Abstract

The thermal stability of virulent hepatitis E virus (HEV) and hepatitis A virus (HAV) was compared. Fecal suspensions of virus were heated to temperatures between 45°C and 70°C, and residual infectivity was determined in a cell culture system that was permissive for both viruses. Although HEV was less stable than was HAV, some HEV would most likely survive the internal temperatures of rare-cooked meat

Hepatitis E virus (HEV) consists of a 7.2-kb positive-sense RNA genome packaged within a nonenveloped capsid. There are 4 genotypes currently recognized as pathogens of humans. The virus is frequently spread via contaminated water supplies and is responsible for epidemic and sporadic cases of acute hepatitis in much of Asia, Africa, and the Middle East; it causes infrequent sporadic hepatitis in most of the industrialized world

As early as 1990, Mikhail Balayan and his colleagues suggested that hepatitis E might be a zoonosis, but it was not until 2003 that direct transmission of HEV from animals to humans was demonstrated. The most compelling evidence for zoonotic spread was obtained from clusters of cases in Japan that were traced to eating a shared meal of raw Sika deer meat [1, 2] or undercooked pork liver [3, 4]. Although antibodies that react with the capsid protein of human strains of HEV have been discovered in numerous species of mammals and in chickens, to date, an HEV-like virus has been isolated only from domestic swine [5], wild boar [2], Sika deer [2], and chickens [6]. The virus infecting chickens shares only 50% amino acid homology with human strains and is unlikely to infect humans, since it was not experimentally transmissible to rhesus macaques [7]. In contrast, viral sequences recovered from Sika deer meat were identical to those recovered from patients who developed hepatitis E after eating a portion of the same deer meat, which suggests that the virus was almost certainly transmitted from the meat to the humans [1, 2]. An HEV genotype 3 strain isolated from swine in the United States was shown to cross species barriers and infect primates after experimental inoculation [8]

Since antibodies indicating previous infection with HEV are present in large numbers of swine around the world as well as in other common food sources, including cattle, sheep, and goats [9], it is prudent to determine how resistant HEV is to heat inactivation, to prevent foodborne hepatitis E. The literature contains a single report of HEV (isolated in Guangzhou, China) being inactivated by heating at 56°C for 30 min, but a time course or range of temperatures tested was not reported, and the results have not been confirmed [10]. In the present study, we compared the thermal stability of wild-type HEV and wild-type hepatitis A virus (HAV) present in suspensions of human feces obtained from patients with hepatitis E and hepatitis A, respectively

Materials and methodsAll virus stocks consisted of fecal suspensions in PBS (pH 7.4), and all except Mex 14 also had 10% monkey or calf serum. The Akluj and Sar55 strains (genotype 1) of HEV were isolated from patients with hepatitis in India and Pakistan, respectively; they both had a 50% monkey infectious dose (MID50) of 106.5/0.5 mL of 10% fecal suspension. The Mex 14 (genotype 2) stock was obtained from a rhesus macaque experimentally infected with virus provided by the Centers for Disease Control and Prevention; its MID50 was unknown. The HM175 strain of HAV was isolated from a patient in Australia; it had an MID50 of 106/mL. All fecal suspensions were stored frozen at ⩽−80°C

For heat inactivation, virus stocks were diluted with PBS (pH 7.4), and 100-μL aliquots were distributed to 1.5-mL conical Sarstadt tubes. In all cases, the final serum concentration was ⩽5%. Duplicate samples were stored on ice before and after 1 h of heating at the indicated temperature in a circulating water bath. Before plating the virus, the tubes were randomly coded by a technician not otherwise involved in the study

For the infectivity assay, HepG2/C3A cells (CRL-10741) obtained from the American Type Culture Collection were grown in MEM with Hanks’ salts, 1.5 g/L sodium bicarbonate, 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and 10% heat-inactivated ultralow-IgG calf serum (Invitrogen), in a CO2 incubator at 37°C. Confluent cells were trypsinized and diluted 1:8 in medium, and 0.1 mL was carefully added to 0.4 mL of medium already in a well of a Nunc Lab Tek 11 CC2 8-well chamber slide (Nalge Nunc International). Slides were incubated overnight at 37°C, to provide a sparse population of attached cells. The medium was aspirated and replaced with 100 μL of virus suspension or PBS. Slides were incubated at 34.5°C for 1 h, and the supernatant was aspirated and replaced with 0.4 mL of culture medium supplemented with 2% dimethyl sulfoxide, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.1 mg/mL gentamicin. Cells were cultured at 34.5°C in 5% CO2 for 5–6 days. Slides were rinsed with PBS, and cells were fixed by immersing the slides in acetone at room temperature. Convalescent-phase serum collected from a chimpanzee infected with either HEV or HAV was diluted with 5% bovine serum albumin in PBS and incubated with air-dried fixed cells at room temperature for 20 min. Slides were rinsed with PBS and flooded with a solution of Alexa Fluor 488–conjugated goat anti–human IgG (Molecular Probes). After 20 min at room temperature, the cells were washed with PBS and covered with Vectashield (Vector Laboratories). Stained cells were counted using a Zeiss fluorescent photomicroscope and a fluorescein isothiocyanate filter set by manually scanning the entire well at 25× magnification and tabulating the number of fluorescent cells. Examples of stained cells are shown in figure 1. Pairs of stained cells that appeared to have just divided were counted as 1 cell. The code was broken only after all samples were counted. Coded controls, in which PBS alone was used as the inoculum, were scored as having, at most, 1 positive cell/well

