Table S1 Each order predicted metabolic pathway class for the collected 5,549 compounds without known metabolic pathway classes. The predicted metabolic pathway class code corresponds to the code in Table 1 . Among the 11 predicted pathway classes, the first 2 order predicted metabolic pathway classes should be paid more attention to. (PDF) Identification and Characterization of a Novel Non-Structural Protein of Bluetongue Virus Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell. Bluetongue is a major infectious disease of ruminants caused by an arbovirus (Bluetongue virus, BTV) transmitted by biting midges (Culicoides spp.) [1] [2] [3] . Historically, bluetongue has been endemic almost exclusively in temperate and tropical areas of the world where the climatic conditions favour both the spread of the susceptible insect vector population and the virus replication cycle within the vector [4] . However, in the last decade BTV has spread extensively in several geographical areas including Southern Europe and also, unexpectedly, in Northern Europe causing a serious burden to both animal health and the economy [5, 6] .