Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/28/2000
Publication Date: N/A
Interpretive Summary: Barley yellow dwarf viruses (BYDVs) cause the most damaging virus disease of cereal crops. The symptoms induced by BYDVs, leaf yellowing and stunting, are similar to those caused by abiotic factors and other vascular diseases. This lack of unambiguous symptomatology necessitates production of reagents for the reliable diagnosis of BYDV infections. The most commonly used diagnostic tool for BYDVs is enzyme-linked immunosorbent assay, which is dependent on the availability of highly specific antisera to differentiate the viruses. While some BYDVs can be purified easily, others remain difficult to extract in sufficient quantities for antiserum production. To test the ability of producing antiserum to BYDVs by expressing the capsid protein directly in the immunized animal, we attempted to produce antibodies against one of the BYDV proteins by DNA-based immunization, thereby eliminating the need for isolation and purification of the virus proteins. The results of our study showed that antibodies could be produced to BYDVs by DNA immunization. This information will be useful to scientists who are interested in producing diagnostic antibodies for plant viruses that are difficult to purify from infected plant tissues. We will attempt to use this approach to produce antisera against the RMV strain of BYDV, which is very difficult to purify.
Technical Abstract: We report here the induction of antibodies to plant and human viral proteins by DNA immunization. In two separate experiments, cDNAs encoding the 22-kDa coat protein (CP) of barley yellow dwarf virus strain PAV (BYDV-PAV) and the human respiratory syncytial virus fusion protein (RSV-F) coding sequence were cloned into mammalian expression vectors (pcDNA22K and pBKRSV-F, receptively). The DNA was injected intramuscularly into BALB/c mice. Three weeks post-immunization, BYDV-PAV and RSV-F specific immune responses were detected in serum. To target the antigen to sites of immune induction and thereby enhance the immune response the coding region for mouse cytotoxic T-lymphocyte antigen 4 (CTLA4) was fused with the CP sequence (pCTLA422K) and the RSV-F sequence (pBKCTLA4RSV-F). Mice injected with pCTLA422K and pBKCTLA4RSV-F DNA showed significantly higher titers than mice injected with pcDNA22K and pBKRSV-F. Increases in antibody titers were observed over time with each subsequent DNA injection. Further, there were large increases in antibody titers when DNA immunized mice were boosted with purified BYDV-PAV virions and gamma-ray-killed RSV antigen. These results suggest that antibodies against BYDV-PAV and RSV-F can be generated using DNA immunization. Taking advantage of this approach it may be possible to produce antisera against other plant viruses, for example BYDV-RMV which are very difficult to purify in sufficient quantities to mediate useful immune response.