H.G. BULTS’

and M.R. BRANDON’*

‘Division of Wildlife Research,

CSIRO, P.O. Box 84, Lyneham,

2602; and ‘Department

of Immun-

ology, John Curtin School of Medical Research, Australian National University, Canberra 2601 A.C.T., Australia (Accepted

18 March 1982)

A technique

is described

Radioimmunoassays show that the soluble suggesting uished.

that

antigens

rabbits

Antigen

for the purification

were used to study

isolated

infected

isolated with

of soluble

the antigenic from

Lu and G.V. strains

Lu and G.V. strains

from the U.F.S.

antigens

differences

strain

of three between

do not cross-react

of the myxoma

cross-reacted

strains of myxoma the three

strains.

virus. Results

in rabbits

tested,

virus can be readily

disting-

with sera from both Lu- and G.V.-infected

rabbits.

myxoma

virus strains

soluble

antigens

radioimmunoassay

INTRODUCTION

The first successful

release of myxoma

virus into populations

of feral rabbits (Olyc-

to&-us cunicuhs (L.)) in Australia was in 1950 at Corowa, N.S.W. (Ratcliffe et al., 1952; Myers, 1954). The initial release was of a highly virulent strain of the virus (Fenner and Marshall, 1957) but attenuated strains now predominate in the field, resulting in a sharp decline in the mortality rate (Fenner and Woodroofe, 1965). Strains causing present epizootics are unidentifiable and it is not known whether the same strains cause subsequent outbreaks in particular areas. Biochemical methods have been sought as an alternative to the biological assay system using test rabbits (Fenner and Marshall, 1957) as a means of measuring virulence. To this end, Reisner et al. (1963) examined the soluble antigens produced by seven strains of myxoma viruses from Brazil and two from California. Serological differences were found between antigens from the different continents but not between those from the same regions. These results were later confirmed by Fenner and Woodroofe (1965).

* Present

address:

3052, Victoria,

Department

of Veterinary

Preclinical

Sciences,

University

Australia.

0166-0934/82/0000~0000/$02.75

@ 1982 Elsevier Biomedical

Press

of Melbourne,

Parkville,

22

Soluble

antigens

obtained

Brazil were examined Glenfield

(Fenner

genie differences

from three strains of myxoma

virus which originated

in this study. These were Lausanne (Fenner

in

and Marshall, 1957)

and Marshall, 1957), and Urana Field Strain (Parer et al., 1981). Antiwere sought among the soluble antigens produced by these three strains,

using affinity chromatography to purify the soluble antigens and isolate specific antibodies to these antigens. A radioimmunoassay was used to study antigenic differences between the three strains. MATERIALS

AND METHODS

Virus Lausanne Laboratories

(Lu) and Glenfield (G.Y.) strains were supplied by Commonwealth Serum (Melbourne , Victoria, Australia) and the Urana Field Strain (U.F.S.) was

isolated from wild rabbits on a property

25 km west of Urana, N.S.W., Australia.

Preparation of soluble antigens Live virus of the Lu and G.V. strains was injected intradermally at 20 sites on the shaven backs of domestic rabbits which were kept isolated from all other rabbits in a small mosquito-proof animal house. After 6-7 days skin lesions were collected, cut into small fragments and stored at -10°C in 0.2 M phosphate buffer, pH 8.0, until required. Skin lesions packed in ice were homogenised for 5 mm in a Virtis 45 homogemser and then sonically disintegrated for 2 min with a Branson B12 sonifier. After sonification each preparation was centrifuged at 24,000 X g in a Spinco refrigerated ultracentrifuge for 20 min at 4°C. The supernatant containing the soluble antigens was stored at -10°C. Rabbits were shot on a grazing property 25 km west of Urana during an epizootic in December, 1979, and following an epizootic in January, 1980. The U.F.S.-soluble antigen was isolated from the eyelids and anal regions of infected rabbits of the December, 1979, and sample and serum prepared from the blood of surviving rabbits from the January, 1980, collection. All material was transported to the laboratory at -10°C. Pveparation of antisera Domestic rabbits which had previously been immunised with live fibroma virus (to give partial immunity to myxoma virus) were injected with large doses of live Lu or G.V. virus and given booster injections at weekly intervals for 3 weeks. Twenty millilitres of blood were taken twice a week from the marginal ear vein and serum prepared. Preparation of antibodies to viral antigens Crude soluble

antigen

preparations

from the three

strains

of myxoma

virus were

23

coupled

to CNBr-activated

Sepharose 4B (Pharmacia

as follows. Six grams of CNBr-activated

Fine Chemicals, Uppsala, Sweden)

