Experimental Research On Culturing Influenza Virus With Human Diploid Cell Lines And Vero Cells

Epidemic influenza is an epidemic disease which has a long history. It is causedby influenza virus. Influenza virus belong to orthomyxoviridae, and were devided intothree type as A type,B type and C type. It could cause pandemic occurrence,localflow and sporadic cases. The antigens of influenza virus are easy to change. Influenzavirus spread fastly and abroudly. The immunal condition of the human is unstable.And the incidence of this disease is very high. The old and the weak have high fidelityand mortality. Clinical manifestations of the flu are fever,headach,myalgia,fatigue,snuffle,angina and caugh. The flu can accentuation potential disease and can causesecondary bacterial pneumonia or essential viral pneumonia. During the pandemicperiod, there is an increase of the hospitalization and the mortality. WHO commendedthat the most effective method for influenza prevention is vaccination. Theconventional influenza vaccine are produced by chick embryo, but the deficiency ofthis method do exist, it can't satisfact the requirement of the vaccine production foranswer the pandemic. Culturing influenza virus by cells has many advantages. Hence,seeking for the new kind of cells for influenza virus culturing can satisfied the need ofthe manufacture of influenza vaccine. At the same time, the flu is a disease which ismonitored at global range, so seeking for the new kind of cells for influenza virusculturing is the cornerstone of all ttie research on influenza, it is helpful for thequickly clinical detection or diagnose.Part 1 Preliminary study on culturing Influenza virus with diploid cell linesCulturing four strain influenza viruses(A/Kunming/1/2005 Va H3N2,A/Wisconsin/67/2005 H3N2,A/New Caledonia/20/1999 H1N1 andB/Malaysia/2506/2004) on KMB₁₇ cells,2BS cells and MRC-5 cells in mediumwithout serum, in different pH and typsin solution, at 35℃. After 72 hours, harvestthe virus and detect the HA titer. On KMB₁₇ cells, the HA titer of the two strainwhich are H3N2 subtype (A/Kunming/1/2005 Va H3N2 and A/Wisconsin/67/2005H3N2) are higher than the other two (A/New Caledonia/20/1999 H1N1 andB/Malaysia/2506/2004). On 2BS ceils, the HA titer of he HA titer of the two strainwhich are H3N2 subtype (A/Kunming/1/2005 Va H3N2 and A/Wisconsin/67/2005H3N2) are undetectable(=0). And the HA titer of the other strain(A/NewCaledonia/20/1999 H1N1 and B/Malaysia/2506/2004) are not very high. On MRC-5cells, the HA titer of A/Wisconsin/67/2005 H3N2 is zero. And the HA titer ofA/Kunming/1/2005 Va H3N2 is very low. And the HA titer of the other strain(A/NewCaledonia/20/1999 H1N1 and B/Malaysia/2506/2004) are just not very high. Aboveall, those three human diploid cell lines are not the best choice to be used in influenzavirus culturing. It also need a deep reaserch. Part 2 Studies on the stability of influenza virus A/Kunming/1/2005 Va H3N2adapted on Vero cells.In this experiment, the HA titer of influenza virus A/Kunming/1/2005 Va H3N2adapted on Vero cells were detected during the continuous passage. It can keep hightiter. And the eight fragment of genome of the influenza virus were detected by PCR.The viron were observed by electron microscopy to verify the integrity of thevirosome. It was confirmed that influenza virus A/Kunming/1/2005 Va H3N2 cankeep its integrity of the virosome and the stability on Vero cells.