Revalidation and genetic characterization of new members of Group C (Orthobunyavirus genus, Peribunyaviridae family) isolated in the Americas

Nunes, Márcio Roberto Teixeira; de Souza, William Marciel; Acrani, Gustavo Olszanski; Cardoso, Jedson Ferreira; da Silva, Sandro Patroca; Badra, Soraya Jabur; Figueiredo, Luiz Tadeu Moraes; Vasconcelos, Pedro Fernando da Costa

Our results showed that the complete SRNA segments of Group C viruses ranged from 1,003 to 1,111 nucleotides (nt) and presents the open reading frame (ORF) of the nucleocapsid (N) protein, with a conserved size of 235 amino acids (aa) and 26.72 to 27.03 kDa for all viruses (Fig 1) . The second ORF in the S segment in these viruses encodes a putative non-structural protein (NSs). Interestingly, the ORF to NSs gene starts at an ATG codon -38 nucleotides downstream of the N ORF start codon, which potentially encodes an NSs protein of 318 nt (105 aa) with a molecular mass of~11.8 kDa (Fig 1) . The NSs protein is common in most orthobunyaviruses that have been isolated from vertebrates, such as the members of Group C viruses [19] . Previously studies have been reported that Caraparu and Madrid from Group C viruses encode an NSs protein with 83 amino acids, and also a 62 aa NSs, predicted to be truncated in the N-terminus of Marituba and Oriboca viruses, and consequently, could not be expressed [6, 7, 9] . Indeed, our analysis demonstrates that possibly the members of Group C viruses present a pre-N coding strategy, as recently described for the Brazoran and Enseada orthobunyaviruses [20, 21] . Therefore, some S segment sequences previously reported for Group C viruses are possibly incomplete, as shown in Fig 2. Whereas that NSs protein has been demonstrated to play crucial roles in virulence and pathogenesis of Orhobunyaviruses [19] , further studies using reverse genetics may elucidate the biological importance of NSs protein found in viruses of Group C. The complete MRNA segments ranged from 4,534 to 4,613 nt in length, which encodes the glycoprotein precursor (GPC), ranging from 1,428 to 1,435 aa in length (159.94 to 161.87 kDa) with a similar topology to other members of the Orthobunyavirus genus (Fig 1) . The GPC is proteolytically cleaved to yield two structural glycoproteins, Gn (204 to 211 aa and 34.01 to 35.13 kDa) and Gc (942 to 947 aa and 100.45 to 102.8 kDa), and a nonstructural protein (NSm) with 281 or 282 nt (24.99 to 26.27 kDa) (Fig 1) . The GPC contains the specific N-terminal signal peptide that is common to all members of the genus, which is responsible for delivering the nascent polypeptide to the endoplasmic reticulum [22] . The conserved Zinc finger region (amino acids position 257 to 295) and fusion peptide (amino acids position 1068 to 1091) in the GPC protein were also identified, which is an important RNA binding site and is probably involved in viral assembly [23] . Furthermore, the GPC was predicted to contain five transmembrane regions (TMDs): a single TMD in Gn (close to the C-terminus); three TMDs in NSm; and a single one, close to the C-terminus in Gc (Fig 1) . The TMDs in Gn and Gc have been shown to play crucial roles in membrane fusion, assembly, and morphogenesis [24] .