Serological studies of two additional Australian blue-tongue virus isolates CSIRO 154 and CSIRO 156

Veterinary Microbiology, 6 (1981) 233--245 Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands

SEROLOGICAL STUDIES OF TWO ADDITIONAL AUSTRALIAN TONGUE VIRUS ISOLATES CSIRO 154 AND CSIRO 156

233

BLUE-

A.J. DELLA-PORTA 1, D.A. McPHEE I, M.C. WARK 2, T.D. ST. GEORGE 3 and

D.H. CYBINSKI s 1 CSIRO Division o f Animal Health, Australian National Animal Health Laboratory, Geelong, at present located at Animal Health Research Laboratory, Private Bag No. 1, P.O., ParkviUe, Vic. 3052 (Australia) 2 Commonwealth Serum Laboratories, 45 Poplar Road, Parkville, Vic. 3052 (Australia) s CSIRO Division o f Animal Health, Long Pocket Laboratories, Private Bag No. 3, P. 0., IndooroopiUy, Qid. 4068 (Australia)

(Accepted 23 February 1981)

ABSTRACT Della-Porta, A.J., McPhee, D.A., Wark, M.C., St. George, T.D. and Cybinski, D.H., 1981. Serological studies of two additional Australian bluetongue virus isolates CSIRO 154 and CSIRO 156. Vet. Microbiol., 6: 233--245. Serological surveys revealed that some cattle in northern Australia possessed bluetongue virus (BTV) group-reactive (agar gel diffusion precipitin, AGDP, and complement-fixing, CF) antibodies, but not serum neutralizing (SN) antibodies, to BTV20, a new type previously found in Australia. A t t e m p t s were made during 1979 to isolate viruses causing these reactions. There was one isolate of a virus (CSIRO 154) and eight isolates of another virus (CSIRO 156) made from the blood of healthy cattle in the Northern Territory. These viruses could not be distinguished from BTV20 by AGDP, CF or fluorescent-antibody tests and hence were designated members of the bluetongue serogroup. Serotyping was carried out using the plaque-inhibition and plaque-reduction SN tests. CSIRO 156 virus could not be distinguished from BTV1 by any of the SN tests and it was concluded that it was an Australian isolate of the BTV1 serotype. CSIRO 154 virus was found to be related to, but not identical with, BTV6. It is probably not one of the known 20 BTV serotypes and may represent a new BTV serotype. None of the three Australian BTV isolates is known to cause clinical disease in sheep or cattle under natural conditions, and biochemical comparisons with the African BTV serotypes may show differences not revealed by these serological studies.

INTRODUCTION Serological surveys of Australian cattle and group antigen reactive sera, using the agar gel complement-fixation (CF) tests, followed the 1 9 ) in A u s t r a l i a ( S t . G e o r g e e t al., 1 9 7 8 ) . T h e

0378-1135/81/0000--0000/$02.50

sheep for bluetongue virus (BTV) diffusion precipitin (AGDP) and identification of BTV20 (CSIRO surveys revealed that there were

© 1981 Elsevier Scientific Publishing Company

234

many cattle with group reactive sere (A.J. Della-Porta, R.F. Sellers, K.A.J. Herniman and others, unpublished data, 1978). Serum neutralization tests carried o u t against BTV1 to 17 and Ibaraki virus using some of these BTV group reactive sera, which were negative for BTV20, suggested that BTV1, BTV15 and Ibaraki viruses, or related orbiviruses may be present in Australia. Also there were many sera which reacted at a low level with many BTV serotypes, making results, in the absence of indigenous viral isolates, difficult to interpret. Further, many of the group reactive sera failed t o neutralize any of the viruses in the study, although some were strongly AGDP and CF positive. It was decided, therefore, to a t t e m p t to isolate viruses from blood samples of sentinel cattle in northern Australia and to use the BTV-AGDP test to monitor their sera for antibody conversion. The studies resulted in the isolation of two additional Australian BTV serotypes (St. George et al., 1980). CSIRO 154 virus was isolated from a blood sample collected from a healthy animal on 23rd March 1979 at the Victoria River Research Station, N.T. Eight isolates were made of CSIRO 156 virus from blood samples of healthy cattle between March and May 1980 at Beatrice Hill and Tortilla, N.T. The results reported in this paper are the serological studies confirming CSIRO 154 and CSIRO 156 as additional Australian bluetongue virus isolates, and the serotyping of these virus isolates. MATERIALS AND METHODS

