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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.

Nanofabrication is a remarkably effective technique to create desirable nanoscale patterns. In this work, the effect of surface nanofabrication on altering virus adhesion to the substrates was examined. Arrays of nanoholes, 50 nm in diameter, 22 nm deep, and 100 nm in pitch distance, were created on silicon (Si) wafers by electron-beam lithography and reactive ion etching. MS2 coliphage, which is 26 ± 2 nm in diameter and is frequently used as a surrogate for human viruses, was applied to investigate the interaction between the virions and smooth or nanostructured Si surfaces. Scanning electron microscopy and atomic force microscopy along with surface wettability analyses revealed that the nanofabrication had the effect of reducing not only the number of viruses attached but also the strength of virus adhesion. These effects were ascribed to the presence of nanoholes, which were inaccessible to the virions due to the unique surface topographical parameters and the surface chemistry, resulting in the decrease of the overall solid contact area for MS2 attachment. The periodic spacing of the nanoholes also limited the unit landing area for MS2 particles, restricting the formation of MS2 aggregates and leading to the reduced amount of MS2 attachment. We anticipate that smart design of a surface’s chemical composition and nanostructure will offer a feasible solution to improve mitigations for controlling viral adhesion and transmission to and from food contact surfaces.

The transmission of foodborne viral gastroenteritis is associated with virus adhesion on food-contact surfaces. In this work, we aimed to identify the key features of a contact surface material that invoke preferential virus adhesion via nanoscopic and quantitative measurements by atomic force microscopy (AFM) on the nano-Newton level. The adhesion strength of MS2 (a virus surrogate) on the surfaces of glass, polyvinyl chloride (PVC), high-density polyethylene (HDPE) and highly ordered pyrolytic graphite (HOPG) was measured; in a neutral (pH 7) aqueous environment, positively charged, hydrophobic surfaces demonstrated stronger interactions. The strength of MS2 adhesion was twice higher on porous than non-porous surfaces. High-resolution imaging of MS2-inoculated PVC surfaces revealed that the 60−80 nm wide and 20−30 nm deep pores on PVC acted as nano-containers to retain and trap the virions, resulting in eight times faster adsorption and longer term retention of MS2 in the porous areas than in the smooth areas of the same substrate. Thus, in addition to choosing a material with low chemical affinity to virus particles, tailoring a material’s physical features with respect to a virion’s dimension can be an alternative way to reduce/interfere virus adhesion.

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.

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 (

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 ~ 8 log10 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 0.63 log10 reduction from tomato 1 to tomato 2 (76% reduction); a 0.07 log10 reduction predicted from tomato 13 to tomato 14 (a 14% reduction); and 0.03 log10 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.

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 ≥0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing

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.

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 (

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.

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°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.

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 levelor = 0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing0.27 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.

Hepatitis A is one of the major infectious diseases epidemiologically associated with worldwide shellfish consumption. Molecular detection using polymerase chain reaction (PCR) to detect hepatitis A virus (HAV) in contaminated shellfish can be hindered by low virus recoveries during the concentration process and by natural PCR inhibitors in shellfish. This study evaluated and modified two major steps of a processing procedure for virus concentration from oysters: acid adsorption-elution and solvent extraction. With the addition of second and third elutions, the acid adsorption-elution step doubled the recovery to 46% of HAV seeded initially. Extraction with chloroform or chloroform-butanol resulted in lower HAV detection limits by reverse transcription-PCR (RT-PCR)-oligoprobing than extraction with the fluorocarbon, Freon. These results led to the following modified procedure: HAV was acid adsorbed at pH 4.8, eluted first with 0.05 M glycine, second with 0.5 M threonine, PEG-precipitated twice, chloroform-extracted twice, RNA-extracted, and RT-PCR (single round) amplified. Using the modified procedure, HAV was detected by RT-PCR in all trials with a seeding density of > or = 1 plaque forming unit (PFU)/g of oyster, and in which the equivalent detection limit was 0.33 PFU of HAV seeded per RT-PCR reaction (corresponding to 111 PCR units). The method developed is capable of detecting low levels of HAV in oysters environmentally contaminated.

In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.

Direct isolation and identification of pathogenic viruses from oysters implicated in gastroenteritis outbreaks are hampered by inefficient methods for recovering viruses, naturally occurring PCR inhibitors, and low levels of virus contamination. In this study we focused on developing rapid and efficient oyster-processing procedures that can be used for sensitive PCR detection of viruses in raw oysters. Poliovirus type 3 (PV3) Sabin strain was used to evaluate the efficacy of virus recovery and the removal of PCR inhibitors during oyster-processing procedures. These procedures included elution, polyethylene glycol precipitation, solvent extraction, and RNA extraction. Acid adsorption-elution in which glycine buffer (pH 7.5) was used was found to retain fewer inhibitors than direct elution in which glycine buffer (pH 9.5) was used. RNA extraction in which a silica gel membrane was used was more effective than single-step RNA precipitation for removing additional nonspecific PCR inhibitors. The final 10-μl volume of RNA concentrates obtained from 2 g of oyster tissue (concentration factor, 200-fold) was satisfactory for efficient reverse transcription-PCR detection of virus. The overall detection sensitivity of our method was 1 PFU/g of oyster tissue initially seeded with PV3. The method was utilized to investigate a 1998 gastroenteritis outbreak in California in which contaminated oysters were the suspected disease transmission vehicle. A genogroup II Norwalk-like virus was found in two of three recalled oyster samples linked by tags to the harvest dates and areas associated with the majority of cases. The method described here improves the response to outbreaks and can be used for rapid and sensitive detection of viral agents in outbreak-implicated oysters.