Production Of Monclonal Antibodies Against Rice Dwarf Virus And Rice Black-streaked Dwarf Virus And Their Application

Monoclonal antibody-based serological methods for plant virus detection are simple, rapid, sensitive, specific, and suitable to detect large-scale field samples. So it has been widely used in plant disease diagnosis, epidemic rule analyses, forecast and breeding for disease resistance. It is also helpful to establishing a prevention and control system to plant virus. Therefore, it is of important significance to develop sensitive and specific monoclonal antibodies(MAbs) against important plant viruses for agricultural production and scientific research. Rice dwarf disease was caused by Rice dwarf virus(RDV). Besides Rice black-streaked dwarf disease, Maize rough dwarf disease and Wheat green dwarf disease were caused by Rice black-streaked dwarf virus(RBSDV). At present, RDV and RBSDV cause great losses of rice production in China. In this study, MAbs against RDV and RBSDV were producted, and several serological methods were set up for rapid and reliable virus detection.(1)MAbs against RDV and its application:The purified RDV virions were used as an immunogen to produce monoclonal antibodies. Three sensitive and specific murine monoclonal antibodies against RDV(1D11,6A8and9B2)were produced with the hybridoma technology. Three serological methods of antigen-coated-plate enzyme-linked immunosorbent assay(ACP-ELISA), dot enzyme-linked immunesor-bent assay(dot-ELISA)and immunocapture reverse transcriptase polymerase chain reaction(IC-RT-PCR)were established for detecting RDV in field rice and leafhopper samples. The ACP-ELISA, dot-ELISA and IC-RT-PCR could detect minimum RDV in infected tissue saps diluted at1:81,920,1:10,240and1:655,360(w/v, g/mL)and in individual viruliferous leafhopper extracts diluted at1:25,600,1:6,400and1:3,276,800(individual leafhopper/uL), respectively. Rice field samples(878)and leafhopper field samples(531)from ten provinces of China were detected. The detection result demonstrated that RDV is only prevalent in Yunnan Province and30%of rice field samples and13.7%of field leafhopper samples from Yunnan Province were RDV-positive.(2)MAbs against RBSDV and its application:The crude purified RBSDV virions were used as an immunogen to produce monoclonal antibodies. Three murine MAbs against RBSDV(5G5,12E10and18F10)were produced with the hybridoma technology. ACP-ELISA and dot-ELISA methods were developed for sensitive, specific and rapid detection of RBSDV in field plant and small brown planthopper samples. The serological detection methods can distinguish RBSDV from SRBSDV, which shares a high degree of similarity in genomic structure and sequence, virion morphology, serology, symptoms and host ranges. The ACP-ELISA could detect minimum RBSDV in infected rice, miaze, wheat and planthopper tissue extracts diluted at1:20,480,1:81,920,1:10,240(w/v, g/mL)and19,200(individual planthopper/μL). The dot-ELISA could detect minimum RBSDV in infected maize, wheat and rice tissue crude extracts diluted at1:320(w/v, g mL-1), and in individual viruliferous planthopper extract diluted at1:1,600(individual planthopper/μL), respectively. Field plant(915)and planthopper samples(594)from five provinces of China were screened for the presence of RBSDV using the two developed serological methods. The field survey demonstrated that RBSDV is prevalent in rice, maize and wheat crops in Jiangsu, Zhejiang, Shandong provinces of China.