Genetic analysis of members of the species Oropouche virus and identification of a novel M segment sequence.

Oropouche virus (OROV) is a public health threat in South America, and in particular in northern Brazil, causing frequent outbreaks of febrile illness. Using a combination of deep sequencing and Sanger sequencing approaches, we determined the complete genome sequences of eight clinical isolates that were obtained from patient sera during an Oropouche fever outbreak in Amapa state, northern Brazil, in 2009. We also report the complete genome sequences of two OROV reassortants isolatd from two marmosets in Minas Gerais state, south-east Brazil, in 2012 that contained a novel M genome segment. Interestingly, all 10 isolates possessed a 947 nt S segment that lacked 11 residues in the S-segment 3' UTR compared with the recently redetermined Brazilian prototype OROV strain BeAn19991. OROV maybe circulating more widely in Brazil and in the non-human primate population than previously appreciated, and the identification of yet another reassortant highlights the importance of bunyavirus surveillance in South America.

Establishment of a minigenome system for Oropouche virus reveals the S genome segment to be significantly longer than reported previously.

Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 3' end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.