Viruses

BTV20 (CSIRO 19) isolated from a mixed pool of C u l i c o i d e s (St. George et al., 1978) and CSIRO 154 and CSIRO 156 viruses isolated from heparinized cattle bloods (St. George et al., 1980) were plaque-purified three times using Veto cells. Attenuated vaccine strains of BTV1 and BTV6 (obtained from B. Erasmus, Veterinary Research Institute, Onderstepoort, South Africa) were three times plaque-purified in L929 cells. All virus stocks were prepared in BHK21 cells. A n tise ra

Typing antisem were prepared against BTV1 to 19 in guinea pigs (Howell, 1970; B. Erasmus, personal communication, 1979; I.M. Parsonson, personal communication, 1979). Ovine antisera were prepared against CSIRO 154 and CSIRO 156 viruses, the sheep being bled 21 days after intravenous inoculation. Ovine antisera were prepared against BTV20 using the blood autograph technique of Luedke et al. (1977; Della-Porta et al., 1981). Hyperimmune mouse ascites t u m o u r fluids were prepared against CSIRO 154 and CSIRO 156 viruses. Rabbit antisera for fluorescent-antibody tests were prepared after hyperimmunizing rabbits with tissue culture grown viruses. Rabbit antisera for

235

AGDP tests were prepared using mouse brain grown virus as antigen. All sera were heat-inactivated at 60°C for 30 min and stored at - 2 0 ° C until used. Sere from outside Australia were treated with 0.4% ~-propriolactone (BPL, Kasei Chemicals) at 37°C for 1 h and then overnight at 4°C, in order to inactivate any residual exotic viruses. The treated sera were dialysed against phosphate buffered saline at pH 7.2 and the volume was adjusted to a final dilution of 1 in 2. Control negative and positive sera were also treated with BPL, and positive controls were treated as for the BPL procedure except that BPL was n o t added.

Agar gel diffusion precipitin (A GDP) test Antigens were prepared b y harvesting virus-infected tissue culture fluids from roller bottles o f Veto cells at an advanced stage of CPE. The fluids were clarified b y centrifugation at 10,000 × g for 10 min and then concentrated by ultrafiltration using an Amicon {Lexington, MA) model 402 stirred cell with a XM50 membrane. The AGDP test was performed with the antigen in the centre well surrounded b y six peripheral wells in a hexagon pattern, each alternative well containing reference antiserum and the test read after incubation at r o o m temperature for 24 h in a humid atmosphere (I.R. Littlejohns, unpublished data, 1977).

Complement-fixation ( CF) test CF and control antigens, prepared as for AGDP antigens and inactivated with 0.2% (v/v) BPL for 2 h at 37°C followed b y overnight at 4°C, were used in a microtitre CF test. Two full units of guinea pig complement were used in the CF test. 2.5% normal calf serum (enhancing serum) was added to the complem e n t when testing bovine antisera and omitted when testing hyperimmune mouse ascites fluids. A mixture of antigen, serum (inactivated at 60°C for 30 min) and complement was incubated overnight at 4°C (Boulanger et al., 1967; D.A. McPhee, E.L. French and W.A. Snowdon, unpublished data, 1977). The test was read, following the overnight incubation, after adding sensitized sheep red blood cells, incubating at 37°C for 1 h, and centrifuging the trays to pellet red blood cells.

Fluorescent antibody (FA ) test A direct fluorescent antibody technique was used with virus-infected BHK21 cells. The immunoglobulin fraction of hyperimmune rabbit anti-BTV20 serum or negative control serum was conjugated with fluorescein isothiocyanate (BBL Cockeysville, MD). Appropriate controls were included in the test to verify the specificity of the reaction.

