Evaluation of Heating Conditions for Inactivation of Hepatitis E Virus Genotypes 3 and 4

Abstract

Hepatitis E virus (HEV) is a causative agent of acute hepatitis throughout the world. HEV genotypes 1 through 4 infect humans, whereas genotypes 3 and 4 (Gt3 and Gt4) also infect other animals. In developed countries, the main HEV infection route is by foodborne transmission, resulting from the consumption of undercooked meat. It is important to know the criteria for HEV control in daily cooking. In this study, we assessed the heat conditions required to inactivate HEV Gt3 and Gt4 in culture supernatants and spiked minced pork meat. HEV inactivation was determined by measuring viral RNA amplification in PLC/PRF/5 cell culture. In our cell culture assay, an inoculum containing HEV titer that is equivalent to >10 genome RNA copies can be determined as infectious. The internal temperature of pork during heating was measured to represent that achieved during cooking. Both HEV Gt3 and Gt4 were inactivated in culture supernatants heated at >65°C for 5 min and at >80°C for 1 min and in minced meat at 70°C for 5 min. Inoculated culture supernatant contained 10 HEV genome RNA copies (10 infectious units [IU]); therefore, it was indicated that HEV titer decreased >3 log IU after heating. In a comparison of Gt3 and Gt4, Gt4 showed slightly greater heat stability than Gt3. Boiling showed superior heating efficacy compared with roasting, and pork liver was slightly easier to heat than pork loin. Heating for 5 min by both boiling and roasting increased the internal temperature of pork products to more than 70°C. Although our data revealed that HEV Gt4 was slightly more heat stable than Gt3, both genotypes were inactivated by the appropriate heating conditions. Therefore, the risk of HEV foodborne infection could be mitigated by the appropriate cooking of pork meat. It is also important that both the supplier and the consumer are cognizant of the risk of HEV foodborne infection from livestock products.

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

“…Some differences have been recently reported by Imagawa et al (2018) who studied HEV inactivation by measuring virus replication in PLC/PRF/5 cell culture. The results showed that exposure to 65°C for 5 min or 75°C for 1 min inactivated HEV-3, while HEV-4 was inactivated at 80°C for 1 min.…”

“…Molecular methods, particularly real-time reverse transcriptase polymerase chain reaction (RT-qPCR), have demonstrated high sensitivity, specificity, and ability to deliver reliable quantitative data in food and environmental samples (Martin-Latil et al, 2014, 2016; Di Bartolo et al, 2015; Mesquita et al, 2016), although such results do not indicate at the infectivity of detected viruses. In addition, alternative strategies to directly study infectivity such as cell culture systems and animal models do not seem to be reliable or practical yet (Ricci et al, 2017; Van der Poel et al, 2018), although promising results have been reported (Emerson et al, 2005; Johne et al, 2016; Imagawa et al, 2018). To enable the differentiation between infectious and inactivated viral particles, different approaches based on capsid integrity have been reported:…”

Clinical Characteristics of Acute Hepatitis E and Their Correlation with HEV Genotype 3 Subtypes in Italy

“…In Italy, pork is generally roasted before eating and dried sausages are rarely consumed raw. Barbequed pork could be undercooked; however, the infectious viral titer should be lower than that in figatelli or paté [ 36 , 37 , 38 ]. In interviews carried out at the hepatology Unit, only two patients in our study admitted to eating dried sausage.…”

The Clinical Perspective on Hepatitis E

“…In contrast, single, non-epidemic zoonotic HEV genotype 3 infections occur in Europe, North- and South America and Australia: contact with swine and consumption of inadequately heated pork are sources of infection with Genotype 3. Pork needs to be cooked for more than 5 min at 70 °C to inactivate the HEV virus [5]. Likewise, genotype 4 infections are also transmitted by pork, but this genotype is prevalent in Asia and only single rare cases have been described to be autochthonously acquired in Europe [6].…”