A serological comparison of the australian isolate of bluetongue virus type 20 (CSIRO 19) with bluetongue group viruses

Veterinary Microbiology, 6 (1981) 9--21 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

9

A SEROLOGICAL COMPARISON OF THE AUSTRALIAN ISOLATE OF BLUETONGUE VIRUS TYPE 20 (CSIRO 19) WITH BLUETONGUE GROUP VIRUSES

A.J. DELLA-PORTA l , K.A.J. HERNIMAN and R.F. SELLERS

The Animal Virus Research Institute, Pirbright, Woking, Surrey GU24 ONF (Great Britain) l Permanent address: CSIRO Division of Animal Health, Animal Health Research Laboratory, Private Bag No. 1, P.O., Parkville, Vic. 3052 (Australia) (Accepted 13 October 1980)

ABSTRACT Della-Porta, A.J., Herniman, K.A.J. and Sellers, R.F., 1981. A serological comparison of the Australian isolate of bluetongue virus type 20 (CSIRO 19) with bluetongue group viruses. Vet. Microbiol., 6: 9---21. Bluetongue virus serotype 20 (BTV20) (CSIRO 19 isolate) was compared with 17 other BTV serotypes using various serum neutralization (type antigen) tests to determine whether any serological relationships existed. Plaque-reduction neutralization tests employing 50% and 80% end-points could not clearly differentiate BTV20 from BTV4. Plaque-inhibition tests and quantal microtitre neutralization tests also showed a relationship between BTV20 and BTV4. Antisera against BTV20 and a Cyprus isolate of BTV4 (A SOT 1) showed a low level of cross-neutralization against BTV17. Investigation of plaque-reduction neutralization of virus--antiserum mixtures, by the calculation of regression curves and comparison of the area under the curves, showed that the BTV4 isolates studied could not be differentiated, and that BTV4 typing antiserum could not distinguish between BTV4 and BTV20, but that BTV20 antiserum could distinguish between BTV20 and BTV4. BTV20 did not show any significant type relationships with any of the other BTV types 1 to 17 using any of the neutralization tests. Our results suggest that BTV20 is closely related to, although not identical with, BTV4 and could be grouped as a subtype of BTV4. BTV17 appears to be distantly related to BTV20 and BTV4, but is clearly a distinct type. INTRODUCTION

In 1975, a virus (CSIRO 19) was isolated from a mixed pool of biting midges (Culicoides spp.) caught at Beatrice Hill, Northern Territory, Australia. This virus was found to be indistinguishable from bluetongue virus (BTV) by complement-fixation (CF) tests (St. George et al., 1978). CSIRO 19 virus was classified as bluetongue virus type 20 by the World Bluetongue Reference Laboratory, Veterinary Research Institute, Onderstepoort, South Africa, and became the prototype strain of the BTV20 sero-

0378-1135/81/0000--0000/$02.50 © 1981 Elsevier Scientific Publishing Company

10 group (B. Erasmus, personal communication, 1978; St. George and McCaughan, 1979). Experimental infection studies with BTV20 (CSIRO 19) in cattle and in sheep indicated that the virus was of low virulence, producing only mild to moderate clinical signs of bluetongue in sheep and none in cattle (St. George and McCaughan, 1979). The question of whether BTV20 in Australia was derived from previously known BTV types, or whether it was a unique new virus, was investigated by serological comparison with other BTV group members. The studies reported in this paper are of the comparisons of virus neutralization (typing) tests. The results of the tests show a strong relationship between BTV20 and and BTV4 but reveal problems with interpretation of some of the typing tests now in use. MATERIALS AND METHODS Viruses

BTV prototype strains for serotypes 1 to 16 were obtained from the Veterinary Research Institute, Onderstepoort, South Africa (Howell, 1970). BTV4 (A SOT 1) had been grown from a blood sample collected in Cyprus during 1969 (Parker et al., 1975). BTV17 (62-45S, Wyoming) and epizootic haemorrhagic disease of deer virus -- New Jersey serotype (EHDV-NJ) -were obtained from the Arthropod-borne Animal Disease Research Laboratory, U.S. Department of Agriculture, Denver, Colorado. Ibaraki virus was obtained from the National Institute of Animal Health, Tokyo, Japan. BTV20 (CSIRO 19) originally isolated from a mixed pool of Culicoides (St. George et al., 1978) was supplied by CSIRO Division of Animal Health, Long Pocket Laboratories, Indooroopilly, Queensland. All virus stocks were grown in baby hamster kidney (BHK-21) cells and stored in liquid nitrogen. Antisera

