Extraction of food-borne viruses from food samples: A review

International Journal of Food Microbiology 153 (2012) 1–9

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Review

Extraction of food-borne viruses from food samples: A review Ambroos Stals a,⁎, Leen Baert b, Els Van Coillie a, Mieke Uyttendaele b a

Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium Ghent University, Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Microbiology and Food Preservation, Coupure Links 653, 9000 Ghent, Belgium

b

a r t i c l e

i n f o

Article history: Received 21 June 2011 Received in revised form 14 October 2011 Accepted 24 October 2011 Available online 6 November 2011 Keywords: Food-borne virus Enteric virus Virus extraction Food Controls

a b s t r a c t Detection of food-borne viruses such as noroviruses, rotaviruses and hepatitis A virus in food products differs from detection of most food-borne bacteria, as most of these viruses cannot be cultivated in cell culture to date. Therefore, detection of food-borne viruses in food products requires multiple steps: first, virus extraction; second, purification of the viral genomic material (RNA for the majority of food-borne viruses); and last, molecular detection. This review is focused on the first step, the virus extraction. All of the numerous published protocols for virus extraction from food samples are based on 3 main approaches: 1) (acid adsorption–) elution–concentration; 2) direct RNA extraction; and 3) proteinase K treatment. This review summarizes these virus extraction approaches and the results obtained from published protocols. The use of process controls is also briefly described. © 2011 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . Detection strategy . . . . . . . . . . . . . . . . . Virus extraction . . . . . . . . . . . . . . . . . . 3.1. (Acid adsorption–) elution–concentration . . . 3.1.1. Virus particle elution . . . . . . . . 3.1.2. Concentration of eluted viral particles 3.2. Direct RNA extraction . . . . . . . . . . . . 3.3. Proteinase K treatment . . . . . . . . . . . 4. Purification virus eluate/concentrate or extracted RNA 5. Quality control . . . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Enteric, human pathogenic food-borne viruses such as noroviruses (NoV), rotaviruses, (RoV), hepatitis A and E viruses (HAV and HEV, respectively) require different detection methods than those appropriate for food-borne bacterial pathogens. Unlike most food-borne bacteria, viruses cannot grow in the environment since they need specific host cells to replicate (Koopmans and Duizer, 2004).

⁎ Corresponding author. Tel.: + 32 9 272 30 26; fax: + 32 9 272 30 00. E-mail address: [email protected] (A. Stals). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.10.014

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However, as most food-borne viruses are unenveloped, they do show a high resistance to environmental stressors, such as heat, high or low pH, drying, light and UV exposure (Baert et al., 2009a; Vasickova et al., 2010). This persistence allows them to remain infective in foods for periods from 2 days to 4 weeks (Bidawid et al., 2001; Hewitt and Greening, 2004; Butot et al., 2008). Additionally, most food-borne viruses have supposedly very low infectious doses of 10–100 infectious viral particles (Teunis et al., 2008). Sensitive methods are therefore needed when screening food products for the presence of food-borne viruses. To date, cultivation of most foodborne viruses is still not possible in cell culture. Therefore, detection of these viruses in foods currently relies upon the use of molecular

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A. Stals et al. / International Journal of Food Microbiology 153 (2012) 1–9

methods. In general, the strategy for detection of food-borne viruses in food samples consists of 3 steps: 1) virus extraction, 2) purification of the viral genomic material and 3) molecular detection. Virus extraction from food matrices can be defined as the separation and concentration of (the genomic material of) virus particles from the food matrix. Despite the great number of published protocols for extraction of viruses from food, only a small number of approaches are used. The current review summarizes these approaches and the results obtained by the various protocols. A review of the strategies for sampling and detection of enteric viruses in food samples was recently published (Bosch et al., 2011) and a number of detection protocols have also been listed (Mattison and Bidawid, 2009). The current review contributes a detailed guide to extracting food-borne viruses from a broad range of food matrices. D'Agostino et al. (2011) reviewed the strategies for correct use and interpretation of process controls when detecting enteric viruses in foods; the current review adds an overview of quality controls specific for virus extraction.

