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Published on: March 29, 2017
Highly sensitive parechovirus CODEHOP PCR amplification of the complete VP1 gene for typing directly from clinical
Jeroen Cremer1, Ursula Morley2, Suzan Pas3,4
1National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
Purpose:
Human parechoviruses (HPeVs), particularly type 3, can cause severe neurological disease and neonatal sepsis in infants. HPeV3 lacks the receptor-binding motif arginine-glycine aspartic acid (RGD), and is proposed to use a different receptor associated with severe disease. In contrast, HPeV1, which contains the RGD motif, is associated with mild disease. Rapid characterization of the presence/absence of this motif is essential for understanding their epidemiology and differential disease profiles. Current HPeV typing assays are based on partial capsid genes and often do not encompass the C-terminus where the RGD region is localized/absent. In addition, these assays lack sensitivity to enable characterization within low viral-load samples, such as cerebral spinal fluid.
Methodology:
We developed a highly sensitive HPeV CODEHOP PCR, which enables typing of parechoviruses directly from clinical samples while generating a complete VP1 gene, including the C-terminus.
Results:
The assay was HPeV-specific and has a sensitivity of 6.3 TCID50 ml-1 for HPeV1 and 0.63 TCID50 ml-1 for HPeV3. Analysis of the complete VP1 gene in comparison to partial VP1 fragments generated by previously published PCRs showed homologous clustering for most types. However, phylogenetic analysis of partial VP1 fragments showed incongruent typing based on the 75 % homology classification rule. In particular, the strains designated as type 17 were found to be either type 3 or 4 when using the (near-) complete VP1 fragment.
Conclusion:
While enabling sensitive characterization of HPeVs directly from clinical samples, the HPeV CODEHOP PCR enables the characterization of RGD and non-RGD strains and correct HPeV typing based on the complete VP1.
Insights
A new PCR method accurately types human parechoviruses (HPeVs) and identifies disease-associated RGD motifs. This sensitive assay improves diagnosis for infants with severe neurological disease or sepsis.
Area of Science:
- Virology
- Molecular Biology
- Infectious Diseases
Background:
- Human parechoviruses (HPeVs), especially type 3, are linked to severe infant neurological disease and sepsis.
- HPeV3 lacks the arginine-glycine-aspartic acid (RGD) motif, suggesting a different receptor and disease mechanism compared to RGD-containing HPeV1.
- Current typing methods use partial viral genes, missing the critical RGD region and lacking sensitivity for clinical samples.
Purpose of the Study:
- To develop a sensitive assay for accurate HPeV typing directly from clinical samples.
- To characterize the presence or absence of the RGD motif for understanding HPeV epidemiology and disease profiles.
- To enable correct HPeV classification using the complete VP1 gene.
Main Methods:
- Development of a highly sensitive HPeV CODEHOP PCR assay.
- Direct typing of parechoviruses from clinical samples.
- Generation of the complete VP1 gene, including the C-terminus.
Main Results:
- The assay demonstrated high specificity and sensitivity (0.63–6.3 TCID50 ml⁻¹).
- Phylogenetic analysis of complete VP1 genes provided more accurate typing than partial fragments.
- Previous typing discrepancies were resolved, correctly classifying strains previously misidentified as type 17.
Conclusions:
- The HPeV CODEHOP PCR enables sensitive and accurate HPeV characterization from clinical samples.
- The assay allows for the identification of RGD and non-RGD strains, crucial for understanding disease severity.
- This method facilitates correct HPeV typing based on the complete VP1 gene, improving diagnostic capabilities.
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