| Previously,a recombinant herpesvirus of turkey(HVT)live vector vaccine named rHVT-H9-HA,which expressed the main protective antigen HA protein of H9 subtype avian influenza virus(AIV)by using the HVT vaccine strain as the vector was constructed in our laboratory.This recombinant virus rHVT-H9-HA can remedy the inadequacies of conventional inactivated avian influenza vaccines,and it also provides immune protection against Marek’s disease and H9 subtype avian influenza at the same time,which makes it has a good potential to be used in clinical.According to the requirements of the national agricultural genetically modified organism’s safety evaluation,genetically modified microorganisms must be subjected to scientific risk analysis based on risk assessment,risk management and risk communication before clinical application.Therefore,this study conducted a biosafety evaluation of the recombinant live vector vaccine rHVT-H9-HA.1.Evaluation of Genetic Stability,Competitiveness and Survival Adaptability of Recombinant Virus rHVT-H9-HAThe recombinant virus rHVT-H9-HA was propagated on chicken embryo fibroblasts(CEF)for 20 passages.The expression of HA protein of H9 subtype AIV was detected by indirect immunofluorescence test(IFA).The monolayer of CEF was co-infected with the equal amount of rHVT-H9-HA and the parent HVT strain FC-126,and propagated continuously for 10 passages.The ratio of total viral genome copy number and rHVT-H9-HA genome copy number was evaluated by fluorescence quantitative PCR method to evaluate the competitiveness of rHVT-H9-HA and HVT strain FC-126.The survival adaptability of rHVT-H9-HA and HVT strain FC-126 was evaluated by the changes of plaque number with viruses under different temperature,pH,ether,chloroform,trypsin,and other conditions.The results showed that the recombinant virus rHVT-H9-HA could stably express the HA protein of the H9 subtypes AIV after 20 passages.There had no obvious difference in the ability of replication when propagated recombinant virus rHVT-H9-HA with parental virus HVT strain FC-126 together.The recombinant virus rHVT-H9-HA and parental HVT strain FC-126 had same characteristics that both of them could be inactivated at 55℃and 65℃ for 15 min,the plaque number was decreased with the extension of time under 37℃ and 45℃,sensitive to ether and chloroform,a certain tolerance to trypsin,no tolerance under pH3,pH5 and pH10,sensitive to ultrasound,ultraviolet rays and common disinfectants.In summary,the recombinant virus rHVT-H9-HA has a good genetic stability,and has a similar reproductive performance and survival adaptability to the parental virus HVT strain FC-126.2.Biosafety Evaluation of Recombinant Virus rHVT-H9-HA to Target AnimalsOne-day-old specific-pathogen-free(SPF)chickens were subcutaneously vaccinated with 5000 PFU of the rHVT-H9-HA from the neck,and the PBS was used in placebo group.The clinical and growth performance of chickens were monitored for 20 weeks.The weight of each group of was measured to analyze the effect of recombinant virus.Three chickens were sacrificed every week from each group,and the heart,liver,spleen,kidney,and bursa of fabricius were collected for common observation,weight measurement and histopathological examination.Serum were collected every week to measure the hemagglutination inhibition(HI)titer against H9 subtype AIV.In order to evaluate the replication and duration of the recombinant virus,the blood was collected every week for the isolation of lymphocytes,which was further inoculated into a single layer of CEF cells to isolate and quantify the virus.The distribution of HA gene of H9 subtype AIV in various tissues was measured by using fluorescent quantitative PCR method.The results showed that the situation of spirit,eating and drinking of all the chickens in each group were normal during the experiment.The body weight and the internal organs showed no difference between different groups,which demonstrated that the recombinant virus rHVT-H9-HA had no side effects to the immunized chickens.No pathological changes of the organs of immunized chickens occurred in the physical.The HI titer against H9 subtype AIV was increased until 8 weeks after immunization,and decreased after that.The number of the recombinant virus rHVT-H9-HA in immunized chickens was increased until 7 weeks,and then decreased after that.The recombinant virus could still be isolated at the 20th week after immunization,indicating that the recombinant virus could be maintained in the chicken for a long time.The HA gene could be detected in various tissues from the second week.It is demonstrated that the recombinant virus rHVT-H9-HA is safety to the chicken,which is the target animal of HVT.3.Biosafety Evaluation of Recombinant Virus rHVT-H9-HA to Non-target AnimalsThe recombinant virus rHVT-H9-HA was inoculated subcutaneously in the neck at a dose of 5000 PFU for each one-day-old meat duck.At the same time,the PBS group was set as placebo group.The experiments were performed for 35 days,and common clinical observation,virus isolation,foreign gene detection,internal organ weighing and pathological tissue examination were used to evaluate the safety of the recombinant virus rHVT-H9-HA to duck,which is not the target animal of H9 subtype AIV.The results showed that the situation of spirit,eating and drinking of all the chickens in each group were normal during the experiment.No recombinant virus was isolated from the blood and no foreign gene was detected in all tissues.The common observation,weight measurement,and histologic section detection of the collected organs showed that the recombinant virus rHVT-H9-HA had no side effects on ducks.It is demonstrated that the recombinant virus rHVT-H9-HA is safety to duck,which is not the target animal of HVT. |
PDF Full Text Request