shellfish digestive system. This phenomenon can be explained by the ability of bivalve shellfish to filter large volumes of water as part of their feeding activities (Le Guyader et al., 2009). A variety of protocols for virus extraction have been described. These can be grouped into 3 main approaches: 1) elution of the viral particles (whether or not preceded by an acid adsorption step) with a subsequent concentration; 2) direct extraction of the viral RNA from the food matrix, which excludes the elution–concentration step; and 3) extraction of viruses from the food via proteinase K treatment (Table 1). These approaches have successfully been applied to detect food-borne viruses in food samples related to viral foodborne outbreaks (Table 2). Tables 3 and 4 provide an overview of detection limits and recovery efficiencies of virus extraction methods obtained for diverse food matrices. Detection limits for NoV when a molecular method was used are expressed as RNA copies or RT-PCR units (RT-PCRU) per analyzed mass (in grams). An RT-PCRU is the lowest amount of viral genomic material that can be detected when using RT-PCR for detection of NoV and can thus differ between different assays.

2. Detection strategy Most food-borne viruses still cannot be cultivated efficiently in vitro (Duizer et al., 2004; Koopmans and Duizer, 2004; Straub et al., 2007). Cloning of food-borne virus genomes in the late 1980s and early 1990s (Cohen et al., 1987; Xi et al., 1990; Tam et al., 1991; Lambden et al., 1992; Matsui et al., 1993; Ketner et al., 1994) led to development of assays for the detection of these viruses based on molecular methods in addition to enzyme immune assays (EIA) and electron microscopy. Although the genome of most food-borne viruses such as NoV, HAV, RoV, Aichivirus and enterovirus consists of single- or double-stranded RNA, adenoviruses (AdV) have a doublestranded DNA genome (Koopmans and Duizer, 2004). This review focuses on food-borne RNA viruses. The general strategy for the detection of food-borne viruses in food samples consists of 3 steps: 1) virus extraction, 2) purification of the viral RNA and 3) molecular detection of the purified RNA. During the first step (virus extraction), viral particles are separated from the food matrix, concentrated to a small volume, and inhibitory compounds are removed. During virus extraction, molecules such as polysaccharides, proteins and fatty acids are removed to prevent inhibition of the subsequent RNA purification and molecular detection (Rijpens and Herman, 2002; Schwab and McDevitt, 2003; Escobar-Herrera et al., 2006; Demeke and Jenkins, 2010). The viruses also need to be concentrated because they generally only occur in very low levels on foods. Naturally contaminated shellfish samples have been known to contain food-borne virus levels ranging between 10 2 and 10 4 viral genomic copies per gram digestive tissue (Costafreda et al., 2006; Le Guyader et al., 2006; Nishida et al., 2007; Le Guyader et al., 2009). During virus detection, false negative results, false positives, or both can occur. False negative results are caused by inhibition and false positives can occur because of cross-over contamination. The risk for cross-over contamination rises when using a highly sensitive molecular method (Rijpens and Herman, 2002). For this reason, appropriate positive and negative controls should be included.

3.1. (Acid adsorption–) elution–concentration Elution–concentration protocols are based on washing the viral particles from the food surface using an appropriate buffer followed by concentration of the eluted viruses. The elution step can be preceded by an acid adsorption step. The principle of acid adsorption as part of a virus extraction method for NoV in foods dates back to Sobsey et al. (1975, 1978). Their technique for the extraction of enteroviruses from oyster tissue involves adsorption of viral particles to the oyster tissue by addition of an acid buffer (pH 5–6) while lowering NaCl concentration under 25 mM. In the next step (elution), the supernatant is discarded and the viruses are eluted from the oyster tissue pellet using either a more acidic or a neutral glycine–PBS buffered solution. The (acid adsorption–) elution–concentration approach is used for extraction of food-borne viruses from a broad range of carbohydrate/ water-based foods, fatty/protein-based foods, and shellfish. An overview of results obtained using different (acid adsorption–) elution– concentration protocols is shown in Table 3. Generally, the elution step is similar in most protocols, as they all use a neutral or basic elution buffer. The concentration methods vary widely, however. They include polyethylene glycol (PEG) precipitation, ultracentrifugation, ultrafiltration, immunoconcentration, and cationic separation. 3.1.1. Virus particle elution In most cases, an alkaline buffer with a pH between 9 and 10.5 is used to elute the viral agents from the food surface. The alkaline environment allows the viral particles to detach from the food matrix. An acidic medium, on the other hand, encourages the viral particles' binding to food surface. The latter can impair virus elution and Table 1 Approaches for the extraction of food-borne viruses from food linked to different food categories. Food categories

