Food Environ Virol (2014) 6:58–61 DOI 10.1007/s12560-013-9131-3

ORIGINAL PAPER

Temperature Effects for High-Pressure Processing of Picornaviruses David H. Kingsley • Xinhui Li • Haiqiang Chen

Received: 23 August 2013 / Accepted: 4 November 2013 / Published online: 23 November 2013 Ó Springer Science+Business Media New York (outside the USA) 2013

Abstract Investigation of the effects of pre-pressurization temperature on the high-pressure inactivation for single strains of aichivirus (AiV), coxsackievirus A9 (CAV9) and B5 (CBV5) viruses, as well as human parechovirus-1 (HPeV) was performed. For CAV9, an average 1.99 log10 greater inactivation was observed at 4 °C after a 400-MPa– 5-min treatments compared to 20 °C treatments. For CBV5, an average of 2.54 log10 greater inactivation was noted after 600-MPa–10-min treatments at 4 °C in comparison to 20 °C treatments. In contrast, inactivation was reduced by an average of 1.59 log10 at 4 °C for HPeV. AiV was resistant to pressure treatments of 600 MPa for as long as 15 min at 4, 20, and 30 °C temperatures. Thus, different pre-pressurization temperatures result in different inactivation effects for picornaviruses. Keywords High-pressure processing  Picornavirus  Temperature  pH

US Department of Agriculture is an equal opportunity provider and employer. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. D. H. Kingsley (&) Food Safety and Interventions Technologies Research Unit, Agricultural Research Service, U. S. Department of Agriculture, James W. W. Baker Center, Delaware State University, Dover, DE 19901, US e-mail: [email protected] X. Li  H. Chen Department of Animal & Food Sciences, University of Delaware, Newark, DE 19716-2150, US

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Introduction In recent years, the potential of high-pressure processing (HPP) to inactivate food-borne viruses has been extensively evaluated (Kingsley 2013). Currently, the two principal viruses that are perceived to pose the greatest threat to consumers of shellfish and produce are human noroviruses (HuNoV) and hepatitis A virus (HAV), which are members of the Caliciviridae and Picornaviridae families, respectively. Pressure treatment of contaminated oysters has demonstrated that it is possible to inactivate HAV within shellfish (Calci et al. 2005; Kingsley et al. 2005; Kingsley et al. 2009; Terio et al. 2010) as well as HuNoV(GI.1 Norwalk strain) and its surrogate virus, murine norovirus (MNV; Kingsley et al., 2007; Leon et al., 2011). Beyond the clear threats of HAV and HuNoV, there are many other viruses that are transmitted by the fecal– oral route which may pose a threat to consumers of uncooked raw shellfish and produce. Among these are other picornaviruses, such as aichivirus (AiV), coxsackievirus A9 (CAV9), coxsackievirus B5 (CBV5), and human parechovirus (HPeV). The potential of high pressure to inactivate a limited number of other potential food-borne human picornaviruses was investigated at room temperature (*21 °C) by Kingsley et al. (2004) using a small custom-built pressure unit that used oil as the pressure medium. Widely variable pressure inactivation thresholds in response to HPP were observed for different picornaviruses. Poliovirus was previously shown to resist 600 MegaPascal (MPa) treatments (Wilkinson et al. 2001; Kingsley et al. 2002) for as long as one hour. Also AiV and CBV5 were resistant to 5-min 600-MPa treatments (Kingsley et al. 2004) at room temperature, while for CAV9, a 5-min HPP treatment in minimum essential growth medium (MEM) supplemented

