be stored frozen at extremely low temperatures such as —78 C. but not at the temperature of the ordinary 

 household freezer, —20° C. An experiment was performed to determine the stability of BT virus at —20 C. 



Materials and Methods 



Virus Strains.— Bluetongue virus strain California BT8, 10th sheep passage was obtained by bleeding an 

 infected sheep 6 DPI. It was supplied 2 in an anticoagulant preservative solution, OPG. 3 California (type 10) BT 

 virus, mouse brain passage was obtained as freeze dried mouse brain. 



Fluorescent Antibody Conjugate. Sheep 877 was inoculated with 2.0 ml of BT virus suspension, strain BT8, 

 blood with OPG. Serum was collected 54 DPI when the complement fixation (CF) titer was 1-40 and a conjugate 

 was prepared as previously described. (8, 13) 



Experimental A nimals.— Sheep were obtained from two closed flocks located in Iowa that had been tested and 

 found negative for BT by the CF test. All sheep were tested before inoculation and found negative. Sheep used for 

 virus detection were maintained in plastic isolation cages maintained under negative air pressure. One group of three 

 sheep employed for long-term studies were kept together in an isolation room free of insect vectors. 



Complement Fixation Test.— The CF test was performed according to the method of Boulanger as previously 

 described (1). 



Cell Cultures.— Primary embryonic bovine kidney (EBK), embryonic ovine testes (EOT), and embryonic ovine 

 kidney cell cultures (EOK); and bovine turbinate (BT) and embryonic bovine tracheal (EBTr) cell lines were 

 propagated on coverslips in Leighton tubes employing appropriate cell culture mediums as previously described 

 (5, 3). The culture medium was decanted and virus inoculum consisting of heparinized whole blood or 10 percent 

 mouse brain suspension was placed on the cell sheets. The cultures were maintained at 37 C. for at least 2 hours, 

 washed three times with Earle's balanced salt solution, and the nutrient medium was replaced. The coverslip cultures 

 were kept at 37 C. and at daily intervals selected coverslips were harvested and strained with the conjugate (4). 



Storage of Frozen Virus.— The virus inoculums were maintained as appropriate at -20° C. and —78° C. s 



Results 



Cell Culture of Bluetongue Virus From Sheep Blood and Mouse Brain.— Heparinized whole blood samples were 

 obtained from infected sheep during the clinical stages of the disease usually 4 - 10 DPI, and isolation attempts were 

 made on cell cultures. Coverslip cultures were stained daily from exposed EBK, EOT, EOK, BT and EBTr cell 

 cultures. Fluorescing cells were detected on coverslips of EOK cell cultures that had been incubated for 5 to 6 days 

 after exposure to virus blood. The fluorescing viral antigen was granular ir, appearance and found in the cytoplasm of 

 the infected cells. However, the detection of infected cells by immunofluorescence was not consistent. A variety of 

 methods were employed to improve the replication of virus recovery in the primary EOK cultures without success. 



Bluetongue virus from infected sheep blood was isolated only once on EBTr cell cultures and was never 

 recovered from viremic blood on EBK, OT, and BT cell cultures by immunofluorescence. 



On the contrary, when mouse brain adapted BT 10 strain of the virus was inoculated on the cell cultures, 

 fluorescing virus infected cells were consistently detected. Numerous large plaques of fluorescing cells were detected 

 in the primary EOT cell cultures. 



Limited attempts to adapt BT virus from sheep blood to cell culture by blind passages were not successful. 



Effect of Freezing and Storage on Bluetongue Virus.— Bluetongue virus in sheep blood mixed with OPG was 

 divided and a portion was maintained by 4 C. while the other was frozen at —78 C. for 48 hours. The two portions 

 were each inoculated into susceptible sheep and the presence of viable BT virus was confirmed by diagnostic 

 increases in CF titers from negative to 1-80 and 1-160 in both sheep. 



To more precisely determine whether there was a loss of virus titer and to study the effect a storage of 

 heparinized sheep blood containing BT virus at the temperature of an ordinary household freezer, a set of virus 



Supplied by Dr. M. M. Jochim, Animal Disease Research Laboratory, ARS, USDA, Federal Center, Denver, Colo. 80225. 

 OPG solution; potassium oxalate, 5 gm., phenol 5 gm., glycerin, 500 ml. and distilled water, 500 ml. 



Supplied by Dr. Paul Boulanger, Animal Pathology Division, Health of Animals Branch, Animal Diseases Research Institute, 

 Hull, Quebec, Canada. 



5 Revco, Inc., Industrial Products Division, Deerfield, Mich. 