3. Virus extraction Methods for the extraction of viruses from food depend on the food composition. According to Baert et al. (2008), 3 main food categories can be distinguished. The first category is composed of carbohydrate and water-based foods, mainly fruits and vegetables. The second category includes fat- and protein-based foods, principally ready-to-eat products such as deli food products. Shellfish are considered as a third and separate food category due to the accumulation and concentration of viral particles and other pathogens in the

Shellfish Fat/protein Water/carbohydrate based foods based foods Virus extraction (Acid approaches adsorption) Elution Concentration Direct RNA extraction Proteinase K treatment

X

X

X

X

X

X

A. Stals et al. / International Journal of Food Microbiology 153 (2012) 1–9

3

Table 2 Viral food-borne outbreaks in which viral agents were recovered from food samples. Food products

Virus

Virus extraction method

Reference

Raspberries (in preparations) Clams Oysters Blueberries Potato stew Buffet food (ham, salami) Buffet food (ham off the bone) Take-out food (sparerib) Raspberries Oysters Oysters Oysters Oysters Oysters (French) Oysters (frozen half shelled) Mussels Raspberries Salad vegetables Lettuce

NoV GIIb NoV GII NoV GI.2 HAV RoV NoV GIIb NoV GII.4 2004 NoV GIIb NoV GII NoV GI.I, GII.3 NoV GII.4 NoV GI, GII.4 NoV GI.I NoV GI.4, GII.4, GII.8 NoV GII NoV GI, GII NoV GI.4 NoV GII NoV GII

Alkaline elution–PEG concentration Alkaline elution –PEG concentration Neutral elution–PEG concentration Elution–concentration (ns) Neutral elution–ultrafiltration Direct RNA extraction Direct RNA extraction Direct RNA extraction Direct RNA extraction (food swabs) Direct RNA extraction Proteinase K treatment Proteinase K treatment Proteinase K treatment Proteinase K treatment nsa Ns Ns Ns Ns

(Le Guyader et al., 2004a) (Kingsley et al., 2002) (David et al., 2007) (Calder et al., 2003) (Mayr et al., 2009) (Boxman et al., 2007) (Boxman et al., 2007) (Boxman et al., 2007) (Verhoef et al., 2008) (Nenonen et al., 2009) (Webby et al., 2007) (Le Guyader et al., 2008) (Le Guyader et al., 2003) (Le Guyader et al., 2006) (Ng et al., 2005) (Prato et al., 2004) (Maunula et al., 2009) (Oogane et al., 2008) (Ethelberg et al., 2010)

a

ns: not specified.

