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Stability of Hepatitis E Virus After Drying on Different Surfaces

Stability of Hepatitis E Virus After Drying on Different Surfaces The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. The zoonotic HEV genotype 3 is mainly transmit- ted by consumption of contaminated food produced from infected animals. However, transmission via contaminated surfaces has also to be considered. Here, the genotype 3c strain 47832c was dried on steel, wood, plastics and ceramics, stored at 23 °C or 3 °C for up to 8 weeks and remaining infectivity was titrated on cell culture. During the drying process, only a mean 0.2 log decrease of HEV infectivity was observed. At 23 °C, remaining infectious virus was detected until week 4 on most surfaces, but HEV was completely inactivated (> 4 log decrease) after 8 weeks. At 3 °C, HEV was detectable up to 8 weeks on most surfaces, with an average 2.3 log decrease. HEV showed the highest stability on plastics, which was lower on ceramics and steel, and lowest on wood. The addition of bovine serum albumin mimicking high protein load had only a slight stabilizing effect. In conclusion, HEV shows a high stability against drying and subsequent storage on differ - ent surfaces. Strict application of hygienic measures during food production is therefore crucial in order to prevent HEV persistence on surfaces and subsequent cross-contamination. Keywords Hepatitis E virus · Drying · Stability · Inactivation · Surfaces Introduction and quasi-enveloped particles with a diameter of ~ 40 nm (Nagashima et al., 2017). Whereas non-enveloped particles The hepatitis E virus (HEV) is an important agent of human are mainly found in feces, quasi-enveloped particles exist in hepatitis. Large outbreaks of hepatitis E were reported from serum and cell culture supernatant (Wolff et al., 2020a; Yin developing countries, whereas sporadic cases are predomi- et al., 2016a). Both particle forms are infectious in cell cul- nant in industrialized countries (Goel & Aggarwal, 2020; ture, with a higher infectivity of the non-enveloped particles Webb & Dalton, 2019). In the last years, increasing numbers compared to the enveloped particles (Capelli et al., 2020; of hepatitis E cases have been notified in Europe (Aspinall Yin et al., 2016b). et al., 2017). The disease is mainly characterized by acute Most of the human-pathogenic HEV strains are grouped hepatitis. However, chronic HEV infections, which can lead into genotypes HEV-1 to HEV-4. HEV-1 and HEV-2 exclu- to life-threatening liver cirrhosis, are increasingly described sively infect humans and their major route of transmission in immunosuppressed transplant patients (Narayanan et al., is via fecally contaminated drinking water (Pallerla et al., 2019). In addition, extrahepatic manifestations like neuro- 2020). In contrast, HEV-3 and HEV-4 are zoonotic and logic disorders have been attributed to HEV infection (Vela- circulate in reservoir animals such as wild boars and pigs van et al., 2021). (Pavio et al., 2017). The main transmission route of these HEV belongs to the family Hepeviridae, which is char- genotypes is foodborne by consumption of undercooked acterized by small single-stranded RNA viruses (Purdy meat or raw meat products from infected animals. Especially, et  al., 2017). Two different particle forms are known for raw liver and sausages containing raw liver have been linked HEV: non-enveloped particles with a diameter of ~ 30 nm to hepatitis E outbreaks in the past (Colson et al., 2010; Mas- uda et al., 2005; Matsuda et al., 2003). Furthermore, RNA of HEV was frequently detected in different meat products * Reimar Johne derived from domestic pigs, wild boars or deer (Pavio et al., Reimar.Johne@bfr.bund.de 2017; Szabo et al., 2015). German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany Vol:.(1234567890) 1 3 Food and Environmental Virology (2022) 14:138–148 139 The distinct stability of HEV in meat and meat products harvest, cells were subjected to a triple freeze/thaw cycle and is mainly unknown because no reliable method for direct the supernatant was collected and stored at − 20 °C until fur- infectivity measurement of HEV in these matrices exists so ther use. Thereafter, the harvested supernatants were pooled, far (Cook et al., 2017). Cell culture systems assessing the cell debris were removed by low-speed centrifugation and infectivity of HEV in solutions have been improved recently virus particles were concentrated via ultracentrifugation (Capelli et al., 2020; Meister et al., 2019; Schemmerer et al., (Wolff et al., 2020a). The resulting pellets were redispersed 2016, 2019; Todt et al., 2020), but are still laborious and in phosphate-buffered saline (PBS, PAN-Biotech GmbH, time-consuming compared to many other viruses. Recent Germany) with 1/10 or 1/100 volume (depending on initial stability studies using the cell culture-adapted HEV strain virus concentration) as compared to the original supernatant 47832c in phosphate-buffered saline (PBS) showed a very volume. After an additional cleaning step by centrifugation, high resistance of HEV against a broad pH range and high the virus concentrates were combined, mixed, aliquoted and salt concentrations (Wolff et al., 2020a, 2020b). Conditions stored at − 20 °C. The resulting HEV stock dispersion had prevailing in raw sausage production could not completely an infectivity of 2.8 × 10 focus-forming units (ffu)/ml. inactivate HEV, indicating that infectious virus can be expected in raw sausages if sufficiently contaminated start- Virus Stability Testing After Drying on Surfaces ing material was used (Wolff et al., 2020a, 2020b). Another scenario of food contamination could involve cross-contam- A stock solution of bovine serum albumin (BSA), was pre- ination via surfaces used during food production and prepa- pared by dissolving 1.65 g BSA (Cell Signaling Technol- ration. However, no data on stability of HEV after drying ogy, USA) in sterile PBS up to a total volume of 50 ml. The on surfaces are available so far, making an assessment of BSA solution was thereafter sterile filtrated (PES membrane, the risk of HEV transmission through this pathway difficult. 0.22 µm, Merck Millipore Ltd., USA), aliquoted and stored In this study, the stability of HEV after drying on differ - at − 20 °C. To prepare an HEV stock preparation with BSA, ent surfaces was assessed, under conditions simulating those 4 ml of the HEV stock dispersion was mixed with 0.4 ml of at food production and storage. Using a cell culture-based the BSA stock solution, leading to a final BSA concentration system for titration of HEV infectivity, the decrease of infec- of 3 g/l, as suggested for virus inactivation studies on non- tivity was analyzed directly after drying and after storage porous surfaces (Rabenau et al., 2012). To prepare a virus at 23 °C or 3 °C for up to 8 weeks. The results should help stock preparation without BSA, 4 ml of the HEV stock dis- to estimate the risk of HEV transmission by cross-contam- persion was mixed with 0.4 ml sterile PBS. The resulting ination during food production and through contaminated virus titer of the preparations (with and without BSA) was surfaces in general. 2.5 × 10 ffu/ml. Four different surface types were selected based on their common use in food production and during food preparation: Materials and Methods steel (a common surface material in slaughter houses, cutting plants or groceries), wood and plastics (common material of Virus and Cells cutting boards), and ceramics (commonly used for dishes). For contamination experiments, the sample carriers con- For all experiments, the HEV genotype 3c strain 47832c sisting of circular steel plates (stainless steel X2CrNi18-9, (GenBank acc. no. KC618403) was used. This strain was surface 2B, YC INOX CO., LTD., Taiwan), wood boards originally isolated from a serum sample of a chronically (European beech, Continenta GmbH, Germany), plastics infected transplant patient (Johne et al., 2014). For produc- boards (polyethylene, IKEA, Sweden) and spot plates of tion of a virus stock, an A549 cell line persistently infected ceramics (glazed porcelain, Roth, Germany) were placed with this virus strain (Johne et al., 2014, 2016) was cul- under a sterile workbench. Thereafter, 275 µl aliquots of tivated. For infectivity titrations, the cell line A549/D3, a the corresponding virus stock with or without BSA were subclone of cell line A549 showing enhanced susceptibility placed on marked sites of the sample carrier of the corre- to HEV strain 47832c (Johne et al., 2016; Schemmerer et al., sponding material. Drying was done at room temperature. 