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with 10 % fetal bovine sera (FBS) resulted in 3.4, 6.5, and 7.6 log10 tissue culture infectious dose 50 % (TCID50) reductions at 400, 500, and 600 MPa, respectively. For HPeV-1, a 5-min treatment in MEM with 10 % FBS resulted in reductions of 1.3-, 4.3-, and 4.6-log10 TCID50 at 400, 500, and 600 MPa, respectively. For the norovirus research surrogates and HAV, subsequent studies investigating the effects of initial temperature and pH at which HPP was performed demonstrated dramatic differences in inactivation rates. For feline calcivirus (FCV), murine norovirus (MNV), and Tulane virus (TV), cooler initial treatment temperatures (ca. 4 °C) are substantially more effective for inactivating these viruses under pressure, as compared to room temperatures or higher temperatures (Chen et al. 2005; Kingsley et al. 2007; Li et al. 2013). Research also suggests enhanced inactivation at cooler temperature for the HuNoV. During a HuNoV volunteer study, it was noted that a 400-MPa pressure treatment at 6 °C protected more subjects than the same treatment at 22 °C (Leon et al. 2011). Thus, to date, all evaluated members of the Caliciviridae family show enhanced inactivation at refrigeration temperatures. Investigation of temperature effects on HPP inactivation of HAV gave opposite results. HAV is more readily inactivated when HPP is performed at temperatures above room temperature, and cooler temperatures were observed to actually inhibit HPP inactivation (Kingsley and Chen 2009; Kingsley et al. 2007). For HAV, lower pH was found to enhance HPP inactivation (Kingsley and Chen 2009), explaining why inactivation of HAV within strawberry puree (pH 3.67) and chopped green onions (pH 5.12) was greater than was observed in neutral pH tissue culture media (Kingsley et al. 2005). However, investigation of MNV inactivation in strawberry puree at pH 4 and 7 revealed that inactivation of MNV was inhibited at pH 4 (Lou et al. 2011). In this study, investigation of temperature and pH effects for CBV5, CAV9, HPeV, and AiV has been performed to characterize the potential of temperature and acidic conditions to influence HPP inactivation of these viruses as well as to investigate whether these effects might be family-specific.

Materials and Methods HPeV-1 stock was obtained from the American Type Culture Collection (ATCC; Manassas, VA) as culture VR52 and propagated in fetal Rhesus monkey kidney cells (FRhK-4). CAV9 and CBV5 were obtained from ATCC as cultures VR-186 and VR-185, respectively, and propagated in buffalo green monkey kidney (BGM) cells provided by Daniel Dahling (Environmental Protection Agency, Cincinnati, OH). The AiV strain A846/88 was obtained from

59 Table 1 Temperature profile of pressure treatment Initial temperature (°C)

Pressure (Mpa)

Maximum temperature (°C)

4

400

11.9

475

13.3

600

15.6

20

30

400

29.2

475

30.4

600

32.4

400 475

40.1 41.6

600

43.0

Susan Matsui (Stanford University, Stanford, CA), and propagated on Vero cells obtained as culture CCL-81 (ATCC). CAV9, CBV5, and HePV stocks were stored in modified Eagle media (MEM) culture media (GibcoBRL, Gaithersburg, MD) with 10 % fetal bovine serum (FBS; GibcoBRL). For pH experiments, virus stocks were dialyzed against 0.1 M solutions of HEPES (N-2-hydroxyethylpiperazine-N’-2-ethane sulfonic acid; Invitrogen) at pH 4.0 and 7.0. For pressure treatments, 2.0 ml virus stocks were transferred into polyester Scotchpak pouches (Kapak 500, Minneapolis, MN) with a second pouch sealed around the first pouch. Heat-sealing was performed using an Impulse Food Sealer (Model MP-8; American International Electric, Whittier, CA). Pressurization of samples was carried out using a high-pressure unit with temperature control (Model Avure PT-1, Avure Technologies, Kent, WA) using water as the hydrostatic medium. A circulating bath surrounded the pressure cell to control temperature. Temperatures for the water bath and samples inside the chamber during pressurization were monitored using K-type thermocouples. Virus samples were subjected to specific pressure–time treatments of 400-MPa–5-min, 475-MPa–5min, and 600-MPa–10-min treatments for CAV9, HPeV, and CBV5, respectively, at specified initial temperatures. AiV was treated at 600 MPa for times ranging from 5 to 15 min. The temperature and pressure data were recorded every 2 s (DASYTEC USA, Bedford, NH). The pressure come-up rate was approximately 22 MPa/s. The pressure release was \4 s. Pressurization time reported in this study does not include the pressure come-up or release times. Temperature increases during pressure treatment due to adiabatic heating were 1.9, 2.5, and 2.9 °C/100 MPa with initial sample temperatures at 4, 20, and 30 °C respectively (Table 1). Virus assays were performed using tenfold, serially diluted stocks in Earle’s balanced salt solution (GibcoBRL) using six wells of a 24-well plate per dilution and 100 ll inocula per well. Tissue culture infectious dose

