水稻条纹病毒纯化

Gang Lu; Min Yao; Yijun Zhou; Xiaorong Tao

doi:10.21769/BioProtoc.3565

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Peer-reviewed

Purification of Rice Stripe Virus

水稻条纹病毒纯化

GL Gang Lu

MY Min Yao

YZ Yijun Zhou

XT Xiaorong Tao email

发布: 2020年03月20日第10卷第6期 DOI: 10.21769/BioProtoc.3565 浏览次数: 3677

评审: Zhibing LaiGuohui ZhouJianxiang Wu

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Cover of PLOS Pathogens, featuring study using the protocol.

Mar 2019

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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 (水稻条纹病毒)

Filamentous virion (丝状病毒)

Density gradient centrifugation (密度梯度离心)

Virion purification (病毒纯化)

Ultracentrifugation (超速离心)

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

Pipette tips
Razor blades
Pasteur pipette
Sterile syringe filters (Millipore, catalog number: SLGV033RB , or equivalent)
Sterile 50 ml conical tubes (Corning, catalog number: 430921 , or equivalent)
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E9884 , or equivalent)
Polyethylene glycol (PEG) 6000 (Sigma-Aldrich, catalog number: 81255 , or equivalent)
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S5886 , or equivalent)
Glycerol (Sigma-Aldrich, catalog number: G5516 , or equivalent)
Na₂HPO₄·12H₂O
NaH₂PO₄·2H₂O
NaOH
Coomassie Brilliant Blue G-250 (Sigma-Aldrich, catalog number: 1.15444 , or equivalent)
Mouse anti-RSV nucleocapsid monoclonal antibody (Provided by Prof.Jianxiang Wu in Zhejiang University)
Goat anti-mouse IgG HRP conjugate (Invitrogen, catalog number: 31430 , or equivalent)
0.1 M phosphate buffer, pH 7.5 (see Recipes)

Equipment

Blender (Philips, catalog number: HR2116 , or equivalent)
1 L glass beaker
Refrigerated centrifuge (Eppendorf, catalog number: 5810R )
4 °C refrigerator (Haier, catalog number: HYC-391 , or equivalent)
Pipettes (Eppendorf, catalog number: R42223G , or equivalent)
Ice machines (Phcbi, catalog number: SIM-F140AY65-PC , or equivalent)
Precision balances (Sartorius, model: Practum313-1CN , or equivalent)
Ultracentrifuge (Beckman Coulter, model: Optima XPN-90 )
Ultra-Clear 26.3 ml tubes for 70Ti rotor (Beckman Coulter, catalog number: 355618 )
Ultra-Clear 10.4 ml tubes for 90Ti rotor (Beckman Coulter, catalog number: 355603 )
Ultra-Clear 10 ml tubes for SW32.1 rotor (Beckman Coulter, catalog number: 344061 )
Formvar and carbon coated 200 mesh Nickel grids (Electron Microscopy Sciences, catalog number: FCF200-Ni )
Transmission electron microscope (Hitachi, model: HT-7700 )
ChemiDoc Touch Imaging System (Bio-Rad, catalog number: 1708370 )