Abstract
Although many spherical and rod-shaped plant virus purification protocols are now available, only a few protocols on filamentous plant virus purification have been published. Here, we report a protocol for large-scale purification of Rice stripe virus (RSV) from RSV-infected rice tissues. RSV virions with high infectivity were first precipitated with polyethylene glycol (PEG) followed by pelleting through primary ultracentrifugation, ultracentrifugation in a glycerol cushion and ultracentrifugation in density gradient. The purified RSV virions can not only be viewed as filamentous particles under an electron microscope, but can also be acquired by insect vector through direct injection into insect body or through membrane feeding prior to transmission to rice plants.
Keywords: Rice stripe virus, Filamentous virion, Density gradient centrifugation, Virion purification, Ultracentrifugation
Background
Many purification protocols for spherical and rod-shaped viruses have been published (André et al., 2002; Balke et al., 2018). These protocols all rely on chemical precipitations or density gradient centrifugations. However, purification protocols for filamentous viruses are currently limited.Rice stripe virus (RSV) is a negative-stranded RNA virus and belongs to the genus Tenuivirus, the order Bunyavirales. RSV often causes severe damages to rice productions in many East Asian countries (Whitfield et al., 2015; Liu et al., 2018). Unlike other members in the order Bunyavirales that produce spherical and enveloped virions, RSV virions are filamentous. However, RSV genome encodes a glycoprotein that is not found in purified RSV virions(Toriyama, 1986; Lu et al., 2019). RSV virions are known to carry several copies of RNA-dependent RNA polymerase (RdRp), necessary for virus replication initiation in host plants and insect vectors. Consequently, establishment of a purification method that allows to maintain active RSV RdRp is crucial for future biological assays using purified virions.Based on a recent report from our laboratory (Lu et al., 2019), we have now developed a purification protocol suitable for producing highly infectious RSV virions. The purified virions can be used for Transmission Electron Microscopy (TEM) and virus transmission assays through insect vectors.
Materials and Reagents
Equipment
Procedure
Data analysis
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Recipes
Acknowledgments
We thank Prof. Tong Zhou (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) for providing RSV-infected rice plants. This work is supported by the National Natural Science Foundation of China (31925032, 31630062 and 31870143), the Fundamental Research Funds for the Central Universities (JCQY201904) and sponsored by K.C.Wong Magna Fund in Ningbo University.
Competing interests
The authors declare no conflicts of interest or competing interests.
References
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