consequently reduce detection sensitivity (Traore et al., 1998; Dubois et al., 2002). The action of the acidic medium can also be used for acid adsorption of viral particles on the food surface. When anionic exchange or ultracentrifugation is used to concentrate the eluted viral particles, a neutral buffer is used (Rutjes et al., 2006; Rzezutka et al., 2008; Fumian et al., 2009; Morales-Rayas et al., 2009; Morales-Rayas et al., 2010). As many food products (particularly fruits and vegetables) contain acidic substances, an alkaline tris-based buffer system is usually applied (Sincero et al., 2006; Baert et al., 2008; Cheong et al., 2009; Scherer et al., 2010; Stals et al., 2011a). However, other elution buffers have been described. First, a phosphate buffer (pH 7.6) has been used to elute HAV from lettuce and fresh strawberries (Bidawid et al., 2000), NoV from rolled cabbage and macaroni (Kobayashi et al., 2004), canine calicicvirus (CaCV) from whipped cream (Rutjes et al., 2006) and HAV and NoV from oysters (Le Guyader et al., 1998). Second, a sodium bicarbonate buffer (1 M) has been used to recover poliovirus from soft fruits and salad vegetables (Kurdziel et al., 2001). Beef extract and glycine are frequently used in combination with elution buffers, as they are able to reduce non-specific virus adsorption to the food matrix during their extraction (Dubois et al., 2002). Furthermore, the high protein concentration of beef extracts facilitates flocculation of NoV on PEG molecules (Kim et al., 2008). Concentrations of 1% beef extract (w/v) (Dubois et al., 2007; Blaise-Boisseau et al., 2010) and 3% (w/v) (Love et al., 2008; de Paula et al., 2010) have been described. The combined use of 1% (w/v) beef extract and alkaline elution resulted in a 7-fold, 3.5-fold and 5.7-fold increase in the recovery of HAV, NoV and RoV from raspberries, respectively (Butot et al., 2007). However, beef extract might also interfere with molecular detection methods, which may result in false negative results (Traore et al., 1998; Schwab et al., 2000; Katayama et al., 2002; Schwab and McDevitt, 2003). Therefore, a virus eluate purification step can be necessary. Regarding the use of glycine, a 0.05 M concentration has been used most often (Baert et al., 2008; Cheong et al., 2009; Scherer et al., 2010; Stals et al., 2011a). Pectinase is also frequently added to the elution buffers when extracting viruses from carbohydrate/water based foods, as it prevents jelly formation in the eluate by breaking the pectin bonds in fruits and vegetable matrices (Dubois et al., 2002). Soya protein powder has been reported to facilitate the liberation of viruses from food surfaces when using ultracentrifugation for extraction of viruses from foods. This improved the recovery efficiency at least 10-fold (Rzezutka et al., 2006). Finally, Cat-floc® has been used to improve flocculation of food solids. The aforementioned components and conditions of the elution buffer are summarized in Table 5.

3.1.2. Concentration of eluted viral particles 3.1.2.1. Polyethylene glycol (PEG) precipitation. Precipitation of the viruses after elution is frequently used to increase the concentration of the virus extract for successful molecular detection. Polyethylene glycol (PEG) is often used for this purpose, as it easily allows the precipitation of these viruses at neutral pH and at high ionic concentrations without precipitation of other organic material (Table 6) (Lewis and Metcalf, 1988; Baert et al., 2008; Kim et al., 2008; Stals et al., 2011a). The molecular weight of the PEG molecules (from 6000 to 20 000 Da) did not significantly influence the recovery of NoV from strawberries or raspberries (Kim et al., 2008). Most studies have described PEG concentrations varying between 8 and 16% (w/v). The use of PEG to precipitate virus particles requires a high NaCl concentration. Early studies suggested final concentrations of 0.4 M NaCl (Yamamoto et al., 1970; Adams, 1973), but most current studies use either 0.3 M (Park et al., 2008; Cheong et al., 2009; Stals et al., 2011a) or 0.525 M (Kingsley and Richards, 2001; Guevremont et al., 2006). After alkaline elution, most authors adjust the pH of the virus eluates to 7.0–7.4, as this is expected to improve the PEG precipitation of the virus particles (Leggitt and Jaykus, 2000; Baert et al., 2007; Cheong et al., 2009; Stals et al., 2011a). Combining alkalic or neutral elution and PEG precipitation protocols with a molecular technique generally results in virus recoveries between 5 and 90%, depending on the food matrix and applied protocols. For soft red fruits, this combination resulted in efficiencies of 7.4%–61.1% and 20.7–47.7% when recovering 10 4 and 10 6genogroups I (GI) and II (GII) NoV genomic copies per 10 g, respectively (Stals et al., 2011a). However, a study comparing different aspects of the NoV alkalic elution–PEG concentration method showed that 85% recovery of 4 × 10 4 GII NoV RT-PCRU from 20 g of fresh strawberry samples was possible (Kim et al., 2008). Cheong et al. (2009) obtained 3.9% to 50% recoveries when extracting 4.8 to 4.8 × 10 3 GII NoV RT-PCRU from 5 g of strawberries, while recoveries of 13% have been observed when extracting 10 to 10 3 RT-PCRU from 30 to 100 g of fresh strawberries and raspberries. By combining acid adsorption, alkaline elution and PEG precipitation, Love et al. (2008) were able to successfully extract 4.2 × 10 3 HAV RT-PCRU from tomato sauce. Alkalic elution combined with PEG precipitation has been selected by the CEN/TC275/WG6/TAG4 working group as the preferred method when extracting NoV and HAV from produce and soft fruits (Croci et al., 2008). Alkalic elution combined with PEG precipitation has been used to confirm raspberries as source of a NoV food-borne outbreak (Le Guyader et al., 2004a) (Table 2).

