David H. Kingsley

Inactivation of a Norovirus by High-Pressure Processing

ABSTRACT Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbeccos modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log10 PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log10 PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log10 PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log10 PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods.

High-Pressure Inactivation of Hepatitis A Virus within Oysters

ABSTRACT Previous results demonstrated that hepatitis A virus (HAV) could be inactivated by high hydrostatic pressure (HHP) (D. H. Kingsley, D. Hoover, E. Papafragkou, and G. P. Richards, J. Food Prot. 65:1605-1609, 2002); however, direct evaluation of HAV inactivation within contaminated oysters was not performed. In this study, we report confirmation that HAV within contaminated shellfish is inactivated by HHP. Shellfish were initially contaminated with HAV by using a flowthrough system. PFU reductions of >1, >2, and >3 log10 were observed for 1-min treatments at 350, 375, and 400 megapascals, respectively, within a temperature range of 8.7 to 10.3°C. Bioconcentration of nearly 6 log10 PFU of HAV per oyster was achieved under simulated natural conditions. These results suggest that HHP treatment of raw shellfish will be a viable strategy for the reduction of infectious HAV.

Rapid and Efficient Extraction Method for Reverse Transcription-PCR Detection of Hepatitis A and Norwalk-Like Viruses in Shellfish

ABSTRACT As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine–PEG–Tri-reagent–poly(dT) method can be performed in less than 8 h on hard-shell clams (Mercenaria mercenaria) and Eastern oysters (Crassostrea virginica) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR50 units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as Ruditapes philippinarum) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.

Detection of both Hepatitis A Virus and Norwalk-Like Virus in Imported Clams Associated with Food-Borne Illness

ABSTRACT Hepatitis A virus (HAV) and Norwalk-like virus (NLV) were detected by reverse transcription-PCR in clams imported into the United States from China. An epidemiological investigation showed that these clams were associated with five cases of Norwalk-like gastroenteritis in New York State in August 2000 (Food and Drug Administration Import Alert 16-50). They were labeled “cooked” but appeared raw. Viral RNA extraction was performed by using dissected digestive tissues rather than whole shellfish meats; this was followed by glycine buffer elution, polyethylene glycol precipitation, Tri-Reagent treatment, and purification of poly(A) RNA with magnetic beads coupled to poly(dT) oligonucleotides. We identified HAV and NLV as genotype I and genogroup II strains, respectively. Both viruses have high levels of homology to Asian strains. An analysis of fecal coliforms revealed a most-probable number of 93,000/100 g of clam meat, which is approximately 300-fold higher than the hygienic standard for shellfish meats.

Temperature and Treatment Time Influence High Hydrostatic Pressure Inactivation of Feline Calicivirus, a Norovirus Surrogate †

Interest in high hydrostatic pressure processing as a nonthermal pasteurization process for foods continues to increase. Feline calicivirus (FCV), a propagable virus that is genetically related to the nonpropagable human noroviruses, was used for detailed evaluation of the high pressure processing parameters necessary for virus inactivation. Pressure inactivation curves of FCV strain KCD in Dulbeccos modified Eagle medium with 10% fetal bovine serum were obtained at 200 and 250 MPa as a function of time at room temperature. Pressure inactivation curves at 200 and 250 MPa also were determined as a function of temperature ranging from --10 to 50 degrees C at treatment times of 4 and 2 min, respectively. Tailing was observed for inactivation as a function of treatment time, indicating that the linear model was not adequate for describing these curves. The two nonlinear models, the log logistic and Weibull functions, consistently produced better fit to inactivation curves than did the linear model. The mean square errors were 0.381 for the log logistic model, 0.425 for the Weibull model, and 1.546 for the linear model. For inactivation as a function of temperature, FCV was most resistant to pressure at 20 degrees C. Temperatures above and below 20 degrees C significantly increased pressure inactivation of FCV. A 4-min treatment of 200 MPa at --10 and 50 degrees C reduced the titer of FCV by 5.0 and 4.0 log units, respectively; whereas at 20 degrees C the same treatment only reduced the titer by 0.3 log units. These novel results point to the potential for using temperatures above and particularly below room temperature to lower the pressure needed to cause the desired level of virus inactivation.

Discrimination between infectious and non-infectious human norovirus using porcine gastric mucin

Human noroviruses (NoVs) are known to bind to human histo-blood group antigens, as well as to chemically-similar porcine gastric mucins. Here, the binding ability of NoV to porcine mucin is shown to be substantially deficient after UV, thermal, and high pressure treatments. Using qRT-PCR, ≥ 68% of GI.1 NoV (Norwalk strain) bound to porcine gastric mucin-conjugated magnetic beads (PGM-MBs). Application of 600-MPa high pressure treatments reduced binding of the virus to PGM-MBs by 4.7-log₁₀, as determined by qRT-PCR, while a 300-MPa pressure treatment, reduced binding to PGM-MBs by only 0.45-log₁₀. This is consistent with a previously reported clinical trial (Leon et al., 2011. Appl. Environ Microbiol. 77:5476-5482.) which demonstrated inactivation of 4-log₁₀ of GI.1 NoV at 600-MPa. After thermal treatment, binding to PGM-MBs decreased when samples were heated from 0 to 80 °C. Ultraviolet treatments of 0.5 and 2 J/cm² reduced observed PGM-MB binding of norovirus to 33% and negligible levels, respectively, from an initially observed 84% binding for untreated NoV. Although thermal and UV treatments are generally recognized to inactivate viruses, verification of NoV inactivation by these treatments may require volunteer studies. In total, these results suggest the loss of NoV binding to porcine mucin as a potential means to preferentially exclude non-infectious virus particles from subsequent RT-PCR detection.