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Food Environ Virol (2014) 6:58–61 7

HPP Log10 Reduction

6

a

5 a b

4

b

3

b

2

4°C 20°C 30°C

a a

b

a

1 0 CAV9

CBV5

HPeV

Virus

Fig. 1 Temperature and high-pressure processing. Comparison of high-pressure inactivation observed at initial temperatures of 4 °C (white bars), 20 °C (gray bars), and 30 °C (black bars). Treatment conditions for coxsackie A9 (CAV9), coxsackie B5 (CBV5), and human parechovirus (HPeV) were 400 MPa for 5 min, 600 MPa for 10 min, and 475 MPa for 5 min, respectively. Like letters indicate no significant differences. Error bars represent SEM. (N = 3; N = 18)

50 % (TCID50) was determined by the method of Reed and Muench (1938). Three independent trials with six replicates per trial were assayed for each sample dilution for all pressure treatments. Statistical analyses was performed by one-way ANOVA following Tukey’s post-hoc comparisons using software IBM SPSS version 21 (IBM, Armonk, NY). A P value of \0.05 was considered statistically significant.

Results Differential temperature effects for high-pressure treatment of CAV9 and CBV5, as well as HPeV were observed (Fig. 1). For the two coxsachieviruses tested, greater inactivation was observed when pressure was applied at 4 °C rather than at room temperature or higher, while for HPeV greater inactivation was observed at warmer temperatures. 400-MPa–5-min treatments of CAV9, performed at an initial temperature of 4 °C, inactivated 5.37 log of virus, while equivalent treatments at 20 and 30 °C resulted in reduced inactivation of 3.39 and 2.46 log, respectively. 600-MPa–10-min treatments of CBV5, performed at 4 °C, resulted in an average reduction of 3.76 log, while 20 and 30 °C treatments resulted in average reductions of 1.22 and 1.38 log, respectively. In contrast, 475-MPa–5–min treatments of HPeV gave average reductions of 2.45, 4.04, and 4.21 at 4, 20, and 30 °C, respectively. For AiV, two separate treatments of 600 MPa for 5 and 15 min applied at 4, 20, and 30 °C did not result in titer reductions for the virus (data not shown). Results observed for AiV were consistent with previous observations (Kingsley et al. 2004).

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Attempts to identify acidic pH-specific effects for the viruses tested herein were unsuccessful, primarily due to lack of tolerance to low pH, as well as a lack of differential pressure sensitivity. Virus stocks were dialyzed against HEPES buffer at pH 4 and 7, and stored approximately 1 week at 4 °C prior to room-temperature pressure treatments (21 °C). For a single trial with AiV, there was no reduction observed after a 600-MPa–10-min treatment at pH 7 or 4 relative to untreated controls at the same pH. However, an approximate one-log decrease was observed for AiV at pH 4 as compared to pH 7, suggesting that AiV is likely sensitive to acidic conditions. For HPeV and CAV9, titer reductions of approximately 3-logs and 4-logs, respectively, were observed between untreated pH 4 and 7 samples indicating that these viruses do not tolerate acidic pH well. For a single trial, CBV5 was found to be tolerant of pH 4, but approximately equivalent 2-log reductions were observed after 600-MPa–10-min treatments at pH 4 and 7, indicating that while CBV5 was tolerant of acidic pH, there was no pH-sensitive effect for the virus.

