Analysis Of Phenotypic And Molecular Characteristics Of Duck- Origin Reoviruses And Identification Of Novel Proteins Encoded By The Genotype 2 Strain

Reoviruses have been associated with three fatal diseases of ducks, locally known as "White spot disease in Muscovy ducks", "New liver disease in Muscovy ducks" and "Pekin duck spleen necrosis disease" based on hosts and lesions. The etiological agents of the diseases were named Muscovy duck reovirus (MDRV), New pathotype of Muscovy duck reovirus (N-MDRV) and Duck reovirus (DRV), respectively. MDRV was first discovered in France in 1972 and is now regarded as a Muscovy duck-origin isolate (ARV-Md) of species Avian orthoreovirus (ARV). As new viruses emerging in China, N-MDRV and DRV were reported in 2009 and 2011 respectively. The precise taxonomic relationships of the three duck-origin reoviruses to one another remain to be resolved.Comparative studies were performed to investigate the phenotypic characteristics of different duck-origin reoviruses, using ARV-Md/815-12, N-MDRV/J18 and DRV/091 as the research material. The data demonstrated that N-MDRV and DRV exhibited similar phenotypic characteristics, including experimental hosts, lattice arrangement of viral particles in cytoplasm, ability to induce cell fusion, antigenicity in agar gel precipitin (AGP) test, and pathogenicity and tissue tropism for Pekin ducklings. Compared with N-MDRV and DRV, ARV-Md showed significant differences in the phenotypic characteristics. All three isolates were pathogenic for Muscovy ducklings, but they also exhibited some differences in terms of lesions and tissue tropism.To investigate the genomic features of the three duck origin reoviruses and its phylogenetic relationship with other waterfowl and chicken origin reoviruses, the genomic electrophoretic patterns of 3 isolates were analysised by SDS-PAGE, the whole genome of ARV-Md/815-12 and the S1 genome segment of N-MDRV/J18 and DRV/091 were sequenced by using RT-PCR and 5TIACE, and the whole genome sequences of 3 duck-origin reovirus isolates resulted. The results of SDS-PAGE showed that N-MDRV and DRV possessed similar genomic electropherotypic patterns. Compared with N-MDRV and DRV, ARV-Md showed significant differences in the electrophoretic pattern of S group genome segments. Comparative genomic sequence analysis revealed that all three viruses possessed genomic features typical of ARV. Notably, N-MDRV and DRV were distinct from ARV-Md in terms of length and structure of polycistronic genome segment. For N-MDRV and DRV, the polycistronic genome segments were the S1 segments, both of which contained three overlapping ORFs encoding for p10, p18 and σC protein. For ARV-Md, the polycistronic genome segment was the S4 segment, which contained two ORFs encoding for p10 and σC. Additionally, the p10 proteins of N-MDRV and DRV were shown to share no sequence identity with the p10 protein of ARV-Md. Phylogenetic analyses revealed that, for the majority of the genome segments (9 out of 10; except M2), all waterfowl isolates formed a host (waterfowl)-associated group different from chicken isolates.In the phylogenetic tree based on the M2 segment, however, a host-independent topology pattern was observed; the recent waterfowl isolates (N-MDRV and DRV) were shown to be distinct from ARV-Md but clustered together with some chicken isolates. Collectively, waterfowl-origin isolates could be classified as members of ARV. However, the species demarcation criteria based on the sequence identity should be reconsidered. Phylogenetic analyses on the basis of nucleotide sequences of the segments encoding for the outer capsid proteins clearly demonstrated the existence of two genotypes:type 1 including ARV-Md, and type 2 including N-MDRV and DRV.The polycistronic genome segments of the genotype 2 waterfowl reoviruses (i.e., N-MDRV and DRV) contained three ORFs, the most remarkable genomic feature of the viruses compared to the genotype 1 viruses. Whether or not the potential p10 and p18 protein are expressed by ORF1 and ORF2 during the process of virus replication is still unclear. A series of experiments were performed to address this question. Antiserum to DRV/091 was observed to react with purified recombinant p18 protein, and the p18-specific polyclonal antibody recognized a protein with a molecular mass of approximately 18 kDa in Vero cells infected with DRV/091 by Western Blot analyses. Using the MALDI-TOF/TOF assay, amino acid sequences specific to p18 were identified in protein samples prepared from Vero cells infected with DRV/091. Collectively, the data supported the view that p18 protein is a newly identified protein encoded by the DRV S1 gene. Examination of the deduced amino acid sequence of p18 revealed the presence of a putative nuclear localization signal (NLS) between residues 118 and 135 (118KRRR121-X10-132KRRR135), implying that the protein is a nuclear targeting protein. To verify the view, the subcellular localization of p18 was investigated using confocal laser scanning microscopy. The investigations revealed that p18 accumulated in nucleus of both infected and transfected cells. Mutagenesis analyses revealed that the sequence between residues 118 and 135 is a functional NLS and that the N-terminal 118KRRR121 sequence is critical for the normal nuclear distribution of p18.Over expression of p18 protein could induce apoptosis in Vero cells.DRV/091 was inoculated onto Vero cells, and the p10-specific monoclonal antibody was used in immunofluorescence assay (IFA) to detect p10 protein expressed in Vero cells. Fluorescent signal was observed in cells, demonstrating that the p10 protein was a mature protein expressed during the process of virus replication. The p10 proteins of N-MDRV and DRV contained fusion-associated motifs, suggesting the p10 proteins had an ability to induce cell-cell fusion. To confirm this view, Vero cells were transfected with plasmids expressing p10 protein, resulting in cell-cell fusion 24 h after transfection. No cell-cell fusion was observed in Vero cells transfected with plasmids expressing all other viral proteins. These investigations suggested that p10 plays a key role in the fusogenic phenotype displayed by genotype 2 waterfowl reoviruses (i.e., DRV).Based on above investigations, the S1 segments of the genotype 2 waterfowl reoviruses (i.e., DRV) are tricistron. To investigate preliminary the strategies for transcription and translation of the S1 segment, Northern Blot analyses were performed using a nucleic acid probe for detecting the S1 segment. A single mRNA was detected in Vero cells 2-8 hours post infection (hpi) with DRV/091. The length of the mRNA was estimated to be 1500-2000 bp, consistent to the length of S1 genome segment (1568 bp). The data showed that the S1 segment can be transcribed to a full-length mRNA in DRV replication. This suggested that the translation of ORF2 and ORF3 might be initiated from the mid AUG codons of polycistronic mRNA.