Typing antisera were prepared against BTV 1 to 17, EHDV-NJ and Ibaraki viruses in guinea pigs (Howell, 1970). Ovine antisera against BTV4 (A SOT 1) was prepared by intravenous inoculation of sheep with eggpassaged virus. Hyperimmune rabbit antisera against BTV20 was prepared using tissue culture grown virus and an immunization procedure similar to that of Barber and Jochim (1974). Bovine and ovine antisera against BTV20 were prepared using tissue culture grown viruses and following the blood autograph technique for Luedke et al. (1977). All sera were heat inactivated at 60°C for 30 min and stored at -20°C until used. Neutralization tests Quantal microtitre assays. The method employed was that of Parker et al. (1975). Briefly, two-fold serial dilutions of sera were prepared in flat-bottom

11 microtitre trays, starting at a dilution of 1/10, 100 TCIDs0 of virus were added to each well and the mixture incubated at 37°C for 1 h and, subsequently, overnight at 4°C. BHK-21 cells were added to the wells' and the plates sealed and incubated at 37°C. The development of cytopathic effect (CPE) was recorded dally from its first appearance at 30 to 36 h. The SNs0 titre was based on the inhibition of CPE, which had developed fully within 4 to 5 days, and was expressed as the reciprocal of the final dilution of serum present at the 50% end point.

Plaque.inhibition tests. The m e t h o d employed was similar to that described previously (Porterfield, 1960), except that 7 mm sterile blotting paper discs, instead of fish spine beads, were soaked in undiluted serum and Super-Veto-Porcine Stable (SVP) cells (Della-Porta and Snowdon, 1979) were used. Confluent monolayers of SVP cells in 90 mm Petri dishes were infected with approximately 50,000 PFU of virus at 37°C for 1 h, and then overlaid with 15 ml of nutrient 1% agar (Medium 199, 30 mM HEPES, 0.05% DEAEdextran, 5% foetal calf serum, 1% of 5.6% sodium bicarbonate solution, 1% noble agar, pH 7.5). Six discs, soaked in the test serum, were placed evenly on the agar surface. The plates were inverted and incubated at 37°C in a CO2 incubator. On day 4 the discs were removed and the plates stained with 1/10,000 neutral red in 5 ml of nutrient 1% agar overlay. The results were read on days 5 and 6. The diameter of the zones of inhibition of cell lysis were measured in mm for at least two replicates for each serum on separate plates. Plaque-reduction tests. Virus-serum mixtures were incubated for 1 h at 20°C and then inoculated, without dilution, onto three monolayers of SVP cells in 50 mm Petri dishes (0.2 ml per dish). The dishes were incubated at 37°C in a CO2 incubator for 1 h, the inoculum removed, the monolayers washed with Hanks' balanced salt solution containing 0.2% bovine serum albumin, and then overlaid with 5 ml o f nutrient 1% agar. The cells were stained after 4 days incubation b y adding 2 ml of 1/10,000 neutral red in overlay. The plaques were counted on days 5 and 6. The results were analysed and regression lines calculated that were defined b y the values of A and B in the equation: Y = A + BX, where the intercept A represents the log10 decrease in virus titre produced b y undiluted serum, and B is the "neutralization slope" (Westaway, 1965). "Area functions" (Table V) were calculated from the regression line analyses, where area = - ~ A 2/B (Westaway, 1965). The standard error (S.E.) in the "area f u n c t i o n " was calculated from the experimental data using the equation, As

S.E. (area)=

2~I

14+(A_2BX)

N

2

B 2- Z (X-~-X) 2

}I/2

12 where N is the number of readings, Yi is a reading of the -log10 surviving virus fraction for X i (-log10 serum dilution) and ~ a n d -'~ are the means for all X and Y readings, and S=

Wt

~

(Yi-Y)2

_ B 2 ~. ( X i _ X ) 2

is the residual standard deviation from fitting the straight line. The 50% and 80% plaque-reduction titres (Table III) were calculated from the regression line analyses. With each serum, comparisons of neutralization curves and areas for the viruses under test were all done using the same batch of SVP cells, on the same day, to avoid variation due to cell-dependent neutralization (Della-Porta and Westaway, 1978). RESULTS