236

Serum neutralization tests Plaque-inhibition (PI) tests The m e t h o d employed was t h a t described by Della-Porta et al. (1981). The test was carried o u t in 90 m m Petri dishes using SVP cells (Della-Porta and Snowdon, 1979) infected with 50,000 PFU of virus. The virus-infected cells were overlaid with a 1% nutrient agar overlay and 7 mm discs soaked in test sera were placed on the overlay to measure the neutralizing activity. This m e t h o d was initially used to screen CSIRO 154 and CSIRO 156 viruses against all the typing sere and then in the confirmatory two-way tests.

Plaque-reduction (PR ) tests The m e t h o d employed was that described by DeUa-Porta et al. (1981) The test was carried o u t in SVP cells and comparisons between viruses were done on the same day with the same batch of cells in order to avoid variation in results due to cell-dependent neutralization (Della-Porta and Westaway, 1978). The results were analyzed and regression lines calculated using the equation: Y = A + BX where the intercept A represents the log~0 decrease in virus titre produced by undiluted serum, and B is the "neutralization slope" (Westaway, 1965). The "area f u n c t i o n s " were calculated from the regression line analyses, where the area = - ~ A 2 / B . The standard error (S.E.) in this "area f u n c t i o n " was calculated from the experimental data using the equation (Della-Porta et al., 1981): s.E. (area)=

As i 4+ (A-2BX')2 }~/2

where N is the number of readings, Yi is areadin_g of the -log10 surviving virus fractions for Xi (-log10 serum dilution); Y and X are the means for all X and Y readings, and

s=

N-2

~(Yi-~)2-B

~ Z (Xi-X) 2

is the residual standard deviation from fitting the straight line. 50% and 80% plaque-reduction titres were calculated from the regression line analysis. RESULTS

Serogrouping o f CSIRO 154 and CSIRO 156 viruses (AGDP, CF, and FA) CSIRO 154 and CSIRO 156 viruses were compared with BTV20 using serological tests which are used to group viruses into the BTV serogroup. None of the viruses could be distinguished from each other by the FA test and all

237

Fig. 1. Agar gel diffusion precipitin comparisons of BTV20 (CSIRO 19; 19), CSIRO 154 (154) virus and CSIRO 156 (156) virus. The AGDP antigens are in the centre well and hyperimmune rabbit antisera in the outer wells. Every second well contains reference homologous serum.

viruses and sera showed complete lines o f identity, for the outer line, by the AGDP test (Fig. 1). In some cases there was a second inner line which appeared identical and in a few cases a third line was seen close to the antigen well, for which n o lines o f identity could be seen. TABLE I Complement-fixing antibody relationships between BTV20, CSIRO 154, and CSIRO 156 viruses Antigen

Antiserum BTV20 a

CSIRO 154 b

CSIRO 156 b

BTV20

40/50 c

30/60

30/60

CSIRO 154

60/30

60/30

30/30

CSIRO 156

40/10

40/30

40/20

aBovine antiserum with 2.5% unheated bovine serum added as enhancing serum. bMouse immune ascites turnout fluids with no added enhancing serum. CAntibody titre/antigen titre.

238

In addition, n o n e of these viruses or their antisera could b e distinguished b y the C F test (Table I). It w a s c o n c l u d e d that C S I R O 1 5 4 and C S I R O 1 5 6 viruses were m e m b e r s of the B T V serogzoup (Borden et al., 1971; J o c h i m et al., 1974).

