Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1–4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin–streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2–3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.

Zika virus (ZIKV) caused a public health threat in the United States in 2016, leading to rapid development and implementation of blood screening assays for ZIKV RNA. Several ZIKV sequences from clinical cases have been reported, but none from asymptomatic/pre-symptomatic infections. We isolated and sequenced ZIKV from asymptomatic/pre-symptomatic blood donor (ABD-ZIKV) samples and compared with reported clinical sequences. Twelve ABD-ZIKV isolates were produced from 67 cultivated samples, and isolates were genetically similar among themselves. Most isolates shared mutations with the clinical isolate PRVABC59 2015, whereas two ABD-ZIKV isolates shared specific mutations with U.S. clinical isolates from 2016. The ABD-ZIKV strains clustered into two distinct subclades: one comprised mostly ABD-ZIKV from Puerto Rico, and another one comprised ABD-ZIKV from Florida and QTX-02 isolate (Puerto Rico). In this study, we showed the circulation of two slightly distinct virus strains among Puerto Rico blood donors, one of which was also reported in Florida.