Isolation,Identification Of Avian Orthoreovirus From Duck And Black Swan

Avian reovirus（ARV）can infect chickens,ducks or geese and other poultry.When infected with chickens,it will cause severe arthritis and tenosynovitis.Waterfowl infected such as ducks and geese will cause spleen enlargement and liver bleeding necrosis.In recent years,the infection of avian reovirus has become increasingly frequent,especially in domestic waterfowl.It has brought serious losses to China’s agriculture.In this study conducted reovirus detection on duck and swan derived from multiple regions in Sichuan,inoculated ARV positive samples with duck embryo to isolated viruses and further identified the strains,studied the biological characteristics and whole genome sequence characteristics of ARV.Firstly,RT-PCR was performed on suspected samples of reovirus infection from various regions.The positive samples were homogenized and inoculated into 9-day-old duck embryos for virus isolation.Two strains of virus were successfully isolated from duck embryos,named CD200801 and DY200518 and the ELD₅₀ of the two strains was determined to be 1×10^-5.33/0.2m L and 1×10^-3.33/0.2m L.Infected 3-day-old ducklings with the isolated virus,which could replicate the same symptoms as clinical symptoms,and the virus could be isolated again from their liver and spleen samples.When the isolated virus was inoculated into DF-1 cells,obvious syncytium formation could be observed.The growth curve showed that the virus titer of CD200801 strain reached the highest at 36 h after inoculation with the duck embryo fibroblasts and TCID₅₀=1×10^-4.69/0.1m L,DY200518 strain reached its highest virus titer at 60 h,with TCID₅₀=1×10^-4.44/0.1m L.After ultracentrifugation of the isolated virus,negative staining electron microscopy was performed.It was found that the virus particles had a diameter of about 80 nm,a spherical,icosahedral symmetric structure,and a double capsid.The high-throughput whole genome sequencing results of ARV showed that there is a 5’-GCUUUUU...UA^U/_CUAUC-3’structure with the characteristic classification of reovirus at the end of the genome segment.Further sequence alignment and phylogenetic analysis were conducted on the ARV genome.The alignment results showed that the nucleic acid similarity between the duck and swan origin strains and the prevalent waterfowl origin novel reovirus coding region was 86.7-98.9%.The phylogenetic analysis results showed that CD200801 and the DY200518 strains were closely related and located in the same branch,aggregated into a group with the novel reovirus of waterfowl origin that has become popular in recent years,but has a distant genetic relationship with other avian reoviruses.In summary,through detection of clinical samples,pathogen isolation and identification,ARV biological characteristics and whole genome sequence analysis,two strains of duck and black swan origin reovirus were successfully isolated,and it is the first report about avian reovirus infection in the black swan.The two ARV strains are different host sources but have a closely relation,and phylogenetic analysis suggests that they may have been recombined from different strains.This study broadens the host source of ARV and provides new insights into the evolutionary relationships between different strains of reovirus,emphasizing the necessity of strengthening reovirus monitoring in poultry and wild birds.