| Since the outbreak of the "1918 Spain Influenza",the fight against influenza has never stopped,and vaccination is the most effective means to prevent influenza.The advances in science and technology have driven the cell culture-based influenza vaccine production to replace egg-based processes.However,due to insufficient understanding and knowledge on viral propagation rules in producer cell line and the interaction between viruses and their production hosts,viral production process are screened based on experiences,which makes it difficult to effectively regulate the virus amplification process to achieve a truly scientific and rational design,development and optimization of the viral vaccine production process.In addition,it is reported that the MDCK-NBL2 cell line commonly used for influenza viral vaccine production comprised a mixed cell population,and low producer cells in the population would deplete nutrients and destroy the culture environment,which could finally impede virus production.Besides,homogeneous cell populations could lead to process instability after a long-term passage.Therefore,how to obtain a stable high producer and how to improve the production efficiency are still difficult problems to be overcome in the current research and development of influenza vaccine production.Consequently,starting from the heterogeneity of the MDCK-NBL2 cell line(parental cell line P),a productive and stable MDCK cell clone(high producer H1)was outstanding after examining the differences of several progeny MDCK clones.Then,the basic characteristics of the high producer,and its viral amplification,the cell maintenance,physiological metabolism,and gene expression characteristics during influenza vaccine production process were studied in depth to dig out the key cellular properties for efficient viral production.Finally,the viral amplification process of the parental cell line P was effectively regulated according to the gene expression features achived from the characterization of the high producer.Firstly,by examining the differences of several progeny clones of the MDCK-NBL2 cell line,we found that the the parental cell line(P)is very heterogeneous.The distribution of the virus producing capacity of different progeny clones spread a wide range,and their cell morphology,the content of sialic acid receptors,and the stability of virus production differed significantly.Among them,a stable and productive clone H1 stood out.Its highest specific virus productivity exceeded 30×103 virions/cell,which was about 2 times that of P cells and was also of the highest level reported.The proportion of infectious particles produced by H1 was comparable to that of P.In addition,the high producer H1 can be continuously and stably passaged,and its 20-day average specific growth rate is(0.95±0.06)d-1,which is fast enough for large-scale culture.Subsequently,the viral amplification process of the high producer H1 and the parental cell line P as well as their material and energy metabolism,cell physiological state,and intracellular gene expression before and after viral infection were deeply studied.Results showed that(1)Regardless of virus infection,H1 cells had higher ribosome and protein translation functions,mitochondrial and energy metabolism-related functions,which are inherent characteristics of the high producer,and H1 cells have higher DNA replication capacity and lower calcium signaling pathway and adenylate cyclase function.These features were beneficial for cell maintenance and viral gene expression.(2)MDCK cells began to release progeny virus particles at about 6 h post virus infection,and virus infection could induce apoptosis,decrease intracellular mitochondrial membrane potential,and cause cell diameter and protein level to increase first and then decrease.The high productivity of H1 cells was mainly due to the higher virus release rate and cell maintenance ability in the middle and late stages of virus production,among which the cell maintenance ability included viable cell density,cell viability,cell diameter,intracellular protein level,mitochondrial content and mitochondrial membrane potential,etc.(3)Virus infection can cause metabolic reprogramming of cells,and H1 cells had a higher level of glucose metabolism after virus infection,and their strong mitochondrial and energy metabolism functions were conducive to alleviating mitochondrial damage caused by virus infection,thereby increased the flow of consumed glucose to the tricarboxylic acid cycle and energy metabolism pathways,providing more material and energy guarantee for the massive proliferation of intracellular viruses.(4)After being infected by the virus,the intracellular viral mRNA can account for more than 25%of the total mRNA level.Among them,five viral genes(PB2,PB1,NP,M1 and M2)were higher expressed in H1 cells,which was not only conducive to the packaging of the progeny influenza virus,but also beneficial to the antagonism of the antiviral effect of the virus on the host cell.(5)H1 and P cells had similar gene expression responses upon influenza virus infection,which were mainly related to host shutoff and inflammatory responses,but the degree of responses varied.The lower antiviral gene expression level of H1 after virus infection favored the amplification of viruses.Combining with its lower expression of endoplasmic reticulum stress-related genes,less amino acid consumption,as well as better ability to maintain intracellular ROS level,it was suggested that H1 had less protein processing pressure,which not only reduced cellular stress and obstacles for virus amplification,but also ensured its survival.According to the above understandings,the intracellular ROS level was decreased by 43.15%,and the virus yield was increased by 46%at 8 hpi by adding 5 mmol/L ROS inhibitor NAC during viral production process in P cells.In addition,the supplement of 10 μmol/L NFκB inhibitor PDTC significantly elevated the mitochondrial membrane potential and increased the virus yield by 76%in P cells as well.These results suggested that intracellular ROS levels and innate immune responses can be used as targets for regulating virus infection and replication.Overall,we presented a deeper understanding of the viral amplification process and its regulation mechanism.Excavated physiological metabolism and gene expression features of the high producer can provide a basis for rational screening and engineering of productive cell line on one hand and provide scientific guidance on the other hand for the future development and establishment of efficient production processes for influenza viral vaccine using the largescale high-density culture technology.At the same time,the results of this thesis can also provide scientific support for the development and production of other viral vaccines. |
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