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Virological, clinical and serological responses of sheep infected with tissue culture adapted bluetongue virus serotypes 10, 11, 13 and 17
Veterinary Microbiology, 10 (1984/85) 179--188 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
179
VIROLOGICAL, CLINICAL AND SEROLOGICAL RESPONSES OF SHEEP INFECTED WITH TISSUE CULTURE ADAPTED BLUETONGUE V I R U S S E R O T Y P E S 10, 11, 13 A N D 17
H.W. GHALIB*, J.M. CHERRINGTON and B.I. OSBURN
Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) (Accepted 17 September 1984)
ABSTRACT
Ghalib, H.W., Cherrington, J.M. and Osburn, B.I., 1985. Virological, clinical and serological responses of sheep infected with tissue culture adapted bluetongue virus serotypes 10, 11, 13 and 17. Vet. Microbiol., 10: 179--188. Sheep were experimentally infected with cloned strains of tissue culture adapted bluetongue virus (BTV) serotypes 10, 11, 13 and 17. All the infected animals developed viremia by Day 2 or 3 post-inoculation (P.I.) and reached maximum viremia on Day 7 P.I. The viremia lasted for 2 to 3 weeks. Animals infected with the different serotypes showed mild clinical bluetongue (BT) responses, characterized by pyrexia and leukopenia, which coincided with the peak of viremia. Antibodies appeared by Day 10 P.I. and reached maximum by Day 28 P.L There was a temporal relationship between the increase in neutralizing antibody titer, the drop in titer and clearance of virus from the peripheral circulation. Recovery from primary infection protected the animals against secondary challenge with homologous virus.
INTRODUCTION
B l u e t o n g u e (BT) disease in sheep is an a c u t e febrile disease m a n i f e s t e d clinically b y a catarrhal i n f l a m m a t i o n o f the m u c o u s m e m b r a n e s o f the digestive and r e s p i r a t o r y tracts and associated with degenerative changes in the skeletal m u s c u l a t u r e . Congenital a b n o r m a l i t i e s such as cerebellar hypoplasia, h y d r a n e n c e p h a l y , or subcortical cerebral cysts and a n o n - s u p p u r a tive meningo~encephalitis were n o t e d in lambs i n f e c t e d in u t e r o ( Y o u n g a n d C o r d y , 1 9 6 4 ; R i c h a r d s and C o r d y , 1 9 6 7 ; O s b u r n and Silverstein, 1972). T h e causative agent is b l u e t o n g u e virus (BTV), w h i c h is the p r o t o t y p e o f the Orbivirus genus within the family Reoviridae ( M u r p h y et al., 1 9 7 1 ; V e r w o e r d et al., 1979). It is an arbovirus t r a n s m i t t e d biologically b y the biting midge, Culicoides variipennis ( F o s t e r et al., 1963). *Present address: Michigan State University, NIH-Sudan Medical Parasitology, Research Project, P.O. Box 8273, Alamarat, Khartoum, Sudan.
0378-1135/85/$03.30
© 1985 Elsevier Science Publishers B.V.
180 Clinical BT disease in sheep was experimentally reproduced by intradermal inoculation of blood obtained from viremic animals {Luedke et ah, 1964; Jochim et al., 1965). Sheep were successfully infected with purified tissue culture adapted BTV 10 (Pini, 1976). The infection was also successfully transmitted by exposure of susceptible sheep to infected art hropod vectors (DuToit, 1944; Foster et al., 1963). Sheep m o u n t a humoral immune response as early as Day 14 P.I. which may last for a year or longer. Typespecific neutralizing antibodies, group-specific precipitating and complem e n t fixing antibodies were observed following experimental infection (Jochim et al., 1965; Klontz et al., 1962). The present study was undertaken to determine the infectious and immune processes associated with 4 plaque-picked, purified BT viruses in sheep. Successful infection of sheep with tissue culture adapted p r o t o t y p e s of the 4 BTV serotypes present in the United States is reported here. Clinical, hematological, virological and serological responses were observed. The temporal relationship between infection, immune responses, virus clearance and protection against secondary challenge are discussed. MATERIALS AND METHODS
Celts Baby hamster kidney (BHK-21) cells and African Green Monkey (Veto) cells were obtained from Naval Biomedical Sciences Labs, Oakland, CA. Cells were grown in Eagle's minimal essential medium (MEM) supplemented with 10% foetal calf serum (FCS) and antibiotics.
Viruses The passage history of the p r o t o t y p e strains of the U.S.A. BTV serotypes (10, 11, 13 and 17) (Table I) were obtained from T.L. Barber, ArthropodBorne Animal Disease Research L a b o r a t o r y , Denver, CO. All virus strains TABLE I History of prototype bluetongue virus strains Strain of virus
Serotypes Original animal isolate
Year of Number of passages isolation Sheep C h i c k Lamb Baby hamster embryo kidney kidney cells cells
Calif8 Station 67-41B 62-455
10 11 13 17
1953 1967 1967 1962
Sheep Sheep Bovine Sheep
3 4 2 3
1 2 1 2
7 7 5 7
5 4 5 7
181 were plaque-picked 3 times in Vero cells. The final plaque-picked clone was used to produce stock virus. Stock viruses were grown in BHK-21 cells with FCS-free MEM medium for 48 h at 37°C in an atmosphere of 5% COs. The cells were harvested and collected by pelleting at 3000 g for 20 min at 4°C, and suspended in 2 mM Tris HC1 buffer at the c o n c e n t r a t i o n of 107 cells m l - ' . Virus was extracted from cells by sonication for 30 s at 4°C in 2 mM Tris-HCl buffer (pH 8.8). The supernatant fluid was collected by centrifugation at 3000 g for 20 min at 4°C. The pellet was extracted in the same way twice. The supernatant fluids were pooled and mixed with an equal volume of buffered lactose p e p t o n e (BLP) (pH 7.1) and aliquots were frozen at --70°C. Viruses were titrated by plaque assay on Vero cells and by TCIDs0 on BHK cells. The plaque-picked viruses were serotyped to confirm the stability of the original strains. These viruses were used for serological assay and for preparative p r o d u c t i o n of pure viruses for animal inoculation.