Quantal microtitre SNso comparisons o f B T V 2 0 with other bluetongue group viruses BTV20 (CSIRO 19) and rabbit anti-BTV20 serum were used for comparisons against BTV 1 to 17, EHDV-NJ, and Ibaraki viruses and their reference antisera using a microtitre neutralization (SNs0) test. The results (Table I) showed a significant two-way cross-neutralization between BTV4 and BTV20. The rabbit anti-BTV20 serum appeared less able to distinguish between BTV4 and BTV20, showing equal titres of 48, than the bovine sera which showed differences of 1:5.6 (630c) and 1:5.5 (637c). The antiBTV20 bovine sera showed weak cross-reactions with a number of BTV serotypes, although the heterologous:homologous titres were only 1:40 (630c) and 1:14 (637c). There were moderately strong cross-reactions with BTV17 and its antisera and the other viruses and their antisera. There was no reaction between any of the other BTV types or their antisera with BTV20. The BTV4 antisera showed very strong reactions with BTV20.

Plaque-inhibition neutralization comparisons o f BTV4, B T V 1 7 and B T V 2 0 In a plaque-inhibition (PI) study (Table II), with two BTV4 isolates and BTV17, anti-BTV20 sera produced in cattle and sheep were used, rather than rabbit serum which is known to be more cross-reactive (Barber and Jochim, 1974). The PI tests were limited to BTV4, BTV17 and BTV20 as these were the only viruses showing any significant serological relationships in the SN test and in order to limit the number of viruses required for the tests. The South African reference strain of BTV4 could n o t be distinguished from a Cyprus isolate obtained in 1969 (A SOT 1). Anti-BTV4 (S. Aft.) and anti-BTV4 (A SOT 1) guinea pig reference sera could not clearly distinguish between BTV4 and BTV20. Ovine anti-BTV4 (A SOT 1) serum did show a difference in neutralization between the BTV4 isolate and BTV20, although

13 TABLE I SNs0 comparisons of BTV20 with bluetongue group viruses using a microtitre serum neutralization test SN,0 titre against (virus) a

Antiserum BTV4 (S. Aft.)

BTV4 (A SOT 1)

BTV17

BTV20

32

11

22

128

11

64

II

32

II

48 320 40

12 15 15

48 1780 220

guinea pig ref.

BTV4 (A SOT 1) ovine ref. BTV17 guinea pig ref. BTV20 rabbit bovine (630c) b bovine (637c) c

aReciprocal of the dilution that inhibited the CPE in 50% of the microtitre tray wells against 100 TCIDs0 units of virus (Parker et al., 1975). SNs0 titres were < 10 for BTV1 to BTV3, BTV5 to BTV16, EHDV-NJ and Ibaraki viruses and antisera. bBovine 630c anti-BTV20 serum, had SNso titres of 20(BTV 1), 20(BTV2), 30(BTV3), 10(BTV6), 30(BTV7), 40(BTV8), 15(BTV9), 20(BTV10), 15(BTVll), and was negative (SNs0 < 10) for remaining BTV1 to BTV17. CBovine 637c anti-BTV20 serum, had SN~0 titres of 15(BTV3), 10(BTV5), 10(BTV6), and was negative (SN~0 < 10) for remaining BTV1 to BTV17. this d i f f e r e n c e was n o t large. A n t i - B T V 2 0 sera s h o w e d a g r e a t e r d i f f e r e n c e b e t w e e n B T V 4 (A S O T 1) a n d B T V 2 0 t h a n b e t w e e n B T V 4 (S. Afr.) a n d B T V 2 0 . T h e r e was also a slight c r o s s - r e a c t i o n o f ovine a n t i - B T V 4 (A S O T 1) s e r u m a n d a n t i - B T V 2 0 sera ( b o t h ovine a n d b o v i n e ) w i t h B T V 1 7 a n d o f t h e a n t i - B T V 1 7 guinea pig s e r u m w i t h B T V 2 0 b u t in s o m e cases t h e results w e r e n o t c o n s i d e r e d significant.

