Development Of Taqman Real-time PCR For The Detection Of EDSV And Studies On Infectious Characteristics Of NE₄Strain

Egg drop syndrome virus (EDSV) belongs to Avian adenovirus Group III and the main clinical signs include egg dropping and incomplete egg shell etc. In1991, Li Gang isolated the EDSV NE4strain and confirmed the existence of the EDS in our country. The virus is highly homologous with bovine adenovirus and Ovine adenovirus, but heterologous with avian adenovirus type I. Thus EDSV may be a virus between mastadenovirus and avian adenovirus.This thesis mainly focuses on the EDSV and consists of the following three parts:1. Development and application of TaqMan real-time PCR for the detection of EDSV;2. Animal experiments of EDSV NE4strain;3. Studies on the infectious characteristics of EDSV NE4strain in mice.1. To establish the fluorescent real-time quantitative PCR assay for rapid detection of egg drop syndrome virus, a pair of primers and a TaqMan probe were designed according to the sequences from highly conserved regions of the hexon protein gene of egg drop syndrome virus. A positive recombinant plasmid was employed as standard template for the construction of standard curve. The detection limit of the assay was10copies DNA/μL and the real-time PCR was reproducible, as shown by its wide dynamic range from101to108copies DNA/μL. This assay was specific and has no cross-reaction with DNA of other avian virus. The quantity of EDSV in eggs of layers infected with NE4strain was detected by the developed real-time PCR method. Compared with common PCR assay, the real-time PCR method is more sensitive and suitable for EDSV detection.2. The EDSV amount in the isolated specimen and the antibody level of layers inoculated with EDSV NE4strain were monitored. Artificial infection was conducted by employing120day-old Roman layers with106TCID50infectious dose of EDSV NE4strain, Natural infection group used no space isolation and control group used space isolation. Through observation of the change in egg laying rate, we found that the natural infection group drops to40%, while the artificial infection group drops to80%, and the clinical signs of the former were more typical than the latter. By monitering the yolk antibodies level and serum antibodies level of layers of test group and control group, we found that artificial infection and natural infection could both result in high level of antibodies up to212. Using FQ-PCR to detect the virus in the layer eggs and feces, we found that7days pi, the test group began to release the virus, and certain amount virus in their eggs and feces could be detected until the end of detection. EDSV could be detected in the soft shelled eggs of natural infection group, which demonstrated that the natural infection group also could spread the virus vertically. During the detection, the control group showed no clinical signs and was negative in antibody against EDSV. This animal experiment showed that NE4strain is infectious and could transmit horizontally, and then result in the fact that natural infected layers produce high level of antibody and release virus to the outside. In addition, there is a further research on the difference of pathogenicity between the artificial infection and natural infection with NE4strain.3. Studies on the characteristics of mice infected with the NE4strain of EDSV:KM mice of4-6weeks old were artificially infected with106TCID50dose of EDSV NE4strain and negative control was employed at the same time by inoculating the mice with the normal allantoic liquid. By observing the clinical signs of mice and monitering the serum antibody, we found that infection experiment had certain influence on the ingestion of mice, and enabled the mice to produce specific antibody, which reach up to212. EDSV could be detected in most parts of mouse tissues by using the FQ-PCR assay, and we found that seven days pi, the mice begin to release virus. EDSV content is different in tissues such as liver, uterus, kidney, lungs, spleen, heart, intestines, esophagus, trachea, brain and feces. We prepared specific antiserum by immunizing rabbits with the purified EDSV. The PAbs were purified by affinity chromatography. By the method of IHC combined with purified PAbs, we found that EDSV could propagate in the tissues of mice. Through the results of research a preliminary couclusion was drawn that EDSV NE4strain virus could infect the KM mice.