Virus production and purification BHK-21 cells were grown in 890 cm 2 roller bottles at 37°C using Eagle's MEM supplemented with 10% FCS and 10% t rypt ose phosphate broth and antibiotics. Confluent monolayers were washed twice with medium supplemented with antibiotics only. Virus was adsorbed to cells at r o o m temperature for 30 min at MOI of 10 PFU/cell. Media w i t hout FCS was then added, and the cells were incubated at 37°C for 48 h. The cells were harvested by scraping, pelleted at 3000 g for 20 min, washed once, and resuspended in 2 mM Tris HCI buffer (pH 8.8). Virus was extracted from infected cells according to Verwoerd as modified by Huismans (Verwoerd, 1969; Huismans, 1979). Briefly, the cells were first homogenized and extracted with 1/3 volume of cold freon after the addition o f one tenth volume of 1% Sephadex G-200. E xt ract i on was at 4°C. The freon phase was washed with 1/2 the original volume of Tris buffer and the c om bi ned freon-free phases were extracted 2 more times in the same way. Bovine serum albumin was added to the last extract to a final 0.5% concentration followed by one tenth volume of 10% Tween 80 solution in 2 mM Tris HCl-buffer pH 8.8. T he final mixture was extracted with 1/2 volume of freshly distilled ether. The salt concent rat i on of the water phase was adjusted to 0.1 M with NaCl. The virus was pelleted through a 5-ml layer of 40% sucrose in an SW 27 r o t o r at 24 000 rpm in a Beckman L-50 ultracentrifuge, for 90 min at 4°C. The virus pellet was resuspended in 2 mM Tris-HCl buffer, pH 8.8, to dilute the sucrose and was pelleted at 50 000 r m i n - ' using an SW 50 r o t o r for 10 min at 4°C. The final virus pellet was resuspended in 2 mM Tris HC1 buffer, pH 8.8, titrated by plaque assay and stored at 4°C for animal inoculation.
182
Animals Ten, 3--6-month old sheep, were included in the experiment. The animals were of the same sex (male) and breed (Corriedale). T h e y were obtained from a BTV-free area and were negative for BT virus. T hey did n o t show precipitating antibodies against BTV. Sheep were housed in a screened facility. Baseline data on blood, serum and peripheral cellular parameters were taken before virus inoculation. The animals were random l y divided into 5 groups. F our groups were infected, each with 1 of the 4 BTV serotypes (10, 11, 13 and 17) present in the U.S.A., and the fifth group was kept as a c o n t a c t control group. Each animal was given 2 ml of pure BTV containing 107 pfu m1-1 in 2 mM Tris HCI buffer, pH 8.8. One ml was given intravenously, 0.5 ml subcutaneously and 0.5 ml intradermally. The animals were kept for periods ranging from 9 to 12 weeks and were rechallenged with the same homologous BTV as described above. Peripheral blood and serum for virus titration and serology were collected daily for the first 10 days and then weekly during the course of the trial. The animals were observed daily for clinical signs and rectal temperature changes. Total white blood cell counts were p e r f o r m e d regularly on collected blood samples.
Virus isolation and titration Peripheral blood was drawn by venipuncture into heparinized tubes. The blood was centrifuged at 1500 r min -1 for 20 min at 4°C. Plasma was discarded. Th e cellular blood pellet including the buffy coat was washed twice in normal saline pH 8.0. The volume was finally adjusted to the original blood volume with normal saline. Blood samples were sonicated for 30 s in an ice bath. Ten-fold dilutions were made in normal saline, stored at 4°C and used for virus isolation and titration within a 24-h period. Virus was isolated and titrated in 10--11-day-old e m b r y o n a t i n g chicken eggs (ECE). Eggs were inoculated intravenously with 0.1 ml of each dilution. Six eggs were used per dilution. E m b r y o deaths between 2 and 7 days P.I. were considered for calculating the 50% end-point of chicken e m b r y o lethal doses per ml (CELDs0 m1-1) (Goldsmit and Brazilai, 1968). Dead embryos were collected, and the virus passaged once more in ECE, and then adapted to tissue culture and re-serotyped.
Serological tests Blood for the antibody tests was collected without anticoagulant, and the serum was separated and stored at --20°C.
t83 S e r u m neutralization t e s t s
The quantal microtiter agsay developed by Parker et al. was employed (Parker et al., 1975; Della-Porta et al., 1981). Briefly, 2-fold dilutions of sera, starting at 1/10, were prepared in flat-bottomed microtiter plates and 100 TCIDs0 of virus were added to each well. The mixture was incubated at 37°C for 1 h and then overnight at 4°C. BHK-21 cells were added to each well and the plates were sealed and incubated at 37°C. Cytopathic effect (CPE) was recorded from its first appearance at 30--36 h. The SNs0 titer was based on the inhibition of CPE, which had developed fully within 4--5 days, and was expressed as the reciprocal of the final dilution of serum present at the 50% end-point. The agar gel immunodiffusion tests (AGIT) were performed as described earlier (Jochim and Chow, 1969). Clin ical o bserva tions
The rectal temperature of infected and control animals was taken daily. Total white blood cell counts were measured. The animals were observed for clinical signs and macroscopic lesions. RESULTS Clinical response
The clinical data are presented in Table II. The animals infected with BTV serotypes 10, 11, and 17 showed transient clinical signs. All the infected animals except those infected with BTV 13 developed pyrexia (105.0--106.2) that lasted between 24 and 48 h. Most of the animals showed excessive salivation and dyspnea coinciding with the pyrexia, but no mouth lesions were observed. T A B L E II Initial clinical responses to primary BTV infection of sheep Sheep No.
