Isolation, Identification And Study On Pathogenicity Of Tembusu Virus

On April2010, an outbreak of a new infectious disease named tembusu virus (TMUV)which mainly caused duck egg-drop emerged in China. The outbreak spread to Shanghai,Zhejiang, Fujian, Jiangsu and other places quickly, bringing huge economic losses to duckindustry. In recent years, the isolation and identification of TMUV have been reported.TMUV not only infected ducks, but also including chickens and geese. In this study, weconfirmed that the isolated virus was TMUV and the virus could cause pathogenesis inducklings which provided a theoretical basis for a better study of the control of TMUVinfection.A strain of virus was obtained from infected duckling characterized paralysis. The studyshowed that the isolated virus is one strain of TMUV which was more closely to TMUV (thehighest similarity could be up to99.7%) than other flaviviruses. The ELD50of the virus is10-3.17/0.2mL. The virus could cause duck embryos died in68h with cutaneous hemorrhagesand allantoic membrane thickening. The study on physical and chemical characteristicsshowed that the isolated virus was inactivated at56℃for20min or60℃for15min andunstable at low pH(pH<5). It was sensitive with chloroform (at4.8%concentration), trypsin(at0.5%concentration) and unstable at50℃for1h despite the protection of Mg2+. Thevirus could not agglutinate the red blood cells of chickens, ducks and pigeons. Pathogencitystudy showed that the virus is highly virulent when experimentally inoculated into ducklingsand the main pathological changes concluded brain edema, liver hemorrhage, the glandularstomach hemorrhage and lung hemorrhage. The clinical manifestations are consistent withnatural incidences. The study provided a theoretical basis for prevention of TMUV.80healthy ducklings were separated into four groups which were infected with0.2mLallantoic fluid by the ways of intracerebral inoculation, oral inoculation and intravenousinoculation at15days old. And at the same time,20ducklings were infected with saline ascontrol group. The clinical symptoms were detected every day. Three ducklings weresacrificed after weighed on3,7,13,15day post-inoculation (dpi) and organs were collected for the study of pathohistological changes. The immune organs including spleen, thymus andbursa were weighed to study the immune organ index. Blood was collected from the calfvein of all experimental ducklings at3,7,9,13and15dpi. The sera were tested for thepresence of TMUV antibodies and serum chemistry values were evaluated, andanticoagulant blood was tested for the CD4+and CD8+level. The results showed that themorbidity was20/20and the mortality was7/20when infected by the way of intravenousinoculation, the morbidity was20/20and the mortality was3/20when infected by the way ofintracerebral inoculation while the morbidity and mortality were not obviously wheninfected by the way of oral injection. The weights of ducklings in infected groups grew moreslowly than control group. The result of CD4+/CD8+was obviously lower than control groupat9dpi. The IFN-γ and IL-6levels were lower than control group at7dpi while higher thancontrol group at9dpi. The values of alanine aminotransferase (ALT), aspartateaminotransferase (AST), creatine kinase (CK), urea nitrogen (UN) and uric acid (UA) levelsshowed an increased trend, aspartate aminotransferase and urea nitrogen changed mostobviously, albumin (Alb) content reduced, indicating that the virus can cause damage to thefunction of liver, kidney and heart. The TMUV antibody levels were increasing at9dpitested by the method of indirect ELISA. The main pathological necropsy changes revealedthat in artificially infected group, the liver, spleen, lung, heart, brain and proventriculuscompletely suffered with hemorrhate along with thymic and bursal atrophy.Histopathological study showed that the brain and lung hyperemia congestion and gliosis.The pancreatic acinar degenerated and necrosis and the liver fatty degenerated, vacuolar andgranular degenerated, accompanied by inflammatory cell infiltration. The results showedthat the TMUV caused serious pathogenicity to ducklings and made an impact on immunefunction of ducklings to a certain extent while the way of intravenous inoculation couldcause more severe pathogenicity than other ways.