Discussion Results indicate that the initial HPP temperature effect for the Picornaviradae is variable and not virus family-specific. Curiously, CAV9 and CBV5 showed enhanced inactivation when HPP was performed at an initial temperature of 4 °C, as opposed to 20 and 30 °C starting temperatures. This behavior under pressure is analogous to observations for the calciviruses such as MNV, HuNoV, TV, and FCV, but counter to the behavior of HAV, the only previously evaluated picornavirus. In contrast, HPP inactivation of HPeV was reduced at 4 °C as compared with 20 and 30 °C in a manner analogous to what was previously observed for HAV (Kingsley et al. 2006). Thus, the picornaviruses HAV and HPeV are more resistant to HPP at cooler temperatures and coxsackie A9 and B5 picornaviruses are more resistant at warmer temperatures. How this different behavior in response to pressure relates to the biology of these viruses is unknown, since biophysical explanations for these HPP temperature effects are lacking. In Kingsley et al. (2004) it was noted that CBV5 and AiV were resistant to pressures up to 600 MPa using a custom-built machine that used oil [bis (2-ethylhexyl) sebacate] as a pressure medium. Subsequently, most pressure sensitivity evaluations for viruses have been performed using a small Avure PT-1 water-based HPP machine for which adiabatic heating is less pronounced. In contrast with previous results, re-evaluation using the water-based PT-1 machine revealed that CBV5 was slightly sensitive to pressure at 600 MPa (10-min treatment) at room temperature with enhanced inactivation

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observed at cooler temperatures. The lack of sensitivity of CBV5 observed previously may be due to higher adiabatic temperatures achieved using oil as the pressure medium. AiV, however, was again found to be resistant to 600 MPa treatments for as long as 15 min at both warm and cooler temperatures. Determination if differential temperature effects might be observed at pressures above 600 MPa for AiV was not possible since 600 MPa is the practical pressure limit for the Avure PT-1 pressure machine. Attempts to determine if acidic pH inhibits or enhances HPP inactivation of the Picornaviradae were generally unsuccessful. As noted in the results, HPeV and CAV9 were intolerant of lower pH, confounding efforts to measure additional inactivation under pressure. AiV appears equally resistant to 600-MPa treatment when applied at pH 4 and 7, although the slight titer reduction was noted for the untreated pH 4 sample as compared to the untreated pH 7 indicated that AiV is somewhat intolerant of pH 4. CBV5 is tolerant of pH 4, but 600-MPa treatments gave similar reductions at pH 4 and 7, suggesting that pH may not be a factor in HPP inactivation of this virus. Lack of pH tolerance by CAV9 and HPeV suggest that these viruses may be less readily transmitted in acidic vegetable products, such as berry or tomato-based foods, and in molluskan shellfish, since the ability of a virus to tolerate low pH is related to a virus’ ability to resist bivalve digestive processes (Provost et al. 2011). In summary, there are no basic generalizations or rules that can be applied to the picornaviruses relative to how matrix pH and pre-pressurization temperatures might affect inactivation under pressure. We find that unlike the Caliciviridae, the Picornaviridae family does not show consistent temperature-specific effects for HPP. In fact, optimal HPP inactivation temperatures may be cooler (CAV9 and CBV5) or warmer (HAV and HPeV). While it was not practically possible to evaluate the effect of pH on CAV9, AiV, and HPeV, we note that no pH effect was found for CBV5, contrasting with HAV which shows enhanced inactivation at lower pH and MNV which shows reduced inactivation at pH 4.

References Calci, K. R., Meade, G. K., Tetzloff, R. C., & Kingsley, D. H. (2005). High pressure inactivation of hepatitis A virus within oysters. Applied and Environmental Microbiology, 71, 339–343.

