Isolation And Indentification And Biological Characteirstics Of Duck-derived Flavivirus In Anhui

Since June2010, an infectious disease outbreaks at parts of the laying duck farms in our country, and was quickly spread in most area of China, which is characterized by sudden and sharp decline in laying rate. The common symptoms is hemorrhagic ovarian inflammation when anatomising the infected ducks. This epidemic has caused tremendous economic losses for China duck industry. In order to understand the biological characteristics of this disease, We isolated the pathogen from the infected ducks in Anhui Province in2010,then detected the pathogen by PCR method, sequenced and analysed the gene. The pathogen are duck Flavivirus, named AH-F10. After that we produced oil emulsion inactivated vaccine by using the AH-F10virus, and take the immunity protective test at the same time. We believe that it can provide technical support and guarantee for the prevention and control of duck Flavivirus disease.First of all, isolation and identification of the pathogen, diseased tissue. Collected ovaries and fallopian tubes of low-laying-rate ducks under sterile conditions, made then into slurry, filter sterilization. Injected the slurry into10days SPF chicken embryo allantoic cavity for virus isolation. Allantoic fluid are collected in96h, and identificated by RT-PCR. The results showed that in the pH7.2conditions, the chick embryo separation had no agglutination for1%chickens and ducks’red blood cell. The RT-PCR results of duck plague virus, duck circovirus, Newcastle disease virus are all negative, while the result of flavivirus NS1gene sequences is positive which amplified size is260bp. The sequencing analysis showed that it has a similar phylogenetic tree with the Bagaza virus of Ntaya group Flaviviridae Flavivirus genus and up to99%nucleotide homology with the domestic ducks. Thus it can be seen that this experiment initially isolated a duck-origin flavivirus strain, which named AH-F10strain. And it proved that there are duck flavivirus infection in Anhui Province.Then, the pathogenicity tests for chicken and duck embryo. Injected the virus into10day-old SPF chicken embryos (0.2ml/embryo) and11-day-old duck embryo (0.2ml/gold) for passage culture. The culture of chick embryo passage results found within96h the first generation of the chick embryo death, the second generation of30%(3/10) chick embryo death, the third generation of60%(6/10) chick embryo death. Necropsy found chorioallantoic membrane thickening, liver spotting, mottled necrosis, and some liver edge white necrotic foci; duck embryo liver khaki-colored, swollen bleeding. The results showed that the AH-F10strain of flavivirus was subcultured in SPF chicken and duck embryos, and its virulence constantly enhanced with the increase in the number of chick embryo passage.Thirdly, pathogenic test for chicken embryo fibroblasts. Take10-day-old SPF chicken embryos, Using conventional methods to product the chick embryo primary fibroblasts. The AH-F10strain of ducks flavivirus were inoculated in chicken embryo fibroblasts. In24h, the cells began to round shrinking circle shrinking cells increased. In48h and72h, cells began to fall in death and refractive index enhancement. The results show that the AH-F10strain of the ducks Flavivirus can be cultured in CEF upload, and will cause cytopathic effect.Finally, Preparation of the AH-F10strain of ducks Flavivirus oil emulsion inactivated vaccine and its immune challenge protection test. Inactivate the AH-F10strain by formaldehyde, and the white oil, Span-80, Tween-80, prepared by a certain proportion of mixed into the AH-F10virus to produce oil emulsion inactivated vaccine, and its physical properties, sterility testing, security testing, stability testing, which pass the inspection. Immune poisoning protection test:28weeks of egg ducks were randomly divided into four groups, each group were5. Group A was immune poisoning group; Group B was attack drug group; Group C was immunohistochemistry; Group D was control group; first day, A and C Groups used subcutaneous and intramuscular immunization the AH-F10emulsion inactivated vaccine, the dose of each ducks was lml; Group B and D injected with sterile saline in the same way and the dose. On14th day when the A and B groups based upon the above-mentioned method of inoculation AH-F10virus of ducks flavivirus solution at a dose of lml. Results the group B after inoculation the first3d egg began to decline in the first6d attack drug group A stop producing eggs, and continue until the20d then laying one. Necropsy results of the attack drug group A that follicle degeneration,atrophy, bleeding. Extraction of the ovary and follicular lesions of viral RNA by RT-PCR, ducks flavivirus results were positive, immune poisoning group A, immunohistochemistry C and the control group D inoculated and necropsy results were normal. The RT-PCR test results of ducks yellow virus were negative. The test proved that the AH-F10strain of ducks flavivirus inactivated oil emulsion vaccine plays a very good protective effect on the disease.