Rapid, Sensitive Bluetongue Virus Serogroup and Serotype Detection Using Polymerase Chain Reaction

Abstract

The objectives of this proposal were to enhance animal health by 1) development of a BTV serogroup diagnostic assay using polymerase chain reaction (PCR) and 2) development of a BTV serotype specific diagnostic PCR assay. A PCR assay for diagnosis of bluetongue virus (BTV) serogroup from clinical samples meeting the criteria of objective 1 was developed. This PCR assay is more sensitive than virus isolation and has been adopted by both the U.S. and Israeli collaborating laboratories of this project, as well as at least one other U.S. laboratory for routine diagnosis of BTV infection in ruminants. The basic BTV PCR protocol has also become an essential tool in BTV molecular research in both collaborating laboratories. During development of the BTV serotype specific PCR we had the opportunity to investigate a nationwide outbreak of abortions and fatal disease in dogs in the U.S. purportedly due to BTV infection via a BTV contaminated canine vaccine. The BTV serogroup PCR was integral in confirming BTV in tissues from affected dogs and in lots of the suspect vaccine. This led to the first published report of BTV infection in dogs. We discovered that BTV can produce silent persistent infection in canine cell culture. This indicated a need for more stringent screening of biologics for occult BTV infection. A novel mixed cell culture method was developed to identify occult BTV and other occult viral infection cell cultures. Serotype specific primers for PCR detection of all U.S. BTV serotypes and two Israel serotypes (BTV-2 and 10) have been evaluated and are available. A subsequent collaboration would logically include sequencing of the L2 genes of Israel BTV-4, 6 and 16, allowing incorporation of these Israel BTV serotypes into a multiplex PCR assay.