Using the ApoH Protein to Capture and Concentrate Human Norovirus Particles and Virus-like Particles from Various Matrices

Introduction: Human noroviruses (HuNoV) are a leading cause of foodborne illness.  The inability to cultivate these viruses in vitro highlights the need for simple and effective methods to concentrate them from relevant sample matrices prior to detection.  This is particularly complicated because HuNoV tend to be present in low concentrations in foods, and commonly used capture ligands (antibodies) lack broad reactivity to all HuNoV strains.   

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.