Biosafety Evaluation Of Recombinant Fowlpox Virus Co-expressing The HA Gene Of H5N1Avian Influenza Virus And Chicken Interleukin6Gene

Avian influenza virus has become one of the most important pathogens, which is harmful to animal husbandry and public health. Although inactivated vaccines play an important role in prevention and control of avian influenza, They only induced humoral immune response with a disadvantage of high cost. Therefore, researchers are working to develop improved genetic engineering vaccines. Recombinant fowlpox vaccine research is one of such genetic engineering vaccines. The bio-safety assessment of recombinant live vector vaccine decides whether the vaccine can be commercialized or not. The immunogenicity of recombinant fowlpox virus co-expressing the HA gene of H5N1avian influenza virus and chicken interleukin6gene has been determined in duck. The bio-safety of rFPVs in waterfowl needs to be evaluated.1.1The physicochemical and biological properties of recombinant FPVIn order to determine the physicochemical and biological properties, rFPV and vaccine strain wild fowlpox (FPV) were treated with different temperatures, pH, chloroform, trypsin, then TCID50s of the viruses were determined. Futhermore, equal quantity of rFPV and FPV were mixed and infected CEF, the ratio of rFPV in the total viruses was determined after several passages. The results showed that rFPV and FPV were sensitive to trypsin, chloroform, heat, but viruses were stability in pH3-9condition. The replication capacity of rFPV was weaker than that of FPV. Therefor the biosafety of rFPV is higher than that of FPV.1.2The biosafety of rFPV in immunized ducksA total of24010-day-old mallard ducks were divided randomly into6groups,40ducks/group. The ducks were immunized with105PFU (0.2mL per duck) of rFPV-HAIL6, rFPV-HA, and FPV,106PFU of rFPV-HAIL6,0.2mL of H5AI killed vaccine and0.2mL of sterile PBS respectively. Five ducks were added in the groups of rFPV or FPV for contact transmission. The physiological or physiological indexes of blood from ducks on days1,7,14 post immunization were determined. Three ducks were killed randomly to analyze the distribution of viruses in ducks and histopathological observation. The weigh of ducks in each group were measured every week to analysis the effect on weight gain of duck during1-21day post immunization. FPV antibody levels in contacted duck were determined by ELISA to evaluate the transmission ability of rFPV. In addition, feed, waste, water, larynx and cloacal swab were collected for virus isolation to evaluate rFPV horizontal transmission. On day60post immunization, muscle in inoculated site of ducks in rFPV-HAIL6group was collected and its genomic DNA was extracted to evaluate the potentiality of foreign gene integration by southern blot. Furthermore,10-day-old duckd were immuned with rFPV-HAIL6105PFU, on day4after immunization, muscles in the vaccination site of ducks were collected and reinoculated to10-day-old duck, The passages were repeated for5generation. The tissue homogenate in vaccination site were inoculated in CEF for virus isolation.The results showed that rFPV caused changes in some of physio-biochemical indexs within two weeks, and the physio-biochemical indexs were returned to normal levels after two weeks. There were no significant pathological changes in histopathological section, and no effect on body weight gain in mallard ducks. The rFPV was only isolated from vaccination site of duck, but not in other organs and tissues. The nucleotide of rFPV could be detected in tissues by PCR and the HA proterin expressed by rFPV could be detected in tissues by immunohistochemistry. The was no horizontal transmission between vaccinated ducks and non-vaccinated ducks. No virus was isolated form feed, waster, water, and swabs. The result of Southern blot showed that the exogenous gene of rFPV didn’t integrate into the genome DNA of ducks. On the other hand, the virus could be isolated from the first generation of passage in ducks, indicating the virus could not transmit constantly in ducks.1.3The biosafety of rFPV in immunized geeseA total of4010-day-old mallard geese were divided randomly into2groups. The geese were immunized with106PFU of rFPV-HAIL6, and the geese in control group were inoculated with PBS. The blood physi-biochemical indexes and histopathological section were measured on days7,14post vaccination.The results showed that the blood physio-biochemical indexes of geese returned to normal after2weeks post vaccination. There was no significant pathological change in histopathological observation. In conclusion, the rFPV is safe to geese.