Screening Of Susceptible Cell Line For Proliferating Newcastle Disease Virus And The Establishment And Application Of Stable Cell Line Overexpressing Sialyl Transferase(ST6Gal)

Newcastle disease virus?NDV?can replicate and proliferate in a variety of primary cells or continuous cell lines,but the cell susceptibility to different viral strains or subtypes differs a lot.In order to determine the cell lines suitable for producing newcastle disease?ND?vaccine,several ND vaccine strains including lineage?and strain Lasota were inoculated into BHK-21?Vero?Hela?LMH and DF-1cells respectively.The best suitable cell line was determined through observing cell pathological change,TCID₅₀ and HA titer.The results showed that bothlineage?strain and strain Lasota can be seen proliferating on all the five cells lines,causing typical cell pathological changes.However,different strain showed different susceptibility to those cell lines:lineage?strain grew better on Hela?BHK-21 and DF-1 cells,while strain Lasota gave higher titers on Hela?LMH and BHK-21 cells.The LMH cell line is proved to be used to proliferate NDV strains including both lineage?and Lasota with good cell pathological effects,especially for lineage?viruses.The first step for NDV infection is the binding between virus protein and host sialic acid receptors on the cell membrane surface.The two sialic acid receptors which can recognize NDV surface proteins are SA?2,3Gal and SA?2,6Gal.The distribution and expression abundance of the two receptors in the host tissues and organs have important effects on the host range and virulence of the virus.In order to compare the preference of strain NDV?and Lasota on different cell lines in molecular level,the distribution of two sialic acid receptors on 5 cells was observed by immunofluorescence staining and visualized by confocal microscope.The results showed that both SA?2,3Gal and SA?2,6Gal receptors are exist in BHK-21?LMH?DF-1?Hela and Vero cells,mainly in both cell membrane and cytoplasm.Only SA?2,3Gal receptor was detected in the nucleus of the BHK-21 cell,while both two receptors were found in the nucleus of the DF-1 cell at the same time.Then,the two sialic acid receptors on the surface of 5 cells were quantitated by flow cytometry.The results displayed that the abundance of SA?2,3Gal receptor is significantly higher than that of SA?2,6Gal recetpor on all the cells'surface,suggesting that the sialic acid receptor of 5 cells is mainly SA?2,3Gal receptor.From high to low,the abundance of SA?2,3Gal receptor in the fivecells are Hela?BHK-21?DF-1?LMH and Vero cell,while that of SA?2,6Gal recetpor are Hela?LMH?BHK-21?Vero and DF-1cell.Therefore,we speculated that NDV?strain may bind to SA?2,3Gal receptor more easily,wihle strain Lasota binds with SA?2,6Gal receptor more easily.Although NDV can proliferate in a variety of cells,its titer is generally low and cannot meet the production requirements.The two kinds of sialic acid transferase:ST3Gal and ST6Gal can mediate formation of SA?2,3Gal and SA?2,6Gal receptor respectively.Therefore,sialic acid transferase plays an important role in expression abundance of sialic acid receptors on cell surface,and determines the invasion and proliferation ability of NDV.Therefore,in order to improve the titer of NDV and further study the influence of different sialic acid receptors,we constructed plasmids PCI-neo-ST3 and PCI-neo-ST6,and transfected them into BHK-21 cells by liposome transfection reagents successfully.22 BHK-ST3 cell clones and 17 BHK-ST6 cell clones were obtained after clonization.Then these cells were inoculated with NDV Lasota strain respectively and the TCID₅₀0 and HA titers of cell cultures were determined seperately.The BHK-ST3-22 and BHK-ST6-15 cell clones were found more suitable for proliferating strain Lasota.By flow cytometry,it showed that the expression abundance of SA?2,3Gal and SA?2,6Gal receptor on the surface of BHK-ST3-22 and BHK-ST6-15 cell strain were significantly increased,andthe BHK-ST6-15 cell clone had better susceptibility to strain Lasota than BHK-ST3-22cell.In addition to virus virulence and cell susceptibility,many other factors such as culture conditions and culture methods are also have great influence on virus proliferation and virus titer.In order to promote virus proliferation and increase the titer of NDV,we firstly optimized the conditions for cultivation of strain Lasota in BHK-ST6-15 adherent cells.Basing on that,the BHK-ST6-15 adherent cells were then domesticated for suspension culture with low serum,and the proliferation conditions of Lasota strain in suspension cultured BHK-ST6-15 cells were also optmized.The results show that the suspension culture of BHK-ST6-15 cells with low serum is successfully achieved.Furthermore,the titer of cell culture is up to 10^8.375TCID₅₀/0.1mL after virus inoculation,which is significantly higher than that on BHK-ST6-15 adherent cell.The results laid a foundation for large-scale production of NDV Lasota strain by bioreactor.In China,The production of poultry vaccines mainly relies on SPF chicken embryo presently.However,the manufacture process is limited by the supply of SPF chicken embryos and biosafety risks such as frequent exogenous virus contamination,which restricts the production of poultry vaccines.In order to replace traditional production process,reduce cost and improve product quality,the study tried to produce NDV strain Lasota live vaccine by suspension culture of BHK-ST6-15 cells.The vaccine was tested according to the requirements of the third part of?Veterinary pharmacopoeia of People's Republic of China?published in 2015.The results showed that all items of NDV live vaccine strain Lasota met the requirements,indicated that this processing technology can replace the traditional production process completely.At the same time,the technology also showed advantages such as high degree of automation,good safety,mature technology,uniform product quality,reduced cost and improved economic benefits,which laid a good foundation for future large scale producing..