Figure 1

Examples of HepG2/C3A cells infected with the Akluj strain of hepatitis E virus (HEV) or the HM175 strain of hepatitis A virus (HAV). Cell monolayers were stained with convalescent-phase serum collected from a chimpanzee infected with either HEV or HAV. Arrows identify cells scored as positive. Negative cells occupy most of each field and display nonspecific nuclear staining

Figure 1

Examples of HepG2/C3A cells infected with the Akluj strain of hepatitis E virus (HEV) or the HM175 strain of hepatitis A virus (HAV). Cell monolayers were stained with convalescent-phase serum collected from a chimpanzee infected with either HEV or HAV. Arrows identify cells scored as positive. Negative cells occupy most of each field and display nonspecific nuclear staining

ResultsDuplicate sets of the Akluj strain of HEV and the HM175 strain of HAV were incubated in parallel at the indicated temperatures before infection of HepG2/C3A cells. The number of virus-positive cells was determined by immunofluorescence microscopy. HAV, which was previously reported to be relatively stable at 60°C [11], was only 50% inactivated by incubation at 60°C for 1 h (figure 2). HAV was, however, almost totally inactivated by incubation at 66°C, a result consistent with a report that 50% of HAV particles disintegrate at 61°C and release their RNA [11]. The Akluj strain of HEV was much more thermally labile than was HAV under identical conditions; the 50% inactivation temperature was between 45°C and 50°C, and almost all of the virus was inactivated at 56°C (figure 2)

Figure 2

Effect of exposure to different temperatures on the infectivity of hepatitis E virus and hepatitis A virus (HAV). Duplicate samples were incubated at the indicated temperatures for 1 h and then inoculated onto HepG2/C3A cells. Infected cells were identified by immunofluorescence microscopy and quantified. Bars indicate the mean number of positive cells in the duplicate samples; asterisks indicate individual results

Figure 2

Effect of exposure to different temperatures on the infectivity of hepatitis E virus and hepatitis A virus (HAV). Duplicate samples were incubated at the indicated temperatures for 1 h and then inoculated onto HepG2/C3A cells. Infected cells were identified by immunofluorescence microscopy and quantified. Bars indicate the mean number of positive cells in the duplicate samples; asterisks indicate individual results

Another genotype 1 HEV strain, Sar55, and a genotype 2 strain, Mex 14, were also tested for thermal stability. The infectivity titer of the Mex 14 stock was apparently low, since a maximum of 47 cells were infected in any 1 well (56°C sample). This strain of virus appeared to be more stable than the Akluj strain and was not inactivated at 56°C, but ∼80% of the virus was inactivated at 60°C (figure 2). The Sar55 strain had a much higher infectivity titer, and as many as 2416 cells were scored as positive after incubation of the virus at 50°C. It appeared to be more thermally labile than the Mex 14 strain, since almost 50% of the virus was inactivated by incubation at 56°C and 96% was inactivated by incubation at 60°C (figure 2). However, it was slightly more resistant to heating than was the Akluj strain, also a genotype 1 strain

To determine the rate of inactivation, the Akluj strain of virus was heated at 56°C for 0, 15, 30, or 60 min before inoculation onto the cells. The mean numbers of HEV-positive cells on day 6 were 204, 11, 3, and 3, respectively. Therefore, inactivation was rapid at this temperature, since, by 15 min, 95% of the virus was already inactivated. However, ∼1% of the virus was still infectious even after heating for 60 min

DiscussionAlthough this test was cumbersome, it was possible to generate meaningful data by performing the assay in duplicate and under code. In these experiments, the virus was diluted in PBS without the addition of stabilizing factors, such as high concentrations of protein or magnesium. Therefore, the temperature that would be required to inactivate virus embedded in a slab of meat is expected to be higher than that determined here

Although there were differences in thermal stability among the 3 HEV strains tested, both the Akluj and Sar55 viruses were almost totally inactivated at 60°C, and the Mex 14 virus was only moderately more resistant. Sixty degrees Celsius is 140°F, a temperature higher than the 135°F internal temperature of a rare steak. In contrast, 50% of the HAV particles tolerated temperatures 5–10°C higher than those tolerated by HEV. However, since HAV infects only primates, it does not pose the same zoonotic threat that HEV does

The HEV samples used in these studies had been collected >10 years ago and were thawed and refrozen more than once, so the virus appears to be relatively stable. We currently have no explanation as to why the Sar55 and Mex 14 (or HAV) samples held on ice (4°C) were less infectious than the samples heated to 50°C or 56°C and then replaced on ice. This phenomenon has not been observed for the Akluj strain

Evidence has been presented suggesting that HEV can replicate in extrahepatic sites in swine [12]. Since HEV has been transmitted by eating naturally infected liver, and since we have shown that the virus is moderately resistant to heat inactivation, it will be important to determine whether the virus is normally harbored in significant concentrations in other tissues or organs commonly used for food and whether the viruses that induce HEV-reactive antibodies in cattle, sheep, and other food sources, can, like the swine virus, be transmitted to humans

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Presented in part: Symposium on Hepatitis E Virus, New Delhi, India, 18 February 2005
Potential conflicts of interest: none reported
Financial support: National Institutes of Health intramural funds appropriated by Congress