Purpose: To evaluate apolipoprotein H (ApoH) as a potential broadly reactive reagent to concentrate HuNoV for detection using quantitative Reverse Transcription PCR (RT-qPCR).
Methods: Experiments were designed to evaluate (i) if ApoH effectively captured HuNoVs or Virus Like Particles (VLPs); and (ii) if so, recovery efficiency. To address the first question, (VLPs) were bound to ApoH coated microplates and evaluated for detection using an ELISA method. To address the second, a representative epidemic GII.4 HuNoV strain obtained as a fecal specimen from an infected individual was used as inoculum. This was serially diluted and mixed with ApoH-conjugated magnetic beads (ApoH Technologies). Subsequent virus detection was done using RT-qPCR targeting the ORF1/ORF2 junction. Capture experiments were done on both 1 and 10 ml sample volumes.
Results: ELISA results indicated significant capture of GII.2 and GII.4 VLPs, as well as the HuNoV surrogate, Tulane virus. Using the ApoH conjugated magnetic beads followed by RT-qPCR, the GII.4 HuNoV strain was effectively concentrated from 1 ml samples at various inoculum levels ranging from 106 to 101 genome equivalent copies (GEC) with virtually 100% efficiency, i.e., no statistically significant difference (P > 0.05) between input and output virus concentrations as per comparison to a standard curve. In 10 ml scale-up experiments, concentration efficiency was about 10%, as evidenced by a 1 log drop in virus titer after concentration relative to the standard curve.
Significance: ApoH represents a novel and efficient method for capture and concentration of HuNoV. Further studies are underway applying this protocol to artificially contaminated food extracts.