Isolation And Molecular Characterization Of A Goose Parvovirus From Swan And Preparation Of Reference Antigen And Antisera Of Gosling Parvovirus

1. Isolation of a goose parvovirus from swan and its molecular characteristicsIn this study, a GPV named GPV-XZ20150328 was isolated from the zoo swan in Jiangsu, China. The complete DNA sequence of GPV-XZ20150328 was amplified by polymerase chain reaction (PCR). The genome of the GPV-XZ20150328 was 5106 nucleotides (nt) in length, same as the strain B. The homology of complete nucleotide sequence was 97.7% and 99.8% with the reference strains B and GPV-SHFX respectively. It is interesting that the 56 bp insertion was found in ITR sequence compared with GPV-SHFX (5050bp).This indicated that GPV may have some differences in different individuals and regions.2. Development of reference antigen of goose parvovirusThe virions from allantoic fluid of goose embryos infected with the goose parvovirus were purified by ultracentrifugation. Then the purified virions were analyzed by SDS-PAGE, direct transmission electron microscopy, hemagglutination test, specific PCR and fumbling inactivated condition. Optimized the inactivated conditions was 0.3% BPL for 48h. The purified virus was used as antigen and compared with the concentrated GPV antigen by PEG6000. Our results showed that the purified virus was high purity and has no other viruses contamination. The titer of the concentrated GPV antigen in AGP test was 1:4. Adding 1% BSA into the concentrated GPV antigen, the reference GPV was made and evaluated by repackaging, freeze-drying, calculating CV, testing physical properties, identifying characteristics of sterility, mycoplasma contamination, uniformity, stability and so on.3. Development of reference antisera to goose parvovirusThe goose sera against GPV were made by oral immunization and injection immunization with GPV for four times. Monoclonal antibody against goose parvovirus were preparated by a hybridoma cell line (GPV-Mab-6C8).The AGP titer of sera was 1:64. The titer of IFA was 1:6400 for sera and 1:1600 for Mab. Moreover, sera against GPV did not show cross-reaction with other avian pathogens such as NDV、AIV、EDS76、ALV、REV、IBV、IBDV、TMUV.The reference antisera to GPV was made by means of adding preservative, repackaging, freeze-drying, calculating CV, testing physical properties, identifying characteristics of sterility, mycoplasma contamination, uniformity, stability, and so on.