Comparison Of Biological Characteristics Of Two Pekin Duck-origin Reoviruses

"Spleen necrosis disease" in Pekin ducklings is a viral disease caused by duck reovirus (DRV). The mortality caused by the disease is generally low in the past. However, the disease occurring in Pekin ducklings in a duck farm located at Henan province in 2013 resulted in losses of about 60% in 1 to 2-week-old birds. In this study, the molecular detection, virus isolation and characterization were performed. Subsequently, the biological characteristics of the reovirus were investigated.Using the DRV-specific reverse transcription (RT)-PCR assays, the μB, σB and σC genes specific to DRV were detected in the seven spleen samples collected from dead ducklings. Following virus isolation, propagation and plaque purification, two DRV clone strains (designated HN5d and HN5c) were obtained. Following nucleotide determination and analysis of the amplified fragment, a unique deletion of 54 nucleotides was found in the σC-encoding region of HN5d when it was compared to the recent duck reoviruses (e.g., DRV 091). Inoculation of Pekin ducklings with the HN5c and HN5d isolates resulted in splenic necrosis typical of "Spleen necrosis disease". Unlike the typical "Spleen necrosis disease", the disease produced experimentally caused severe hemorrhagic and/or necrotic lesions in the liver of all fatal cases and most survivors. Particularly, in experimentally exposed birds, mortalities of between 20% and 40% occurred during the 7 day observation period. Histopathological examination results demonstrated the presence of severe lesions in liver and spleen. In immunohistochemistry experiments, a large number of brown positive signals were detected in liver and spleen tissues. Using the S1-based DRV-specific RT-PCR assay, DRV RNA was easily detected from all tissues of dead ducklings and from the spleen and liver samples of survivors. These investigations suggest that the HN5c and HN5d are new variants possessing a higher pathogenicity in Pekin ducklings compared with classical 091-like reovirus. The mortality caused by infection with HN5c strain was slightly higher than that of HN5d strain. There was no significant difference in pathogenicity between the twd isolates.On the basis of above studies, the molecular features and influence of the 54-bp deletion in the aC-encoding region on biological characteristics of the virus were further investigated. SDS-PAGE analysis showed that the genome of the HN5c and HN5d strains contained 10 genomic fragments. Following genome sequencing and phylogenetic analysis, the HN5c and HN5d strains were shown to be closely related to the recently determined DRV 091 strain, belonging to waterfowl reovirus genotype 2. The 18 aa deletion in the σC protein of HN5d strain was confirmed. Both HN5c and HN5d possessed good reproductive performance in Vero cells. However, the titer of HN5d strain was higher. Cross-neutralization tests showed that antigen correlation coefficient between HN5c and HN5d was 56.2%. These investigations suggest that the 18-aa deletion in the σC protein of HN5d may is correlated to the replication ability of the virus and could affect the viral antigenicity. Peptide scanning showed that there is at least one B cell linear epitope in the 18-aa sequence, which causes antigenic differences between the two DRVstrains.Using real time RT-PCR and western blotting analyses, the expression levels of both σC mRNA and σC protein of HN5d in infected Vero cells were higher than those of HN5c. Using fluorescence/immunofluorescence and immunoblotting analyses, the aC proteins of both viruses were shown to be expressed in Vero cells transfected by eukaryotic expression plasmids containing corresponding σC genes, respectively. Under confocal fluorescence microscopy, the σC proteins were observed in cytoplasm. Using an Annexin V-FITC/PI Apoptosis Assay Kit, apoptosis was observed in both infected and transfected Vero cells. In viral infected cells, the apoptosis induced by HN5d was higher than those by HN5c (P<0.05). In transfected cells, there was no significant difference in apoptosis induced by aC proteins of the two viruses (P>0.05). These investigations suggest that the 18-aa deletion may have no significant influence on the degree of apoptosis induced by the two reoviruses and their aC proteins.Taken together, the diversity of Pekin duck-origin reovirus strains in terms of molecular and biological characteristics was highlighted in this study. Both DRV and its σC protein were shown to induce apoptosis in Vero cells. Our study contributes to the understanding of "Spleen necrosis disease" in Pekin ducklings and the features of its etiological agent, and also provides more data for pathogenesis of duck reovirus.