Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

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.

A novel highly divergent enteric calicivirus in a bovine calf, India

“…In the month of May of 2015, 14 cross-bred calves less than a year old reared in the temperate high-altitude Himalayan region (Mukteshwar, Uttarakhand, India), were presented with severe diarrhea, weight loss, and dehydration. To identify the causative pathogen, fecal samples (n = 14) were collected aseptically from the infected calves, all of which tested negative for genome of rotavirus group A (RVA) (7), Picobirnavirus (PBV) (8), Bovine Coronavirus (BCoV) (9), and Astrovirus (AstV) (10). However, the RIDASCREEN Norovirus ELISA kit (R-Biopharm, Darmstadt, Germany) detected the possible presence of Norovirus in one of the samples, UK-B6.…”

Advances in Diagnostic Approaches for Viral Etiologies of Diarrhea: From the Lab to the Field

“…The concept of this assay was based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. This assay has already been validated on the fecal samples of four hosts (bovine, porcine, poultry, and human) and also showed a high concordance with diagnostic RT-PCR (Kumar et al, 2016). Recently, a sensitive sandwich ELISA for the detection of NoV genogroup II has been developed that provided an improved detection limit of 13.2 copies/mL fecal suspension for Norovirus as compared to gold-immunoassay (1000-fold) and horseradish peroxidase-based ELISA (100-fold).…”

Genomic Analysis of an Indian G8P[1] Caprine Rotavirus-A Strain Revealing Artiodactyl and DS-1-Like Human Multispecies Reassortment

“…During an investigation of the outbreak of diarrhea in goat population located in an unorganized sector, reared by local people randomly, a sample K-98 was collected from a goat kid in the Northern state of Uttar Pradesh, India was found positive using an antigen-capture sandwich ELISA for RVA infection ( 33 ).…”

“…Structural genes VP2, VP3, VP4, VP6, VP7, and non-structural genes NSP1, NSP2, NSP3, NSP4, and NSP5 were amplified using earlier reported primers ( 18 , 37 , 38 ) whereas two new pair of overlapping primers for VP1 gene were designed to amplify its partial gene for nearly ~1,400 bp ( Supplementary Data 1 ). Standalone software like MEGA 7.0 ( 39 ) and online primer designing tool IDTs Oligoanalyzer ( https://eu.idtdna.com/calc/analyzer ) were used to design and verify the primers sets ( 33 ).…”