61 Chen, H., Hoover, D. G., & Kingsley, D. H. (2005). Temperature and treatment time influence high hydrostatic pressure inactivation of feline calicivirus, a norovirus surrogate. Journal of Food Protection, 68, 2389–2394. Kingsley, D. H. (2013). High pressure processing and its application to the challenge of virus-contaminated foods. Food and Environmental Virology, 5, 1–12. Kingsley, D. H., Calci, K. R., Holliman, S., Dancho, B. A., & Flick, G. J. (2009). High pressure inactivation of HAV within oysters: Comparison of whole-in-shell with shucked oyster meats. Food and Environmental Virology, 1, 137–140. Kingsley, D. H., & Chen, H. (2009). Influence of pH, salt, and temperature on pressure inactivation of hepatitis A virus. International Journal of Microbiology, 130, 61–64. Kingsley, D. H., Chen, H., & Hoover, D. G. (2004). Inactivation of selected picornaviruses by high hydrostatic pressure. Virus Research, 102, 221–224. Kingsley, D. H., Guan, D., & Hoover, D. G. (2005). Pressure inactivation of hepatitis A virus in strawberry puree and sliced green onions. Journal of Food Protection, 68, 1748–1751. Kingsley, D. H., Guan, D., Hoover, D. G., & Chen, H. (2006). Inactivation of hepatitis A virus by high pressure processing: The role of temperature and pressure oscillation. Journal of Food Protection, 69, 2454–2459. Kingsley, D. H., Holliman, D. R., Calci, K. R., Chen, H., & Flick, G. J. (2007). Inactivation of a norovirus by high pressure processing. Applied and Environmental Microbiology, 73, 581–585. Kingsley, D. H., Hoover, D. G., Papafragkou, E., & Richards, G. P. (2002). Inactivation of hepatitis A virus and a calicivirus by high hydrostatic pressure. Journal of Food Protection, 65, 1605–1609. Leon, J. S., Kingsley, D. H., Montes, J. S., Richards, G. P., Lyon, G. M., Abdulhafid, G. M., et al. (2011). Randomized, doubleblinded clinical trial for human norovirus inactivation in oysters by high hydrostatic pressure processing. Applied and Environmental Microbiology, 77, 5476–5482. Li, X., Ye, M., Neetoo, H., Golovan, S., & Chen, H. (2013). Pressure inactivation of Tulane virus, a candidate surrogate for human norovirus and its potential application in food industry. International Journal of Food Microbiology, 162, 37–42. Lou, F., Neetoo, H., Chen, H., & Li, J. (2011). Inactivation of a human norovirus surrogate by high-pressure processing: effectiveness, mechanism, and potential application in the fresh produce industry. Applied and Environmental Microbiology, 77, 1862–1871. Provost, K., Dancho, B. A., Ozbay, G., Anderson, R., Richards, G. P., & Kingsley, D. H. (2011). Hemocytes are sites of persistence for enteric viruses within oysters. Applied and Environmental Microbiology, 77, 8360–8369. Reed, L. J., & Muench, H. A. (1938). A simple method of estimating fifty percent endpoints. American Journal of Hygiene, 27, 493–497. Terio, V., Tantillo, G., Martella, V., Di Pinto, P., Buonavoglia, C., & Kingsley, D. H. (2010). High pressure inactivation of HAV within mussels. Food and Environmental Virology, 2, 83–88. Wilkinson, N., Kurdziel, A. S., Langton, S., Needs, E., & Cook, N. (2001). Resistance of poliovirus to inactivation by high hydrostatic pressure. Innovative Food Science and Emerging Technologies, 2, 95–98.

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Temperature Effects for High-Pressure Processing of Picornaviruses.

Investigation of the effects of pre-pressurization temperature on the high-pressure inactivation for single strains of aichivirus (AiV), coxsackieviru...
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