The Molecular Basis Of Pathogenesis Of Duck Tembusu Virus In Mice

Since April2010, a newly emerged viral disease, firstly broken out in Southeast China,caused decreased egg production, loss of appetite, retarded growth and death in ducks, andresulted in a great financial loss in duck industry. Several laboratories subsequently isolatedand identified this causative agent of the disease. The result of sequence analysis indicated thatthe causative agent was duck tembusu virus, belonging to the Ntaya virus group within thegenus of Flavivirus, in the family of Flaviviridae. Many viruses in the genus of flavivirus cancause zoonoses, and so far, we cannot rule out the possibility that DTMUV will infect humans.In order to study the DTMUV pathogenicity in mammals, we used mouse as animal model andFX2010DTMUV isolate as representative strain. After25passages in mouse brain by directlyinoculated intra-cerebrally, FX2010was then passed3times in mouse brain by inoculatedintranasally. By comparing the pathogenicities of the viruses with different passages(including FX2010, P7, P14, P21and P28) in mice, we found that FX2010was non-pathagenicin mice inoculated intranasally. However, P7, P14, P21and P28viruses which were theoffspring virus passed7,14,21and28times in mouse brain became lethal in mice inoculatedintranasally.To further investigate the pathogenicities of DTMUV and its offsprings with differentpassages in mice and ducks, we designed a specific reverse transcription primer, nucleotideprobe and amplification primers according to conserved region on the E gene of DTMUV andestablished a TaqMan real-time PCR assay for the detection of DTMUV in tissue. Then, wecompared the influence of different viral doses and different inoculating routes on thepathogenicities in mice. No disease symptom or death was detected in the mice after injectedintramuscular with FX2010, P7or P28. However the pathogenicities of FX2010, P7and P28were significantly different in mice inoculated intranasally.103ELD50of P7and P28could killthe mice, while105ELD50of FX2010couldn’t kill mice. We further tested the replication ofFX2010, P7and P28in the tissues of mice inoculated intranasally by real-time PCR. Theresults showed that all of these viruses could replicate in the lungs and nasal turbinates, and thecopies of FX2010ware higher than P7and P28. None of these viruses could replicte in thespleens and kidneys. FX2010couldn’t replicate in the mouse brain, however, P7and P28could replicate in mouse brain and higher neuleotide copies were detected. The results of duckexperiments showed that both FX2010and P28caused systemic infection and no evidentdifference between these two viruses. The complete genome sequences of the viruses withdifferent passages (including FX2010, P7, P14, P21and P28) were compared. There were onlytwo amino acid changes, E597K and A2001T in E protein and NS3protein respectively, fromFX2010to P7. There were twelve nucleotide encoding only six amino acid changes fromFX2010to P28. The results showed that E597K and A2001T are important factors determinedthe DTMUV pathogenesis in mice.To study the viral diffusion path from nose to brain in mice, we inoculated intra-nasallywith5%of zinc sulphate (ZnSO4) solution to destruct the olfactory epithelium of the mice, andthe P7virus were inonculated intranasally. The mortality was desceeased and the death wasdelayed in the olfactory epithelium destructed group comparing with the control group.The study found that FX2010was so easy to adapt to replicate in mice. The resultsindicated that E597K and A2001T play a key role in the pathogenicity of DTMUV in mice.Meanwhile, we found that DTMUV used the olfactory epithelium to reach the beain.