Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

Walter Randazzo; Andrea Vasquez-García; Rosa Aznar; Gloria Sánchez

doi:10.3389/fmicb.2018.01973

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

Front Microbiol. 2018 Aug 24:9:1973. doi: 10.3389/fmicb.2018.01973. eCollection 2018.

Authors

Walter Randazzo^{1

2}, Andrea Vasquez-García³, Rosa Aznar^{1

2}, Gloria Sánchez²

Affiliations

¹ Department of Microbiology and Ecology, University of Valencia, Valencia, Spain.
² Department of Preservation and Food Safety Technologies, Instituto de Agroquímica y Tecnología de Alimentos - Consejo Superior de Investigaciones Científicas, Valencia, Spain.
³ Faculty of Animal Science and Food Engineering, University of São Paulo, São Paulo, Brazil.

Abstract

The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To this end, different concentrations of viability markers (PMAxx and platinum chloride, PtCl₄) were screened firstly on purified viral RNA using different RT-qPCR assays. Reductions of HEV RNA signals of >17.5, >15.0, and >15.5 quantification cycles (Cq) were reported for PtCl₄ and 1.6, 2.9, and 8.4 Cq for PMAxx, clearly indicating a better performance of PtCl₄ than PMAxx irrespective of the RT-qPCR assay used. The most efficient viability pretreatment (500 μM PtCl₄ incubated at 5°C for 30 min) was then assessed on native, heat-, and HPP-treated HEV suspension. The optimized viability RT-qPCR discriminated successfully between native, heat-, and HPP-treated HEV, to different extents depending on the experimental conditions. In particular, approximately 2-log₁₀ reduction was reported by PtCl₄-RT-qPCR at both 72 and 95°C compared to the control. Additionally, both viability pretreatments were tested for HPP-treated HAV without success, while PtCl₄-RT-qPCR completely eliminated (>5.6-log₁₀ reduction) the RT-qPCR signals of HPP-treated HEV. Although this viability procedure may still overestimate infectivity, the PtCl₄ pretreatment represents progress to better interpreting the quantification of intact HEV, and it could be included in molecular procedures used to quantify enteric viruses in food and environmental samples.

Keywords: HAV; HEV; food safety; foodborne virus; intercalating dye; viability RT-qPCR.