SUMMARYThe relationship between serotype specificity and protein structure was studied by polyacrylamide gel electrophoresis, peptide mapping and radioimmune precipitation (RIP) of structural and non-structural proteins of the five U.S. serotypes of bluetongue virus (BTV). The surface proteins, VP2 and VP5, showed the most variation in size among the serotypes. Peptide mapping of the proteins showed that VP2 is unique for each of the U.S. serotypes. The nucleocapsid and non-structural proteins showed a high degree of conservation, whereas the other surface protein, VP5, showed intermediate conservation among the serotypes. Monospecific neutralizing antiserum produced in rabbits against each serotype was used in cross-RIP against cytoplasmic extracts prepared from cells infected with each BTV serotype. There were extensive crossreactions among those proteins which showed a high degree of structural conservation, whereas VP2 was immunoprecipitated best in the homologous RIP system. Thus, a correlation between serotype specificity and protein structure was shown among the five U.S. serotypes of BTV.
Abstract. An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1-and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.Epizootic hemorrhagic disease of deer viruses (EHDVs) are members of the orbivirus genus of the family Reoviridae. These viruses cause an often fatal hemorrhagic disease of white-tailed deer and can cause a bluetongue-like illness in cattle. 8 Two serotypes of EHDV, designated EHDV-1 (New Jersey) and EHDV-2 (Alberta), are enzootic in the US. The agar gel immunodiffusion (AGID) test is the primary groupspecific test used for detection of antibodies to either bluetongue viruses (BTVs) or EHDVs; however, serological cross-reactivity between these two groups of viruses can result in false-positive results. Serum neutralization is used for serotype determination. 13 Enzyme-linked immunosorbent assay (ELISA) procedures that are highly sensitive and specific have been developed for the detection of antibody to BTV. 10 Because of their disease potential, the development of improved diagnostic tests for EHDV is needed. ELISA procedures showing various degrees of specificity and sensitivity with both polyclonal and monoclonal antibody-based formats have been described for the detection of antibody to EHDV. 1,3,7,[14][15][16] This paper describes the development of a monoclonal antibody-based ELISA for the detection of antibody to EHDV.Hybridoma cultures were prepared from the spleens of adult female BALB/c mice that had been immunized by a series of intraperitoneal and intravenous inoculations with EHDV-2 (Alberta). Monoclonal antibody production in the hybridoma cultures was determined in an indirect binding ELISA with antigen produced from EHDV-2-infected cell cultures 6 and anti-mouse IgG conjugated to horseradish peroxidase. The specificity of positive hybridoma culture fluids
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