Validation of Synthetic Peptide Enzyme Immunoassays in Differentiating Two Subgroups of Ruminant Respiratory Syncytial Virus

Abstract

Abstract. Subgroup-specific peptide-based enzyme immunoassays from each respective G-glycoprotein of the ovine and the bovine respiratory syncytial virus (RSV) were developed to detect RSV-specific IgG responses in cattle. Antigenic peptides from the respective G-glycoprotein were identified from the extracellular central hydrophobic region (amino acids 158-189) located between 2 mucin-rich regions. These antigenic peptides identified by epitope mapping from each G-glycoprotein were synthesized and used to develop the subgroupspecific enzyme immunoassays. The negative cutoff for each enzyme immunoassay was established as the mean optical density of indirect immunofluorescent antibody-negative bovine sera plus 3 SDs. The sensitivity (82.9%) and specificity (100%) of the bovine enzyme immunoassay and the specificity (95.8%) of the ovine enzyme immunoassay were determined by comparison with indirect immunofluorescence (used as the ''gold standard''). The negative and positive predictive values were calculated for each assay. The presence of serum antibody to ovine RSV in cattle implies that this virus infects cattle and may contribute to the pathogenesis of bovine respiratory disease.Economic losses due to respiratory disease in the US cattle industry approximate $1 billion annually. 10 Respiratory syncytial virus (RSV) has been isolated from people (human RSV [HRSV]) and from several animal species, including cattle (bovine RSV [BRSV]), sheep (ovine RSV [ORSV]), and goats (caprine RSV). These viruses are members of the genus Pneumovirus in the family Paramyxoviridae. They are enveloped, single-stranded, nonsegmented, negativesense RNA viruses. 11 The genome encodes 10 viral proteins: 1B, 1C, N, P, M, SH, M2, L, the major glycoprotein (G), and the fusion (F) protein. 3,8 The G and F proteins are present in the membrane of infected cells and in the virus envelope. 16 The attachment protein G has been the most variable antigen among viral strains. 2 The proposed 3-dimensional model for the Gglycoprotein of BRSV contains a hydrophobic globular region (residues 158-189) bordered by 2 hydrophilic densely carbohydrate-coated mucin-like regions. 15 The G-glycoprotein gene from an ORSV strain has been cloned and sequenced and had greater homology to the BRSV gene (and its encoded protein) than to the HRSV gene (and its encoded protein). 2,4 However, the degree of base sequence differences between BRSV and ORSV suggests that 2 subgroups of an ungulate RSV exist. 2,15 Recent RNase protection assays, Received for publication October 26, 1999. reverse transcription polymerase chain reaction amplification studies, and a phosphoprotein profile analysis support the existence of 2 ruminant RSV subgroups. 1,9,17,18 Current knowledge concerning interspecies transmission of RSV strains is limited. Antibodies to BRSV were found in cattle, goats, and horses but were not detected in sheep. 14,20 Recently, serologic evidence of RSV infection in swine has been reported. 5 Respiratory syncytial virus is recognized as an important pathog...

Analysis and solution of false-positives when testing CVA16 sera using an antibody assay against the EV71 virus

“…Absorbance was read at 450 nm using an ELISA plate reader. The cut-off value was set at the mean optical density (OD) value using the healthy children's sera (n = 92) plus three standard deviations (SD) (Grubbs et al, 2001).…”

Prevalence of Ovine and Bovine Respiratory Syncytial Virus Infections in Cattle Determined with a Synthetic Peptide-Based Immunoassay

“…The BRSV peptide ELISA was done as described elsewhere, with modifications. 12,15,18 The optimum concentration of peptide antigens was determined over the range of 0.075-20 g/ml using negative and positive sera. As a consequence, each well of a 96-well ELISA plate j was coated with 0.125 g of peptide in 100 l of phosphate-buffered saline (PBS, pH 7.2) and incubated overnight at 4 C. The plates were washed 6 times with PBS (200 l/well) containing 0.05% polyoxyethylenesorbitan monolaurate (PBSTW) and were treated with PBSTW (280 l/ well) for 60 min at room temperature.…”

“…20 The RNA contains 10 genes that are transcribed to 10 unique mRNAs, each of which encodes a unique protein. 4,[10][11][12] The fusion (F) protein is broadly cross-reactive among strains, whereas the attachment protein G has been the most variable among viral strains. 11,20 Considerable progress has been made concerning the nature of interspecies transmission of RSV strains.…”

“…9,11,22 Subgroup-specific ELISAs developed from peptides corresponding to extracellular regions of the respective bovine and ovine G-glycoprotein could be used to evaluate the epidemiology of ruminant RSV strains. 12,18,25 The estimates of economic losses due to respiratory tract disease in the US cattle industry may approach $1 billion. 11 BRSV is a major respiratory tract pathogen of cattle and is one of few viruses that can cause acute respiratory disease without significant interaction with other pathogens.…”

Development of a 1-Step Enzyme-Linked Immunosorbent Assay for the Rapid Diagnosis of Bovine Respiratory Syncytial Virus in Postmortem Specimens

“…The cut-off value was set at the mean OD value of this negative population (n 5 93) plus 3 SD. 6 Clearview RSV enzyme immunoassay (EIA) h . The Clearview RSV test is a lateral flow immunogold assay that allows direct visual detection of RSV antigen in clinical samples.…”