BTV strain
inoculated
Rectal temperature Maximum
Duration
Minimum WBC X 10 3
Day of o n s e t P.I.
Duration
temperature
105.8 105.2 105.2 104.6 106.2 105.0 Normal Normal
5th 4th 5th 9th 5th 4th ---
2 days 1 day 1 day 1 day 7 days 2 days ---
7.4 7.9 7.9 6.3 4.6 8.6 4.4 Normal
5th 7th 7th 7th 4th 4th 5th --
3 5 3 3 2 5 1 --
(°F)
959 969 962 968 242 279 244 281
II II 17 17 10 10 13 13
ap.I. = post infection.
hemogrdm: leukopenia
Day o f o n s e t P.I. a
days days
days days
days days day
184
All the infected animals developed marked leukopenia on Day 3 P.I. which lasted for 5--7 days. The 2 control animals remained normal. All the animals remained clinically normal following challenge with homologous virus. Virem ia
All the infected animals developed viremia as early as Day 3 P.I. which lasted in some animals up to Day 14 P.I. The maximum viremia was reached by Day 7 P.I. The virus titer dropped considerably by Day 14 P.I. in the animals infected with BTV 11 and 17, while sheep infected with BTV 10 and 13 showed a noticeable drop by Day 10 P.I. The animals cleared BTV 10 and 13 by Day 14 P.I., while sheep infected with BTV 11 and 17 cleared the virus by Day 21 P.I. The control animals did not develop viremia during the experimental period. Viremias were not observed following secondary challenge with homologous virus. The viruses recovered from dead embryos used in virus isolation and titration were confirmed to be of the same serotype used for inoculation. Serological responses Precipitating an tibodies All the infected animals developed detectable agar gel immunodiffusion test (AGIT) antibodies by Day 10 P.I. These antibodies lasted for the 12week duration of the triM. The control animals did not develop AGIT antibodies. TABLE III S e r u m n e u t r a l i z a t i o n t i t e r of s h e e p i n f e c t e d w i t h d i f f e r e n t B T V s e r o t y p e s against h o m o l o g o u s viruses. SNs0 a t i t e r against h o m o l o g o u s v i r u s Sheep number
BTV strain inoculated
Experimental period 0
7
10
14
21
28
Days p o s t - i n f e c t i o n 0b
959 969 962 968 242 279 244 281
11 11 17 17 10 10 13 13
7
<10 <10 <10 <10 10 40 10 80 <10 20 <10 20 <10 4<10 <10 tu<10
63
70
77
91
Days p o s t ~ h a U e n g e
10
14
21
28
0c
7
40 40 40 80 80 40 20 20
160 160 160 80 160 160 20 80
320 320 160 160 320 640 40 ND
640 320 160 160 640 640 40 40
640 320 320 320 160 320 160 320 640 1280 640 1280 8 0 r e 160 40 80
14
28
640 320 640 640 1280 1280 160 160
640 320 640 640 1280 1280 160 160
a R e c i p r o c a l o f t h e d i l u t i o n t h a t i n h i b i t e d t h e CPE in 50% of t h e m i c r o t i t e r t r a y wells a g a i n s t 100 TCIDs0 u n i t s o f virus. b s e r u m c o l l e c t e d on Day 0 b e f o r e i n o c u l a t i o n . CSerum c o l l e c t e d on Day of challenge b e f o r e i n o c u l a t i o n . d p r i m ary i n f e c t i o n . e S e c o n d a r y challenge with h o m o l o g o u s virus.
