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

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.

Evaluation of High-Pressure Processing in Inactivation of the Hepatitis E Virus

“…to report the presence of intact viral particles post treatment (Randazzo et al, 2018). The inactivation by HPP of other foodborne viruses, including norovirus, hepatitis A virus (HAV), and surrogates for foodborne viruses has been investigated (Kingsley et al, 2002;Kingsley, 2013;Lou et al, 2015;Lou et al, 2016).…”

“…This indicates that replication of virus occurred, and therefore the elimination of HEV infectivity was not complete. In another study, it was shown that the treatment of HEV solution at 500 MPa for 15 min did not result in complete inactivation assayed by using viability markers (PMAxx and platinum chloride, PtCl4-RT-qPCR) (Randazzo et al, 2018). Knowing that the virus is capable of replication (and therefore has the potential to cause illness) is important for interpretation of surveillance and inactivation studies on HEV to inform risk assessment and mitigation (Cook et al, 2017).…”

Evaluation of High-Pressure Processing in Inactivation of the Hepatitis E Virus

“…To the best of our knowledge, this is the first published study to quantify the inactivation of HEV following HPP treatment. HEV response to 500 MPa for 15 min has been previously investigated with RT-qPCR viability markers (PMAxx and platinum chloride, PtCl4-RT-qPCR), but was only able to report the presence of intact viral particles post treatment (43). The inactivation by HPP of other foodborne viruses, including norovirus, Hepatitis A virus (HAV), and surrogates for foodborne viruses has been investigated (26, 44-46).…”

“…This indicates that replication of virus occurred, and therefore the elimination of HEV infectivity was not complete. In another study, it was shown that the treatment of HEV solution at 500 MPa for 15 min did not result in complete inactivation assayed by using viability markers (PMAxx and platinum chloride, PtCl4-RT-qPCR) (43). Knowing that the virus is capable of replication (and therefore has the potential to cause illness) is important for interpretation of surveillance and inactivation studies on HEV to inform risk assessment and mitigation (28).…”

Hepatitis A infections from food

“…Viability RT-qPCR assays have been used to estimate infectious HAV exposed to thermal treatments. The have been optimized in viral suspensions, tested in inoculated food concentrates (including lettuce, spinach, parsley, cockles, coquina clams, mussels, oysters and berries) and successfully applied in naturally contaminated river, sewage and reclaimed waters (Moreno et al 2015;Fuster et al 2016;Randazzo et al 2018bRandazzo et al , 2018cChen et al 2020) (Table 3).…”