2016), was used. After 1 h, which resulted in a 2- to 3-fold volume reduction without complete drying, two aliquots were aspirated from Preparation of Virus Stock each surface material with or without BSA using a pipette, put into a 1.5 ml tube and filled up to 0.5 ml with sterile Virus stock preparation was performed as described (Wolff PBS. Virus left on the surface was then picked up with a et al., 2020a). Briefly, the persistently HEV-infected A549 sterile PBS-moistened cotton swab (Boettger GmbH & Co. cell line was cultivated in a humidified incubator for 7 days KG, Germany), which was placed into the tube containing and thereafter split 1:2 for culture expansion. For virus the corresponding aspirated virus aliquots (sample “before 1 3 140 Food and Environmental Virology (2022) 14:138–148 drying”). Each tube was vortexed for 1 min, and the cotton show a normal distribution, the Kruskal–Wallis test was swab was squeezed and removed from the tube. All tubes used to analyze the general differences between all condi- were centrifuged at 2000×g for 10 min at 4 °C and 450 µl of tions for each experiment. In case of a significant result a each supernatant was transferred into a new tube and stored pairwise comparison of the conditions was performed using at 4 °C until virus titration, which was performed at the the Wilcoxon test for unpaired samples, to identify respec- same day. When the drying process was complete, the dried tive significantly different pairs. For all statistical tests aliquots from each surface material were picked up with performed, a p value < 0.05 was defined as significant. All sterile PBS-moistened cotton swabs, placed into 1.5 ml tubes statistical tests were conducted with the software R 3.6.1 containing 0.5 ml sterile PBS and processed as described (R Core Team, 2020). For the descriptive analyses and the above (sample “after drying”, t ). For storage experiments, plots, the software MS Excel was used. the sample carriers were removed from the bench after complete drying and immediately placed into a plastic box together with a data logger (OM-24, OMEGA, USA) record- ing temperature and relative humidity (RH) every hour. For Results the 23 °C experiment, the closed box was stored in the dark at room temperature. For the 3 °C experiment, two open Eec ff t of Drying on HEV Infectivity water bowls (diameter: 20 cm) filled with aqua bidest. were placed within the box beside the sample carriers in order to The effect of drying on HEV infectivity was analyzed using maintain high humidity. After adding the data logger, the four different surface materials and the presence or absence box was closed and stored in a dark cooling room. Samples of BSA mimicking high protein load. HEV aliquots were were taken by swabbing as described above at 1 day, 1 week, added to the surface materials and the infectivity before 4 weeks and 8 weeks after contamination. All experiments and immediately after drying was assessed. The mean tem- were performed with two biological replicates. perature during the drying process was 22 °C. As shown in Fig. 1, only minor differences in the mean HEV infectivity Titration of HEV Infectivity before and after drying are evident. In line with this, sta- tistical analyses indicated no significant differences within HEV infectivity titrations were performed as described all analyzed condition pairs. However, direct comparison (Johne et al., 2016). Briefly, confluent A549/D3 cell layers of mean values might indicate a general trend of slightly were infected with tenfold dilution series of the samples in decreased infectivity after drying. a 96-well plate format. Each of the two biological replicates In detail, the HEV mean infectivity titers decreased after derived from a specific experimental condition (see above) drying for almost all conditions tested, in the range from was titrated in four technical replicates, resulting in eight 0.1 log ffu/ml for plastics with BSA to 0.7 log ffu/ml for 10 10 titrated subsamples for each experimental condition. After wood without BSA. Only for steel with BSA, an increase infection, the cells were incubated for 2 weeks and subse- in mean infectivity of 0.5 log ffu/ml was determined after quently stained by immunofluorescence with an HEV capsid drying; however, with overlapping standard deviations. The protein-specific rabbit antiserum and a fluorescein isothio- calculated arithmetic mean of the decrease after drying with cyanate (FITC)-conjugated secondary antibody. Fluorescent and without BSA on all surfaces was 0.2 log ffu/ml. cell foci were manually counted using a confocal fluores - By comparing the differences with and without BSA for cence microscope (Opera Phenix, PerkinElmer). A focus each material, a general trend of slightly decreased infectiv- was defined as at least 2 contiguous cells showing a clear ity without BSA as compared to the condition with BSA is intraplasmatic fluorescence. The resulting foci numbers were likely. These mean differences range from 0.3 log ffu/ml multiplied by the dilution factors of the corresponding wells, for plastics and ceramics to 0.4 log ffu/ml for wood. Steel in order to calculate ffu/ml, which were log -transformed. showed a mean difference of 0.6 log ffu/ml, including the 10 10 Arithmetic means from the eight titrated subsamples of each mentioned increase of infectivity after drying with BSA. distinct experimental condition and the respective standard By comparing different materials in case of the absence deviations were calculated using MS Excel. of BSA, steel showed the smallest inactivation effect with 0.1 log ffu/ml, followed by plastics with 0.3 log ffu/ml 10 10 Data Analysis and ceramics with 0.6 log ffu/ml, whereas wood showed the strongest inactivation effect with 0.7 log ffu/ml. In the All three experiments were analyzed according to the differ - presence of BSA, steel showed the least inactivation effect of ences between the conditions. As a prerequisite, all experi- HEV with − 0.5 log ffu/ml, followed by plastics with 0.1 mental results were tested for normal distribution using the log ffu/ml, and the strongest inactivation effect was found Shapiro–Wilk test and q–q plots. As most results did not by ceramics and wood with 0.3 log ffu/ml. 1 3 Food and Environmental Virology (2022) 14:138–148 141 Fig. 1 Stability of HEV against drying at 22 °C on different surfaces. The paired columns show infectivity before (gray) and after (white) drying. Each pair of columns represents a specific surface material in the presence or absence of bovine serum albumin (BSA) as load- ing substance. The arithmetic mean of two replicates, which were titrated in 4 replicates each, is shown. Scaled in log focus-forming units (ffu)/ml. Error bars indicate the standard deviations ceramics (4.0 log ffu/ml decrease) and plastics (3.9 log Long‑Term Stability of Dried HEV on Different 10 10 ffu/ml decrease), whereas no infectivity could be detected Surfaces at 23 °C on steel at this time-point. In order to investigate the long-term stability of dried HEV Long‑Term Stability of Dried HEV on Different at ambient conditions usually present in groceries or kitch- Surfaces at 3 °C ens, a storage experiment for up to 8 weeks at room tem- perature and low RH was performed. Briey fl , HEV with and In order to investigate the long-term stability of dried HEV without BSA was dried on four different surface materials at low temperature conditions usually present in refrigerators and stored in a plastic box in the dark. The recorded data and cooling facilities, a storage experiment for 8 weeks in a for temperature and RH are shown in Supplementary Data cooling room at high RH was performed. Briefly, HEV with S1, indicating for the whole experiment arithmetic means and without BSA was dried on four different surface materi- of 23 °C and 26% RH. The results generated without BSA als and stored together with open water reservoirs in a plastic (Fig.  2A) and with BSA (Fig.  2B) indicate a continuous box placed in a cooling room in the dark. The recorded data decline of infectivity, with minor differences between the for temperature and RH are shown in Supplementary Data surface materials. S2, indicating for the whole experiment arithmetic means of Whereas the mean values at t (immediately after drying) 3 °C and 98% RH. After 40 days, the box with the samples and after one day storage were nearly identical, an almost had to be transferred to another cooling room, which resulted linear decrease of mean infectivity was evident from 1 day in a slight temperature increase of about 3 °C (Supplemen- to 4 weeks storage for all materials without BSA (Fig. 2A). tary Data S2). The results generated without BSA (Fig. 3A) The mean infectivity on wood declined to