185 Neutralizing an tibodies Table III includes the serum neutralizing (S.N.) antibody profile which appeared by Day 10 P.I. The titer continued to increase reaching a maximum by Day 28 P.I. There was a noticeable increase in the S.N. titer following secondary challenge in the infected animals. Sheep infected with BTV 13 induced a lower S.N. titer following both primary and secondary challenge compared to BTV serotypes 10, 11 and 17. The highest S.N. titer developed in response to BTV-10. The 2 sheep infected with BTV 17 had an S.N. titer of 10 before inoculation and these animals reached a significant titer by Day 7 P.I. The control animals did not develop 8.N. antibodies during the course of the trial. DISCUSSION
Tissue culture adapted and cloned BTV prototypes found in the U.S.A. were capable of producing experimental BT in sheep. It is clearly demonstrated that cloning does not affect the virus infectious process and its ability to induce an immune response. This confirms the previous observation when BT was successfully reproduced in Merino sheep using tissue culture adapted BTV (Pini, 1976). Cloning of the virus guards against contamination with other serotypes or related viruses and provides a homogeneous population of virus inoculum. Multiple serotypes were frequently isolated from naturally infected animals (Stott et al., 1982), and were suspected in an original inoculum of BTV obtained from sick sheep used to infect a Hereford bull which led to persistent BTV infection. The original inoculum was thought to be only BTV 13, but was later shown to have included BTV 11 as well (Leudke et al., 1982). Cloning is essential for the assessment of the serological response such as group specific and type specific neutralizing antibodies. These assays cannot be determined with confidence if the exact strain of the virus inoculum is not carefully determined. Recent observations on antigenic epitopes and genetic reassortants make it clear that cloning of the virus is essential to avoid introduction of new variables. Genetic recombinants or reassortants were detected between BTV serotypes (Sugiyama et al., 1981). The genetic changes were in segments that are not detected by conventional serologic methods, yet may play an important role in pathogenesis (Collison and Roy, 1983). Also, monoclonal antibodies were shown to identify serotype-restricted antigenic epitopes within the same serotype (Letchworth and Appleton, 1983). Animals inoculated with the 4 different BTV serotypes, except 1 animal inoculated with BTV 13, developed mild transient clinical responses with no apparent complications. It is interesting that BTV 13, which is an original calf isolate (Barber and Jochim, 1973), did not cause a severe clinical re-
186 sponse in temperature or a leukopenia; it induced a shorter viremia and was less antigenic than the other serotypes. This may be a character of the virus not being adapted to sheep. BTV 13 is also not c o m m o n l y isolated from sheep as compared to the other serotypes (Osburn et al., 1981; Barber, 1979). There was a close association noted between the appearance of clinical responses and the peak of viremia. All the infected animals recovered, cleared the virus and were protected against secondary challenge with homologous virus. There was a temporal relationship between the rise in serum antibody titer and the drop in virus titer and the clearance of virus from the peripheral circulation. This correlation indicates the importance of the humoral immunity to BT. All 4 serotypes were immunogenic inducing both neutralizing and AGIT antibodies. Again, BTV 13, which had the least clinical manifestation, was also less immunogenic in comparison to the other serotypes. The immunity induced following primary challenge was also protective against secondary challenge. Although all the animals were AGIT negative on their pre-infection sera, one of the animals infected with BTV 17 had a low S.N. titer when the titer was determined at the end of the experimental period. The AGIT used for pre-inoculation screening was not sensitive enough to detect these low S.N. titers. However, this animal contracted infection following primary inoculation and this low S.N. titer was not protective. The mild clinical BT observed could well represent clinical BT in nature. BTV has been isolated from sheep without apparent clinical BT disease (Osburn et al., 1984). Animals infected with BTV serotype 20 showed similar mild clinical signs (Groocock et al., 1982). The outcome of infection and the severity of the disease is probably dependant on the nature of the virus strain encountered and the host susceptibility. Classical clinical BT disease may be naturally selected against in endemic areas, where vectors prevail and animals are continuously exposed to the virus. The mild nature of the disease in sheep, the high prevalence of vector and the unique BTV genome structure could be potential factors for virus perpetuation and viral persistence. The inapparent or mild nature of BTV in sheep may create problems for eradication and control of the disease. ACKNOWLEDGMENTS This work was supported in part by USDA Special Grant No. 59-20631-2-055. The authors would like to thank J o y Boswell for technical assistance, Carrie Shore for manuscript preparation and Sheryl Flocchini for typing the manuscript.
REFERENCES
Barber, T.L., 1979. Temporal appearance, geographic distribution, and species of origin of bluetongue virus serotypes in the United States. Am. J. Vet. Res., 40: 1654-1656.
187 Barber, T.L. and Jochim, M.M., 1973. Serologic characterization of selected bluetongue virus strains from the United States. Proc. Annu. Meet. U.S. Anim. Health Assoc., 77: 352--359. Collison, E. and Roy, P., 1983. Analysis of the genomes of bluetongue virus serotype 10 vaccines and a recent BTV-10 isolate from Washington. Am. J. Vet. Res., 4 4 : 235--237. 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. Du Toit, R.M., 1944. The transmission of bluetongue and horse-sickness by Culicoides. Onderstepoort J. Vet. Sci. Anim. Ind., 19: 7--16. Foster, N.M., Jones, R.H. and Mc Crory, B.R., 1963. Preliminary investigations on insect transmission of bluetongue virus in sheep. Am. J. Vet. Res., 24: 1195--1200. Goldsmit, L. and Brazilai, E., 1968. An improved method for the isolation and identification of bluetongue virus by intravenous inoculation of embryonating chicken eggs. J. Comp. Pathol., 78: 477--487. Groocock, C.M., Parsonson, I.M. and Campbell, C.H., 1982. Bluetongue virus serotype 20 and 17 infection in sheep: Comparison of clinical and serological responses. Vet. Microbiol., 7 : 189--196. Huismans, H., 1979. Protein synthesis in bluetongue virus-infected cells. Virology, 92: 385--396. Jochim, M.M. and Chow, T.L., 1969. Immunodiffusion of bluetongue virus. Am. J. Vet. Res., 30: 33--41. Jochim, M.M., Luedke, A.F. and Bowne, J.G., 1965. The clinical and immunogenic response of sheep to oral and intradermal administrations of Bluetongue virus. Am. J. Vet. Res., 26: 1254--1260. Klontz, G.W., Svehag, S.E. and Gorham, J.R., 1962. A study of the agar diffusion technique of precipitating antibody directly against bluetongue virus and its relation to homotypic neutralizing antibody. Arch. Gesamte Virusforsch., 12: 259--268. Letchworth, G.J. and Appleton, J.A., 1983. Heterogeneity of neutralization-related epitopes within a bluetongue virus serotype. Virology, 124: 300--307. Luedke, A.J., 1969. Bluetongue in sheep: viral assay and viremia. Am. J. Vet. Res., 30: 499--509. Luedke, A.J., Bowne, J.G. and Jochim, M.M., 1964. Clinical and pathologic features of Bluetongue in sheep. Am. J. Vet. Res., 25: 963--970. Luedke, A.J., Jochim, M.M. and Barber, T.L., !982. Serologic and virologic responses of a Hereford bull persistently infected with bluetongue virus for eleven years. Proc. Annu. Meet. Am. Assoc. Vet. Lab. Diag., 25: 115--133. Murphy, F.A., Borden, E.C., Shope, R.E. and Harrison, A., 1971. Physiochemical and morphological relationships of some arthropod-borne viruses to bluetongue virus -a new taxonomic group. Electron microscopic studies. J. Gem Virol., 13: 273-288. Osburn, B.I. and Silverstein, A.M., 1972. Animal model for human disease: Bluetonguevaccine-virus infection in fetal lambs. Am. J. Pathol., 67: 211--214. Osburn, B.I., Mc Gowan, B., Heron, B., Loomis, E., Bushnell, R., Stott, J. and Utterback, W., 1981. Epizootiologic study of bluetongue: virologic and serologic results. Am. J. Vet. Res., 42: 884--887. Osburn, B.I., Stott, J.L. and Howard, M., 1984. A review of the bluetongue program in California: studies of sheep. Proc. Annu. Meet. U.S. Anita. Health Assoc., 87: 527-532. 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. Pini, A., 1976. A study on the pathogenesis of bluetongue: replication of the virus in the organs of infected sheep. Onderstepoort J. Vet. Res., 43: 159--164.
