| In 1987,a disease caused by avian adenovirus(FADVs)caused by Hydropericardial hepatitis syndrome(HHS)was found in the Ankara area of Karachi,Pakistan,so the disease It is called Ankara disease,Angola’s disease(ADV),and lychee disease.The pathogen is identified as Fowl adenovirus serotype-4(FAdV-4).Since June 2015,the disease has exploded in parts of Anhui,Henan,Liaoning,Shandong,Jilin,and Hebei,with a mortality rate of 20% to 30%,which has caused great losses to the poultry industry in China.In recent years,the main epidemic strains reported in China are serotype 4 and 8b avian adenoviruses,in which serum type 4strains dominate,and there is no formal commercial vaccine on the market for targeted prevention.Therefore,the development of serum type 4 vaccine is particularly important to prevent and control the disease.1.Selection of culture methods for Fowl adenovirus serotype-4(FAV-4):In order to speed up the trial production process and avoid the obstacles in the intermediate trial production process,preliminary research on the culture characteristics of FAdV-4is required before the start of trial production to select an optimal culture expansion carrier.At present,there are two main ways for vaccine companies to cultivate avian viruses in large production processes.One is chicken embryo expansion and the other is cell expansion.Both methods have their own advantages and disadvantages.However,FAdV-4 is suitable for which method to cultivate the drug,which needs to be learned through experiments.In this experiment,FAdV-4 was cultured by inoculation of specific pathogen free(SPF)chicken embryos and chicken liver cancer cells(LMH cells).It was found that from the perspective of culture effect,FAdV-4was cultured in SPF chicken embryos.As the number of passages increases,the titer of the virus will gradually decrease.By inoculation of the yolk sac,a higher titer canbe obtained,and the titer of the FAdV-4 virus obtained by inoculation of the allantoic cavity is not high.However,the actual large-scale production,inoculation through the yolk sac is less efficient than inoculation through the allantoic cavity,which is not conducive to large-scale production in the later stage.Through LMH cell culture and expansion,not only can continuous culture,the increase in the number of passages has little effect on the virus titer,and the LMH cell culture can expand the virus to obtain higher virus titer.2.Screening for poisoning methods: The program for culturing FAdV-4 in LMH cells has been established.Which of the several methods of toxicology currently used is more suitable for vaccination of FAdV-4? In this study,a comparative experiment was conducted on three kinds of poisoning methods(adsorption inoculation method,non-adsorption inoculation method,and simultaneous inoculation method),and it was finally determined that the non-adsorption inoculation method is more suitable for the cultivation of FAdV-4,and the simultaneous inoculation method is suitable.Determination of virus content in FAdV-4.3.Comparison of two kinds of serum: serum plays a key role in the cultivation of cells.Therefore,serum is also the largest cost item for enterprises that use cell production lines to produce vaccines.The most suitable and economical culture serum is selected,which is beneficial for later scale.To produce FAdV-4 vaccine,this trial study conducted a comparative test on the sera of two of the best-selling companies in the market(Jinyuankang and Wickson),and the results obtained showed that Wickson serum is more suitable for the culture of LMH cells;Jinyuankang serum is more suitable for liquid exchange and poisoning of LMH cells.4.Preparation,conservation,and detection of production toxicants: Since this experiment requires the use of LMH cells to culture FAdV-4,the amount of toxic species required is large,so it is necessary to prepare a sufficient amount of production toxicants in the early stage,and to prevent The toxic potency decreases with the prolongation of the storage time,so it is necessary to expand the seed toxicant,and at the same time,the high-potency batch is screened as the production toxicant,and the batch is also lyophilized and preserved.In this experiment,FAdV-4was cultured in LMH cells.In this experiment,FAdV-4 was inoculated with LMH cells by non-adsorption inoculation method,and a total of 6000 mL of poison was obtained,and 100 bottles were lyophilized.The virus content,foreign virus,bacteria,mycoplasma,etc.were tested according to the biological product inspection protocol,and all the detection indexes met the requirements.5.Preparation and testing of semi-finished products: In this trial study,FAdV-4production seed poisons were inoculated into LMH cells according to the ratio of0.1%,and were produced by spinner flask culture to prepare antigenic liquid.A total of 5 batches of semi-finished products were prepared.After testing,all the test indicators of the five batches of semi-finished products obtained in this trial production meet the requirements,indicating that the process can stably produce qualified semi-finished products and can be extended to large-scale production.6.Preparation and quality evaluation of the finished product: This experiment was carried out by adding mineral oil adjuvant to the obtained 5 batches of semi-finished products,and mixing and emulsifying to obtain 5 batches of group I type 4 avian adenovirus inactivated vaccine for preventing group I 4 Heterocardial Hepatitis Syndrome(HHS)caused by avian adenovirus,according to the biological product inspection protocol,the quality,quantity,sterility,safety,potency,formaldehyde residue and other indicators of the quality of the 5 batches of finished products are tested.,each evaluation index meets the requirements.At the same time,a batch of new inactivated three-way inactivated vaccine was prepared,which was compared with other manufacturers’ new inactivated vaccines.The comparison showed that the quality of the new inactivated vaccines of the new gland was compared with the domestic first-class vaccine.The quality of the same vaccine produced by the company is comparable.Conclusion: This study successfully produced a group A type 4 avian adenovirus inactivated vaccine,indicating that the production process is reasonable,stable and can be promoted.It laid the foundation for the subsequent large-scale production and promoted the progress of the I group 4 avian adenovirus inactivated vaccine on the market. |
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