Establishment of plaque assay titration for GFP-labeled recombinant human metapneumovirus / 中华微生物学和免疫学杂志

ABSTRACT

Objective To establish the plaque assay for the titration of GFP-labeled recombinant human metapneumovims(rhMPV). Methods Vero-E6 cells were selected as host cells for titration. GFP-labeled hMPV was serially diluted and added to each well to infect the cells. The plates were covered with low melting agarose overlay and incubated for different days in incubator. The plates were then observed under fluorescence microscope for plaques with green flourecence, at the same time, the number of plaques was counted by blue plaque-forming. Results Under the low melting agarose overlay, Vero-E6 cells grew well until the CPE caused by hMPV was seen. Clear green flourescence could be observed the first day post infection, much clearer on the third day post infection but showed somewhat fusion between plaques later on.Blue plaques on the fifth day after infection were large and easy to observe. The recombinant GFP-labeled hMPV could replicate up to 1 × 106 PFU in the Vero-E6 cells. Conclusion Plaque assay for titration of recombinant GFP-rhMPV has been sucessfully established. This methodology will offer a solid base for further studies on pathogenesis and vaccine development of this virus.