188 Richards, W.P.C. and Cordy, D.R., 1967. Bluetongue virus infection: pathologic response of nervous systems in sheep and mice. Science, 156: 530--531. Stott, J.L., Osburn, B.I. and Barber, T.L., 1982. Recovery of dual serotypes of bluetongue virus from infected sheep and cattle. Vet. Microbiol., 7: 197--207. Sugiyama, K., Bishop, D.H.L. and Roy, P., 1981. Analysis of the genome of bluetongue viruses recovered in the United States. 1. Oligonucleotide fingerprint studies that indicate the existence of naturally occurring reassortent BTV isolates. Virology, 114: 210--217. Verwoerd, D.W., 1969. Purification and characterization of bluetongue virus. Virology, 38: 203--212. Verwoerd, D.W., Huismans, H. and Erasmus, B.J., 1979. Orbiviruses. In: H. FraenkelConrat and R.R. Wagner (Editors), Comprehensive Virology. Vol. 14. Plenum Press, New York, pp. 285--345. Young, S. and Cordy, D.R., 1964. An ovine foetal encephalopathy caused by bluetongue vaccine virus. J. Neuropathol. Exp. Neuroi., 23: 635--659.
179
VIROLOGICAL, CLINICAL AND SEROLOGICAL RESPONSES OF SHEEP INFECTED WITH TISSUE CULTURE ADAPTED BLUETONGUE V I R U S S E R O T Y P E S 10, 11, 13 A N D 17
H.W. GHALIB*, J.M. CHERRINGTON and B.I. OSBURN
Department of Pathology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) (Accepted 17 September 1984)
ABSTRACT
Ghalib, H.W., Cherrington, J.M. and Osburn, B.I., 1985. Virological, clinical and serological responses of sheep infected with tissue culture adapted bluetongue virus serotypes 10, 11, 13 and 17. Vet. Microbiol., 10: 179--188. Sheep were experimentally infected with cloned strains of tissue culture adapted bluetongue virus (BTV) serotypes 10, 11, 13 and 17. All the infected animals developed viremia by Day 2 or 3 post-inoculation (P.I.) and reached maximum viremia on Day 7 P.I. The viremia lasted for 2 to 3 weeks. Animals infected with the different serotypes showed mild clinical bluetongue (BT) responses, characterized by pyrexia and leukopenia, which coincided with the peak of viremia. Antibodies appeared by Day 10 P.I. and reached maximum by Day 28 P.L There was a temporal relationship between the increase in neutralizing antibody titer, the drop in titer and clearance of virus from the peripheral circulation. Recovery from primary infection protected the animals against secondary challenge with homologous virus.
INTRODUCTION
B l u e t o n g u e (BT) disease in sheep is an a c u t e febrile disease m a n i f e s t e d clinically b y a catarrhal i n f l a m m a t i o n o f the m u c o u s m e m b r a n e s o f the digestive and r e s p i r a t o r y tracts and associated with degenerative changes in the skeletal m u s c u l a t u r e . Congenital a b n o r m a l i t i e s such as cerebellar hypoplasia, h y d r a n e n c e p h a l y , or subcortical cerebral cysts and a n o n - s u p p u r a tive meningo~encephalitis were n o t e d in lambs i n f e c t e d in u t e r o ( Y o u n g a n d C o r d y , 1 9 6 4 ; R i c h a r d s and C o r d y , 1 9 6 7 ; O s b u r n and Silverstein, 1972). T h e causative agent is b l u e t o n g u e virus (BTV), w h i c h is the p r o t o t y p e o f the Orbivirus genus within the family Reoviridae ( M u r p h y et al., 1 9 7 1 ; V e r w o e r d et al., 1979). It is an arbovirus t r a n s m i t t e d biologically b y the biting midge, Culicoides variipennis ( F o s t e r et al., 1963). *Present address: Michigan State University, NIH-Sudan Medical Parasitology, Research Project, P.O. Box 8273, Alamarat, Khartoum, Sudan.
0378-1135/85/$03.30
© 1985 Elsevier Science Publishers B.V.