Sepharose 4B were washed with 2 litres of 1mM

HCl at room temperature on a 15 cm diameter sintered glass funnel. The washed Sepharose 4B was added to 50 ml of crude soluble antigen together with 35 ml of 0.1 M NaHCOa buffer in 0.5 M NaCl at pH 8.3. This mixture was gently stirred at 4°C overnight using a multipurpose rotator (Model 15 1, Scientific Industries Inc., Bohemia, NY) at 20 r.p.m. in a 200 ml round bottom flask. After coupling, the gel was washed with 0.1 M NaHCOa buffer in 0.5 M NaCl until the absorbance of the supernatant was less than 0.003 at 280 nm to ensure the removal of unbound protein. Any remaining active groups on the gel were blocked by adding 10 ml of 0.1 M ethanolamine @H 8 5) and rotating for 2 h at room temperature. The gel was then washed alternately with 0.1 M acetate buffer in 0.5 M NaCI, pH 4.0, and 0.1 M borate buffer in 0.5 M NaCl, pH 8.5, for a total of six times. All buffer solutions used were at room temperature. The gel was then stored in 0.2 M Tris-HCl buffer in 0.5 M NaCI, pH 8.0, in equal quantities in six 20 ml quick fit test tubes. Six millilitres of serum containing antibodies were added to the crude soluble antigens coupled to 6 g of CNBr-activated Sepharose 4B gel. After gently rotating the mixture for 2 h to facilitate coupling of the antibodies to the soluble antigen, it was centrifuged and the supernatant discarded. Following six washings with 0.2 M Tris-HCl buffer in 0.5 M NaCl, pH 8.0, or until the absorbance of the supematant was less than 0.020 at 280 nm, 60 ml of 2 M NaSCN, pH 8.0, were added and gently rotated at 4°C for 30 mm. The supernatant containing the purified antibodies was immediately dialysed against 2 litres of 0.01 M phosphate buffer in 0.9% NaCl, pH 8.0. After dialysis the antibodies were concentrated to 5 ml in an Amicon stirred cell (Amicon Corporation, Lenington, MA) fitted with a PM10 membrane.

This was repeated for each of the strains of virus.

Purification of soluble antigens Twelve milligrams of the antibodies isolated by affinity chromatography to each of the three virus strains were coupled to six grams of CNBr-activated Sepharose 4B. Six millilitres of the respective crude soluble antigen were then added to the gel containing specific antibodies and purified antigens eluted in a similar manner to that described for obtaining viral antibodies. After concentrating in an Amicon stirred cell the antigens were suitable for labelling with 12’I. Iodination of soluble antigens Purified soluble antigens of the Lu, G.V. and U.F.S. strains were labelled with “‘1 (The Radiochemical Centre, Amersham, U.K.), carrier free in NaOH solution at pH 8-l 1, 100 mCi/ml) by the chloramine T procedure (Hunter and Greenwood, 1962). Briefly, 6 pg of purified antigen were dissolved in 75 ~1 of 0.05 M phosphate buffer of pH 7.6. To this were added 200 pg of 12’1, 100 pg of chloramine T in 25 /*I of distilled Hz0 followed

24

immediately by 280 /+I of sodium metabisulphite dissolved in 100 ~1 of distilled H,O. The reaction was carried out in a small stirred cell. A G-25 Sephadex column (1.0 X 20.0 cm) was washed with 2 ml of normal rabbit serum before the reaction mixture was passed through the column. The labelled antigen that was collected in one ml fractions had a specific activity of between 10 and 20 &i/pg.

Antigen binding assay The ‘251-labelled soluble antigens were diluted in 0.01 M borate buffer, pH 8.4, containing 1% normal rabbit serum to a concentration of 37.5 ng per 2.5 ~1. Twenty-five microlitres of test antisera were serially diluted with neat normal rabbit serum to a dilution of l/32. All tests were run in duplicate. To each well of a V-bottom microtitre tray (Cooke Engineering Co., Alexandria, VA), containing the serially diluted antisera were added 25 ~1 of the 1251-labelled antigen solution. After incubation for 1 h at 37°C and overnight at 4”C, 25 ~1 of a sheep anti-rabbit IgG serum were added to each well followed by a second incubation for 4 h at 4°C. The mixture was centrifuged at 4°C and 50 ~1 of supernatant were removed for counting in a Packard automatic well-type gamma scintillation counter. All results are expressed as the percentage of total radioactivity bound in complexes at a serum dilution of l/2. Positive rabbit sera had titres between l/4 to l/16. Non-specific binding of antigens by normal rabbit serum was less than 3%. RESULTS

Representative antisera against the three strains of myxoma virus were tested by radioimmunoassay with radioiodinated soluble antigens of Lu, G.V. and U.F.S. From Table 1 it can be seen that the soluble antigen prepared by affinity chromatography from the G.V. strain possesses unique antigenic determinants; however, both Lu and U.F.S. strain antibodies recognise the soluble antigens isolated from the Lu strain of virus. The soluble antigen isolated from the U.F.S. strain reacted with both Lu and G.V. sera, whichsuggests that in the preparation of U.F.S.-soluble antigen no unique antigenic molecules were isolated and soluble antigens in common with Lu and G.V. made up the U.F.S. strain of soluble antigen used here. The soluble antigens isolated from G.V. and Lu strains are clearly distinguishable by radioimmunoassay (show no cross-reactivity in the rabbits tested), and by using these antigens it should be possible to distinguish rabbits infected with G.V. and Lu strain myxoma virus. The observation that the test antisera for each strain of virus did not behave similarly is most likely due to the heterogeneous nature of the immune response to the virus by individual rabbits. DISCUSSION

Earlier less sensitive

serological

techniques

(Reisner

et al., 1963; Fenner

and Wood-

25

TABLE

I

Reaction

of ‘ZSI-labelled

infected

with the different

soluble strains

antigens

from

of myxoma

the Lu, G.V. and U.F.S.

