To enable comparative sequence analysis and phylogenetic investigations, we determined almost full-length S-, M-, and L-segment sequences for 9 Simbu serogroup viruses belonging to 5 species (Table 1): SHAV, Peaton virus, and Sango virus, species Shamonda virus; DOUV and SATV, species Sathuperi virus; Aino virus and Shuni virus, species Shuni virus; Sabo virus, species Akabane virus; and Simbu virus, species Simbu virus. Sample preparation and sequencing were done by using the Genome Sequencer FLX (Roche, Mannheim, Germany) as described (10). Sequence data obtained in this study are archived in the International Nucleotide Sequence Database Collaboration databases (www.insdc.org; accession nos. HE795087–HE795110 and HE800141–HE800143). In addition to the newly determined sequences, published full-genome sequences of AKAV and Oropouche virus (OROV) from the National Center for Biotechnology Information reference genome database (www.ncbi.nlm.nih.gov/sites/genome) were used for sequence comparisons and the reconstruction of phylogenetic relationships. Coding sequences of each genome segment were aligned by using ClustalW (www.clustal.org) for codons, and phylogenetic analyses were performed by using maximum-likelihood methods in MEGA5 (11). For the N and L gene analysis Tamura-Nei parameter, the M gene analysis Tamura 3-parameter was used. The robustness of the trees was tested by bootstrap analysis by using 1,000 replications. Sequence identities were calculated by using BioEdit version 7.0.9.0 (12).

SBV N gene nucleotide sequence identities to other viruses ranged from 69.8% (OROV; 69.9% aa identity) to 97.7% (SHAV; 100% aa) (Table 2). The L gene sequence of SBV had the lowest identity to OROV (60.4% nt; 57.5% aa) and highest identity to SHAV (92.9% nt; 98.4% aa); the SBV M gene showed the highest sequence identity to SATV (82.1% nt; 90.1% aa), whereas identity of SBV and SHAV M gene was low (48.2% nt; 36.5% aa). In general, identity of the SHAV M gene to the other Simbu serogroup viruses was low, from 45.6% nt (33.4% aa; OROV) to 55.0% nt (47.9% aa; Sango virus), which indicates that its M segment belongs to another virus, as previously suggested (8). On the other hand, the high sequence identity of all SBV genes to SATV and DOUV indicates that SBV belongs to the species Sathuperi virus.

A phylogenetic tree derived from the M gene (Figure, panel A) demonstrates that SBV and DOUV cluster closely with SATV, whereas SHAV was placed distantly from all other viruses. In contrast, phylogenetic trees of the L (Figure, panel B) and N (Figure, panel C) genes showed a close relationship between SBV and SHAV. Results for all genome segments show that SBV should be classified within the species Sathuperi virus and is likely to be the ancestor of SHAV, which is in contrast a reassortant virus comprising the S and L segments from SBV and the M segment from another virus, as proposed previously (8).

To further clarify the placement of SBV within the Simbu serogroup, we determined the ability of SBV antibodies to neutralize other Simbu serogroup viruses. We performed titer reduction assays of the 9 other viruses with anti-SBV serum; the viruses were propagated in BHK-21 cells clone CT (L164, Collection of Cell Lines in Veterinary Medicine, Friedrich-Loeffler-Institute, Riems, Germany). Neutralizing potencies of SBV antibodies against Simbu serogroup virus strains were tested by titrating virus in the presence of anti-SBV serum (neutralizing titer 32); results were expressed as percentage titer reduction in relation to a parallel test without antiserum. Although DOUV and SATV were well neutralized, SHAV titers were not reduced at all (Figure, panel A), which supports the M gene phylogeny. Thus, phylogeny and cross-reactivity identified SHAV, but not SBV, as a reassortant within the Simbu serogroup.

Although our results do not support the suggestions of Yanase et al. (9), they are fully consistent with the conclusions of Saeed et al. (8). On the basis of our results and those of Saeed et al. (8), we suggest that SHAV should be reclassified into the species Sathuperi virus and that the species Shamonda virus should be renamed Peaton virus or Sango virus.

In addition to showing that SBV belongs to the species Sathuperi virus, our results show that the virus is most likely not a reassortant and is likely to be one of the ancestors of SHAV, whereas SHAV is a reassortant comprising the SBV S and L genomic segments and the M segment from an unclassified virus. These detailed insights into the phylogeny of SBV could be the basis for the development of efficient, cross-protective vaccines. Our results also highlight the importance of full-genome analyses to identify potential genetic reassortments and to investigate the evolutionary history of viruses with segmented genomes.