The diagnosis and successful replication of a clinical case of Duck Spleen Necrosis Disease: An experimental co-infection of an emerging unique reovirus and Salmonella indiana reveals the roles of each of the pathogens

•We identified a new Orthoreovirus reassortment strain, (DRV/GX-Y7), and along with S. Indiana have evaluated their roles in clinical DSND.•DRV/GX-Y7 can cause serious damage to the immune organs in ducklings and the co-infection with the Salmonella indiana isolate could increase the severity of the disease.•There are two potential genetic reassortment events found in the M2 and S1 segments of the viral genome of DRV/GX-Y7.•The genetic reassortments among different gene fragments might play important roles in the recent emergence of this new Orthoreovirus, especially as it relates to recent outbreaks of DSND in the duck production industry. Duck spleen necrosis disease (DSND) is an emerging infectious disease that causes significant economic loss in the duck industry. In 2018, a duck reovirus (named DRV/GX-Y7) and Salmonella indiana were both isolated from the spleens and livers of diseased ducks with DSND in China. The DRV/GX-Y7 strain could propagate in the Vero, LMH, DF-1 and DEF cells with obvious cytopathic effects. The genome of DRV/GX-Y7 was 23,418 bp in length, contained 10 dsRNA segments, ranging from 3959 nt (L1) to 1191 nt (S4). The phylogenetic analysis showed that the DRV/GX-Y7 strain was in the same branch with the new waterfowl-origin reovirus cluster, but was obviously far distant from the clusters of other previous waterfowl-origin reoviruses Muscovy duck reovirus (MDRV) and goose-origin reovirus (GRV), broiler/layer-origin reovirus (ARV) and turkey-origin reovirus (TRV). The RDP and SimPlot program analysis revealed that there were two potential genetic reassortment events in the M2 and S1 segments of the genome. In order to have a clear insight into the pathogenic mechanism of DRV/GX-Y7 and S. Indiana in clinical DSND, an infection experiment was further conducted by challenging commercial ducklings with the two isolates individually and with both. The results showed that DRV/GX-Y7 produced severe hemorrhagic and/or necrotic lesions in the immune organs (thymus, spleen, and bursae) of experimentally infected ducklings. And, that the co-infection of DRV/GX-Y7 and S. Indiana could greatly enhance the pathogenesis by increasing the morbidity and mortality in ducklings whose clinical symptoms and lesions were similar to the natural clinical DSND cases. In summary, the results suggested that the pathogen causing duck spleen necrosis was an emerging unique genetic reassortment strain of duck Orthoreovirus that was significantly different from