Cultivation Of Influenza Viruses Using Vero Cells As Substrate

Influenza is a kind of infectious disease with the typical characteristics ofbreaking out suddenly and spreading rapidly that can not be controlled by humanefficiently up to the present. Except human, some animals such as. pigs, chicken andhorses can also be infected by influenza viruses and die from this disease. A few ofantivirus medicines have shown effect to prevent influenza infection, but they can noteliminate all of the calamities which influenza brings. Therefore vaccine inoculationbecame the best way to deal with this problem. At present, WHO encouraged themanufacturers using mammal cells as the substrate to produce influenza vaccine. Sowe investigated the proliferation of the influenza virus vaccine strains recommendedby WHO during 2006 and 2007 in Vero cell line in this experiment. The resultsproved that it is not feasible to use the influenza virus vaccine strains recommendedby WHO directly in the vaccine producing.Part 1 The proliferation of the different influenza virus strains in Vero cell lineThere are three types of influenza virus strains recommended by WHO used forvaccine production in northern hemisphere during 2006 and 2007. In the experiment,three types of the influenza virus vaccine strain were inoculated to Vero cellsrespectively and they were cultivated in the optimal condition which is most suitablefor their proliferation. The lytic liquid from cells was obtained three days afterinoculation and their hemagglutinintiters were determined by haemagglutinationassay. Then we used the lytic liquid of the cells from the first inoculation to infectVeto cells again. The process of the second inoculation was just the same as the firsttime and then followed by the third inoculation, the fourth inoculation and so on. Wefound that the hemagglutinin titers of the lytic liquid obtained from the cell woulddecrease gradually during the series passages and we can not detect any influenzaviruses after the fourth time of inoculation. The result of the experiment proved thatthe influenza virus vaccine strains recommended by WHO had poor adaptability and sensitivity to the Vero cell line. They can not grow in Vero ceils efficiently andcontinually. So we must look for another way to improve the proliferation abilities ofthe influenza virus strains recommended by WHO if we used the Vero cell line as asubstrate to produce influenza vaccines.Part 2 Veto cells cultivation on the surface of microcarrier and the optimal cultivatedconditions of the influenza A viruses(H3N2) Vero-adapted strain when used Vero cellsgrown on the microcarrier as the substrateNowadays microcarrier suspension culture technique combined with bioreactorhas been widely used in many field of bio-engineering. It has become the mostconvenient way to get large sum of cells. Furthermore cells cultured in microcarriercan avoid de-differentiation occurred if we use general cultivation methods. Thephenotype of the cells can be maintained and the quality of the cells will be improvedin the cultivation when the microcarrier suspension culture technique applied. Theexperiment optimized the growth condition of the Veto cells adhered in the surface ofthe microcarrier. Then the influenza A viruses(H3N2 Va) were inoculated to Vero cellsgrown on microcarrier. Finally we identified the cultivated condition which was mostsuitable for the growth of influenza A viruses(H3N2 Va).