Isolation,Identification And Pathogenicity Of Duck Tembusu Virus LJ-20 Strain

In April 2010,acute infectious diseases broke out in many duck farms in southeast coastal areas of China.After detection,the pathogen was duck Tembusu virus,which was identified as the pathogen of duck egg drop syndrome for the first time in China.Within a few months,the disease spread to the inland areas of Chinese mainland,and has become one of the main diseases that threaten our poultry breeding industry.A strain of duck Tembusu virus was isolated and identified from 19-days-old sheldrake of a farm in Anhui Province,mice were used as animal models to study its pathogenicity to mammals:1.Isolation and identification of duck Tembusu virusThe Ducklings Infected with Tembusu virus in nature showed depression,yellow green feces,mouth breathing,and some of the ducks developed catarrh.After autopsy,the samples of liver,spleen and brain were collected,and the RNA was extracted from the samples.The samples were identified as Tembusu virus by RT-PCR.The 9-days-old duck embryos were inoculated with the homogenate of the diseased material through the allantoic cavity,and the dead embryos were bleeding,and there were bleeding spots in the liver.BHK-21 cells were infected with allantoic fluid.After 72 hours of infection,there were cytopathy and morphological changes.The TCID₅₀ was 10^-5/0.1m L.These results confirmed that the isolate was Tembusu virus,and named LJ-20 strain.2.Whole genome determination of duck Tembusu virusWe design primers covering the full length of Tembusu virus c DNA,then amplify it to get ten fragments.After the fragments were recovered,they were connected with p MD-19T vector and transformed into DH-5?competent cells.The positive clones were screened and sequenced.The whole genome sequence of the isolate was 10986 bp.It contains an open reading frame encoding 3426 amino acids.Upload to NCBI,Gen Bank login number is MW367213.3.Pathogenicity of LJ-20 strain duck Tembusu virus to ICR miceThree-week-old ICR mice were challenged intracranially with the LJ-20 strain DTMUV.Five days after the challenge,mice developed neurological symptoms,hind limbs were paralyzed,and yellow sticky secretion was seen in the eyes.After grinding,c DNA was extracted and RT-PCR was used to identify duck Tembusu virus.Formalin was used to fix the brain,liver,spleen and other pathological materials.The pathological section showed that there were vascular cuff,neuron and satellite phenomenon in the brain tissue of the experimental group,and the gap of liver tissue increased.In conclusion,the duck Tembusu virus was isolated and identified in this experiment.The whole length of the virus was 10896bp,and it was named LJ-20.The results of pathological section showed that the brain tissue of the mice in the group of the attack group was damaged,indicating that LJ-20 strain of the virus had neuropathogenicity in mice.