Serotyping of CSIRO 156 virus (PI and PR) Initially CSIRO 156 virus was tested using antisera against BTV1 to BTV20, CSIRO 154 and CSIRO 156 viruses. PI zones were seen with anti-CSIRO 156 and anti-BTV1 sera, but n o t with any of the other BTV reference antisera or anti-CSIRO 154 serum. A two-way cross-neutralization study was carried out using a three times plaque-purified attenuated vaccine strain of BTV1 (Table II). The results indicated t h a t CSIRO 156 virus could n o t be distinguished from BTV1 using the PI test. The results also showed t h a t treating the sera with BPL produced a slight reduction of antibody activity. T A B L E II Plaque-inhibitionneutralizationcomparison of B T V 1 and CSIRO 156 viruses Antiserum

Anti-BTV1, guinea pig, BPL b Anti-CSIRO 156, ovine (E960), Anti~-CSIRO 156, ovine (E960), Anti-CSIRO 156, ovine (E961), Anti-CSIRO 156, ovine (E961), Negative control,ovine, BPL

Plaque-inhibition zone (mean ± S.E. in m m ) againsta

BPL NI c BPL NI

BTV1

CSIRO 156

17.3±1.1 17.3±1.1 19.9±1.7 18.9±2.5 21.4±2.0 0±0

16.3±1.1 16.6±1.0 i9.3±1.5 16.9±1.5 19.6±1.0 0±0

aMean and standard error of seven determinations made on three separate occasions. bBPL, sera treated with 0.4% O-propriolactone(BPL) to inactivateany residualvirus in the antiserum. The sera were then dialyzed and adjusted to a dilutionof 1 in 2 with respect to the originalserum volume. CNI, sera treated as for b except not inactivated.Tested at a dilutionof 1 in 2.

The 50% and 80% plaque-reduction (PR) neutralization titres were determined for BTV1 and CSIRO 156 virus and indicated that the viruses could not be distinguished from each other. If anything, the heterologous titres are slightly higher than the homologous titres. Further analysis of the dose-response relationships in the plaque-reduction assay of neutralization mixtures of BTV1 and CSIRO 156 virus was made by calculation of the regression line equations (Fig. 2), and comparison of the area under the curves (Fig. 2) by calculation of the "area f u n c t i o n " . The standard errors in the "area f u n c t i o n s " indicate that again these viruses could n o t be differentiated, as differences less than 2~/S.E.~ 2 + S.E.~ 2 are n o t statistically significant at the 5% significance level.

239 A

o

g, m,,,

Z O ffi

::)

I SERUM

2 3 DILUTION

4 (-log,o)

Fig. 2. Linear regression curves of dose-response relationships for (a) guinea pig anti-BTV1 and (b) ovine anti-CSIRO 156 virus serum (E960) from the surviving fractions of BTV1 (•) and CSIRO 156 virus (e). Both sera had been treated with 0.4% BPL as described in materials and methods.

Serotyping o f CSIRO 154 virus (PI and PR) Initially CSIRO 154 virus was tested using antisera against BTV1 to BTV20, CSIRO 154 and CSIRO 156 viruses. PI zones were recorded with anti-CSIRO 1 5 4 a n d a n t i - B T V 6 sera, b u t n o t w i t h a n y o f t h e o t h e r B T V r e f e r e n c e a n t i s e r a or a n t i - C S I R O 1 5 6 s e r u m . A t w o - w a y c r o s s - n e u t r a l i z a t i o n s t u d y was carried out using a three times plaque-purified attenuated vaccine strain of BTV6 (Table III). T h e r e f e r e n c e a n t i - B T V 6 s e r u m s t r o n g l y n e u t r a l i z e d B T V 6 , b u t TABLE III Plaque-inhibition neutralization comparison of BTV6 and CSIRO 154 virus Antiserum

Anti-BTV6, guinea pig, BPL b Anti-CSIRO 154, ovine (E957), Anti-CSIRO 154, ovine (E957), Anti-CSIRO 154, ovine (E958), Anti-CSIRO 154, ovine (E958), Anti-CSIRO 154, rabbit, BPL

Plaque-inhibition Zone (mean +_ S.E. in mm) against a

BPL NI c BPL NI

BTV6

CSIRO 154

23.0 _+ 1.9 7.0 +_ 2.0 15.6 + 1.5 1.6 +_ 3.6 0+- 0 0+- 0

15.4 _+ 0.8 17.9 +_ 1.7 20.4 _+ 1.7 17.4 _+ 1.3 19.9+_ 1.1 12.4+ 1.3

aMean and standard error of five to seven determinations done on two to three separate occasions. bBPL, sera treated with 0.4% BPL to inactivate any residual virus in the antiserum. The sera were then dialyzed and adjusted to a dilution of 1 in 2 with respect to the original serum volume. CNI, sera treated as for b except not inactivated. Tested at a dilution of 1 in 2.

