Identification And Sequence Determination Of Novel Duck Reovirus

In2011, an unidentified disease in Peiking duck was reported in China. The infectioncaused40%morbidity at different age and35-40%mortality in different flocks. Clinicalsigns included unstable gait, weakness in legs and diarrhea. At necropsy, large necroticfoci were consistently observed in the spleens of the dead ducks. All classical endemic andemerging viruses, such as Duck enteritis virus （DEV）, Duck hepatitis virus （DRV）, Duckflavivirus （DFV）, Duck parvovirus （DPV） and Bird flu, could be excluded as the causativeagent.To identify the cause of the disease, we analyzed tissue from affected ducks and anovel duck-pathogenic orthoreovirus subsequently was isolated from liver and spleen ofaffected ducks. All of the embryos inoculated with the virus were killed at2–5d.p.i, andthe isolated virus was named TH11. The titer of TH11was105.5ELD₅₀/0.2mL.To determine the taxonomic position of TH11strain, it was purified with sucrosedensity gradient centrifugation and was observed under electron microscopy. The results ofelectron microscopy revealed that TH11was nonenveloped, with icosahedral symmetryand a double capsid containing10double-stranded RNA segments which can be separatedby polyacrylamide gel electrophoresis （PAGE） into three size classes: large （L1–L3）,medium （M1–M3） and small （S1-S4）.For phylogenetic analyses, the partial S2gene was amplified by reversetranscription–PCR with avian reovirus–specific primers. The obtained sequence wasaligned with20published ARVs sequences including newly obtained all of the threesequence data from Peiking in China in2008and2011.The phylogenetic tree shows thatthe S2segment sequence is distinct but clusters closely with the three duck isolates withinthe ARVs serogroup, which suggests that the novel virus is a ARVs-like virus within thegenus Orthoreovirus.Following inoculation with TH11, CPEs were observed in BHK-21cells within2days. As the severity of the CPEs increased, the cells became round and weaking andaggregated. Additional adjoining cells became granular and vacuolated within3days. Inaddition, the culture supernatant of DRV-infected BHK-21cells could re-infect othernormal BHK-21cells. The DRV titers in BHK-21cells were determined using media collected at12,24,36,48,60,72,84,96,108and120h p.i... The BHK-21cells wereinfected with DRV initially propagated in BHK-21cells. The DRV titers increaseddramatically to between102.5and106.3from24to72h p.i., reaching maximal levels at84hp.i., after which point the titer declined.To assess expression of DRV TH11in BHK-21cells, IFA and Western blot analyses were undertaken with protein extracts of passage8BHK-21cells. The IFA results showed that sigma C was present at a high level in theBHK-21cytoplasm. The results of the western blot assay showed that viral proteinexpressed in BHK-21cells could be recognized by the specific antibody against TH11.In order to understand the molecular genetics information of DRV TH11, severaloverlapping cDNA clones, covering the entire genome of DRV TH11strain, were obtainedby primer walking RT-PCR. The full-length genome of DRV TH11is23,418bp, and thesizes of the10segments are as follows: S1,1,568bp; S2,1,326bp; S3,1,202bp; and S4,1,191bp M1,2,283bp; M2,2,158bp; M3,1,996bp; L1,3,958bp; L2,3,829bp andL3,3,907bp. The sizes of the deduced structural/nonstructural proteins are as follows: λA,1,293amino acids （aa）; λB,1,259aa; λC,1285aa; μA,732aa; μB,676aa; μNS,635aa;p10,97aa; p18,162aa; σC,321aa; σA,416aa; σB,367aa and σNS,367aa.To explore the sequence homology and phylogenetic relationship between NDRVTH11and other ARVs, the sequence of the S-class, M-class and L-class segment genes ofTH11were compared with its counterpart in the other ARV or DRV strains,respectively.Based on homology analysis of the deduced S-, M-and L-amino acidsequences of TH11and reference strains, TH11, NP03, GZ and HC were clustered intonovel duck reovirus（NDRV）. Based on the phylogenetic analysis and sequence identity atthe nucleotide and protein level, it is proposed that the novel reovirus strains from Chineselocal ducks are different species from the DRVs isolated from Muscovy ducks.The present study provides additional insights into the evolutionary relationshipamong different members of Reoviridae. In addition, studies of sequence diversity of duckorthoreovirus and other viruses should increase our understanding of the disease of ducksand contribute to the development of vaccines against these viral pathogens.