Replication Kinetics Of Human Metapneumovirus In Different Cell Lines

PART 1 REPLICATION KINETICS OF HUMAN METAPNEUMOVIRUS IN DIFFERENT CELL LINES Objiective To find a cell line suitable for the culture and isolation of hMPV.Method hMPV A subtype type syain hMPV/NL/1/00 and B subtype type stain hMPV/NL/1/99 were inoculated in Vero cell, Vero-E6 cell, LLC-MK2 cell, A549 cell and HEp-2 cell. After inoculation, CPE was observed every day, and RNA was extracted from cells every other day for 20 days. Then cDNA was synthesized and used for hMPV subtype specificity real-time PCR.Result After inoculated 3 days, CPE was observed in Vero cell, Vero-E6 cell, LLC-MK2 cell and A549 cell. There was no difference between different subtype, but CPE was observed easier in Vero-E6 cell. hMPV could replicate efficiently in Vero cell, Vero-E6 cell and LLC-MK2 cell, but could not in A549 cell and HEp-2 cell. The highest hMPV viral load of two stains was detected in Vero cell. Conclusion Vero cell, LLC-MK2 cell and Vero-E6 cell were allsuitable for the culture and isolation of hMPV. A549 cell and HEp-2 cell weren't suitable for the virus culture.PART 2 ISOLATION OF HUMAN METAPNEUMOVIRUS FROM NASOPHARYNGEAL ASPIRATE IN DIFFERENT CELL LINESObjective Clinical specimen were used for study to evaluate the conclusion of part 1.Method Nasopharyngeal aspirate which were confirmed by sequenceing existence of hMPV infection(2 A subtype and 2 B subtype) were inoculated in Vero cell, Vero-E6 cell, LLC-MK2 cell, A549 cell and HEp-2 cell. After inoculation, CPE was observed every day, and RNA was extracted from cells every other day for 8 days. Then cDNA was synthesized and used for hMPV subtype specificity real-time PCR.Conclusion (1)CPE was not observed in five cell lines after inoculation. (2)One A subtype specimen was detected in Vero-E6 cell and HEp-2 cell after inoculated 2 days, but wasn't detected in all cells after inoculated 8 days; another A subtype specimen was detected in five cell lines after inoculated 2 days, the highest viral load was in Vero-E6 cell, after inoculated 8 days it could be detected in three cell lines, the highest viral load was also in Vero-E6 cell. (3)Two B subtype specimen were detected in five cell lines after inoculated 2 days, the highest viral load were in HEp-2 cell and LLC-MK2 cell; after inoculated 8 days it could be detected in four cell lines, the highest viral load were in LLC-MK2 cell and Vero cell.Conclusion Vero-E6 cell was suitable for the isolation of hMPV A subtype, LLC-MK2 cell was suitable for the isolation of hMPV B subtype. HEp-2 cell was not suitable for the isolation of hMPV.