no infectivity and with BSA (Fig. 3B) indicate a trend of a lower decline (3.8 log ffu/ml decrease) after 4 weeks, whereas residual of infectivity as compared to the storage at 23 °C, with more infectivity was detected at this time-point on the other sur- obvious differences between the surface materials. faces, with mean infectivity decreases of 3.1 log ffu/ml The data from the experiment without BSA (Fig.  3A) for ceramics, 3.0 log ffu/ml for plastics and 3.2 log ffu/ 10 10 indicated similar mean infectivity values for t and one day ml for steel. After 8 weeks, no residual infectivity could be of storage. Whereas for plastics only a slight mean decrease detected for all materials. of about 1.0 log ffu/ml was observed within 4 weeks, the The diagram in Fig. 2B, which shows the results for the values for the other materials dropped more pronounced: experiment with BSA addition, is largely similar to that in for ceramics (2.6 log ffu/ml decrease), steel (3.1 log ffu/ Fig. 2A. Again, no residual infectivity was detected on all 10 10 ml decrease) and wood (3.5 log ffu/ml decrease). Between surfaces after 8 weeks. After 4 weeks, residual infectivi- weeks 4 and 8, the mean decrease of infectivity for plastics ties were determined on wood (2.8 log ffu/ml decrease), 1 3 142 Food and Environmental Virology (2022) 14:138–148 Fig. 2 Time-course analysis of HEV infectivity after drying on different surface materi- als at 23 °C and 26% relative humidity (RH) for 8 weeks A without and B with adding of bovine serum albumin (BSA) as loading substance. Each data point represents the mean HEV infectivity on a specific surface material at the indicated time- point of storage. The arithmetic mean of two replicates, which were titrated in 4 replicates each, is shown. Scaled in log focus-forming units (ffu)/ml. Error bars indicate the standard deviations was minimal with 0.2 log ffu/ml, whereas no residual especially at 4 weeks of storage. By comparing the inacti- infectivity (> 3.5 log ffu/ml decrease) could be detected vation curves shown in Fig. 3A, HEV shows a trend of the after 8 weeks storage for steel and wood. Ceramics showed highest stability on plastics, followed by ceramics and steel, a mean increase of infectivity of 1.2 log ffu/ml between whereas HEV stability was lowest on wood. 4 and 8  weeks; however, with large standard deviations 1 3 Food and Environmental Virology (2022) 14:138–148 143 Fig. 3 Time-course analysis of HEV infectivity after drying on different surface materi- als at 3 °C and 98% relative humidity (RH) for 8 weeks A without and B with adding of bovine serum albumin (BSA) as loading substance. Each data point represents the mean HEV infectivity on a specific surface material at the indicated time- point of storage. The arithmetic mean of two replicates, which were titrated in 4 replicates each, is shown. Scaled in log focus-forming units (ffu)/ml. Error bars indicate the standard deviations 1 3 144 Food and Environmental Virology (2022) 14:138–148 By analyzing the data for the conditions with added BSA a low effect of adding BSA. When comparing the surface (Fig. 3B), no differences of mean infectivity between t and materials, wood showed the highest number of significant one day of storage was observed for plastics and ceramics, differences between t and the other time-points, followed by whereas a mean decrease of infectivity of about 0.5 log steel and ceramics, whereas plastics showed the lowest num- ffu/ml was detected for steel and wood. During storage ber. This indicates that HEV inactivation is fastest on wood, for 8 weeks, HEV infectivity on plastics showed a mean followed by steel and ceramics, and slowest on plastics. decrease of only 0.4 log ffu/ml. At the same time, the mean HEV infectivity decreased on ceramics by 1.6 log ffu/ml, and on steel by 2.4 log ffu/ml. The largest mean Discussion decrease of infectivity showed HEV on wood with 3.9 log ffu/ml after 8 weeks of storage. The HEV inactivation curves Knowledge about the stability of viruses under differ- shown in Fig. 3B indicate the trend that HEV was most sta- ent environmental conditions is essential to uncover their ble on plastics, followed by ceramics and steel, and had the transmission pathways, to develop concepts for prevention lowest stability on wood, under these conditions. of virus transmission and to establish effective methods for virus inactivation. Although the assessment of HEV stability Statistical Analyses is still hampered by the lack of rapid and easy-to-perform methods for HEV infectivity determination, significant pro- The result of statistical analysis for the first experiment gress has been made in the evaluation of the stability of HEV (effect of drying) is described in chapter 3.1., indicating no against various physico-chemical treatments recently. Gener- significant differences between the analyzed conditions. The ally, a high stability of HEV against pH, salts, chlorine, UV p values calculated from the storage experiments are sum- light and high hydrostatic pressure was assessed in previ- marized in Table 1. ous studies (Girones et al., 2014; Guerrero-Latorre et al., As evident from Table 1, the frequency of significant dif- 2016; Imagawa et al., 2018; Johne et al., 2021; Wolff et al., ferences between t and the other time-points (bold p val- 2020a, 2020b), which is in line with the major transmission ues) increases with longer time intervals, indicating a time- pathways of HEV through contaminated food and water. In dependent inactivation of HEV. In addition, more significant contrast, the stability of HEV after drying on surfaces is differences were found at 23 °C as compared to 3 °C, indicat- not known so far. However, contaminated surfaces may be ing faster inactivation at higher temperature. Only minor dif- involved in HEV transmission, e.g., by cross-contamination ferences between the numbers of significant values derived of food or by direct contact resulting in smear infections. with or without adding BSA are evident, indicating only Table 1 Statistical analyses Conditions t vs. 1 day t vs. 1 week t vs. 4 week t vs. 8 week 0 0 0 0 of data from the experiments investigating storage of dried Plastics, without BSA, 23 °C 0.183 0.570 0.009 0.002 HEV on different surfaces Ceramics, without BSA, 23 °C 0.346 0.037 0.004 0.002 Steel, without BSA, 23 °C 0.167 0.115 0.003 0.002 Wood, without BSA, 23 °C 0.869 0.003 0.002 0.002 Plastics, with BSA, 23 °C 0.103 0.200 0.002 0.002 Ceramics, with BSA, 23 °C 0.776 0.126 0.002 0.002 Steel, with BSA, 23 °C 0.254 0.007 0.002 0.002 Wood, with BSA, 23 °C 0.223 0.033 0.003 0.002 Plastics, without BSA, 3 °C 0.803 0.296 0.058 0.011 Ceramics, without BSA, 3 °C 0.460 0.115 0.004 0.004 Steel, without BSA, 3 °C 0.734 0.083 0.003 0.002 Wood, without BSA, 3 °C 0.427 0.008 0.002 0.002 Plastics, with BSA, 3 °C 0.803 0.633 0.699 0.306 Ceramics, with BSA, 3 °C 1.000 0.234 0.020 0.003 Steel, with BSA, 3 °C 0.103 0.183 0.004 0.006 Wood, with BSA, 3 °C 0.356 0.059 0.003 0.002 Time point t was compared with all other time-points for all conditions. The calculation methods used are described in detail in chapter 2.5. Results are presented as p values. Bold p values indicate significant dif- ferences (p < 0.05) 1 3 Food and Environmental Virology (2022) 14:138–148 145 To investigate the HEV stability after drying, a cell cul- loading substance, had only a minor stabilizing effect. Other ture system using the cell culture-adapted HEV subtype enterically transmitted viruses like hepatitis A virus (HAV), 3c strain 47832c was used (Johne et  al., 2014). Subtype norovirus, rotavirus or astrovirus have also been shown to 3c represents one of the predominant subtypes detected be highly stable against the drying process (Mahl & Sadler, in humans and animals in Europe (Adlhoch et al., 2016; 1975; Keswick et al., 1983; Sattar et al., 1986; Sobsey et al., Anheyer-Behmenburg et al., 2017). In addition, this cell cul- 1988; Abad et al., 1994; Abad et al., 2001). By comparison ture system was used previously in several studies assessing of HAV, rotavirus, poliovirus and adenovirus during dry- the stability of strain 47832c (Johne et al., 2016, 2021; Wolff ing on different smooth surfaces, HAV showed the high- et al., 2020a, 2020b), enabling direct comparison of the data. est stability (Abad et al., 1994). According to the results of In our study, the cell culture system enabled an initial assess- our study, HEV inactivation during drying showed similar ment of HEV stability after drying on surfaces. However, characteristics as described for HAV. For example, in the as the method is still laborious and time-consuming, only a absence of a loading substance during drying on