Thermal Inactivation of Hepatitis E Virus in Pork Products Estimated with a Semiquantitative Infectivity Assay

Abstract

Hepatitis E virus genotype 3 (HEV-3) is a food-borne pathogen causative of hepatitis E infections in humans. In Europe, HEV-3 is mainly transmitted through the consumption of raw or undercooked pork. In order to determine the effectiveness of control measures that can be taken in the industry or by the consumer, it is pivotal to determine the infectivity of HEV present in pork products after thermal food-processing steps. First, we implemented a method for the detection of infectious HEV-3c and HEV-3e in a cell culture medium and in extracts from inoculated pork products. Next, we investigated the effect of the thermal inactivation of HEV by mimicking food-processing steps specific for dried sausage and liver homogenate matrices. After four weeks, HEV-inoculated dried sausage subjected to 21 °C or lower temperatures was still infectious. For the liver homogenate, the highest HEV-3c/e inactivation of the conditions tested was observed at 71 °C for five min or longer. Finally, our method was able to successfully detect and estimate viral loads of infectious HEV in naturally infected pig livers. Our data provide a basis for the future use of the quantitative microbial risk assessment of infectious HEV in pork products that are subjected to thermal food processing steps.

Keywords: HEV infectivity; HEV-3c; HEV-3e; cell culture method; food processing; food-borne; immunofluorescence; pork matrices.

Figure 1

Inactivation of HEV-3c/e in cell culture medium subjected to different temperatures. HEV-3c/e inactivation factors (log₁₀) were estimated using the Bayesian MPN method. Data are representative of 2 or 3 experiments performed on different days depending on the time–temperature combination (Table 1). At least four different wells of inoculated A549/D3 cells per dilution were examined. The 95% confidence intervals are indicated by colored bands. Red squares indicate the first data point, for which the 89% HDI was outside of the ROPE interval (−0.5, 0.5), and denote a meaningful reduction in infectious HEV.

Figure 2

Inactivation of HEV-3c/e in dried sausage subjected to different temperatures. HEV-3c/e inactivation factors (log₁₀) were estimated using the Bayesian MPN method. Data are representative of 2 or 3 experiments performed on different days depending on the time–temperature combination (Table 1). At least four different wells of inoculated A549/D3 cells per dilution were examined. The 95% confidence interval is indicated by colored bands. Red squares indicate the first data point, for which the 89% HDI was outside of the ROPE interval (−0.5, 0.5), and denote a meaningful reduction in infectious HEV.

Figure 3

Inactivation of HEV-3c/e in liver homogenate subjected to different temperatures. HEV-3c/e inactivation factors (log₁₀) were estimated using the Bayesian MPN method. Data are representative of 2 or 3 experiments performed on different days depending on the time–temperature combination (Table 1). At least four different wells of inoculated A549/D3 cells per dilution were examined. The 95% confidence interval is indicated by colored bands. Red squares indicate the first data point, for which the 89% HDI was outside of the ROPE interval (−0.5, 0.5), and denote a meaningful reduction in infectious HEV.