180 Clinical BT disease in sheep was experimentally reproduced by intradermal inoculation of blood obtained from viremic animals {Luedke et ah, 1964; Jochim et al., 1965). Sheep were successfully infected with purified tissue culture adapted BTV 10 (Pini, 1976). The infection was also successfully transmitted by exposure of susceptible sheep to infected art hropod vectors (DuToit, 1944; Foster et al., 1963). Sheep m o u n t a humoral immune response as early as Day 14 P.I. which may last for a year or longer. Typespecific neutralizing antibodies, group-specific precipitating and complem e n t fixing antibodies were observed following experimental infection (Jochim et al., 1965; Klontz et al., 1962). The present study was undertaken to determine the infectious and immune processes associated with 4 plaque-picked, purified BT viruses in sheep. Successful infection of sheep with tissue culture adapted p r o t o t y p e s of the 4 BTV serotypes present in the United States is reported here. Clinical, hematological, virological and serological responses were observed. The temporal relationship between infection, immune responses, virus clearance and protection against secondary challenge are discussed. MATERIALS AND METHODS
Celts Baby hamster kidney (BHK-21) cells and African Green Monkey (Veto) cells were obtained from Naval Biomedical Sciences Labs, Oakland, CA. Cells were grown in Eagle's minimal essential medium (MEM) supplemented with 10% foetal calf serum (FCS) and antibiotics.
Viruses The passage history of the p r o t o t y p e strains of the U.S.A. BTV serotypes (10, 11, 13 and 17) (Table I) were obtained from T.L. Barber, ArthropodBorne Animal Disease Research L a b o r a t o r y , Denver, CO. All virus strains TABLE I History of prototype bluetongue virus strains Strain of virus
Serotypes Original animal isolate
Year of Number of passages isolation Sheep C h i c k Lamb Baby hamster embryo kidney kidney cells cells
Calif8 Station 67-41B 62-455
10 11 13 17
1953 1967 1967 1962
Sheep Sheep Bovine Sheep
3 4 2 3
1 2 1 2
7 7 5 7
5 4 5 7
181 were plaque-picked 3 times in Vero cells. The final plaque-picked clone was used to produce stock virus. Stock viruses were grown in BHK-21 cells with FCS-free MEM medium for 48 h at 37°C in an atmosphere of 5% COs. The cells were harvested and collected by pelleting at 3000 g for 20 min at 4°C, and suspended in 2 mM Tris HC1 buffer at the c o n c e n t r a t i o n of 107 cells m l - ' . Virus was extracted from cells by sonication for 30 s at 4°C in 2 mM Tris-HCl buffer (pH 8.8). The supernatant fluid was collected by centrifugation at 3000 g for 20 min at 4°C. The pellet was extracted in the same way twice. The supernatant fluids were pooled and mixed with an equal volume of buffered lactose p e p t o n e (BLP) (pH 7.1) and aliquots were frozen at --70°C. Viruses were titrated by plaque assay on Vero cells and by TCIDs0 on BHK cells. The plaque-picked viruses were serotyped to confirm the stability of the original strains. These viruses were used for serological assay and for preparative p r o d u c t i o n of pure viruses for animal inoculation.
Virus production and purification BHK-21 cells were grown in 890 cm 2 roller bottles at 37°C using Eagle's MEM supplemented with 10% FCS and 10% t rypt ose phosphate broth and antibiotics. Confluent monolayers were washed twice with medium supplemented with antibiotics only. Virus was adsorbed to cells at r o o m temperature for 30 min at MOI of 10 PFU/cell. Media w i t hout FCS was then added, and the cells were incubated at 37°C for 48 h. The cells were harvested by scraping, pelleted at 3000 g for 20 min, washed once, and resuspended in 2 mM Tris HCI buffer (pH 8.8). Virus was extracted from infected cells according to Verwoerd as modified by Huismans (Verwoerd, 1969; Huismans, 1979). Briefly, the cells were first homogenized and extracted with 1/3 volume of cold freon after the addition o f one tenth volume of 1% Sephadex G-200. E xt ract i on was at 4°C. The freon phase was washed with 1/2 the original volume of Tris buffer and the c om bi ned freon-free phases were extracted 2 more times in the same way. Bovine serum albumin was added to the last extract to a final 0.5% concentration followed by one tenth volume of 10% Tween 80 solution in 2 mM Tris HCl-buffer pH 8.8. T he final mixture was extracted with 1/2 volume of freshly distilled ether. The salt concent rat i on of the water phase was adjusted to 0.1 M with NaCl. The virus was pelleted through a 5-ml layer of 40% sucrose in an SW 27 r o t o r at 24 000 rpm in a Beckman L-50 ultracentrifuge, for 90 min at 4°C. The virus pellet was resuspended in 2 mM Tris-HCl buffer, pH 8.8, to dilute the sucrose and was pelleted at 50 000 r m i n - ' using an SW 50 r o t o r for 10 min at 4°C. The final virus pellet was resuspended in 2 mM Tris HC1 buffer, pH 8.8, titrated by plaque assay and stored at 4°C for animal inoculation.
