Development and characterization of secondary standards for nucleic acid amplification technology (NAAT) assays for detection of hepatitis E virus

Background: To harmonize assays for detection of HEV RNA, a World Health Organization International Standard (WHO IS) was established. The WHO IS represents the highest order standard for HEV RNA but is limited in quantity. Secondary standards are needed to limit the use of WHO IS and minimize the need to replace it. Objective: Establish secondary standards for HEV NAAT assays and to calibrate these against the WHO IS.Methods: Stocks of genotype 3 HEV were prepared using both cell lysates and cell culture supernatants to produce non-enveloped and quasi-enveloped virus stocks, respectively. Both stocks were heat-inactivated, diluted in negative human plasma, and lyophilized to produce two candidate secondary standards: HEV-RR (non-enveloped virus) and HEV-RR.1 (quasi-enveloped virus). Both candidate standards were characterized and calibrated against the WHO IS for HEV RNA in an international collaborative study.Results: The collaborative study returned a total of 15 data sets, with different RNA extraction and ampli-fication methods. The estimated mean values relative to the WHO IS (250,000 IU/ml) are 229,000 IU/ml and 355,000 IU/ml for HEV-RR and HEV-RR.1, respectively. Conclusion: We have established two secondary standards for HEV RNA calibrated against the WHO IS. These standards are non-infectious and stable under different storage temperatures.