Antiviral activity of copper contact surfaces against MS2 coliphage and hepatitis a virus.

AIMS: Viral diseases can be indirectly transmitted by contaminated non-food contact surfaces to final food products by cross-contamination. The interaction of metal surfaces and viruses, MS2 coliphage and hepatitis A virus (HAV), was investigated for strategy development in decreasing this transmission risk. METHODS AND RESULTS: MS2 deposited onto stainless-steel surface was stable but inactivated at 0.95 log10 PFU min-1 on 99.9% copper surfaces. Greater copper-inactivation of MS2 was observed in (a) simple media (phosphate buffered saline, PBS) than protein-rich media (beef extract buffer), and (b) acidic than pH ≥ 6.8 environments. Among food matrices (strawberry juices and beef broth), the greatest MS2 inactivation by copper occurred in filtered strawberry juice at pH 3.5. At a reduction of 0.17 log10 PFU min-1, HAV survived longer than MS2 on copper by FRhK-4 cell infectivity assay. CONCLUSIONS: The inactivation of virus on copper surfaces was greater in acidic viral surrounding environments and in simple PBS medium. In the same 99% PBS medium, MS2 may not be an appropriate surrogate for HAV when assessing viral inactivation on copper surfaces.

Comparison of RNA extraction kits for the purification and detection of an enteric virus surrogate on green onions via RT-PCR.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) offers a rapid and sensitive molecular method for detection of enteric viruses. Unfortunately, these assays are often hampered by the low virus titre found in foods and PCR inhibition due to matrix carryover during RNA extraction. Four commercial RNA extraction kits (Qiagen's QIAamp Viral RNA Mini and UltraSens Virus kits, MoBio UltraClean Tissue & Cells RNA Isolation kit, and Ambion MagMAX Viral RNA Isolation kit) were evaluated for their ability to extract and purify MS2 bacteriophage RNA, an enteric virus surrogate, from inoculated green onions, a food which has been associated with viral gastroenteritis outbreaks. Inoculated green onion wash concentrates and green onion pieces with and without Qiagen QIAshredder homogenization were assayed in the kit comparison. MS2 detection and PCR inhibition were evaluated using a duplex real-time RT-PCR for MS2 and an exogenous internal amplification control (IAC) assay. Without homogenization, MS2 inoculated at 40pfu/g was detected in at least 4 lots of green onion wash concentrates using the silica-membrane spin-column kits. Inhibition was a factor for the magnetic silica-based MagMAX kit, which resulted in detection of MS2 in 1 of 5. Addition of QIAshredder homogenization prior to extraction did not adversely affect the silica-membrane kit results but improved the MS2 detection by MagMAX to 5 of 5 lots. Use of a 1:10 dilution of primary RNA extracts also improved detection. The QIAamp Viral RNA Mini and MagMAX kits were further compared for detection of MS2 from green onion pieces inoculated at 20 and 5pfu/g. Using homogenization, the MagMAX kit detected 20pfu/g in only 1 of 2 green onion lots, whereas the QIAamp Viral RNA kit detected 2 of 2 lots at 5 pfu/g without homogenization.

Effect of Process Temperature on Virus Inactivation during High Hydrostatic Pressure Processing of Contaminated Fruit Puree and Juice.

High pressure processing (HPP) can inactivate pathogens and retain fruit qualities. Elevated HPP pressure or time increases virus inactivation, but the effect of temperature is not consistently observed for norovirus and hepatitis A virus. In the present study, the effectiveness of HPP holding temperatures (<40°C) and pressures were evaluated for inactivating surrogates (murine norovirus [MNV] and MS2 coliphage) in pomegranate and strawberry juices and strawberry puree using a 24-liter HPP system. The holding temperature was established by setting the HPP initial temperature via pretrials. All trials were able to arrive at the designated holding pressure and holding temperature simultaneously. MNV inactivation in juices was conducted at 300 MPa for 3 min with various holding temperatures (10 to 30°C). A regression equation was derived, Y = -0.08 × X + 2.6 log PFU, R2 = 0.96, where Y is the log reduction and X is the holding temperature. The equation was used to predict a 2.6-log reduction in juices at 0°C holding temperature and indicated that MNV inactivation was inversely proportional to temperature increase. MNV survival during HPP did not differ significantly in pomegranate and strawberry juices. However, MS2 coliphage inactivation was greater as the holding temperature increased (from 15 to 38°C) at 600 MPa for 3 min. The increased inactivation trend is presumably similar to that for hepatitis A virus, but the holding temperature was not correlated with the reduction of HPP-resistant MS2 in strawberry puree. When the HPP holding pressure was evaluated independently in strawberry puree, a 5-log reduction of MNV was predicted through regression analysis at the holding pressure of 424 MPa for 3 min at 20°C. These parameters should inactivate >5 log PFU of MNV in juices, based upon a greater inactivation in berry juice than in puree (1.16-versus 0.74-log reduction at 300 MPa). This research illustrates use of predictive inactivation and a feasible means for manipulating HPP parameters for effective virus inactivation in fruit juices and puree.

Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

Tracking and modeling norovirus transmission during mechanical slicing of globe tomatoes.

Recent epidemiological evidence indicates that preparation of fresh produce for use as ingredients in ready-to-eat food in commercial settings has been a significant source of the norovirus (NoV) infections in the U.S. This research investigated the dissemination of NoV from a single tomato to many others via the use of an 11-horizontal blade slicer commonly found in restaurants or sandwich shops. A total of eight trials were conducted. The source of contamination in each trial was a soak-inoculated, air-dried globe tomato containing ~8log10 murine norovirus (MNV). Each trial began by slicing a single un-inoculated tomato in the slicer, followed by slicing an inoculated tomato. This was then followed by slicing 9 to 27 un-inoculated tomatoes. A similar and constant hand pressure on the slicer was used in every trial. Three slices from each tomato were collected for virus elution, concentration, and extraction before RT-PCR detection of MNV. The change in MNV per sliced tomato was averaged over all eight trials, and two mathematical models were fit to the average data using a logarithmic model or a power model. Regression analysis determined that the equation that best fit the data was y=-0.903∗ln(x)+7.945, where y=log10 MNV per slicing and x=tomato slicing number. An acceptable fit (R(2)=0.913) was indicated. The MNV levels transferred (y) generally decreased as the number of tomatoes sliced (x) increased, with some exceptions. Infrequent but erratic transfers, where the MNV level of a subsequent tomato was higher than that of a preceding tomato, occurred in later transfer of some trials. In contrast, the first and second transfers of each trial were always shown to have sharply decreased levels of MNV from the inoculum. The MNV log10 reduction per slicing event changes throughout the process: with a predicted 0.63log10 reduction from tomato 1 to tomato 2 (76% reduction); a 0.07log10 reduction predicted from tomato 13 to tomato 14 (a 14% reduction); and 0.03log10 reduction predicted from tomato 27 to tomato 28 (a 7% reduction). Virus transfer is clearly variable even given the consistent slicing procedure used throughout each trial. This study illustrates the complex nature of risk prediction associated with NoV cross-contamination during food preparation in commercial establishments.

Effective hepatitis A virus inactivation during low-heat dehydration of contaminated green onions.

Preserving fruits and vegetables by dehydration is common; however, information is limited concerning viral survival on the produce during the process. This work demonstrated the effects of low heat dehydration on inactivating hepatitis A virus (HAV) on contaminated green onions. Inoculated and uninoculated onion samples were dehydrated at target temperatures of 45-65 °C for 20 h. HAV from artificially contaminated onions (fresh or dehydrated) was eluted by shaking at 145 rpm at 20 °C for 20 min with 3% beef extract, pH 8, and followed by 0.2 µM-membrane filtration before plaque assay and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. Dilutions of the filtrates were made for obtaining countable plaques on FRhK-4 cell monolayers in 6-well plates, and also for eliminating inhibitors in qRT-PCR. Average water activity of the onions after 20 h-dehydration was 0.227, regardless of temperature used (47.9 °C or 65.1 °C). Eight dehydration trials resulted in a linear relationship between HAV inactivation and dehydration temperature, with HAV log reduction = 0.1372x(°C) - 5.5572, r(2) = 0.88. Therefore, the 20 h-heating at 47.8, 55.1, and 62.4 °C reduced infectious HAV in onions by 1, 2, and 3 logs respectively, the Z value being 7.3 °C. It was concluded that low heat dehydration using 62.5 °C or above could effectively inactivate HAV on contaminated onions by >3 logs.

Survival of hepatitis A virus in spinach during low temperature storage.

