Establishment Of A Mouse Model Of Duck Tembusu Virus Neurophilic Infection

Duck Tembusu virus(Duck Tembusu Virus,DTMUV)is a member of flaviridae and flavivirus genus.Most flaviviruses have a strong tropism to the host central nervous system(CNS),and cause central nervous system injury,viral encephalitis and even death.DTMUV infected ducks showed neurological symptoms such as paralysis,ataxia and dilatation of angular arch,which was proved to have strong CNS tropism.In this study,a real-time fluorescence quantitative PCR(qRT-PCR)method for the detection of DTMUV was established,and the pathogenicity of DTMUV to ICR mice was preliminarily studied by using the model of DTMUV perineural infection in ICR mice.Firstly,according to the NS gene sequence of DTMUV,a pair of specific primers were designed by Primer6.0 and amplified by RT-PCR.Then the purified amplification product was ligated into pMD19-T vector to construct recombinant plasmid pMD19-NS.The positive plasmids were used as templates to establish the SYBRGreenⅠqRT-PCR standard curve,and the specificity,sensitivity and repeatability tests were carried out.The results show that the linear relationship R~2 of the standard curve of SYBRGreenⅠqRT-PCR of DTMUV is more than 0.99,the average coefficient of variation between groups is 0.235%,and the detection sensitivity can reach 1×10~1copies/μL.In this study,a SYBR-GreenⅠqRT-PCR method for the detection of Tembusu virus was successfully established,which is faster,more sensitive and accurate than the conventional PCR detection method.In order to establish a mouse model of DTMUV perineural infection,three-week-old ICR mice were used as animal models to infect DTMUV through intracranial,subcutaneous,abdominal,muscular and nasal routes.At the time of 5 dpi,the mice in the intracranial group began to develop typical clinical symptoms such as severe hindlimb paralysis,tears and weight loss,and began to die on the 6th day,while all the other groups were normal.qRT-PCR was used to detect the virus in the tissue samples of each group of 6 dpi.The results showed that the virus proliferated in the intracranial infection group,but no virus was detected in the other groups.Then qRT-PCR was used to detect the viral load in different organs and brain tissues of mice in intracranial infection group,and the brain tissue was fixed and made into paraffin sections,and the histopathological changes of mice were observed by HE staining.The results showed that the titer of DTMUV in the brain tissue was the highest,and the characteristics of encephalitis induced by neurotoxic virus were observed in the infected group,and the dissolution of nerve cells,the invasion of microglia into necrotic nerve cells and the proliferation of glial cells were also observed.To sum up,we established a mouse model of DTMUV neurotropic infection in ICR mice,which provides an experimental animal model for further study of the mechanism of DTMUV pathogenicity in mammals.