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A. Stals et al. / International Journal of Food Microbiology 153 (2012) 1–9

Table 3 Detection limits and recovery efficiencies of (acid adsorption–) elution–concentration virus extraction methods for diverse food matrices. Protocol Elution Alkaline buffer

Concentration PEG

a

Food matrix (weight) Raspberries, forest fruit mix, strawberry puree (10 g) Mixed lettuce, fruit salad, raspberries (10 g) Strawberries, lettuce (25–100 g) Berries & vegetables (30–100 g)

Green onions (25 g) Strawberries (20 g) Tomato sauce, strawberries (30 g)b Lettuce (50 g) Lettuce (8 g) Oysters, clams (25 g) Mussels (2 g) Mussels (2 g) Whipped cream (5 g) Ham (10 g) Hamburger (50 g)

Alkaline buffer

Neutral buffer

Alkaline/neutral buffer

Ultrafiltration

Various berries/vegetables (15 g)

Ultracentrifugation

Ham (10 g) Lettuce (10 g) Raspberries (10 g) Raspberries, strawberries (60 g)

Cationic separation

Immunoconcentration a b c d

Lettuce (5 g) Oysters (25 g) Roast pork chop, salami, gammon (20 g) Lettuce, strawberries, green onions (25 g) Oysters (5 g) Deli turkey (25 g) Cake (25 g) Lettuce, strawberries, green onions, raspberries (50 g) Lettuce (15 g) Minas Cheese (15 g) Lettuce, green onions, strawberries (25 g) Deli ham, lettuce, green onions, strawberries (25 g)d

Viral agent

Recovery

NoV GI NoV GII NoV GII

7.4%–61.1% 20.7–47.7%

NoV HAV PV NoV HAV NoV GII NoV GI NoV GII HAV NoV NoV NoV HAV NoV GII HAV CaCV NoV GII NoV HAV PV NoV HAV RoV NoV GII

2.9%–3.4% 17% 45% 13%

NoV GI HAV CaCV HAV FCV

NoV

4

(Stals et al., 2011a)

48 RT-PCRU 40 TCID50 44 TCID50 1.2 × 103 RT-PCRU 1 TCID50 1 RT-PCRU

(Cheong et al., 2009) (Dubois et al., 2002)

(Baert et al., 2008)

(Guevremont et al., 2006) (Park et al., 2008)

14–33 PFU 1.5 × 103 RT-PCRU 10 RT-PCRU 22.4 RT-PCRU50c 0.015 PFU 20–100 RT-PCRU 10 RT-PCRU

(Love et al., 2008) (Leggitt and Jaykus, 2000) (Le Guyader et al., 2004b) (Kingsley and Richards, 2001)

