Abstract
Severe fever with thrombocytopenia syndrome virus (SFTSV) can infect multiple cells, such as Vero, RD, BHK21, HUVEC and 293T. Vero cells are highly susceptible, so they were used to isolate virus from sera of patients, as well as for producing virus in the laboratory. Other cells can be used for additional functional analysis.
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Standard curve of quantitative real time-PCR for SFTSV NP at 102 to 106 copies. When quantitative PCR was performed, the plasmid NP- pCI-neo was diluted from 102 to 106. Next, 2 μl was added in 20 μl reaction using SYBR Premix Ex Taq as per the user manual, the reaction was duplicated, and PCR was performed on the 7500 fast Real Time PCR System. X axis is the quantity of the NP copies. The Y axis is the value of CT of PCR at different copies. Sample copies will be automatically generated by the program depending on the standard curve. The right figure is the amplification plot. Figure 2. HUVEC cells were infected with SFTSV virus produced in Vero cells (vero-105 batch). Cells infected with different amounts of virus (2 μl, 5 μl, 10 μl, 20 μl and 40 μl) as indicated in the figure. Cells were stained with antibodies against NP (green), nuclei were stained using 4',6-diamidino-2-phenylindole (DAPI) in mounting media (blue). Images were acquired using a Zeiss LSM510 Meta laser scanning confocal microscope at 20 times.
Notes
Recipes
Acknowledgments
We thank Zhaoling Sun from Yiyuan County Chinese Medical Hospital for providing us with sera samples from SFTSV patients. This work was supported by the Chinese Ministry of Science and Technology (2010CB530101, 2011CB812501) and the Beijing Municipal Government.
References
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