Identification of Simbu, California and Bunyamwera serogroup bunyaviruses by nested RT-PCR

Abstract

We describe a reverse transcription-polymerase chain reaction (RT-PCR) with primers that anneal to the 5′ and 3′ ends and amplify the Bunyavirus S RNA segments. The RT-PCR was done on the fluids of C6/36 cells infected with each of 21 bunyaviruses. The bunyaviruses studied, with the exception of Catu virus, produced amplicons having 700 to 1300 base pairs and probably contained the whole S RNA segment sequence. A nested PCR performed with these amplicons distinguished California and most Bunyamwera serogroup viruses from other bunyaviruses by use of BBC specific internal primers for the S RNA segment, and distinguished Simbu serogroup viruses from others by use of BS specific internal primers. The nested-PCR amplicons of Guaroa, Maguari, California encephalitis, Bunyamwera, and Oropouche viruses were sequenced. The sequences were aligned with previously known sequences of the S RNA segment of the same viruses, showing a high degree of homology and thus confirming the specific origin of these amplicons. The nested RT-PCR is suitable as a specific screening for most California and Bunyamwera serogroup and Simbu serogroup viruses depending on the use of BBC or BS internal primers. Oropouche virus is an important public health problem in Brazil and the nested PCR with BS primers could be used for the detection of this virus in tissue culture and mouse brain isolates as well as in clinical samples.