‘ZSI-labelled

Test antisera

strains with sera from rabbits

virus soluble

antigen

Lu (% of total radioactivity

G.V. bound

U.F.S. in complexesa)

Control

1.3

2.3

2.2

G.V. 31

2.4

1.5

26.28

G.V. 38

1.2

30.36

2.4

G.V. 39

1.1

2.3

37.39

Lu 3

16.20

1.2

3.4

Lu 4

43.45

2.3

40.46

Lu 405

20.24

3.3

2.4 38.40

U.F.S.

103

1.5

2.3

U.F.S.

104

34.34

2.3

1.3

U.F.S.

109

39.41

1.4

2.4

U.F.S.

128

17.21

3.4

2.3

a

% Non-specific

binding

not subtracted.

roofe, 1965) were unable to differentiate between myxoma virus strains of South American origin. In this study it was possible to isolate soluble antigens from two strains of myxoma virus that appear antigenically distinct. This distinction is best highlighted by the G.V. and Lu strains of virus. Table 1 shows that the G.V.-soluble antigens are only recognised by serum from a rabbit infected with G.V. virus, whereas sera from rabbits infected with Lu strain virus did not recognise the soluble antigens prepared from the G.V.

strain. Sera from all of the Lu-infected

rabbits recognised the Lu-soluble

antigens but not

those from the G.V. strain. These results suggest that it should be possible to distinguish between different virus strains. That the U.F.S. strain virus shares antigenic determinants with the Lu strain suggests that U.F.S. may have been derived from the Lu strain which was released in 1954 10 km form the collection site of U.F.S. (Fenner et al., 1957) and again in 1977, 1978 and 1979 (Parer, I., personal communication). Further support for this is provided by the reaction characteristics of rabbit serum Lu 4 (Table 1). This serum recognises with equal avidity the Lu- and U.F.S.-soluble antigens. It was expected that G.V. and U.F.S. would be closely related as they were both collected from the field in Australia. Further testing of field strains is needed to determine whether other field strains from areas where Lu has not been released show affinity to Lu. Large areas of central N.S.W. and northern Victoria have annual summer epizootics and the source of the virus is unknown (Fenner and Ratcliffe, 1965). Long-distance mosquito transmission is one possibility, but the virus could be of local origin (Williams et

26

al., 1972). If field strains from the annual epizootics may be possible to determine

at selected sites are tested, then it

whether local antigen& variants persist from year to year

or whether the same virus is present over large areas. Recent studies by Wiktor and Koprowski (1980) have shown that field strains of rabies virus, previously thought to be antigenically identical, can be clearly distinguished as antigenically different, using monoclonal antibodies directed against either nucleocapside or glycoprotein antigens. By use of soluble antigens isolated on affinity chromatography columns or by immunising mice with killed myxoma virus, monoclonal antibodies could possibly be made that distinguish strain differences. Certainly, the results obtained in the present experiment suggest that the soluble antigens of G.V. and Lu strain viruses possess unique antigenic determinants. REFERENCES

Fenner, F.and I.D. Marshall, 1957, J. Hyg. 55, (2) 151-190. Fenner, F. and F.N. Ratcliffe, 1965, Myxomatosis (Cambridge Fenner,

F. and G.M. Woodroofe,

University

Press).

1965, Aust. J. Exp. Biol. Med. Sci. 43,359-370.

Fenner,

F., Poole, W.E., Marshall,

Hunter,

W.M. and F.C. Greenwood,

I.D. and Dyce, A.L., 1957, J. Hyg. 55, (2) 192-206. 1962, Nature

194,495-496.

Myers, K., 1954, J. Hyg. 42,47-59. Parer, I., Conolly, Ratcliffe, Reisner, Wiktor, Williams,

D. and Sobey,

W.R., 1981, Aust. Wildl. Res. 8, 613-626.

F.N., Myers, K., Fennessy, A.H. Sobey,

B.V. and Calaby,

W.R. and Conolly,

T.J. and H. Koprowski, R.T., Dunsmore,

J.H., 1952,

D., 1963,Virology

Nature

20,359-541.

1980, J. Exp. Med. 152,99-112.

J.D. and Parer, I., 1972, Nature

238,99-101.

170,7-11.