Plaque-reduction neutralization titres T h e 50% a n d 80% p l a q u e - r e d u c t i o n n e u t r a l i z a t i o n titres (Table I I I ) w e r e c o m p a r e d f o r a n t i s e r a p r e p a r e d against B T V 4 (S. Afr.), B T V 4 (A S O T 1), B T V 1 7 a n d B T V 2 0 . T h e a n t i - B T V 4 (S. Afr.) g u i n e a pig s e r u m r e a c t e d to give higher titres in t h e h e t e r o l o g o u s r e a c t i o n s w i t h B T V 4 (A S O T 1) a n d B T V 2 0 t h a n w i t h B T V 4 (S. Aft.). T h e r e was a v e r y l o w level r e a c t i o n w i t h B T V 1 7 , giving a 5070 p l a q u e - r e d u c t i o n titre o f 34 b u t n o m e a s u r a b l e 8070 p l a q u e - r e d u c t i o n titre. T h e a n t i - B T V 4 (A S O T 1) ovine s e r u m r e a c t e d s t r o n g l y in t h e h e t e r o l o g o u s r e a c t i o n s w i t h B T V 4 (S. Afr.) and B T V 2 0 . Again t h e r e was a l o w level c r o s s - r e a c t i o n w i t h B T V 1 7 . A n t i - B T V 1 7 guinea

14 T A B L E II

Cross-reactions w i t h t h e p l a q u e - i n h i b i t i o n assay o f n e u t r a l i z a t i o n m i x t u r e s Zone o f i n h i b i t i o n ( m e a n ± S.E. in m m ) against a Antiserum

BTV1 (S. A f t . )

BTV4 (S. A f t . )

BTV4 ( A S O T 11

BTV17

BTV20

0

BTVI (S. Aft.) g u i n e a pig ref.

21 -+ 2

0

0

0

B T V 4 (S. Afr.) g u i n e a pig ref.

0

20 -+ $

18 ± 2

0

16 ± 4

B T V 4 ( A S O T 1) g u i n e a pig ref.

0

21 + 0

19 ± 2

0

18 +- 2

B T V 4 ( A S O T 11 o v i n e ref.

0

28 ± 1

26 +- 3

14 ± 1

20 ± 1

B T V 4 ( A S O T 1) ovine 385

0

20 ± 1

20 ± 1

0

7 ± 7

BTV17 (Wyoming) g u i n e a pig ref.

0

0

0

20 ± 1

4 ± 5

BTV20 (CSIRO 19) ovine E425

0

15 ± 1

10 ± 1

5 ± 5

19 ± 2

B T V 2 0 ( C S I R O 19) bovine 637c

0

18 +- 2

13 ± 3

6 ± 6

20 + 1

B T V 2 0 ( C S I R O 191 bovine 630c

NT b

NT

14 +- 1

10-+ 1

21 ± 1

aSize o f p l a q u e - i n h i b i t i o n z o n e ( m e a n ± s t a n d a r d e r r o r i n r a m ) f o r 2 t o 7 d e t e r m i n a t i o n s . b N T = n o t tested.

pig serum did not react significantly with BTV4 (S. Aft.) or BTV4 (A SOT 1) and showed a significant 50% plaque-reduction titre (200) but an undetectable 80% plaque-reduction titre against BTV20. Anti-BTV20 serum reacted most strongly in the homologous reactions. Heterologous titres were higher against BTV4 (A SOT 1) than against BTV4 (S. Aft.), both showing strong cross-reactions. There was a low level cross-neutralization of BTV17, giving 50% and 80% plaque-reduction titres of 55 and 8 respectively.

Analyses of dose--response relationships in the plaque-reduction assay of neutralization mixtures Linear regression line analyses were performed on the plaque-reduction assay results (Table IV) and the results plotted (Fig. 1). In these analyses, the slope of the line (B) and the intercept on the Y-axis (A) are always

15 TABLE Ill 50% and 80% plaque-reduction neutralization titzes Neut~ralization titre a against BTV4 (S. Afr.)

BTV4 (A SOT 1)

BTV17

BTV20

50% redn.

redn.

50% redn.

17,800

1,780

34

0.1

Antiserum 50% redn. BTV4 (S. Afr.) guinea pig ref. BTV4 (A SOT 1) ovine ref. BTV 17 guinea pig ref. BTV20 bovine 680c

80%

redn.

365

15....O0

80%

80% redxL

50% redn.

80% redn.

9,200

780 2,150

8,050

2,700

27,900

9,200

86

4

10,000

0

0

I0

0

41__._0

12_.._0

200

280

90

1,300

250

55

8

0.02

1,750

92..__0

aReciprocal of the dilution of antiserum that produced either a 50% or 80% reduction in plaque counts, calculated from linear regression line equations (Table IV).

