Isolation, Identification And Fluorescence Quantitative RT-PCR Assay Of Tembusu Virus

Tembusu virus infection is a contagious disease which caused by the tembusu virus(TMUV). The virus is mainly caused by a severe drop in egg production of the laying ducksand neurological symptoms of the ducklings. It is characterized by its high prevalence,morbidity and mortality. It is going to serious threat to the development of duck industry. Thestudy of the disease has great significance to protect and promote the development of China’sduck industry.Tembusu virus infection was first reported by Cao Zhenzhen and Zhang Dabing in2010,after that it was prevalent in most of the ducks regions. In order to efficiently prevent andcontrol TMUV, it’s essential to establish simple, rapid and sensitive methods for diagnosis anddetection of TMUV. In the study we isolated one stain of TMUV from the ducklings.Establishing quantitative PCR was used to detect the TMUV distribution in different tissuesof ducklings and hens, exploring pathogenic mechanism of TMUV.Part1: Isolation and identification of TMUV SDLZ strainA strain virus (SDLZ) isolated from a suspected case of TMUV infection through duckembryo allantoic cavity was identified as Tembusu virus (TMUV) by PCR identification andanimal experimental infection. It was identified by PCR, and the amplified fragments weresequenced. The gene of TMUV SDLZ has the homology100%in nucleotide sequence withother TMUV in GenBank. The ducklings infected artificially showed the same or similarclinical symptoms and pathological changes as the natural infections in the animal regressiontest. Toxicity tests showed that DELD50of SDLZ strain is10-4.5/0.2mL. The TMUV couldproliferate in the chicken embryo, duck embryo, duck embryo fibroblasts and Vero cells. Allthe results show that the isolated virus is TMUV. And it was named SDLZ stain.Part2: Development and application of fluorescence quantitative RT-PCR assay for the rapiddetection of Tembusu virusTo establish a method detected Tembusu virus by SYBR GreenⅠ relative fluorescence quantitative RT-PCR. Special primers based on Tembusu virus NS5and E gene were designedand a pair of primers of house-keeping gene β-actin was chosen. Then these amplifiedfragments were cloned into pMD18-T. Using the plasmids NS5-pMD18-T, E-pMD18-T andβ-actin-pMD18-T as standard products, a real-time quantitative reversetranscription-polymerase chain reaction (RT-PCR) was performed to construct the standardcurves of NS5, E and β-actin gene and detected the sensitivity, specificity and repeatability.The results showed a precise linear relationship with a correlation coefficient of R2>0.99; thedetection limits was10copies of DNA plasmid reaction. The amplification curve showed asingle peak could only been detected for Tembusu virus. The variation coefficient was lessthan0.5%by within and between the groups of repeatability tests. The clinical samples weredetected3times by this method, and all results were positive. The developed Real-Time PCRassay was highly specific, sensitive, and reproducible and could be an available tool fordiagnosis and monitoring of Tembusu virus in duck farms.Part3: Detection of TMUV in the tissues of artificially infected ducklingsThree-day-old ducklings were infected with Tembusu virus (TMUV), slaughtered indifferent period. We used the fluorescence quantitative RT-PCR assay to detect TMUV of theinfected ducklings and the results showed that: At three day post-infection (PI), the TMUVcould be detected from pancreas, brain, heart, liver, spleen, bursa of Fabricius and lungs. TheTMUV reached the period of maximum quantity at seven day PI, and the TMUV could bedetected from all organs at fifteen day PI. The highest relative expression of the TMUV wasthe pancreas, brain and heart, relative expression between0.5-0.8, which indicated that theseorgans were the major target organs after the TMUV infection. The relative expression of theliver, spleen was between0.4-0.6. The lowest relative expression of the TMUV was the bursaof Fabricius and lung, relative expression between0.2-0.4.Part4: Detection of TMUV in the tissues of artificially infected hensWe used the fluorescence quantitative RT-PCR assay to detect TMUV of the infectedhens and the results showed that: At three day post-infection (PI), the TMUV could bedetected from the heart, liver, spleen, lungs, pancreas, follicle and oviduct. The relativeexpression level of the TMUV was higher in all organs. And the highest organs were thefollicle and oviduct. At five day PI, relative expression level of the liver, spleen, lung, pancreas were reduced, while the follicle and oviduct were not change. At nine day PI, wecould detect the TMUV in the follicle only. The highest relative expression of the TMUV wasfollicle and oviduct. The lowest relative expression of the TMUV was the pancreas and lung.The results showed that though the TMUV had not caused significant clinical symptoms andpathological changes of laying hens, but it could be detected in the tissues of the laying hens.The TMUV could infect and proliferate in the hens.