Abstract
We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings.
Highlights
Rotaviruses (RVs) belong to the family Reoviridae and are a leading cause of diarrhea in humans and animals worldwide
We identified six regions of high antigenic index within bovine RVA VP6 protein and correspondingly synthesized peptides by solid phase peptide synthesis over Wang resin
The Circular Dichroism (CD) spectra analysis revealed that all six peptides in polar solvent had a negative minimum below 200 nm in a far UV range
Summary
Rotaviruses (RVs) belong to the family Reoviridae and are a leading cause of diarrhea in humans and animals worldwide. RVs account for approximately one fourth of global mortality in Indian children annually [1,2,3]. RVs are classified into at least nine distinct serological species or groups (A to I), of which group A rotaviruses (RVAs) are frequently associated with acute diarrhea [1,2,5]. Used dual classification system for RVAs designate P- and G-genotypes to the genes coding for outer most proteins VP4 and VP7, respectively. At least 27 G and 37 P genotypes of RVAs have been identified in humans and animals, reflecting huge genetic diversity among RVs [2,6]
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