T A B L E IV A n a l y s e s o f d o s e - - r e s p o n s e r e l a t i o n s h i p s in t h e p l a q u e - r e d u c t i o n a s s a y o f n e u t r a l i z a t i o n mixtures Virus

Antiserum

BTV4 (S. A f t . )

BTV4 ( A S O T 1)

BTV4

BTV20

Aa

Ba

A

B

A

B

A

B T V 4 (S. A f r . )

2.94

-1.03

2.00

-0.40

0.56

-0.17

1.51

-0.28

B T V 4 ( A S O T 1) o v i n e ref.

3.62

-0.84

3.99

-0.83

0.88

-0.3

2.70

-0.60

BTV17 g u i n e a pig ref.

0.06

0.4

-0.1

.2.23

-0.74

0.53

-0.10

BTV20 bovine 630c

2.26

2.07

-0.57

1.12

-0.47

4.97

-1.44

0.01 -0.80

a A a n d B v a l u e s are f r o m t h e r e g r e s s i o n line e q u a t i o n Y = A + BX, w h e r e Y r e p r e s e n t s t h e -log10 s u r v i v i n g f r a c t i o n o f virus a f t e r n e u t r a l i z a t i o n a n d X t h e -log10 o f t h e antiserum dilution which produced the neutralization. After Westaway (1965).

B

16 J

~) B4A antiserum"

•

.

o

.

B4S antiserum

..= r3 Z B

1 SERUM

DILUTION

2 3 (-log,o)

4

Fig. 1. Linear regression curves of dose--response relationships (Table IV) of neutralization mixtures of BTV4 (A SOT 1; B4A), e, BTV4 (S. Aft.; B4S), o, BTV17 (B17), A, and BTV20 (B20), m, and of (a) anti-BTV4 (A SOT 11 ovine reference serum, (b) antiBTV4 (S. Aft./ guinea pig reference serum, (c) anti-BTV17 (62-45S, W y o m i n g / g u i n e a pig reference serum and (d) anti-BTV20 (CSIRO 191 serum.

greatest for the homologous antiserum--virus reactions. These results illustrate why the 50% and 80% plaque-reduction neutralization titres distinguish less clearly between these virus isolates. The most dramatic illustration of this is Fig. 1 (d) where the slope for the homologous BTV20 reaction and the intercept are much greater than for the heterologous BTV4 reactions and clearly show greater differences than the 50% and 80% plaque-reduction neutralization titres (Table III). In order to allow direct comparisons between different antiserum--virus mixtures, the area under ~he curves was calculated and the results normalized with respect to the homologous antiserum--virus reaction (Table V). This enabled direct comparison of the results. The areas were significantly different at the 5% level (for the same antiserum) if the difference between their area values I AI - A21 > 2 ~/S.E.12 + S.E.22. The anti-BTV4 (S. AFT.) serum could not distinguish between BTV4 (S. AFT.), BTV4 (A SOT 1), but found BTV20 closely related but distinct. Again there was a low level crossneutralization of BTV17. Anti-BTV17 serum showed no detectable crossneutralization of BTV4 (S. AFT.) but did weakly neutralize BTV4 (A SOT 1) and BTV20. Anti-BTV20 serum showed cross-reactions with BTV4 (S. Aft.) and BTV4 (A SOT 1) and a low level neutralization of BTV17 but in all cases the homologous reaction (BTV20) was significantly different (at the 5% level) from the heterologous reactions. The normalized area func-

7.7 ± 0.5

0.2 ± 0.6

3.2 ± 1.0

BTV4 (A SOT 1) ovine ref.

BTV17 guinea pig ref.

BTV20 bovine 630c 37

0

80

100

Normalized a value

3.8 ± 0.8

0.8 ± 0.1

9.6 ± 1.2

4.9 ± 0.5

Area -+ S.E.

BTV'4 (A SOT 1)

44

24

100

117

Normalized value

1.3 ± 0.7

3.3 ± 0.4

1.3 ± 0.1

0.9 ± 0.1

Area ± S.E.

BTV 17 (Wyoming)

15

10__O0

14

22

Normalized value

8.6 ± 0.4

1.4 ± 0.2

6.1 ± 0.6

4.1 ± 0.1

Area ± S.E.