ceram- limited number of conditions and replicates could be inves- ics, the mean infectivity was reduced by 0.5 log for HAV tigated. Future studies using improved methods are therefore (Abad et al., 1994) and by 0.6 log for HEV. In the presence desirable in order to broaden the conditions to other surface of a loading substance, the mean infectivity during drying on materials and temperatures, and to gain more precise data ceramics decreased by 0.5 log for HAV (Abad et al., 1994) regarding variation of mean errors. and by 0.3 log for HEV. In the tested preparation, both non-enveloped and quasi- The persistence of infectious HEV after drying on dif- enveloped particles are present (Wolff et al., 2020a). It has ferent surfaces and subsequent storage was assessed in our been shown that non-enveloped particles mainly occur in study at two different conditions, which simulated typical feces and quasi-enveloped particles in serum of patients, scenarios during food production and preparation. First, although non-enveloped HEV particles have also been ambient conditions usually present in groceries or kitchens identified in human sera in a recent study (Costafreda et al., (or in hospitals, or generally in rooms) with a temperature 2021). The particle form present in pig liver, meat and meat of 23 °C and a low RH of 26% were tested. Second, low products is unknown so far. However, the presence of a mix- temperature conditions usually present in refrigerators and ture of enveloped particles (budded from cells or originating cooling facilities with a temperature of 3 °C and a high RH from residual serum in the meat) and non-enveloped parti- of 98% were chosen. This approach enabled the testing of cles (released from damaged cells or generated by removal typical scenarios, but the distinct contributions of tempera- of the envelope by environmental factors) is most proba- ture and RH to HEV inactivation could not be differenti- ble. Therefore, particle mixtures similar to that used in our ated and should therefore be analyzed in future experiments. experiments may reflect those occurring in meat in practice, Generally, stability of HEV was lower at the ambient condi- although the distinct combination of particle forms in certain tions compared to the low temperature conditions. Whereas meat products may vary. A higher infectivity of non-envel- at ambient conditions, HEV infectivity was mostly destroyed oped particles as compared to quasi-enveloped particles after 4 weeks and totally absent after 8 weeks, remaining has been described using cell culture studies (Capelli et al., infectious HEV could be detected at 8 weeks in 5/8 sam- 2020; Yin et al., 2016b), and a removal of the envelope due ples at low temperature conditions. Higher stability at 4 °C to environmental factors may therefore increase infectivity. as compared to 20 °C has also been described for HAV, In order to analyze the distinct contribution of inactivating poliovirus and adenovirus after drying on surfaces (Abad and infectivity-increasing processes during drying and stor- et al., 1994), although the effect was less pronounced than age, additional experiments with preparations of separated for HEV. non-enveloped and enveloped particles should be performed Especially at the low temperature condition, a marked in future studies. effect of the distinct surface material was observed in our The resistance of viruses against the drying process rep- study. Here, HEV showed an exceptionally high stability on resents the major factor for their survival in dried condition plastics, with only 0.4 log ffu/ml infectivity decrease after on surfaces (Sánchez & Bosch, 2016). In our experiments, 8 weeks in the experiment with addition of BSA. Ceramics only slight decreases of HEV infectivity have been found and steel showed moderate inactivation rates, and HEV was during the drying process, with no statistical significance almost completely inactivated (3.9 log ffu/ml decrease) between the titers before and after drying. This indicates that on wood under the same conditions. The reasons for the HEV is highly stable against drying, and the persistence of differences are not known, but it could be speculated that infectious virus has to be expected on surfaces after contact the porous surface of wood absorbs water from the virus to contaminated meat or to excretions from infected ani- particles with higher efficiency as compared to non-porous mals or humans. The high stability was evident for all tested surfaces, which may lead to a faster virus inactivation, as surface types and the addition of BSA, which was used as recently hypothesized for SARS-CoV-2 (Corpet, 2021). This 1 3 146 Food and Environmental Virology (2022) 14:138–148 is in line with results from experiments with HAV, showing a more complete risk assessment on HEV transmission via a higher stability on smooth surfaces (aluminium, ceramics) surfaces. as compared to porous surfaces (paper) (Abad et al., 1994). Supplementary Information The online version contains supplemen- Generally, the stability of HEV and HAV at low temperature tary material available at https://doi. or g/10. 1007/ s12560- 022- 09510-7 . and high RH turns out to be rather similar, at least for sur- face materials where data are available for both viruses. For Acknowledgements We want to thank Silke Apelt, Stefanie Prosetzky example, in the presence of a loading substance after drying and Anja Schlosser (BfR, Berlin, Germany) for excellent technical assistance, Chris-Tina Höfer and Norman Violet (BfR, Berlin, Ger- on ceramics for 8 weeks, the mean infectivity was reduced many) for excellent support with the Opera Phenix device and scientific by 1.1 log for HAV (Abad et al., 1994) and by 1.6 log for 10 10 discussions, and Dirk Meyer (BfR, Berlin, Germany) for providing HEV, and on metal by 1.5 log for HAV (aluminium) (Abad storage facilities. et al., 1994) and by 2.4 log for HEV (steel). Our study has some limitations. Because of the necessary Funding Open Access funding enabled and organized by Projekt use of the laborious and time-consuming titration system, DEAL. This research was funded by a Grant of the Federal Food Safety and Veterinary Office of the Swiss Confederation (Project number only a low number of samples could be analyzed, thus limit- 4.18.01). ing the analyzed conditions and replications of the experi- ments. As already mentioned, the distinct influence of tem- Data Availability Data are available in Supplementary Materials and perature and humidity could not be differentiated because additional data can be retrieved upon request by R.J. (Reimar.Johne@ bfr.bund.de). of the specifically selected experimental conditions. Also, a discrimination of the inactivation profile of non-enveloped Open Access This article is licensed under a Creative Commons Attri- vs. quasi-enveloped particles could not be assessed. In addi- bution 4.0 International License, which permits use, sharing, adapta- tion, only one strain has been investigated and other strains tion, distribution and reproduction in any medium or format, as long may show different behaviors. The use of PBS and addition as you give appropriate credit to the original author(s) and the source, of BSA may not completely reflect the complex composition provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are of blood, feces or meat juice, which are suspected to be the included in the article's Creative Commons licence, unless indicated most probable matrices containing HEV in the field. Gener - otherwise in a credit line to the material. If material is not included in ally, the transmission rate of HEV to surfaces and from the the article's Creative Commons licence and your intended use is not surfaces to food and humans has to be determined in the permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a future to better assess the transmission probability under copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . field conditions. It can be concluded that HEV is highly resistant against the process of drying and shows a high stability against long-term storage on several surfaces. The highest stability References was determined at low temperature and high RH resulting Abad, F. X., Pintó, R. M., & Bosch, A. (1994). Survival of enteric in detection of infectious virus for as long as 8 weeks in viruses on environmental fomites. Applied and Environmental most cases. 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Therapeutic Advances in Infectious Disease, 6, 2049936119837162. https://doi. or g/10. 1177/ 20499 36119 837162 Wolff, A., Günther, T., Albert, T., Schilling-Loeffler, K., Gadicherla, A. K., & Johne, R. (2020a). Stability of hepatitis E virus at different 1 3 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Food and Environmental Virology Springer Journals

