Establishment of a Minigenome System for Oropouche Orthobunyavirus 1 Reveals the S Genome Segment to Be Significantly Longer than Previously 2 Reported 3 Running Title: Oropouche Virus Minigenome 23 Animal -negative-strand Rna Viruses 24

(2015) Establishment of a minigenome system for oropouche virus reveals the S genome segment to be significantly longer than reported previously, Abstract 33 34 Oropouche virus (OROV) is a medically important orthobunyavirus, which causes 35 frequent outbreaks of a febrile illness in the Northern parts of Brazil. However, despite 36 being the cause for an estimated half a million human infections since its first isolation 37 in Trinidad, 1955, details of the molecular biology of this tripartite, negative-sense RNA 38 virus remain limited. We have determined the complete nucleotide sequence of the 39 Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences 40 compared to sequences in the database. Most notable were that the S segment 41 contains an additional 204 nucleotides at the 3' end and that there is a critical 42 nucleotide mismatch at position 9 within the base-paired terminal panhandle structure 43 of each genome segment. In addition, we obtained the complete sequence of the 44 Trinidadian prototype strain TRVL 9760 that showed similar characteristics to the 45 BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we 46 demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed 47 functional proteins and also that the newly determined terminal untranslated 48 sequences acted as functional promoters in the minigenome assay. By co-transfecting 49 a cDNA to the viral glycoproteins, virus-like particles (VLP) were generated that 50 packaged a minigenome and were capable of infecting naive cells.