240 TABLE IV 50% and 80% plaque-reduction neutralization titres for neutralization mixtures of BTV6 and CSIRO 154 virus Antiserum

Neutralization 'titres against a BTV6

BTV6 b CSIRO 154 c

CSIRO 154

50% reduction

80% reduction

50% reduction

80% reduction

2042

525

48

0

81

6

214

25

aReciprocal of dilution of antiserum that produced either a 50% or 80% reduction in plaque counts. bBTV6 guinea pig reference antiserum treated with 0.4% BPL. cCSIRO 156 virus ovine antiserum (E957) treated with 0.4% BPL.

TABLE V Analyses of dose-response relationships in the plaque-reduction assay of neutralization mixtures of BTV6 and CSIRO 154 virus Antiserum

Virus CSIRO 154

BTV6 Aa

Ba

A

B

BTV6 b

2.52

--0.67

0.69

--0.24

CSIRO 154 c

0.95

--0.34

1.31

--0.43

aA and B values are from the regression line equation Y = A + BX. Y represents the --log10 surviving fraction of virus after neutralization and X the --log10 of the antiserum dilution which produced the neutralization. See Westaway (1965). bBTV6 guinea pig reference antiserum treated with 0.4% BPL. cCSIRO 156 virus ovine antiserum (E957) treated with 0.4% BPL. the reaction with CSIRO 154 virus was much weaker. In the reverse direction, the anti-CSIRO 154 sera only weakly neutralized BTV6 whilst the homologous reaction was stronger. Of note was the animal to animal variation and the effect BPL t r e a t m e n t h a d o n r e d u c i n g t h e cross-reaction of sera tested; this was seen w i t h a n t i - C S I R O 1 5 4 o v i n e ( E 9 5 7 ) s e r u m a g a i n s t B T V 6 (see T a b l e I I I ) . T h e 50% a n d 80% P R n e u t r a l i z a t i o n titres were d e t e r m i n e d for B T V 6 and C S I R O 154 virus (Table IV) and showed that these viruses could be readily differentiated. The 50% PR neutralization titres indicated more cross-relatedness than the 80% PR neutralization titres. Further analysis of the dose-response

241 m

I--

o£ u.

ntis

um

°i z_ _>

1

2

3

4

SERUM DILUTION (-Ioglo) Fig. 3. Linear regression curves of dose-response relationships (Table V) for (a) guinea pig anti-BTV6 serum and (b) ovine anti-CSIRO 154 virus serum (E957) (BPL treated sera) from the surviving fractions of BTV6 (m) and CSIRO 154 virus (o).

relationships in t h e PR assay of neutralization m i x t u r e s of antisera of BTV6 or

CSIRO 156 virus was made by calculating the regression line equations (Table V, Fig. 3) a n d b y c o m p a r i n g the area u n d e r the curves (Fig. 3) and calculation o f t h e " a r e a f u n c t i o n " ( T a b l e V I ) . A n a l y s e s o f t h e s t a n d a r d e r r o r s in t h e " a r e a f u n c t i o n s " i n d i c a t e d t h a t these viruses, a l t h o u g h related, can be readily distinguished from each other. Normalizing the "area function", with respect to the homologous virus-antiserum reaction allowed direct comparison of the relatedness o f B T V 6 a n d C S I R O 1 5 4 virus. TABLE VI Activity of cross-reacting antisera in the plaque-reduction assay of neutralization mixtures of BTV6 and CSIRO 154 virus expressed as an "area function" derived from the regression line equation Y = A + BX Antiserum

Virus BTV6

CSIRO 154

Area a function ± S.E. b

Normalized c value

Area function ± S.E.

