Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors

Wu, Jiajing; Ni, Yuequn; Liu, Huan; Ding, Ming; Zhou, Xueping

doi:10.1016/j.jviromet.2013.09.013

Cited by 25 publications

(18 citation statements)

References 22 publications

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“…For enzyme-linked immunosorbent assay (ELISA), insects were used chilled at Ϫ20°C for 5 min and then mashed using a tissue grinder in 40 l 0.01ϫ PBS for each sample. ELISA detection was then performed as described previously (45).…”

Section: Methodsmentioning

confidence: 99%

Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

et al. 2015

J Virol

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The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCEWe show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome via direct interaction with the 26S proteasome subunit RPN3. Our results imply that the molecular arms race observed in plant hosts can be extended to the insect vector that transmits those viruses. Rice stripe virus (RSV), one of the most destructive pathogens of rice production, has been responsible for numerous epidemics since it was first described in Japan in 1897 (1-3). RSV is the type member of the Tenuivirus genus, and the viral genome consists of four single-stranded RNA segments which range in size from approximately 8.9 to 2.1 kb (4, 5). RNA 1 is negative sense and encodes a putative RNA-dependent RNA polymerase. RNAs 2, 3, and 4 are ambisense, and each contains two open reading frames (ORFs), with one on the viral RNA strand (vRNA) and the second on the viral cRNA strand (vcRNA) (6). RSV vRNA 2 encodes a membrane-associated protein that reportedly is an RNA silencing suppressor and interacts with suppressor of gene silencing 3 (SGS3) (7). vcRNA 2 encodes a glycoprotein (NSvc2) which when expressed in insect cells is displayed on the membrane su...

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Section: Methodsmentioning

confidence: 99%

Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

et al. 2015

J Virol

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“…The purified recombinant CYVCV-CQ CP protein was used to immunize BALB/c mice as described previously (Wu et al 2011). After the forth immunization, spleen cells of the immunized mice were obtained and used for hybridoma production as described (Wu et al 2014). Isotypes of MAbs were discriminated using a mouse monoclonal antibody isotyping kit as instructed (Sigma-Aldrich, St. Louis, MO, USA).…”

Section: Preparation Of Polyclonal and Monoclonal Antibodiesmentioning

confidence: 99%

“…Isotypes of MAbs were discriminated using a mouse monoclonal antibody isotyping kit as instructed (Sigma-Aldrich, St. Louis, MO, USA). Specificity and sensitivity of the antibodies were determined respectively by Western blot and triple antibody sandwich(TAS)-ELISA-ELISA as described previously (Wu et al 2007(Wu et al , 2014Shang et al 2011).…”

Section: Preparation Of Polyclonal and Monoclonal Antibodiesmentioning

confidence: 99%

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Liu

Sunzhu

Zhou

et al. 2017

Journal of Integrative Agriculture

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Citrus yellow vein clearing virus (CYVCV) is considered as the causal agent of Citrus yellow vein clearing disease and belongs to the genus Mandarivirus in the family Alphaflexiviridae. Capsid protein (CP) of CYVCV Chongqing isolate (CYVCV-CQ) was produced using a prokaryotic expression system and used as the immunogen for monoclonal antibody (MAb) production. Four highly specific and sensitive murine MAbs and one polyclonal antibody were prepared in this study. Titers of the four MAbs in ascites fluids ranged from 10-6 to 10-7 as determined by indirect enzyme-linked immunosorbent assay (ELISA). Three serological assays, including dot enzyme-linked immunosorbent assay (dot-ELISA), tissue blot-ELISA, and double-antibody sandwich (DAS)-ELISA, were developed for quick and reliable detections of CYVCV in citrus samples. The developed dot-ELISA and DAS-ELISA methods could detect CYVCV in the infected citrus leaf crude extracts diluted at 1: 2 560 and 1:10 240 (w/v, g mL-1), respectively. The detection result of 125 citrus leaf samples collected from citrus groves in the Yunnan Province and Chongqing Municipality of China showed that approximately 36% samples were positive for CYVCV. This virus was, however, not detected in any sample collected from the Zhejiang or Jiangxi Province, China.

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“…In addition, the results of electron microscopy usually need to be confirmed by other methods [12]. Serological methods, such as enzyme-linked immunosorbent assay, are economical for detection of high throughput samples [14], but they are limited by the specificity and availability of antibodies against the virus. Deep sequencing and qRT-PCR and are of high sensitivity and specificity [15–17], but expensive.…”

Section: Introductionmentioning

confidence: 99%

Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén)

Liu

Yuan

et al. 2017

Virol J

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BackgroundThe small brown planthopper (SBPH) is an important pest of cereal crops and acts as a transmission vector for multiple RNA viruses. Rapid diagnosis of virus in the vector is crucial for efficient forecast and control of viral disease. Reverse transcription polymerase chain reaction (RT-PCR) is a rapid, sensitive and reliable method for virus detection. The traditional RT-PCR contains a RNA isolation step and is widely used for virus detection in insect. However, using the traditional RT-PCR for detecting RNA virus in individual SBPHs becomes challenging because of the expensive reagents and laborious procedure associated with RNA isolation when processing a large number of samples.ResultsWe established a simplified RT-PCR method without RNA isolation for RNA virus detection in a single SBPH. This method is achieved by grinding a single SBPH in sterile water and using the crude extract directly as the template for RT-PCR. The crude extract containing the virus RNA can be prepared in approximately two minutes. Rice stripe virus (RSV), rice black streaked dwarf virus (RBSDV) and Himetobi P virus (HiPV) were successfully detected using this simplified method. The detection results were validated by sequencing and dot immunobinding assay, indicating that this simplified method is reliable for detecting different viruses in insects. The evaluation of the sensitivity of this method showed that both RSV and HiPV can be detected when the cDNA from the crude extract was diluted up to 103 fold. Compared to the traditional RT-PCR with RNA isolation, the simplified RT-PCR method greatly reduces the sample processing time, decreases the detection cost, and improves the efficiency by avoiding RNA isolation.ConclusionsA simplified RT-PCR method is developed for rapid detection of RNA virus in a single SBPH without the laborious RNA isolation step. It offers a convenient alternative to the traditional RT-PCR method.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-017-0732-6) contains supplementary material, which is available to authorized users.

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Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors

Cited by 25 publications

References 22 publications

Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

Monoclonal antibody-based serological detection of Citrus yellow vein clearing virus in citrus groves

Development of a simplified RT-PCR without RNA isolation for rapid detection of RNA viruses in a single small brown planthopper (Laodelphax striatellus Fallén)

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