BTV 20 (CSIRO 19)

100

42

64

98

Normalized value

aArea = _ 1 A2/B. The normalized value has been obtained by assigning a value of 100 to the homologous area function of each antiserum and adjusting the functions in the cross-reactions by proportion. Calculations are after Westaway (1965). bS.E., standard error of the area function, calculated from the formula shown in the materials and methods.

4.2 ± 1.4

Area a ± S,E. b

BTV4 (S. AFT.) guinea pig ref.

Antiserum

BTV4 (S. Afr. ref)

Activity of cross-reacting antisera in the plaque-reduction assay of neutralizing mixtures expressed as an "area function" derived from the regression line equation: Y = A + BX

TABLE V

18 tions (Table V) can also be analysed vertically for each virus. The results confirmed that BTV4 (S. AFT.) is most closely related to BTV4 (A SOT 1) and more distantly to BTV20, whereas cross-comparison indicates that BTV20 cannot be clearly distinguished from BTV4 (S. AFT.), is closely related to BTV4 (A SOT 1), and shows significant cross-reactions with BTV17. The normalized values represent the proportional cross-~reaction between these virus isolates and their antisera. DISCUSSION The studies reported in this paper raise many questions about the current methods of serotyping of BTV isolates. Neitz (1948) used cross-protection .tests in sheep to compare 10 BTV isolates, and found they could be grouped into a number of serotypes instead of just one serotype. Howell (1960) introduced a serological basis to typing when he compared isolates using a neutralization index test. At that time the BTV isolates were grouped into 12 serotypes, with strong cross-reactions between viruses in the same serogroup but little between serogroups. These serotypes matched Neitz's (1948) groups and it was concluded that neutralization tests could replace cross-protection tests for typing BTV isolates. Since Howell's (1960) study, various plaque neutralization tests have been introduced (Barber and Jochim, 1974, 1976; Howell, 1970; Howell et al., 1970; Thomas and Trainer, 1971) as well as microtitre quantal neutralization tests (Parker et al., 1975; St. George et al., 1978). Further, neutralization tests employing guinea pig antisera (Howell, 1970) have been substituted for the crossprotection test in sheep (Neitz, 1948) and neutralization tests employing ovine antisera (Howell, 1960) for typing. The number of serotypes identified is now at least 20 (B. Erasmus, personal communication, 1978), but no serotyping data have been published for BTV17 to BTV20. Plaque-reduction tests employing 50% (Thomas and Trainer, 1971) or 80% (Barber and Jochim, 1974, 1976) end points have been used for typing BTV isolates. Whilst more sensitive than the quantal neutralization tests (Table I and Table III), the results are not always as specific. Thomas and Trainer (1971) using the 50% plaque-reduction test could not separate the United States BTV isolates in their studies into three serotypes, as could Barber and Jochim (1974), using the 80% plaque-reduction test. In Table III the 50% plaque-reduction test showed a strong relationship between BTV4 and BTV20; BTV20 was weakly neutralized by anti-BTV17 serum; and anti-BTV20 serum weakly neutralized BTV17. Similar results were obtained in the quantal assay (Table I) and in the plaque-inhibition test (Table II). Our observations also suggest species to species variation in the formation of cross-reactive antibodies, rabbits producing sera that are markedly cross-reactive (Table I; Barber and Jochim, 1974, 1976), as well as varying from animal to animal. The plaque-inhibition test (Table II) proved both simple to use and

