BLUETONGUE 



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BLUETONGUE VIRUS STUDIES^ L PROPAGATION IN CELL CULTURES AND THE EFFECTS OF 

 FREEZING. .Carbrey, E. A., Gustafson, G. A. and Lee, L. R. (Project Report) 



Summary 



Coverslip cell cultures were inoculated with blood from bluetongue (BT) infected sheep in the acute stages of 

 the disease. Infected cells were detected by immunofluorescence in embryonic ovine kidney and embryonic bovine 

 tracheal cells 5 to 7 days later. Negative results were obtained following inoculation of embryonic bovine kidney, 

 embryonic ovine testes, and bovine turbinate cells. However, propagation of BT virus was successful on all primary 

 and cell line cultures when the inoculum was mouse brain adapted virus in 10 percent brain tissue suspension. 



There was a hundredfold loss of titer in BT virus infected sheep blood that was stored at —20° C. for one year. 



Introduction 



Isolation and identification of bluetongue virus in the laboratory has been hampered by the lack of a suitable 

 cell culture system. Bluetongue (BT) virus obtained directly from the blood of an infected sheep does not readily 

 propagate in cell cultures. Some passages of the virus in the chick embryo (CE) or mouse brain seem to be required 

 and the virus becomes cytopathogenic after adaptation. 



Haig et al (7) 1 were unable to grow BT virus on lamb kidney cell cultures from the serums of infected sheep. 

 However, after one passage in the CE, a cytopathic effect was detected by the fifth day after inoculation. If four 

 passages were made in the CE, then the cytopathic effect was well marked by the third day of growth in the lamb 

 kidney cells. Fernandez (6), however, was able to culture BT virus on lamb kidney cells directly from the viremic 

 blood of acutely ill sheep. A cytopathic effect was detected as early as 24 to 36 hours. 



Pini et al (11) were the first to describe a fluorescent antibody technique for detecting BT virus infected cells 

 in coverslip cultures. The degree of fluorescence was high with concentrated cell culture adapted virus but quite low 

 when the virus source was blood from infected sheep. Baby hamster kidney cells were satisfactory when horse serum 

 was used in the medium after the cells were exposed to the virus. They (10) also found that the fluorescent antibody 

 reaction was group specific since cells infected with six different antigenic types of the virus were stained with the 

 same conjugate. 



Bowne and Jochim (2) obtained specific viral fluorescence in lamb kidney cell cultures 28 hours after 

 inoculation. However, the BT virus was isolated initially by the intravenous inoculation of the CE and then passaged 

 six times in lamb kidney cell culture. The conjugate was prepared from goat serum. 



Ruckerbauer et al (12) described the use of the fluorescent antibody technique for the detection of BT virus in 

 bovine fetal kidney cell cultures inoculated with blood and tissues from infected sheep. The technique was also 

 applied to frozen cultures and tissue impressions with some degree of success. 



After preliminary passage of BT virus six times in lamb kidney cells, Ohder et al (9) were able to detect the 

 fluorescence of infected baby hamster kidney cells in 26 hours. A cytopathic effect was detected at 72 hours when 

 30 percent of the cells were fluorescing. The fluorescent antigen appeared granular and was found in the cytoplasm. 



It was considered of value to investigate the use of cell cultures for the initial isolation and detection of BT 

 virus by immunofluorescence. 



A related diagnostic problem is the lack of information on the stability of BT virus at temperatures below 

 C. Since an effort is made to have all specimens for virus isolation frozen and shipped to the laboratory packed in 

 dry ice, it is important to know if an exception should be made for BT virus. It was reported that the BT virus could 



Number in parentheses refers to References at the end of this report. 