1.5 × 103 RT-PCRU 1 × 103 PFU 2 × 102 PFU 54 RT-PCRU 1.2 TCID50 0.21 TCID50 2 × 102 RT-PCRU 2 × 102 RT-PCRU 2 × 103 RT-PCRU

(Leggitt and Jaykus, 2000)

10% 10%

7% 9% 3% 10%

Reference

3.99 × 10 RNA copies 9.63 × 104 RNA copies 102 RT-PCRU

29.5% 14.1%

103 RT-PCRU 10% 9.9% 3.4–12.5% 2 × 100–102 RT-PCRU

HAV HAV HAV HAV NoV GII HAV MNV-1 NoV GII NoV GII NoV

Detection limit

(Baert et al., 2007) (Sincero et al., 2006) (Rutjes et al., 2006)

(Butot et al., 2007)

(Scherer et al., 2010)

(Rzezutka et al., 2005; Rzezutka et al., 2006) (Rutjes et al., 2006) (Casas et al., 2007) (Rzezutka et al., 2008) (Papafragkou et al., 2008)

1

5 - 16% 12–33% 5.2–72.3% 6.0–56.3%

2 × 10 RT-PCRU 2 × 100 RT-PCRU 2 × 102 RT-PCRU 102 RT-PCRU 102 RT-PCRU 10 PFU 9.5 × 103 RNA copies 4.1 × 104 RNA copies 10 RNA copies 102–104 RNA copies

(Morales-Rayas et al., 2009)

(Fumian et al., 2009) (Morton et al., 2009) (Morton et al., 2009)

PEG: polyethylene glycol. Elution preceded by an acid adsorption step. Level whereby 50% of inoculations could be recovered. Immunoconcentration step preceded by an acid adsorption step.

For fat/protein based foods, successful recovery of 10 3 to 104 HAV, PV and NoV RT-PCRU from hamburger was possible using the concentration approach in combination with an alkaline or neutral elution buffer, and recovery efficiencies of 10 and 24% have been observed when detecting canine calicivirus (CaCV) and NoV in whipped cream and ham, respectively (Leggitt and Jaykus, 2000; Rutjes et al., 2006; Scherer et al., 2010). Moreover, a study comparing 2 concentration methods after neutral elution showed significantly better recovery of NoV from 10 g ham samples when using PEG precipitation (24% recovery; detection limit 2 × 101 RT-PCRU/10 g) compared to ultrafiltration (7% recovery; detection limit 2 × 102 RT-PCRU/10 g) (Morales-Rayas et al., 2010). In shellfish, the elution–PEG precipitation approach has resulted in varying recoveries when detecting NoV from either whole shellfish or shellfish digestive tissue. Some studies report successful recovery

of 5 to 22.4 NoV RT-PCRU in 1.5 g to 25 g of shellfish tissue (Atmar et al., 1995; Kingsley and Richards, 2001) while other authors report higher detection limits: up to 3 × 10 3 RT-PCRU per 1.25 g of oyster digestive tissue (Hafliger et al., 1997). For HAV, successful recovery of 10 RT-PCRU has been reported from oyster digestive tissue (Sincero et al., 2006). This approach has been used to show the prevalence of AdV and NoV in Moroccan mussels and French oysters (Beuret et al., 2003; Karamoko et al., 2005). Alkaline and neutral elution combined with PEG precipitation has been applied for confirmation of oysters and clams as the sources of 2 food-borne NoV outbreaks (Kingsley et al., 2002; Calder et al., 2003) (Table 2). 3.1.2.2. Ultracentrifugation. Ultracentrifugation has been used to concentrate HAV and NoV particles eluted from shellfish, carbohydrate/ water based foods and fat/protein based foods, although fewer assays

A. Stals et al. / International Journal of Food Microbiology 153 (2012) 1–9 Table 4 Detection limits and recovery efficiencies of non elution–concentration virus extraction methods for diverse food matrices. Protocol Direct RNA extraction

Viral agent

Detection limit

Reference

Concentration method

Advantage

Disadvantage

Penne salad (10 g)

NoV GI

(Stals et al., 2011b)