182
Animals Ten, 3--6-month old sheep, were included in the experiment. The animals were of the same sex (male) and breed (Corriedale). T h e y were obtained from a BTV-free area and were negative for BT virus. T hey did n o t show precipitating antibodies against BTV. Sheep were housed in a screened facility. Baseline data on blood, serum and peripheral cellular parameters were taken before virus inoculation. The animals were random l y divided into 5 groups. F our groups were infected, each with 1 of the 4 BTV serotypes (10, 11, 13 and 17) present in the U.S.A., and the fifth group was kept as a c o n t a c t control group. Each animal was given 2 ml of pure BTV containing 107 pfu m1-1 in 2 mM Tris HCI buffer, pH 8.8. One ml was given intravenously, 0.5 ml subcutaneously and 0.5 ml intradermally. The animals were kept for periods ranging from 9 to 12 weeks and were rechallenged with the same homologous BTV as described above. Peripheral blood and serum for virus titration and serology were collected daily for the first 10 days and then weekly during the course of the trial. The animals were observed daily for clinical signs and rectal temperature changes. Total white blood cell counts were p e r f o r m e d regularly on collected blood samples.
Virus isolation and titration Peripheral blood was drawn by venipuncture into heparinized tubes. The blood was centrifuged at 1500 r min -1 for 20 min at 4°C. Plasma was discarded. Th e cellular blood pellet including the buffy coat was washed twice in normal saline pH 8.0. The volume was finally adjusted to the original blood volume with normal saline. Blood samples were sonicated for 30 s in an ice bath. Ten-fold dilutions were made in normal saline, stored at 4°C and used for virus isolation and titration within a 24-h period. Virus was isolated and titrated in 10--11-day-old e m b r y o n a t i n g chicken eggs (ECE). Eggs were inoculated intravenously with 0.1 ml of each dilution. Six eggs were used per dilution. E m b r y o deaths between 2 and 7 days P.I. were considered for calculating the 50% end-point of chicken e m b r y o lethal doses per ml (CELDs0 m1-1) (Goldsmit and Brazilai, 1968). Dead embryos were collected, and the virus passaged once more in ECE, and then adapted to tissue culture and re-serotyped.
Serological tests Blood for the antibody tests was collected without anticoagulant, and the serum was separated and stored at --20°C.
t83 S e r u m neutralization t e s t s
The quantal microtiter agsay developed by Parker et al. was employed (Parker et al., 1975; Della-Porta et al., 1981). Briefly, 2-fold dilutions of sera, starting at 1/10, were prepared in flat-bottomed microtiter plates and 100 TCIDs0 of virus were added to each well. The mixture was incubated at 37°C for 1 h and then overnight at 4°C. BHK-21 cells were added to each well and the plates were sealed and incubated at 37°C. Cytopathic effect (CPE) was recorded from its first appearance at 30--36 h. The SNs0 titer was based on the inhibition of CPE, which had developed fully within 4--5 days, and was expressed as the reciprocal of the final dilution of serum present at the 50% end-point. The agar gel immunodiffusion tests (AGIT) were performed as described earlier (Jochim and Chow, 1969). Clin ical o bserva tions
The rectal temperature of infected and control animals was taken daily. Total white blood cell counts were measured. The animals were observed for clinical signs and macroscopic lesions. RESULTS Clinical response
The clinical data are presented in Table II. The animals infected with BTV serotypes 10, 11, and 17 showed transient clinical signs. All the infected animals except those infected with BTV 13 developed pyrexia (105.0--106.2) that lasted between 24 and 48 h. Most of the animals showed excessive salivation and dyspnea coinciding with the pyrexia, but no mouth lesions were observed. T A B L E II Initial clinical responses to primary BTV infection of sheep Sheep No.
BTV strain
inoculated
Rectal temperature Maximum
Duration
Minimum WBC X 10 3
Day of o n s e t P.I.
Duration
temperature
105.8 105.2 105.2 104.6 106.2 105.0 Normal Normal
5th 4th 5th 9th 5th 4th ---
2 days 1 day 1 day 1 day 7 days 2 days ---
7.4 7.9 7.9 6.3 4.6 8.6 4.4 Normal
5th 7th 7th 7th 4th 4th 5th --
3 5 3 3 2 5 1 --
(°F)
959 969 962 968 242 279 244 281
II II 17 17 10 10 13 13
ap.I. = post infection.
hemogrdm: leukopenia
Day o f o n s e t P.I. a
days days
days days
days days day
184
All the infected animals developed marked leukopenia on Day 3 P.I. which lasted for 5--7 days. The 2 control animals remained normal. All the animals remained clinically normal following challenge with homologous virus. Virem ia
All the infected animals developed viremia as early as Day 3 P.I. which lasted in some animals up to Day 14 P.I. The maximum viremia was reached by Day 7 P.I. The virus titer dropped considerably by Day 14 P.I. in the animals infected with BTV 11 and 17, while sheep infected with BTV 10 and 13 showed a noticeable drop by Day 10 P.I. The animals cleared BTV 10 and 13 by Day 14 P.I., while sheep infected with BTV 11 and 17 cleared the virus by Day 21 P.I. The control animals did not develop viremia during the experimental period. Viremias were not observed following secondary challenge with homologous virus. The viruses recovered from dead embryos used in virus isolation and titration were confirmed to be of the same serotype used for inoculation. Serological responses Precipitating an tibodies All the infected animals developed detectable agar gel immunodiffusion test (AGIT) antibodies by Day 10 P.I. These antibodies lasted for the 12week duration of the triM. The control animals did not develop AGIT antibodies. TABLE III S e r u m n e u t r a l i z a t i o n t i t e r of s h e e p i n f e c t e d w i t h d i f f e r e n t B T V s e r o t y p e s against h o m o l o g o u s viruses. SNs0 a t i t e r against h o m o l o g o u s v i r u s Sheep number
BTV strain inoculated
Experimental period 0
7
10
14
21
28
Days p o s t - i n f e c t i o n 0b
959 969 962 968 242 279 244 281
11 11 17 17 10 10 13 13
7
<10 <10 <10 <10 10 40 10 80 <10 20 <10 20 <10 4<10 <10 tu<10
63
70
77
91
Days p o s t ~ h a U e n g e
10
14
21
28
0c
7
40 40 40 80 80 40 20 20
160 160 160 80 160 160 20 80
320 320 160 160 320 640 40 ND
640 320 160 160 640 640 40 40
640 320 320 320 160 320 160 320 640 1280 640 1280 8 0 r e 160 40 80
14
28
640 320 640 640 1280 1280 160 160
640 320 640 640 1280 1280 160 160
a R e c i p r o c a l o f t h e d i l u t i o n t h a t i n h i b i t e d t h e CPE in 50% of t h e m i c r o t i t e r t r a y wells a g a i n s t 100 TCIDs0 u n i t s o f virus. b s e r u m c o l l e c t e d on Day 0 b e f o r e i n o c u l a t i o n . CSerum c o l l e c t e d on Day of challenge b e f o r e i n o c u l a t i o n . d p r i m ary i n f e c t i o n . e S e c o n d a r y challenge with h o m o l o g o u s virus.