BTV6 d

4.7

100

1.02 ± 0.3

CSIRO 154 e

1.34 ± 0.04

± 0.7

68

2.02 ± 0.005

Normalized value 22 100

aArea function = -~A2/B. Calculation after Westaway (1965). bS.E., standard error in area function (see materials and methods). Calculation after DellaPorta et al. (1981). CThe normalized value has been obtained by assigning a value of 100 to the homologous area function of each antisera and adjusting the functions in the cross reactions by proportion. Calculations after Westaway (196 5). dBTV6 guinea pig reference antiserum treated with 0.4% BPL. eCSIRO 154 virus ovine antiserum (E957) treated with 0.4% BPL.

242 DISCUSSION Serotyping of CSIRO 156 virus showed that it could not be distinguished from BTV1 by plaque-inhibition (Table II) or plaque-reduction neutralization tests (Fig. 2). In fact, the 50% and 80% PR neutralization titres for the heterologous reactions were higher than for the homologous reactions. Similar observations on other serotypes of BTV have been made by Barber and Jochim (1973) using an 80% PR test, by Thomas and Trainer (1971) using a 50% PR test and by Howell (11960) using a neutralization index test. In both the plaque-inhibition test (Table II) and analyses of the dose-response relationships (Fig. 2), the results suggest that the heterologous reactions may be slightly greater but this difference was not statistically significant. BTV1 has been isolated in South Africa, Egypt and India (Howell, 1970). It is possible that the virus distribution extends through South East Asia into Australia and this is worthy of investigation by serological testing. Serological surveys in Australia (A.J. DeUa-Porta, R.F. Sellers, K.A.J. Hemiman and others, unpublished data, 1978) have suggested that BTV1, or a virus capable of producing antibodies in cattle that would cross-react strongly with BTV1, was present in Australia. CSIRO 156 virus may be the virus that produced the serological response to BTV1 in these cattle. Studies on the pathogenicity of CSIRO 156 for cattle and sheep have shown that no clinical signs are induced in cattle and only mild to moderate clinical signs in sheep (T.D. St. George, personal communication, 1979), similar to the clinical response observed with BTV20 (St. George and McCaughan, 1979). We concluded from this and our studies that CSIRO 156 virus ~vas an Australian isolate of the BTV1 serotype and is of low pathogenicity. Serotyping of CSIRO 154 Virus indicated it was related to BTV6 but distinct from it and from the other BTv serotypes 1 to 20. The plaque-inhibition tests (Table III) showed that reference guinea pig anti-BTV6 serum would neutralize CSIRO 154 virus with a zone of inhibition of 15.4 mm. Zones ~ 10 mm in diameter have been considered significant (Davies and Blackburn, 1971; Davies, 1978; Della-Porta et al., 1981) and zones ~ 15 mm have been considered as strong positive reactions. However, inhibition of homologous BTV6 was significantly greater (23.0 mm diameter) than that of CSIRO 154 virus. Further, some anti-CSIRO 154 virus sera weakly neutralized BTV6. Thus, the plaqueinhibition neutralization test indicates that BTV6 and CSIRO 154 virus are not identical serotypes. Cross-reactions of this nature have been observed, between different BTV serotypes, cf. Nigerian isolates (Davies and Blackburn, 1971), and between the Australian BTV20 and BTV4 (Della-Porta et al., 1981). Another observation of interest was made when using BPL to inactivate any possible residual exotic viruses in the reference antisera obtained from Onderstepoort (Tables II and III). Whereas with anti-CSIRO 156 sera there was only a slight reduction in antibody titres (Table II), with anti-CSIRO 154 sera (Table III), especially from sheep E957, there was a significant reduction in the heterologous antibody titres, but only a slight reduction in homologous ~ titres.