19

economical on antisera while being relatively specific. It confirmed the relationship between the South African BTV4 (S. Aft.) and the 1969 Cyprus BTV4 (A SOT 1) isolates, whilst also showing that BTV20 was closely related to these B T V 4 isolates b u t distinguishable from them. Of interest was the variation between individual animals in the production of cross-reacting antibodies, the anti-BTV4 (A SOT 1) ovine reference serum showing a significant cross-neutralization of BTV17 as did an anti-BTV20 bovine (630 c) serum. L o w level cross-reactions between serotypes have been observed by Davies and Blackburn (1971) for Kenya BTV isolates which reacted strongly with anti-BTV1 serum and weakly with anti-BTV4 serum, whereas Howell et al. (1970) found that South African BTV isolates reacted specifically in the plaque-inhibition test. The 50% and 80% plaque-reduction neutralization titres represent only the portions of plaque-reduction neutralization curves where either 50% or 20% of the plaques are n o t neutralized or there is a surviving fraction of - 0 . 3 log10 or - 0 . 7 log10 respectively (or - l o g V/Vo of 0.3 or 0.7, Fig. 1). An examination of the plaque reduction neutralization curves (Fig. 1) illustrates w h y the 50% plaque reduction titres lack specificity and w h y neutralization indices (value for - l o g V/Vo of undiluted or 1/10 diluted serum) are more specific (Howell, 1960). However, it is the whole plaquereduction neutralization curve which best represents the reaction o f virus and antiserum. The reaction of an individual serum with different viruses can be evaluated b y comparing the coefficients from linear regression lines (Table IV). To allow comparison between sera, it was necessary to calculate the area under the neutralization curves and normalize the areas with respect to the homologous antiserum-virus reaction (Westaway, 1965). The calculation of the standard errors in the areas under the neutralization curves allowed us to evaluate statistically the relationships between various neutralization mixtures. The normalized values were then compared for all antisera-virus reactions (Table V). Using anti-BTV4 (S. Aft.) guinea pig typing serum, BTV4 (S. Afr.), BTV4 (A SOT 1) and BTV20 could not be distinguished (normalized values of 100, 117 and 98 respectively). The cross comparison in the reverse direction using anti-BTV20 bovine (630c) serum showed that BTV20 was n o t identical with BTV4 (S. A f t . ) a n d BTV4 (A SOT 1)(normalisedvalues of 100, 37 and 44 respectively). Further, the results confirmed a distant relationship between BTV4, BTV20 and BTV17. There was no significant difference (at the 5% level) in the areas for BTV4 (S. Aft.) and BTV4 (A SOT 1) in b o t h directions and hence they are isolates of the same serotype. BTV4 (S. Aft.), BTV4 (A SOT 1) and BTV20 were indistinguishable (at the 5% level) in one direction (using BTV4 antisera) b u t could be distinguished (at the 5% level) in the other direction (using BTV20 antiserum). Whether BTV20 is confirmed as a new serotype or as a s u b t y p e of the BTV4 serotype is an important question, as certainly there is a close serological relationship between the two.

20

Cross-protection studies in sheep further challenge the conclusion that CSIRO 19 virus should be typed as the prototype of BTV20. Sheep inoculated with BTV20 are protected against challenge with both virulent BTV4 (B. Erasmus, personal communication, 1978) and with virulent BTV17 (I.M. Parsonson, C.H. Campbell and C.M. Groocock, unpublished data, 1979). Considering Neitz's (1948) typing of BTV isolates by crossprotection tests, BTV20 would appear to be a member of both the BTV4 and BTV17 serotypes. Cross-protection studies in two directions were not possible because of the mild clinical response to BTV20 in sheep (St. George and McCaughan, 1979). Our serological results would support the sheep cross-protection test results, and also indicate that BTV4 (S. AFT.) and BTV17 are members of different serotypes. It may be t h a t BTV20 shares common antigenic determinants with BTV4 and BTV17 but that BTV4 and BTV17 do not share many common determinants involved in the neutralization reaction. The results further indicate that the distribution of antigenic determinants on the surface of bluetongue virions and the reaction of antibodies to form a critical pattern is important for the neutralization of the infectivity of orbiviruses as has been described for other animal viruses (Della-Porta and Westaway, 1978). In contrast to the serological evidence which shows a close relationship between BTV20 and BTV4, biochemical studies (Huismans and Bremer, 1980) indicate that these viruses are very different at the gene level. Huismans and Bremer used the methods of total RNA gene hybridization (Verwoerd and Huismans, 1969) and analyses of RNA hybrids by polyacrylamide gel electrophoresis (Huismans and Howell, 1973). Whereas there was a 60%--70% total RNA homology between BTV types 1 to 19 (Verwoerd and Huismans, 1969) there was less than a 30% RNA homology between BTV20 and other BTV serotypes. Further, analyses b y polyacrylamide gel electrophoresis, of the RNA hybrids from BTV1 to 19 showed 6 or 7 of the 10 RNA segments were closely related (Huismans and Howell, 1973) in all, whereas only two related segments between BTV20 and BTV4 were detected. The segments that were related were RNA segments 7 and 10 possibly coding for the virus protein associated with the group CF antigen. These biochemical results would appear to agree with serological results which indicate that BTV20 contains the group antigen involved in the CF test for BTV (St. George et al., 1978) but are in contrast to the close antigenic relationships between BTV20 and BTV4 reported in this study. It would appear that further biochemical and antigenic studies are required to clearly define the position of BTV20. ACKNOWLEDGEMENT

