| A kind of acute infectious disease broke out successively in partial duck industries in some provinces of southeastern China since April2010, mainly caused a sharp drop in egg production. The incidence was about60%～100%. Once suffering from the disease after about4～5days, ducks led to decrease in egg production rapidly from the range of80%～85% to10%～30%, even didn’t lay eggs any more. There were some typical pathological changes in the infected ducks such as visible shrunken oaries, follicular bleeding, degradation and white tip shape necrosis of liver and so on. Originally the disease occurred in Jiangsu and Zhejiang provinces, soon continued in most duck industries in Hebei, Shandong, Anhui, Fujian, Guangdong province and so on. Besause of that, there was huge economic loss in duck industries in China. The viruses which caused the disease were isolated and identified as Duck Tembusu Viruses (DTMUV) by gene sequencing and virus cultures.This study aimed at establishing a rapid, sensitive and specific Real-Time RT-PCR diagnostic method for DTMUV. First of all, according to the the E gene sequence (GenBank login number:JQ957513)of DTMUV AH-10strains, the specific primers were designed. Then the E gene was RT-PCR, and cloned into pBSK vector with a T7promotor.The plasmid confirmed by sequencing was as a template, the E gene RNA was in vitro synthesized under the T7RNA polymerase and purified as the standard templates of Real-Time RT-PCR. Then a pair of specific primers was designed for PCR amplification among E gene. Finally, a high specificity and sensitivity one-step Real-Time RT-PCR detection method for DTMUV was established with a chimeric fluorescence dye SYBR Green I through the optimization of reaction system and reaction conditions. The method could be used to detect a minimum of20copies of the viral nucleic acids, and was1000times more sensitive than the conventional RT-PC without nonspecific amplification of pathogen nucleinic acid from other common poultry diseases.168clinical samples were detected. The results showed that the positive rate was73.8%(124/168) by Real Time RT-PCR compared with the positive rate35.1%(59/168) by the conventional RT-PCR. Animal regression test results displayed the positive rate was30.2%(29/96) by the conventional RT-PCR, but the positive rate as64.6%(62/96) by Real-Time RT-PCR. So it indicated that the experimental Real-Time RT-PCR detection method established was significantly better than the conventional RT-PCR. This established detection method was a rapid, sensitive and specific clinical diagnostic method For DTMUV, as well as provided a necessary technical support for DTMUV epidemiology and molecular biology investigation.In order to explore wether the new virus DTMUV would have the ability to infect human by cross-species and some possible pathogenicity, for the first time105,104,103TCID5doses of viruses infected Balb/c mice by abdominal cavity and intracerebral inoculation as mammal models, respectively. The weight of infected mice and the incidences were observed and recorded every day until the animals died or recovered. The infecting experiment was repeated a few times. the half of the mice of each group were executed at the vesting period and separated the sera to detect the total level of antibody-DTMUV and the level of specific IgG and IgG subclass. And we measured the virus titers of liver, kidney, spleen, brain in order to analyse natural immune responses after DTMUV infecting mice and virus proliferation situation in the tissures. In addition, in order to explore wether DTMUV might have antibody-dependent enhancement (ADE) effect on mammals, The mice were repeatly infected by DTMUV14days in intraperitoneal inoculation way, observed their clinical symptoms, and executed the mice that were the worst. We detected the specific IgG from sera and the contents of DTMUV in viscera and analysed DTMUV ADE effect on mice. Finally, we verified the experiments again.The results showed that mice were infected by DTMUV in intracerebral inoculation way, the virus had proliferation in tissues, especially the highest level of virus in brain tissue. The virus nucleic acid were up to3000～4000copies·mg-1in brain tissue, more than that of spleen, but the minimum of about800～600copies·mg-1in liver. Moreover,the mice infected by DTMUV had the loss of weight and appetites with the symptoms of slight bleeding in brain.At the same time, the infected mice produced higher levels of neutralizing antibodies.The concentration of IgG2a and IgG in the group104TCID50reached up to100μg·mL-1, but104TCID50and103TCID50groups were significantly less than105TCID50group. Besides, DTMUV had obvious ADE effect on mice. There were severe morbidity symptoms four days after the second infection in mice, and about350-1000copies·mg-1virus nucleic acid was detected in mice tissue, specific IgG level was significantly higher than that of the secondary infection before.It indicated that the new viruses DTMUV had a certain ability of cross-species transmission to infect animals and had ADE on mammals. Therefore, whether the new virus DTMUV might have a certain ability of cross-species transmission and have the potential possibility to infect human would required to be detailly investigated. Once the viruses in invasion of host cell could meet with proper viruses receptors,or the amino acids of some key site of the viruses in the process of natural evolution that might have specific variations, it would be likely to lead to that the viruses will come across species spread barrier and infect human. |
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