Isolation,Identification,and Pathogenicity Study Of The Duck Tembusu Virus Sansui

In 2010,the Duck Tembusu virus（DTMUV）broke out in many duck farms along the southeastern coast of China,and was identified as the pathogen causing duck egg drop syndrome for the first time.It then spread to inland areas of China within a few months,causing huge economic losses to the poultry industry.To investigate the prevalence and pathogenicity of DTMUV in Sansui County,Guizhou Province,our laboratory conducted epidemiological investigations and pathogen detection analysis by collecting blood samples and specimens from multiple breeding farms in Sansui County from 2020 to 2021.We successfully isolated and identified a strain of DTMUV from clinical samples,named GZSS2022.In addition,we also studied the pathogenicity of this virus on ducklings using an animal model.1.Clinical seroepidemiological investigation and pathogen detection of duck Tembusu virus infectionTo investigate the prevalence and infection status of Duck Tembusu virus in Sansui County,Guizhou Province,our laboratory collected 1170 blood samples and366 suspected clinical samples from seven duck farms that had not been vaccinated against Tembusu virus during 2020-2021.Pathological sections were made from naturally infected cases,and ELISA was used to detect antibodies to Duck Tembusu virus,while RT-PCR was used to detect its pathogen.The results showed that naturally infected laying ducks exhibited congested and hemorrhagic egg follicles,and typical vascular loop phenomenon was observed in brain tissue pathology sections.Severe fatty degeneration,expansion of bile ducts filled with bile pigment,and infiltration of inflammatory cells were observed in the liver.Antibody testing revealed positive results in three duck farms,with positivity rates of 7.4%,4.8%,and3.6%,respectively.Positive samples were mainly found in laying ducks,and most positive ducks were of Guangdong origin.RT-PCR detected 12 positive samples for Duck Tembusu virus,with a positivity rate of 3.3%,and there were also cases of infection in the local Sansui Ma duck breed.These findings indicate the presence of Duck Tembusu virus infection in Sansui County,Guizhou Province,laying a solid foundation for subsequent isolation and identification of Duck Tembusu virus.2.Isolation and identification of Duck Tembusu virus and complete genome sequencing of GZSS2022 strain of Duck Tembusu virusIn order to isolate,identify,and sequence the full genome of the Duck Tembusu virus in Sansui County,Guizhou Province,positive samples obtained from an investigation were homogenized and inoculated into 10-day-old SPF chicken embryos for virus isolation.The isolated virus was identified through RT-PCR,infection of BHK-21 cells,and preparation of transmission electron microscope sections from infected cells.The virus titer of the isolated strain was determined,and the full genome was amplified and subjected to genetic evolution analysis.The results showed that some chicken embryos died after inoculation,exhibiting systemic bleeding and developmental abnormalities,but stable death times were observed after four passages.Cytopathic effects were observed in cells 48 hours after virus inoculation,with a peak at 60 hours,characterized by significant rounding,shrinkage,and reduced density of cells.Transmission electron microscopy revealed the presence of virus particles ranging from 40 to 60 nm in size,enclosed in vesicles in the cytoplasm.The TCID₅₀ of the isolated strain was 10^-4.84/0.1 m L,and the ELD₅₀ was10^-3.5/0.2 m L.The full genome sequence of the isolated strain was obtained by splicing 11 sequenced fragments,with a total length of 10,249 bp,showing a high homology of 99.4%with the Duck Tembusu virus,and was named the Sansui strain（GZSS2022 strain）.Genetic evolution analysis showed that the GZSS2022 strain and the Thai strain belonged to the same major branch,while the Beijing strain in China belonged to the same minor branch.3.Pathogenicity of GZSS2022 strain of Duck Tembusu Virus on duck embryos and ducklingsThe separated GZSS2022 strain was inoculated into duck embryos and 7-day-old ducklings.The virus successfully replicated in the duck embryos and ducklings.The pathological changes of the duck embryos were observed,and the clinical manifestations of the ducklings were recorded.The ducklings were dissected and their pathological changes were observed,and pathological sections were made.RT-PCR was used to detect viral nucleic acid in the affected organs.The results showed that the duck embryos died 5 days after inoculation,exhibiting systemic bleeding and developmental abnormalities,and the liver showed bleeding and necrosis.The animal experiment showed that the inoculated ducklings had elevated body temperature,yellow watery feces,and neurological symptoms.Heart bleeding,spleen enlargement,and liver bleeding were observed in the inoculated ducklings.The pathological sections showed typical features of non-suppurative encephalitis,splenic sinus bleeding and congestion,disordered arrangement of pancreatic cells,and lymphocyte infiltration.RT-PCR amplification produced target bands of the expected size.In summary,this study conducted an epidemiological investigation on multiple duck farms in Sansui County,Guizhou Province and successfully isolated and identified a strain of duck Tembusu virus.This strain was named GZSS2022 and its complete genome sequencing showed a length of 10249 bp.Animal experiments demonstrated that the GZSS2022 strain of duck Tembusu virus is pathogenic to ducklings.