Pressure inactivation of hepatitis A virus in strawberry puree and sliced green onions.

Hepatitis A can be acquired by ingesting contaminated produce. To investigate the potential of high-pressure processing as an intervention strategy for virus in produce, strawberry puree and sliced green onions were inoculated with > 10(6) PFU of hepatitis A virus and treated with pressures ranging from 225 to 375 megapascals (MPa) in 25-MPa increments at ambient temperature. Subsequent virus extraction and plaque assay determined that hepatitis A virus was inactivated in strawberry puree and sliced green onions after 5-min exposures to pressures of 375 MPa with log PFU reductions of 4.32 and 4.75, respectively. Hepatitis A virus was equally sensitive in puree and onions at pressures > or = 350 MPa. For treatments of < 325 MPa, the virus was more sensitive to pressure in strawberry puree than in sliced onions with log reductions of 1.2, 2.06, and 3.13 observed for strawberries and 0.28, 0.72, and 1.42 observed for onions after 5-min treatments at 250, 275, and 300 MPa, respectively. Although high-pressure processing may cause some organoleptic alterations to strawberries and onions, results show high-pressure processing will inactivate hepatitis A virus in these foods.

Persistence of hepatitis A virus in oysters.

We investigated the ability of hepatitis A virus (HAV) to persist for up to 6 weeks in Eastern oysters (Crassostrea virginica). Viral RNA was detected by reverse transcription-polymerase chain reaction 6 weeks after 16 h of exposure to 90,000 PFU (180 PFU/ml of seawater) of HAV. Assaying for infectious virus in oysters that received a daily feeding of phytoplankton recovered 3,800, 650, and 500 PFU of HAV 1, 2, and 3 weeks after contamination with 90,000 PFU of HAV, respectively. However, no infectious HAV was isolated from oysters 4, 5, or 6 weeks after contamination. These results support the position that shellfish depuration is insufficient for the complete removal of infectious viruses. Extended relay times (in excess of 4 weeks) may be required to produce virologically safe shellfish.

Influence of pH, salt, and temperature on pressure inactivation of hepatitis A virus

The effects of pH (3-7), NaCl (0-6%), and temperature on pressure inactivation of hepatitis A virus (HAV) were determined. The HAV samples were treated at 400 MPa for 1 min at 5, 20, and 50 degrees C. Decreasing solution pH enhanced pressure inactivation of HAV. This enhanced inactivation effect was most evident at 20 degrees C. A baroprotective effect was observed for NaCl concentrations from 1 to 6%. For example, a treatment of 400 MegaPascals (MPa) for 1 min at 50 degrees C reduced the HAV titers by 4.0, 4.1, 1.3 and 0.4 log plaque forming units (PFU)/ml for NaCl concentrations of 0, 1, 3, and 6%, respectively. Overall, increasing the treatment temperature enhanced pressure inactivation of HAV in the solutions. The pressure resistance of HAV in oysters was also examined. Temperature in the range of 5 to 50 degrees C did not significantly affect the pressure inactivation of HAV within oyster homogenates. It is concluded that HPP treatment of oysters at temperatures above room temperature would not provide any additional benefit for inactivation of HAV. However, the observation that HAV inactivation is enhanced in acidic matrices is information that may be useful for designing product formulations and processing parameters for high pressure processing of products such as low pH fruit juices and salsa.

Inactivation of Hepatitis A Virus by High-Pressure Processing: The Role of Temperature and Pressure Oscillation

Inactivation of hepatitis A virus (HAV) in Dulbeccos modified Eagle medium with 10% fetal bovine serum was studied at pressures of 300, 350, and 400 MPa and initial sample temperatures of -10, 0, 5, 10, 20, 30, 40, and 50 degrees C. Sample temperature during pressure application strongly influenced the efficiency of HAV inactivation. Elevated temperature (> 30 degrees C) enhanced pressure inactivation of HAV, while lower temperatures resulted in less inactivation. For example, 1-min treatments of 400 MPa at -10, 20, and 50 degrees C reduced titers of HAV by 1.0, 2.5, and 4.7 log PFU/ml, respectively. Pressure inactivation curves of HAV were obtained at 400 MPa and three temperatures (-10, 20, and 50 degrees C). With increasing treatment time, all three temperatures showed a rapid initial drop in virus titer with a diminishing inactivation rate (or tailing effect). Analysis of inactivation data indicated that the Weibull model more adequately fitted the inactivation curves than the linear model. Oscillatory high-pressure processing for 2, 4, 6, and 8 cycles at 400 MPa and temperatures of 20 and 50 degrees C did not considerably enhance pressure inactivation of HAV as compared with continuous high-pressure application. These results indicate that HAV exhibits, unlike other viruses examined to date, a reduced sensitivity to high pressure observed at cooler treatment temperatures. This work suggested that slightly elevated temperatures are advantageous for pressure inactivation of HAV within foods.