The visit of one of us (A.J.D.P.) to the Animal Virus Research Institute was supported by CSIRO. We wish to thank Richard Jarrett from the CSIRO Division of Mathematics and Statistics for statistical advice and

21

Hank Huismans for making available data on his biochemical studies before publication. REFERENCES Barber, T.L. and Jochim, M.M., 1974. Serological characterization of selected bluetongue virus strains from the United States. Proc. U.S. Anim. Health Assoc. 77th, 1973: 352--359. Barber, T.L. and Jochim, M.M., 1976. Serotyping bluetongue and epizootic hemorrhagic disease virus strains. Proc. Am. Assoc. Vet. Lab. Diagnost., 18th: 1975: 149--162. Davies, F.G. and Blackburn, N.K., 1971. The typing of bluetongue virus. Res. Vet. Sci., 12: 181--183. Della-Porta, A.J. and Snowdon, W.A., 1979. An experimental inactivated virus vaccine against bovine ephemeral fever. 1. Studies of the virus. Vet. Microbiol., 4: 183--195. Della-Porta, A.J. and Westaway, E.G., 1978. A multi-hit model for neutralization of animal viruses. J. Gem Virol., 38: 1--19. Howell, P.G., 1960. A preliminary antigenic classification of strains of bluetongue virus. Onderstepoort J. Vet. Res., 28: 357--363. Howell, P.G., 1970. The antigenic classification and distribution of naturally occurring strains of bluetongue virus. J.S. Aft. Vet. Med. Assoc., 41: 215--223. Howell, P.G., Kiimm, N.A. and Botha, M.J., 1970. The application of improved techniques to the identification of strains of bluetongue virus. Onderstepoort J. Vet. Res., 37 : 59--66. Huismans, H. and Bremer, C.W., 1980. A comparison of an Australian bluetongue virus isolate (CSIRO 19) with other bluetongue virus serotypes by cross hybridization and cross immune precipitation tests. Onderstepoort J. Vet. Res. (in press). Huismans, H. and Howell, P.G., 1973. Molecular hybridization studies on the relationships between different serotypes of bluetongue virus and o n the difference between the virulent and attenuated strains of the same serotype. Onderstepoort J. Vet. Res., 40: 93--104. Luedke, A.J., Jones, R.H. and Walton, T.E., 1977. Overwintering mechanism for bluetongue virus: biological recovery of latent virus from a bovine by bites of Culicoides varriipennis. Am. J. Trop. Med. Hyg., 26: 313--325. Neitz, W.O., 1948. Immunological studies on bluetongue in sheep. Onderstepoort J. Vet. Sci. Anita. Ind., 23: 93--136. Parker, J., Herniman, K.A.J., Gibbs, E.P.J. and Sellers, R.F., 1975. An experimental inactivated vaccine against bluetongue. Vet. Rec., 96: 284--287. Porterfield, J.S., 1960. A simple plaque-inhibition test for the study of arthropod-borne viruses. Bull. W.H.O., 22: 373--380. St. George, T.D. and McCaughan, C.I., 1979. The transmission of the CSIRO 19 strain of bluetongue virus type 20 to sheep and cattle. Aust. Vet. J., 55: 198--199. St. George, T.D., Standfast, H.A., Cybinski, D.H., Dyce, A.L., Muller, M.J., Doherty, R.L., Carley, J.G., Filippich, C. and Frazier, C.L., 1978. The isolation of a bluetongue virus from Culicoides collected in the Northern Territory of Australia. Aust. Vet. J., 54: 153--154. Thomas, F.C. and Trainer, D.O., 1971. Bluetongue virus: some relationships among North American isolates and further comparisons with EHD virus. Can. J. Comp. Med., 35: 187--191. Verwoerd, D.W. and Huismans, H., 1969. On the relationship between bluetongue, African horse sickness and reoviruses: hybridization studies. Onderstepoort J. Vet. Res., 36: 175--180. Westaway, E.G., 1965. The neutralization of arboviruses. II. Neutralization in heterologons virus--serum mixtures with four group B arboviruses. Virology, 26: 528--537.