243

A more detailed investigation of the serological relationships between CSIRO 154 virus and BTV6 was carried o u t using plaque-reduction neutralization tests. The 50% and 80% plaque-reduction neutralization titres (Table IV) confirmed that CSIRO 154 virus and BTV6 were n o t identical viruses. The 50% endpoints showed much greater cross-reactivity than did the 80% endpoints. This would justify the use of 80% endpoints in preference to 50% endpoints (Thomas and Trainer, 1971; Barber and Jochim, 1973; Della-Porta et al., 1981), although. we believe the plaque-inhibition test and an analysis of plaque-reduction assays in terms o f dose-relationships and area functions to be superior for typing BTV isolates (Howell et al., 1970; Davies and Blackburn, 1971; Della-Porta et al., 1981). Analyses of the dose-response relationships in the plaque-reduction assay of neutralization mixtures of BTV6 or CSIRO 154 virus (Tables V and VI, Fig. 3) show clearly that the regression line equations (Table V and Fig. 3) and the area under these curves (Fig. 3, Table VI) are significantly different. The normalization o f the area function allowed us to compare the crossreactions between BTV6 and CSIRO 154 virus. It was concluded that, provided all reference anti-BTV1 to 19 sera were active, CSIRO 154 virus was n o t an isolate of one of the recognized 20 serotypes, b u t was probably a new BTV serotype. The virus was related to BTV6 or is possibly a subtype of BTV6, in a similar way to BTV20 being a possible subtype of BTV4 (Della-Porta et al.,

1981). BTV6 has been isolated only in Africa (Howell, 1970) and serological surveys in Australia (A.J. Della-Porta, R.F. Sellers, K.A.J. Herniman and others, unpublished data, 1978) showed very few cattle sera with significant neutralizing antibody titres against BTV6. Thus, it would appear that CSIRO 154 virus infectior~s in cattle have n o t been detected by serum neutralization tests using BTV6 as the test virus. CSIRO 154 virus could be one of the viruses responsible for the BTV-AGDP positive bovine sera which did n o t react with BTV1 to 17, BTV20 or Ibaraki virus in serum neutralization tests (A.J. Della-Porta, R.F. Sellers, K.A.J. H e m i m a n and others, unpublished data, 1978). Studies on the pathogenicity of CSIRO 154 virus for cattle and sheep have shown that there are no clinical signs in cattle and only mild to moderate clinical signs are produced in sheep (T.D. St. George, unpublished data, 1979), similar to those observed with BTV20 (St. George and McCaughan, 1979). In conclusion, our serological studies have demonstrated that CSIRO 154 and CSIRO 156 viruses are Australian BTV isolates additional to BTV20 (St. George et al., 1978). CSIRO 156 virus is a member of the BTV1 serotype, whilst CSIRO 154 virus is related to the BTV6 serotype b u t is probably distinct from all 20 serotypes. No clinical disease has been seen in field animals and only mild to moderate clinical signs in sheep experimentally infected with either virus. Analyses of the viral proteins and nucleic acids may reveal differences between these isolates and the virulent BTV isolates; studies aimed at defining these differences are being undertaken. N o t withstanding the results of such analyses, the serological results meet the presently accepted requirements for the classification of CSIRO 156 virus as BTV1 and CSIRO 154 virus as probably a n e w BTV serotype.

244 ACKNOWLEDGEMENTS We wish t o t h a n k t h e D i r e c t o r o f t h e C o m m o n w e a l t h S e r u m L a b o r a t o r y , Dr. N.J. M c C a r t h y , f o r p e r m i s s i o n t o u n d e r t a k e t h e s e r o t y p i n g in t h e Bazeley M a x i m u m S e c u r i t y L a b o r a t o r y . Dr. B. Erasmus, V e t e r i n a r y Research I n s t i t u t e O n d e r s t e p o o r t , supplied us w i t h t h e anti-BTV1 t o 16 a n d 18 t o 19 g u i n e a pig reference sera a n d Dr. I.M. P a r s o n s o n t h e a n t i - B T V 1 7 guinea pig r e f e r e n c e serum. T h e c o l l e c t i o n o f t h e b l o o d samples f r o m t h e sentinel animals, f r o m w h i c h t h e t w o s e r o t y p e s o f b l u e t o n g u e virus were isolated, was carried o u t b y officers o f t h e N o r t h e r n T e r r i t o r y D e p a r t m e n t o f P r i m a r y Industries.

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