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

Reassortment is an important evolutionary mechanism of segmented RNA viruses in which co-infection of a host cell with multiple viruses may result in the shuffling of genomic segments [30] . This fact has been shown to be involved in virus emergence and interspecies transmission, which include Schmallenberg and Oropouche virus [31] [32] [33] . In Group C viruses, a classic and informative study using standard antigenic tests showed cross-reactivity in Marituba and Murutucu by hemagglutination-inhibition and neutralization, Murutucu and Oriboca by complement-fixation, Oriboca and Itaqui by hemagglutination-inhibition and neutralization, Itaqui and Caraparu by complement-fixation, Caraparu and Apeu by hemagglutination-inhibition and neutralization and Apeu and Marituba by complement-fixation [27] . However, some these viruses cross-reacted completely by one test, but not by another test and still are related. Thus, these and other data indicated that the Group C viruses include many natural reassortants [34] . To elucidate this point, we compared the topology of both S and L trees with the phylogenetic tree generated using the M segment sequences and observed some discrepancies, which were supported by higher bootstrap values and significantly different likelihood scores, which suggested natural reassortment. Also, after combining these branching inconsistencies in the phylogenetic trees with RDP4 analyses of concatenated segments, we confirmed four reassortment events with different genome segment organization than previously described [1, 7] . The first reassortment event was identified in APEUV strain BeAn848 contains S and L segments from MTBV strain BeAn15 and an M segment that is probably unique. Also, ITQV strain BeAn 12797 has S and L segments from CARV strain BeAn3994 and a possibly unique M segment. Interestingly, based on the classification of International Committee on Taxonomy of Viruses (ICTV), the reassortment events described in this study occurs between different viral species, as the Apeu virus that is classified into Caraparu orthobunyavirus, but the S and L this virus is from Marituba orthobunyavirus. Also, the Itaqui and Oriboca viruses are classified into Oriboca orthobunyavirus, but the S and L segments of both viruses are from viruses of Caraparu orthobunyavirus. Despite, previous studies based on serological tests and partial genome sequences indicated that APEUV and ITQV are potential reassortments, the first complete coding sequences for three segments were reported in this study [1, 7, 9, 34] . The other reassortment event was observed in ORIV strain BeAn17, which possesses the S and L segments from MURV strain BeAn974, and a unique M segment (Fig 4) . Interestingly, all reassortments observed in this study have been reported cross-reactions only by the complement-fixation method [27] . This fact, suggests that cross-reactivity observed by complement-fixation test was determined by S segment because serologic makers identified by the L segment remains unidentified, as well as hemagglutination-inhibition, and neutralization assay that is determined by M segment, therefore, these viruses probably are unique [34] . Also, we confirmed the reassortments in ITYV identified in Amazon Region of Peru in 1999, both strains of this virus have S and L segments from Caraparu virus and a unique M segment [4] . On the other hand, MURV and RESV were previously reported to be reassortments, but we did not find any evidence to s