Stability of Hepatitis E Virus After Drying on Different Surfaces

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Abstract

The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. The zoonotic HEV genotype 3 is mainly transmit- ted by consumption of contaminated food produced from infected animals. However, transmission via contaminated surfaces has also to be considered. Here, the genotype 3c strain 47832c was dried on steel, wood, plastics and ceramics, stored at 23 °C or 3 °C for up to 8 weeks and remaining infectivity was titrated on cell culture. During the drying process, only a mean 0.2 log decrease of HEV infectivity was observed. At 23 °C, remaining infectious virus was detected until week 4 on most surfaces, but HEV was completely inactivated (> 4 log decrease) after 8 weeks. At 3 °C, HEV was detectable up to 8 weeks on most surfaces, with an average 2.3 log decrease. HEV showed the highest stability on plastics, which was lower on ceramics and steel, and lowest on wood. The addition of bovine serum albumin mimicking high protein load had only a slight stabilizing effect. In conclusion, HEV shows a high stability against drying and subsequent storage on differ - ent surfaces. Strict application of hygienic measures during food production is therefore crucial in order to prevent HEV persistence on surfaces and subsequent cross-contamination. Keywords Hepatitis E virus · Drying · Stability · Inactivation · Surfaces Introduction and quasi-enveloped particles with a diameter of ~ 40 nm (Nagashima et al., 2017). Whereas non-enveloped particles The hepatitis E virus (HEV) is an important agent of human are mainly found in feces, quasi-enveloped particles exist in hepatitis. Large outbreaks of hepatitis E were reported from serum and cell culture supernatant (Wolff et al., 2020a; Yin developing countries, whereas sporadic cases are predomi- et al., 2016a). Both particle forms are infectious in cell cul- nant in industrialized countries (Goel & Aggarwal, 2020; ture, with a higher infectivity of the non-enveloped particles Webb & Dalton, 2019). In the last years, increasing numbers compared to the enveloped particles (Capelli et al., 2020; of hepatitis E cases have been notified in Europe (Aspinall Yin et al., 2016b). et al., 2017). The disease is mainly characterized by acute Most of the human-pathogenic HEV strains are grouped hepatitis. However, chronic HEV infections, which can lead into genotypes HEV-1 to HEV-4. HEV-1 and HEV-2 exclu- to life-threatening liver cirrhosis, are increasingly described sively infect humans and their major route of transmission in immunosuppressed transplant patients (Narayanan et al., is via fecally contaminated drinking water (Pallerla et al., 2019). In addition, extrahepatic manifestations like neuro- 2020). In contrast, HEV-3 and HEV-4 are zoonotic and logic disorders have been attributed to HEV infection (Vela- circulate in reservoir animals such as wild boars and pigs van et al., 2021). (Pavio et al., 2017). The main transmission route of these HEV belongs to the family Hepeviridae, which is char- genotypes is foodborne by consumption of undercooked acterized by small single-stranded RNA viruses (Purdy meat or raw meat products from infected animals. Especially, et  al., 2017). Two different particle forms are known for raw liver and sausages containing raw liver have been linked HEV: non-enveloped particles with a diameter of ~ 30 nm to hepatitis E outbreaks in the past (Colson et al., 2010; Mas- uda et al., 2005; Matsuda et al., 2003). Furthermore, RNA of HEV was frequently detected in different meat products * Reimar Johne derived from domestic pigs, wild boars or deer (Pavio et al., Reimar.Johne@bfr.bund.de 2017; Szabo et al., 2015). German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany Vol:.(1234567890) 1 3 Food and Environmental Virology (2022) 14:138–148 139 The distinct stability of HEV in meat and meat products harvest, cells were subjected to a triple freeze/thaw cycle and is mainly unknown because no reliable method for direct the supernatant was collected and stored at − 20 °C until fur- infectivity measurement of HEV in these matrices exists so ther use. Thereafter, the harvested supernatants were pooled, far (Cook et al., 2017). Cell culture systems assessing the cell debris were removed by low-speed centrifugation and infectivity of HEV in solutions have been improved recently virus particles were concentrated via ultracentrifugation (Capelli et al., 2020; Meister et al., 2019; Schemmerer et al., (Wolff et al., 2020a). The resulting pellets were redispersed 2016, 2019; Todt et al., 2020), but are still laborious and in phosphate-buffered saline (PBS, PAN-Biotech GmbH, time-consuming compared to many other viruses. Recent Germany) with 1/10 or 1/100 volume (depending on initial stability studies using the cell culture-adapted HEV strain virus concentration) as compared to the original supernatant 47832c in phosphate-buffered saline (PBS) showed a very volume. After an additional cleaning step by centrifugation, high resistance of HEV against a broad pH range and high the virus concentrates were combined, mixed, aliquoted and salt concentrations (Wolff et al., 2020a, 2020b). Conditions stored at − 20 °C. The resulting HEV stock dispersion had prevailing in raw sausage production could not completely an infectivity of 2.8 × 10 focus-forming units (ffu)/ml. inactivate HEV, indicating that infectious virus can be expected in raw sausages if sufficiently contaminated start- Virus Stability Testing After Drying on Surfaces ing material was used (Wolff et al., 2020a, 2020b). Another scenario of food contamination could involve cross-contam- A stock solution of bovine serum albumin (BSA), was pre- ination via surfaces used during food production and prepa- pared by dissolving 1.65 g BSA (Cell Signaling Technol- ration. However, no data on stability of HEV after drying ogy, USA) in sterile PBS up to a total volume of 50 ml. The on surfaces are available so far, making an assessment of BSA solution was thereafter sterile filtrated (PES membrane, the risk of HEV transmission through this pathway difficult. 0.22 µm, Merck Millipore Ltd., USA), aliquoted and stored In this study, the stability of HEV after drying on differ - at − 20 °C. To prepare an HEV stock preparation with BSA, ent surfaces was assessed, under conditions simulating those 4 ml of the HEV stock dispersion was mixed with 0.4 ml of at food production and storage. Using a cell culture-based the BSA stock solution, leading to a final BSA concentration system for titration of HEV infectivity, the decrease of infec- of 3 g/l, as suggested for virus inactivation studies on non- tivity was analyzed directly after drying and after storage porous surfaces (Rabenau et al., 2012). To prepare a virus at 23 °C or 3 °C for up to 8 weeks. The results should help stock preparation without BSA, 4 ml of the HEV stock dis- to estimate the risk of HEV transmission by cross-contam- persion was mixed with 0.4 ml sterile PBS. The resulting ination during food production and through contaminated virus titer of the preparations (with and without BSA) was surfaces in general. 2.5 × 10 ffu/ml. Four different surface types were selected based on their common use in food production and during food preparation: Materials and Methods steel (a common surface material in slaughter houses, cutting plants or groceries), wood and plastics (common material of Virus and Cells cutting boards), and ceramics (commonly used for dishes). For contamination experiments, the sample carriers con- For all experiments, the HEV genotype 3c strain 47832c sisting of circular steel plates (stainless steel X2CrNi18-9, (GenBank acc. no. KC618403) was used. This strain was surface 2B, YC INOX CO., LTD., Taiwan), wood boards originally isolated from a serum sample of a chronically (European beech, Continenta GmbH, Germany), plastics infected transplant patient (Johne et al., 2014). For produc- boards (polyethylene, IKEA, Sweden) and spot plates of tion of a virus stock, an A549 cell line persistently infected ceramics (glazed porcelain, Roth, Germany) were placed with this virus strain (Johne et al., 2014, 2016) was cul- under a sterile workbench. Thereafter, 275 µl aliquots of tivated. For infectivity titrations, the cell line A549/D3, a the corresponding virus stock with or without BSA were subclone of cell line A549 showing enhanced susceptibility placed on marked sites of the sample carrier of the corre- to HEV strain 47832c (Johne et al., 2016; Schemmerer et al., sponding material. Drying was done at room temperature. 