Production of polyclonal antisera for diagnosis of rice yellow stunt virus(RYSV)in Vietnam

HA Viet-cuong;TRAN Thi-nhu-hoa;TRAN Nguyen-ha;DO Tan-dung;NGUYEN Duc-huy;WEI Shan-fu;QIN Wu;LYU Rong-hua;Department of Plant Pathology,Faculty of Agronomy,Vietnam National University of Agriculture;Research Center for Tropical Plant Diseases,Vietnam National University of Agriculture;Plant Protection Research Institute,Guangxi Academy of Agricultural Sciences;Division of International Cooperation,Guangxi Academy of Agricultural Sciences;

rice yellow stunt virus;;polyclonal antibody;;enzyme-linked immunosorbent assay(ELISA);;N protein;;expression;;virus purification

【Objective】Polyclonal antibodies specific to rice yellow stunt virus(RYSV)was prepared to provide technique support for diagnosis of this virus in rice plants and green leafhopper vectors(Nephotettix spp.).【Method】Two sources of the RYSV antigens were prepared for the rabbit immunization. The first one was the RYSV virion purified from the infected rice leaf tissue. The second one was the complete N protein of one RYSV isolate from Vietnam,which was cloned in the pET28 a vector and expressed in Escherichia coli strain Rosetta(DE3). The N protein antigen was used in two forms,the eluted fraction of purified N protein and the homogenate of polyacrylamide slices containing N protein band. The specificity and sensitivity of the antisera and antibodies for detection of RYSV from rice leaves and rice green leafhopper vector individuals were evaluated by a plate trapped antigen enzyme linked immunosorbent assay(PTA-ELISA)method.【Result】In this study the N gene of the RYSV isolate from Vietnam was composed of 1542 nucleotides,shared sequence identities of 98.1 % and 97.9%(nucleotide level)and 83.4 % and 99.4 %(amino acid level)with the two RYSV isolates from China and Japan,respectively,available in the GenBank. All three antisera produced in rabbits immunized with the virion,purified N protein and homogenate of polyacrylamide slices containing N protein band were able to detect RYSV in infected rice plants with the PTA-ELISA absorbance values(OD405)were 1.449,2.337 and 1.649,respectively,while those values in healthy plants were 0.375,0.294 and 0.283,respectively. The antisera were able to detect RYSV in a single viruliferous green leafhopper vector in PTA-ELISA test as confirmed by reverse transcriptase PCR(RT-PCR)test.【Conclusion】In conclusion,the rabbit polyclonal antisera produced in this study had high specificity and sensitivity again RYSV,and therefore they can be used for diagnosis of this virus from rice plants and green leafhopper vectors. This study also indicates that the polyacrylamide slices containing a viral protein band can be used for direct injection of animal for production of diagnostic polyclonal antibody.