Spinach leaves are frequently consumed raw and have been involved with past foodborne outbreaks. In this study, we examined the survival of hepatitis A virus (HAV) on fresh spinach leaves in moisture- and gas-permeable packages that were stored at 5.4 +/- 1.2 degrees C for up to 42 days. Different eluents including phosphate-buffered saline (PBS), pH 7.5 (with and without 2% serum), and 3% beef extract (pH 7.5 and 8) were compared for how efficiently they recovered viruses from spinach by using a simple elution procedure (<1 h). The recoveries were compared and determined by a plaque assay with FRhK-4 cells. Culture grade PBS containing 2% serum was found to be appropriate for HAV elution from spinach leaves, with an average recovery of 45% +/- 10%. Over 4 weeks of storage at 5.4 +/- 1.2 degrees C, HAV in spinach decreased slightly more than 1 log, with 6.75% of the original titer remaining. HAV survived under refrigerated temperatures on spinach leaves with a D-value of 28.6 days (equivalent to an inactivation rate of -0.035 log of HAV per day, r(2) = 0.88). In comparison, HAV in PBS containing 2% serum under the same storage conditions remained constant throughout 7 weeks. The inactivation rate of -0.035 log each day for HAV on spinach leaves was possibly due to the interaction of the virus and the leaf.

Use of molecular epidemiology to confirm a multistate outbreak of hepatitis A caused by consumption of oysters.

The 39 oyster consumption-related cases of hepatitis A reported in 2005 represent the first large outbreak of hepatitis A associated with shellfish consumption in the United States in >15 years. This is the first outbreak investigation in which an identical hepatitis A virus sequence was obtained from both the implicated food product and case patients.

Molecular surveillance of enterovirus and norwalk-like virus in oysters relocated to a municipal-sewage-impacted gulf estuary.

An 18-month survey was conducted to examine the prevalence of enteric viruses and their relationship to indicators in environmentally polluted shellfish. Groups of oysters, one group per 4 weeks, were relocated to a coastal water area in the Gulf of Mexico that is impacted by municipal sewage and were analyzed for enteroviruses, Norwalk-like viruses (NLV), and indicator microorganisms (fecal coliform, Escherichia coli, and male-specific coliphages). The levels of indicator microorganisms were consistent with the expected continuous pollution of the area. Fourteen of the 18 oyster samples were found by reverse transcription (RT)-PCR to harbor NLV and/or enterovirus sequences. Of the four virus-negative oysters, three had exposure to water temperatures of >29 degrees C. Concomitant with these findings, two of these four oysters also accumulated the lowest levels of coliphages. PCR primers targeting pan-enteroviruses and the NLV 95/96-US common subset were utilized; NLV sequences were detected more frequently than those of enteroviruses. Within the 12-month sampling period, NLV and enterovirus sequences were detected in 58 and 42%, respectively, of the oysters (67% of the oysters tested were positive for at least one virus) from a prohibited shellfish-growing area approximately 30 m away from a sewage discharge site. Eight (4.6%) of the 175 NLV capsid nucleotide sequences were heterogeneous among the clones derived from naturally polluted oysters. Overall, enteric viral sequences were found in the contaminated oysters throughout all seasons except hot summer, with a higher prevalence of NLV than enterovirus. Although a high percentage of the oysters harbored enteric viruses, the virus levels were usually less than or equal to 2 logs of RT-PCR-detectable units per gram of oyster meat.

Detection of enteroviruses in shellfish by fluorogenic polymerase chain reaction integrated with 96-well microplate scanning.

A one-step procedure was developed to confirm viral targets by using a fluorometric 96-well microplate scanner following polymerase chain reaction (PCR). The fluorogenic PCR, integrated with fluorometric scanning, measured the end point fluorescence of viral PCR amplicon/probe hybrids and permitted the use of nonfluorogenic PCR conditions with addition of a Cy3 fluorophore-labeled linear probe for viruses. This linear probe generated higher ratios of viral signal-to-noise than a comparative beacon probe. Detection efficiency with a Cy3/quencher linear probe was comparable with Southern analysis at the level > or = 0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing < 0.27 PFU, the fluorometric measurements of the first-round PCR viral amplicon were not as sensitive as Southern analysis; however, equivalent sensitivities were achieved with fluorogenic nested PCR. Concentrates of 11 oyster samples exposed to municipal sewage were tested for enteroviruses; the fluorogenic detection correlated 100% with Southern analysis. This method using fluorometric scanning of viral amplicon is simple; it requires neither continuously monitoring equipment nor redesigning PCR primers; and it accurately detects enteroviruses in oyster sample concentrates in less time than classic spectrophotometry or Southern analysis.