2016), was used. After 1 h, which resulted in a 2- to 3-fold volume reduction without complete drying, two aliquots were aspirated from Preparation of Virus Stock each surface material with or without BSA using a pipette, put into a 1.5 ml tube and filled up to 0.5 ml with sterile Virus stock preparation was performed as described (Wolff PBS. Virus left on the surface was then picked up with a et al., 2020a). Briefly, the persistently HEV-infected A549 sterile PBS-moistened cotton swab (Boettger GmbH & Co. cell line was cultivated in a humidified incubator for 7 days KG, Germany), which was placed into the tube containing and thereafter split 1:2 for culture expansion. For virus the corresponding aspirated virus aliquots (sample “before 1 3 140 Food and Environmental Virology (2022) 14:138–148 drying”). Each tube was vortexed for 1 min, and the cotton show a normal distribution, the Kruskal–Wallis test was swab was squeezed and removed from the tube. All tubes used to analyze the general differences between all condi- were centrifuged at 2000×g for 10 min at 4 °C and 450 µl of tions for each experiment. In case of a significant result a each supernatant was transferred into a new tube and stored pairwise comparison of the conditions was performed using at 4 °C until virus titration, which was performed at the the Wilcoxon test for unpaired samples, to identify respec- same day. When the drying process was complete, the dried tive significantly different pairs. For all statistical tests aliquots from each surface material were picked up with performed, a p value < 0.05 was defined as significant. All sterile PBS-moistened cotton swabs, placed into 1.5 ml tubes statistical tests were conducted with the software R 3.6.1 containing 0.5 ml sterile PBS and processed as described (R Core Team, 2020). For the descriptive analyses and the above (sample “after drying”, t ). For storage experiments, plots, the software MS Excel was used. the sample carriers were removed from the bench after complete drying and immediately placed into a plastic box together with a data logger (OM-24, OMEGA, USA) record- ing temperature and relative humidity (RH) every hour. For Results the 23 °C experiment, the closed box was stored in the dark at room temperature. For the 3 °C experiment, two open Eec ff t of Drying on HEV Infectivity water bowls (diameter: 20 cm) filled with aqua bidest. were placed within the box beside the sample carriers in order to The effect of drying on HEV infectivity was analyzed using maintain high humidity. After adding the data logger, the four different surface materials and the presence or absence box was closed and stored in a dark cooling room. Samples of BSA mimicking high protein load. HEV aliquots were were taken by swabbing as described above at 1 day, 1 week, added to the surface materials and the infectivity before 4 weeks and 8 weeks after contamination. All experiments and immediately after drying was assessed. The mean tem- were performed with two biological replicates. perature during the drying process was 22 °C. As shown in Fig. 1, only minor differences in the mean HEV infectivity Titration of HEV Infectivity before and after drying are evident. In line with this, sta- tistical analyses indicated no significant differences within HEV infectivity titrations were performed as described all analyzed condition pairs. However, direct comparison (Johne et al., 2016). Briefly, confluent A549/D3 cell layers of mean values might indicate a general trend of slightly were infected with tenfold dilution series of the samples in decreased infectivity after drying. a 96-well plate format. Each of the two biological replicates In detail, the HEV mean infectivity titers decreased after derived from a specific experimental condition (see above) drying for almost all conditions tested, in the range from was titrated in four technical replicates, resulting in eight 0.1 log ffu/ml for plastics with BSA to 0.7 log ffu/ml for 10 10 titrated subsamples for each experimental condition. After wood without BSA. Only for steel with BSA, an increase infection, the cells were incubated for 2 weeks and subse- in mean infectivity of 0.5 log ffu/ml was determined after quently stained by immunofluorescence with an HEV capsid drying; however, with overlapping standard deviations. The protein-specific rabbit antiserum and a fluorescein isothio- calculated arithmetic mean of the decrease after drying with cyanate (FITC)-conjugated secondary antibody. Fluorescent and without BSA on all surfaces was 0.2 log ffu/ml. cell foci were manually counted using a confocal fluores - By comparing the differences with and without BSA for cence microscope (Opera Phenix, PerkinElmer). A focus each material, a general trend of slightly decreased infectiv- was defined as at least 2 contiguous cells showing a clear ity without BSA as compared to the condition with BSA is intraplasmatic fluorescence. The resulting foci numbers were likely. These mean differences range from 0.3 log ffu/ml multiplied by the dilution factors of the corresponding wells, for plastics and ceramics to 0.4 log ffu/ml for wood. Steel in order to calculate ffu/ml, which were log -transformed. showed a mean difference of 0.6 log ffu/ml, including the 10 10 Arithmetic means from the eight titrated subsamples of each mentioned increase of infectivity after drying with BSA. distinct experimental condition and the respective standard By comparing different materials in case of the absence deviations were calculated using MS Excel. of BSA, steel showed the smallest inactivation effect with 0.1 log ffu/ml, followed by plastics with 0.3 log ffu/ml 10 10 Data Analysis and ceramics with 0.6 log ffu/ml, whereas wood showed the strongest inactivation effect with 0.7 log ffu/ml. In the All three experiments were analyzed according to the differ - presence of BSA, steel showed the least inactivation effect of ences between the conditions. As a prerequisite, all experi- HEV with − 0.5 log ffu/ml, followed by plastics with 0.1 mental results were tested for normal distribution using the log ffu/ml, and the strongest inactivation effect was found Shapiro–Wilk test and q–q plots. As most results did not by ceramics and wood with 0.3 log ffu/ml. 1 3 Food and Environmental Virology (2022) 14:138–148 141 Fig. 1 Stability of HEV against drying at 22 °C on different surfaces. The paired columns show infectivity before (gray) and after (white) drying. Each pair of columns represents a specific surface material in the presence or absence of bovine serum albumin (BSA) as load- ing substance. The arithmetic mean of two replicates, which were titrated in 4 replicates each, is shown. Scaled in log focus-forming units (ffu)/ml. Error bars indicate the standard deviations ceramics (4.0 log ffu/ml decrease) and plastics (3.9 log Long‑Term Stability of Dried HEV on Different 10 10 ffu/ml decrease), whereas no infectivity could be detected Surfaces at 23 °C on steel at this time-point. In order to investigate the long-term stability of dried HEV Long‑Term Stability of Dried HEV on Different at ambient conditions usually present in groceries or kitch- Surfaces at 3 °C ens, a storage experiment for up to 8 weeks at room tem- perature and low RH was performed. Briey fl , HEV with and In order to investigate the long-term stability of dried HEV without BSA was dried on four different surface materials at low temperature conditions usually present in refrigerators and stored in a plastic box in the dark. The recorded data and cooling facilities, a storage experiment for 8 weeks in a for temperature and RH are shown in Supplementary Data cooling room at high RH was performed. Briefly, HEV with S1, indicating for the whole experiment arithmetic means and without BSA was dried on four different surface materi- of 23 °C and 26% RH. The results generated without BSA als and stored together with open water reservoirs in a plastic (Fig.  2A) and with BSA (Fig.  2B) indicate a continuous box placed in a cooling room in the dark. The recorded data decline of infectivity, with minor differences between the for temperature and RH are shown in Supplementary Data surface materials. S2, indicating for the whole experiment arithmetic means of Whereas the mean values at t (immediately after drying) 3 °C and 98% RH. After 40 days, the box with the samples and after one day storage were nearly identical, an almost had to be transferred to another cooling room, which resulted linear decrease of mean infectivity was evident from 1 day in a slight temperature increase of about 3 °C (Supplemen- to 4 weeks storage for all materials without BSA (Fig. 2A). tary Data S2). The results generated