Development and validation of a Q-PCR based TCID₅₀ method for human herpesvirus 6

For titer assessment of human herpesvirus 6 (HHV-6), IFA targeting viral proteins or a TCID₅₀ method with ocular inspection for CPE can be used. These methods rely on the subjective decision of the assessor, obstructing the ability to obtain unanimous results.

We have developed and validated an alternative TCID₅₀ read-out approach where infection in the titration culture plate is assessed by viral DNA load change by quantitative PCR. A ten time increase in viral DNA load was determined as cut point for infection since that yielded a maximum correlation with viral protein expression (93%). The average intra-assay CV was 9% for quantitative PCR read-out of TCID₅₀ compared to 45% for ocular inspection read-out of TCID₅₀, 14% for IFA read-out of TCID₅₀, and 43% for an infectious units approach using IFA. The average inter-assay CV for quantitative PCR read-out of TCID₅₀ was 73%, compared to 66%, 25% and 77% for the ocular inspection read-out for TCID₅₀, IFA read-out of TCID₅₀ and infectious unit approaches respectively.

The quantitative PCR based read-out of TCID₅₀ proved to be more robust and easier to interpret than traditional TCID₅₀ assessment approaches for HHV-6, and therefore it might be considered as an alternative method.