RESUMO
Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.
Assuntos
Anticorpos Antivirais/química , Bioensaio , Western Blotting/métodos , Coloide de Ouro/química , Herpesvirus Bovino 1/imunologia , Rinotraqueíte Infecciosa Bovina/diagnóstico , Animais , Anticorpos Antivirais/isolamento & purificação , Benchmarking , Western Blotting/instrumentação , Bovinos , Cães , Ensaio de Imunoadsorção Enzimática , Herpesvirus Bovino 1/isolamento & purificação , Soros Imunes/química , Imunoconjugados/química , Rinotraqueíte Infecciosa Bovina/sangue , Rinotraqueíte Infecciosa Bovina/virologia , Células Madin Darby de Rim Canino , Nanopartículas Metálicas/química , Sensibilidade e EspecificidadeRESUMO
The selected dodecapeptide (1)DRALYGPTVIDH(12) from a phage-displayed peptide library and the crystal structure of the envelope glycoprotein B (Env gB) from Herpes Simplex Virus type 1 (HSV-1) led us to the identification of a new discontinuous epitope on the Bovine herpesvirus type 1 (BoHV-1) Env gB. In silico analysis revealed a short BoHV-1 gB motif ((338)YKRD(341)) within a epitope region, with a high similarity to the motifs shared by the dodecapeptide N-terminal region ((5)YxARD(1)) and HSV-1 Env gB ((326)YARD(329)), in which the (328)Arg residue is described to be a neutralizing antibody target. Besides the characterization of an antibody-binding site of the BoHV-1 Env gB, we have demonstrated that the phage-fused peptide has the potential to be used as a reagent for virus diagnosis by phage-ELISA assay, which discriminated BoHV-1 infected serum samples from negative ones.