without BSA (Fig. 3A) The mean infectivity on wood declined to no infectivity and with BSA (Fig. 3B) indicate a trend of a lower decline (3.8 log ffu/ml decrease) after 4 weeks, whereas residual of infectivity as compared to the storage at 23 °C, with more infectivity was detected at this time-point on the other sur- obvious differences between the surface materials. faces, with mean infectivity decreases of 3.1 log ffu/ml The data from the experiment without BSA (Fig.  3A) for ceramics, 3.0 log ffu/ml for plastics and 3.2 log ffu/ 10 10 indicated similar mean infectivity values for t and one day ml for steel. After 8 weeks, no residual infectivity could be of storage. Whereas for plastics only a slight mean decrease detected for all materials. of about 1.0 log ffu/ml was observed within 4 weeks, the The diagram in Fig. 2B, which shows the results for the values for the other materials dropped more pronounced: experiment with BSA addition, is largely similar to that in for ceramics (2.6 log ffu/ml decrease), steel (3.1 log ffu/ Fig. 2A. Again, no residual infectivity was detected on all 10 10 ml decrease) and wood (3.5 log ffu/ml decrease). Between surfaces after 8 weeks. After 4 weeks, residual infectivi- weeks 4 and 8, the mean decrease of infectivity for plastics ties were determined on wood (2.8 log ffu/ml decrease), 1 3 142 Food and Environmental Virology (2022) 14:138–148 Fig. 2 Time-course analysis of HEV infectivity after drying on different surface materi- als at 23 °C and 26% relative humidity (RH) for 8 weeks A without and B with adding of bovine serum albumin (BSA) as loading substance. Each data point represents the mean HEV infectivity on a specific surface material at the indicated time- point of storage. The arithmetic mean of two replicates, which were titrated in 4 replicates each, is shown. Scaled in log focus-forming units (ffu)/ml. Error bars indicate the standard deviations was minimal with 0.2 log ffu/ml, whereas no residual especially at 4 weeks of storage. By comparing the inacti- infectivity (> 3.5 log ffu/ml decrease) could be detected vation curves shown in Fig. 3A, HEV shows a trend of the after 8 weeks storage for steel and wood. Ceramics showed highest stability on plastics, followed by ceramics and steel, a mean increase of infectivity of 1.2 log ffu/ml between whereas HEV stability was lowest on wood. 4 and 8  weeks; however, with large standard deviations 1 3 Food and Environmental Virology (2022) 14:138–148 143 Fig. 3 Time-course analysis of HEV infectivity after drying on different surface materi- als at 3 °C and 98% relative humidity (RH) for 8 weeks A without and B with adding of bovine serum albumin (BSA) as loading substance. Each data point represents the mean HEV infectivity on a specific surface material at the indicated time- point of storage. The arithmetic mean of two replicates, which were titrated in 4 replicates each, is shown. Scaled in log focus-forming units (ffu)/ml. Error bars indicate the standard deviations 1 3 144 Food and Environmental Virology (2022) 14:138–148 By analyzing the data for the conditions with added BSA a low effect of adding BSA. When comparing the surface (Fig. 3B), no differences of mean infectivity between t and materials, wood showed the highest number of significant one day of storage was observed for plastics and ceramics, differences between t and the other time-points, followed by whereas a mean decrease of infectivity of about 0.5 log steel and ceramics, whereas plastics showed the lowest num- ffu/ml was detected for steel and wood. During storage ber. This indicates that HEV inactivation is fastest on wood, for 8 weeks, HEV infectivity on plastics showed a mean followed by steel and ceramics, and slowest on plastics. decrease of only 0.4 log ffu/ml. At the same time, the mean HEV infectivity decreased on ceramics by 1.6 log ffu/ml, and on steel by 2.4 log ffu/ml. The largest mean Discussion decrease of infectivity showed HEV on wood with 3.9 log ffu/ml after 8 weeks of storage. The HEV inactivation curves Knowledge about the stability of viruses under differ- shown in Fig. 3B indicate the trend that HEV was most sta- ent environmental conditions is essential to uncover their ble on plastics, followed by ceramics and steel, and had the transmission pathways, to develop concepts for prevention lowest stability on wood, under these conditions. of virus transmission and to establish effective methods for virus inactivation. Although the assessment of HEV stability Statistical Analyses is still hampered by the lack of rapid and easy-to-perform methods for HEV infectivity determination, significant pro- The result of statistical analysis for the first experiment gress has been made in the evaluation of the stability of HEV (effect of drying) is described in chapter 3.1., indicating no against various physico-chemical treatments recently. Gener- significant differences between the analyzed conditions. The ally, a high stability of HEV against pH, salts, chlorine, UV p values calculated from the storage experiments are sum- light and high hydrostatic pressure was assessed in previ- marized in Table 1. ous studies (Girones et al., 2014; Guerrero-Latorre et al., As evident from Table 1, the frequency of significant dif- 2016; Imagawa et al., 2018; Johne et al., 2021; Wolff et al., ferences between t and the other time-points (bold p val- 2020a, 2020b), which is in line with the major transmission ues) increases with longer time intervals, indicating a time- pathways of HEV through contaminated food and water. In dependent inactivation of HEV. In addition, more significant contrast, the stability of HEV after drying on surfaces is differences were found at 23 °C as compared to 3 °C, indicat- not known so far. However, contaminated surfaces may be ing faster inactivation at higher temperature. Only minor dif- involved in HEV transmission, e.g., by cross-contamination ferences between the numbers of significant values derived of food or by direct contact resulting in smear infections. with or without adding BSA are evident, indicating only Table 1 Statistical analyses Conditions t vs. 1 day t vs. 1 week t vs. 4 week t vs. 8 week 0 0 0 0 of data from the experiments investigating storage of dried Plastics, without BSA, 23 °C 0.183 0.570 0.009 0.002 HEV on different surfaces Ceramics, without BSA, 23 °C 0.346 0.037 0.004 0.002 Steel, without BSA, 23 °C 0.167 0.115 0.003 0.002 Wood, without BSA, 23 °C 0.869 0.003 0.002 0.002 Plastics, with BSA, 23 °C 0.103 0.200 0.002 0.002 Ceramics, with BSA, 23 °C 0.776 0.126 0.002 0.002 Steel, with BSA, 23 °C 0.254 0.007 0.002 0.002 Wood, with BSA, 23 °C 0.223 0.033 0.003 0.002 Plastics, without BSA, 3 °C 0.803 0.296 0.058 0.011 Ceramics, without BSA, 3 °C 0.460 0.115 0.004 0.004 Steel, without BSA, 3 °C 0.734 0.083 0.003 0.002 Wood, without BSA, 3 °C 0.427 0.008 0.002 0.002 Plastics, with BSA, 3 °C 0.803 0.633 0.699 0.306 Ceramics, with BSA, 3 °C 1.000 0.234 0.020 0.003 Steel, with BSA, 3 °C 0.103 0.183 0.004 0.006 Wood, with BSA, 3 °C 0.356 0.059 0.003 0.002 Time point t was compared with all other time-points for all conditions. The calculation methods used are described in detail in chapter 2.5. Results are presented as p values. Bold p values indicate significant dif- ferences (p < 0.05) 1 3 Food and Environmental Virology (2022) 14:138–148 145 To investigate the HEV stability after drying, a cell cul- loading substance, had only a minor stabilizing effect. Other ture system using the cell culture-adapted HEV subtype enterically transmitted viruses like hepatitis A virus (HAV), 3c strain 47832c was used (Johne et  al., 2014). Subtype norovirus, rotavirus or astrovirus have also been shown to 3c represents one of the predominant subtypes detected be highly stable against the drying process (Mahl & Sadler, in humans and animals in Europe (Adlhoch et al., 2016; 1975; Keswick et al., 1983; Sattar et al., 1986; Sobsey et al., Anheyer-Behmenburg et al., 2017). In addition, this cell cul- 1988; Abad et al., 1994; Abad et al., 2001). By comparison ture system was used previously in several studies assessing of HAV, rotavirus, poliovirus and adenovirus during dry- the stability of strain 47832c (Johne et al., 2016, 2021; Wolff ing on different smooth surfaces, HAV showed the high- et al., 2020a, 2020b), enabling direct comparison of the data. est stability (Abad et al., 1994). According to the results of In our study, the cell culture system enabled an initial assess- our study, HEV inactivation during drying showed similar ment of HEV stability after drying on surfaces. However, characteristics as described for HAV. For example, in the as the method is still laborious and time-consuming, only