| Avian influenza (AI) is a fowl disease caused by type A influenza virus. H9-subtype avian influenza was recently circulating in some areas in the country which caused great economic losses in poultry industry. Now there is still no ideal vaccine for preventing AI, only inactivated oil vaccines available. But the use of inactivated oil vaccines is limited by the potential risk of spilling out AIV and by the disadvantages of high cost and interference of serological surveillance. A better alternative is recombinant virus vaccine, several different recombinant fowlpox virus (FPV) vaccines expressing AIV HA gene demonstrated good protective efficacy against homologous challenge in chickens.A recombinant FPV vaccine expressing H9 AIV HA gene has been developed by Animal Infectious Diseases Laboratory, (AIDL, PRCMA) Yangzhou University. In the thesis study, its technological process of production, genetic stability and immune protective efficacy are under research.1 technological process of large-scale production4 major parts of technological process have been deplored: large-scale cell culture, ratio of virus seed to cell, post-harvest processing and lyophilization.1.1 large-scale cell cultureBy continuous Trypsin digestion of SPF chicken embryos, 200-million cells with good growing activity could be produced from each embryo. Only 5 embryos are needed for each 3000ml-roller bottle to produce a monolayer.1.2 Ratio of virus to cellTo get an optimum ratio of virus to cell for a maximum production number is made certain quantity of virus seed was changed serially. The production was quantitated byplaque technique, when a virus/cell ratio is proper, a good production would be achieved within 72 hours.1.3 Processing after harvestTo make the product homogeneous, three procedures were adopted: homogenization, freeze/thaw and supersonic processing. Using magnetic revolver to agitate at a max speed in the liquid product for about 10 min would lead to a more homogeneous solution. The process of freeze/thaw and supersonic processing would disrupt cell membrane and release a great part of virion, which also made the product homogeneous. Plaque-counting results showed that 1 to 3 times of freeze/thaw process would lead to a good result, but 4 times or more would make the plaque number decrease.1.4 LyophilizationThaw virus solution and mix it with a protective agent at a proper ratio. Distribute the mixture into small bottles, 2 ml each, and take these bottles into the lyophilizing machine for lyophilization. After lyophilization, sample from the batch for plaque counting. The virus titer of the lyophilized product was a little higher than that of original virus solution.2 Genetic stabilityA recombinant fowlpox virus vaccine expressing H9-subtype avian influenza hemaglutinin (HA) gene was serially passaged to passage 30 on specific-pathogen-free (SPF) chicken embryo fibroblast (CEF) monolayers. The HA genes from passage 10, 20 and 30 were cloned by polymerase chain reaction (PCR) and sequenced. The expression of HA genes from passage 10, 20 and 30 were identified by indirect immunofluorescence assay (IF). These three passages were administered to SPF chickens at 5 days of age and challenged by parental strain of H9-subtype avian influenza virus three weeks later. Specific 1.7 Kb fragments were cloned, identified and sequenced from the three passages respectively, and there was no change in the amino acid sequences. Specific fluorescence could be detected from plaques formed by each passage. Even though the mean hemagglutination-inhibition antibody titer of each rFPV-vaccinated group was lower than that of inactivated oil vaccine group beforepassage groups had over 95% protection from virus shedding at 5 days postchallenge while oil vaccine group had 88%, and there was no difference of protection among different passage groups. These findings demonstrated that this recombinant fowlpox virus vaccine could provide enough protection against H9-subtype avian infl...
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