185 Neutralizing an tibodies Table III includes the serum neutralizing (S.N.) antibody profile which appeared by Day 10 P.I. The titer continued to increase reaching a maximum by Day 28 P.I. There was a noticeable increase in the S.N. titer following secondary challenge in the infected animals. Sheep infected with BTV 13 induced a lower S.N. titer following both primary and secondary challenge compared to BTV serotypes 10, 11 and 17. The highest S.N. titer developed in response to BTV-10. The 2 sheep infected with BTV 17 had an S.N. titer of 10 before inoculation and these animals reached a significant titer by Day 7 P.I. The control animals did not develop 8.N. antibodies during the course of the trial. DISCUSSION
Tissue culture adapted and cloned BTV prototypes found in the U.S.A. were capable of producing experimental BT in sheep. It is clearly demonstrated that cloning does not affect the virus infectious process and its ability to induce an immune response. This confirms the previous observation when BT was successfully reproduced in Merino sheep using tissue culture adapted BTV (Pini, 1976). Cloning of the virus guards against contamination with other serotypes or related viruses and provides a homogeneous population of virus inoculum. Multiple serotypes were frequently isolated from naturally infected animals (Stott et al., 1982), and were suspected in an original inoculum of BTV obtained from sick sheep used to infect a Hereford bull which led to persistent BTV infection. The original inoculum was thought to be only BTV 13, but was later shown to have included BTV 11 as well (Leudke et al., 1982). Cloning is essential for the assessment of the serological response such as group specific and type specific neutralizing antibodies. These assays cannot be determined with confidence if the exact strain of the virus inoculum is not carefully determined. Recent observations on antigenic epitopes and genetic reassortants make it clear that cloning of the virus is essential to avoid introduction of new variables. Genetic recombinants or reassortants were detected between BTV serotypes (Sugiyama et al., 1981). The genetic changes were in segments that are not detected by conventional serologic methods, yet may play an important role in pathogenesis (Collison and Roy, 1983). Also, monoclonal antibodies were shown to identify serotype-restricted antigenic epitopes within the same serotype (Letchworth and Appleton, 1983). Animals inoculated with the 4 different BTV serotypes, except 1 animal inoculated with BTV 13, developed mild transient clinical responses with no apparent complications. It is interesting that BTV 13, which is an original calf isolate (Barber and Jochim, 1973), did not cause a severe clinical re-
186 sponse in temperature or a leukopenia; it induced a shorter viremia and was less antigenic than the other serotypes. This may be a character of the virus not being adapted to sheep. BTV 13 is also not c o m m o n l y isolated from sheep as compared to the other serotypes (Osburn et al., 1981; Barber, 1979). There was a close association noted between the appearance of clinical responses and the peak of viremia. All the infected animals recovered, cleared the virus and were protected against secondary challenge with homologous virus. There was a temporal relationship between the rise in serum antibody titer and the drop in virus titer and the clearance of virus from the peripheral circulation. This correlation indicates the importance of the humoral immunity to BT. All 4 serotypes were immunogenic inducing both neutralizing and AGIT antibodies. Again, BTV 13, which had the least clinical manifestation, was also less immunogenic in comparison to the other serotypes. The immunity induced following primary challenge was also protective against secondary challenge. Although all the animals were AGIT negative on their pre-infection sera, one of the animals infected with BTV 17 had a low S.N. titer when the titer was determined at the end of the experimental period. The AGIT used for pre-inoculation screening was not sensitive enough to detect these low S.N. titers. However, this animal contracted infection following primary inoculation and this low S.N. titer was not protective. The mild clinical BT observed could well represent clinical BT in nature. BTV has been isolated from sheep without apparent clinical BT disease (Osburn et al., 1984). Animals infected with BTV serotype 20 showed similar mild clinical signs (Groocock et al., 1982). The outcome of infection and the severity of the disease is probably dependant on the nature of the virus strain encountered and the host susceptibility. Classical clinical BT disease may be naturally selected against in endemic areas, where vectors prevail and animals are continuously exposed to the virus. The mild nature of the disease in sheep, the high prevalence of vector and the unique BTV genome structure could be potential factors for virus perpetuation and viral persistence. The inapparent or mild nature of BTV in sheep may create problems for eradication and control of the disease. ACKNOWLEDGMENTS This work was supported in part by USDA Special Grant No. 59-20631-2-055. The authors would like to thank J o y Boswell for technical assistance, Carrie Shore for manuscript preparation and Sheryl Flocchini for typing the manuscript.
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