a absence of a loading substance during drying on ceram- limited number of conditions and replicates could be inves- ics, the mean infectivity was reduced by 0.5 log for HAV tigated. Future studies using improved methods are therefore (Abad et al., 1994) and by 0.6 log for HEV. In the presence desirable in order to broaden the conditions to other surface of a loading substance, the mean infectivity during drying on materials and temperatures, and to gain more precise data ceramics decreased by 0.5 log for HAV (Abad et al., 1994) regarding variation of mean errors. and by 0.3 log for HEV. In the tested preparation, both non-enveloped and quasi- The persistence of infectious HEV after drying on dif- enveloped particles are present (Wolff et al., 2020a). It has ferent surfaces and subsequent storage was assessed in our been shown that non-enveloped particles mainly occur in study at two different conditions, which simulated typical feces and quasi-enveloped particles in serum of patients, scenarios during food production and preparation. First, although non-enveloped HEV particles have also been ambient conditions usually present in groceries or kitchens identified in human sera in a recent study (Costafreda et al., (or in hospitals, or generally in rooms) with a temperature 2021). The particle form present in pig liver, meat and meat of 23 °C and a low RH of 26% were tested. Second, low products is unknown so far. However, the presence of a mix- temperature conditions usually present in refrigerators and ture of enveloped particles (budded from cells or originating cooling facilities with a temperature of 3 °C and a high RH from residual serum in the meat) and non-enveloped parti- of 98% were chosen. This approach enabled the testing of cles (released from damaged cells or generated by removal typical scenarios, but the distinct contributions of tempera- of the envelope by environmental factors) is most proba- ture and RH to HEV inactivation could not be differenti- ble. Therefore, particle mixtures similar to that used in our ated and should therefore be analyzed in future experiments. experiments may reflect those occurring in meat in practice, Generally, stability of HEV was lower at the ambient condi- although the distinct combination of particle forms in certain tions compared to the low temperature conditions. Whereas meat products may vary. A higher infectivity of non-envel- at ambient conditions, HEV infectivity was mostly destroyed oped particles as compared to quasi-enveloped particles after 4 weeks and totally absent after 8 weeks, remaining has been described using cell culture studies (Capelli et al., infectious HEV could be detected at 8 weeks in 5/8 sam- 2020; Yin et al., 2016b), and a removal of the envelope due ples at low temperature conditions. Higher stability at 4 °C to environmental factors may therefore increase infectivity. as compared to 20 °C has also been described for HAV, In order to analyze the distinct contribution of inactivating poliovirus and adenovirus after drying on surfaces (Abad and infectivity-increasing processes during drying and stor- et al., 1994), although the effect was less pronounced than age, additional experiments with preparations of separated for HEV. non-enveloped and enveloped particles should be performed Especially at the low temperature condition, a marked in future studies. effect of the distinct surface material was observed in our The resistance of viruses against the drying process rep- study. Here, HEV showed an exceptionally high stability on resents the major factor for their survival in dried condition plastics, with only 0.4 log ffu/ml infectivity decrease after on surfaces (Sánchez & Bosch, 2016). In our experiments, 8 weeks in the experiment with addition of BSA. Ceramics only slight decreases of HEV infectivity have been found and steel showed moderate inactivation rates, and HEV was during the drying process, with no statistical significance almost completely inactivated (3.9 log ffu/ml decrease) between the titers before and after drying. This indicates that on wood under the same conditions. The reasons for the HEV is highly stable against drying, and the persistence of differences are not known, but it could be speculated that infectious virus has to be expected on surfaces after contact the porous surface of wood absorbs water from the virus to contaminated meat or to excretions from infected ani- particles with higher efficiency as compared to non-porous mals or humans. The high stability was evident for all tested surfaces, which may lead to a faster virus inactivation, as surface types and the addition of BSA, which was used as recently hypothesized for SARS-CoV-2 (Corpet, 2021). This 1 3 146 Food and Environmental Virology (2022) 14:138–148 is in line with results from experiments with HAV, showing a more complete risk assessment on HEV transmission via a higher stability on smooth surfaces (aluminium, ceramics) surfaces. as compared to porous surfaces (paper) (Abad et al., 1994). Supplementary Information The online version contains supplemen- Generally, the stability of HEV and HAV at low temperature tary material available at https://doi. or g/10. 1007/ s12560- 022- 09510-7 . and high RH turns out to be rather similar, at least for sur- face materials where data are available for both viruses. For Acknowledgements We want to thank Silke Apelt, Stefanie Prosetzky example, in the presence of a loading substance after drying and Anja Schlosser (BfR, Berlin, Germany) for excellent technical assistance, Chris-Tina Höfer and Norman Violet (BfR, Berlin, Ger- on ceramics for 8 weeks, the mean infectivity was reduced many) for excellent support with the Opera Phenix device and scientific by 1.1 log for HAV (Abad et al., 1994) and by 1.6 log for 10 10 discussions, and Dirk Meyer (BfR, Berlin, Germany) for providing HEV, and on metal by 1.5 log for HAV (aluminium) (Abad storage facilities. et al., 1994) and by 2.4 log for HEV (steel). Our study has some limitations. Because of the necessary Funding Open Access funding enabled and organized by Projekt use of the laborious and time-consuming titration system, DEAL. This research was funded by a Grant of the Federal Food Safety and Veterinary Office of the Swiss Confederation (Project number only a low number of samples could be analyzed, thus limit- 4.18.01). ing the analyzed conditions and replications of the experi- ments. As already mentioned, the distinct influence of tem- Data Availability Data are available in Supplementary Materials and perature and humidity could not be differentiated because additional data can be retrieved upon request by R.J. (Reimar.Johne@ bfr.bund.de). of the specifically selected experimental conditions. Also, a discrimination of the inactivation profile of non-enveloped Open Access This article is licensed under a Creative Commons Attri- vs. quasi-enveloped particles could not be assessed. In addi- bution 4.0 International License, which permits use, sharing, adapta- tion, only one strain has been investigated and other strains tion, distribution and reproduction in any medium or format, as long may show different behaviors. The use of PBS and addition as you give appropriate credit to the original author(s) and the source, of BSA may not completely reflect the complex composition provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are of blood, feces or meat juice, which are suspected to be the included in the article's Creative Commons licence, unless indicated most probable matrices containing HEV in the field. Gener - otherwise in a credit line to the material. If material is not included in ally, the transmission rate of HEV to surfaces and from the the article's Creative Commons licence and your intended use is not surfaces to food and humans has to be determined in the permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a future to better assess the transmission probability under copy of this licence, visit http://cr eativ ecommons. or g/licen ses/ b y/4.0/ . field conditions. It can be concluded that HEV is highly resistant against the process of drying and shows a high stability against long-term storage on several surfaces. The highest stability References was determined at low temperature and high RH resulting Abad, F. X., Pintó, R. M., & Bosch, A. (1994). Survival of enteric in detection of infectious virus for as long as 8 weeks in viruses on environmental fomites. Applied and Environmental most cases. 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Journal

Food and Environmental VirologySpringer Journals

Published: Jun 1, 2022

Keywords: Hepatitis E virus; Drying; Stability; Inactivation; Surfaces

References