Polyethylene glycol precipitation Ultracentrifugation

Cheap Easy to perform Consistent results

Ultrafiltration

Removal of RT-PCR inhibitors

Immunoconcentration

High specificity

Cationic separation

Automatization

pH neutralization of virus eluate necessary Requires virus eluates free of vegetable matter Need for specialized equipment/personnel Requires virus eluates free of vegetable matter Long-term use unsure due to immunogenetic drift Need for specialized equipment Inconsistent results

Oysters (0.15 g)

NoV

104 RNA copiesa 104 RNA copiesa 10 RT-PCRU

Oysters (5–50 g)

HAV

8 PFU

Hamburger, turkey, roast beef (30 g) Penne tagliatelle salad (10 g) Deli ham (10 g)

NoV/ HAV NoV

102 RT-PCRU 102 RT-PCRU

NoV/PV

1 RT-PCRU

Proteinase K Oysters (n ≥ 6) treatment Oysters (1.5 g)

NoV NoV GI NoV GII

b

Table 6 Advantages and disadvantages of diverse methods used to concentrate eluted virus particles.

Food matrix (weight)

NoV GII

a

5

102 RNA copiesb 102 RNA copiesb

(de Roda Husman et al., 2007) (Cromeans et al., 1997) (Schwab et al., 2000) (Baert et al., 2008) (Boxman et al., 2007) (Jothikumar et al., 2005) (Le Guyader et al., 2009)

Observed recovery efficiencies: 1.0–49.0% (NoV GI) and 3.3–11.8% (NoV GII). Observed recovery efficiencies: 20.5 ± 14.7% (NoV GI) and 33.6 ± 5.3% (NoV GII).

have been described compared to PEG precipitation. For fresh strawberries and raspberries, ultracentrifugation resulted in somewhat lower recoveries of 0.1% (NoV) and 2.5% (HAV) in comparison to PEG precipitation (Rzezutka et al., 2005, 2006). Additionally, ultracentrifugation has been proposed for detection of viruses from lettuce as a 10% recovery of CaCV was observed on this food matrix (Rutjes et al., 2006). Similarly, recovery efficiencies of 10% for HAV were observed in 25 g of oysters (Casas et al., 2007), along with recovery efficiencies of 3.4%, 5.9% and 12.5% for feline calicivirus (FCV) in salami, smoked ham, and roast pork chop, respectively (Rzezutka et al., 2008). Ultracentrifugation protocols require additional purification of virus eluates, as debris and other components originating from the food samples can interfere with the ultracentrifugation protocols. This purification can include either high speed conventional centrifugation or 0.22/0.45 μm pore filtration. During ultracentrifugation the viral particles are precipitated by centrifugational forces up to 120 000 × g and 235 000 × g of the filtered virus eluates (Rutjes et al., 2006; Rzezutka et al., 2006; Croci et al., 2008). This concentration technique has the advantage of being very consistent but has several disadvantages as well. The equipment is expensive; (hard) fruit/vegetable matter must be eliminated from the virus eluates (Table 6) (Croci et al., 2008); and it may result in Table 5 Elution buffers and additional components used for virus extraction. Component/condition

Detail

Function

Alkaline–neutral pH

pH 9.5 to 10.5 (alkaline) or pH 7 (neutral) Tris(− HCl) buffer Phosphate buffer

Detaches viral particles from food surface Prevents pH reduction caused by acidic fruit/ vegetable juices Facilitates flocculation of NoV on PEG Reduces non-specific adsorption of protein or virus Prevents jelly formation of the eluate Improves flocculation of food solids Facilitates liberation of viruses from food surfaces

pH buffer system

Beef extract

1% to 3% (w/v)

Glycine

0.05 M to 0.5 M

Pectinase

180 U/300 ml to 570 U/30 ml

Cat-floc® TL Soya protein powder

1% (w/v)

voluminous pellets that can be difficult to dissolve (Rutjes et al., 2006; Rzezutka et al., 2006). 3.1.2.3. Ultrafiltration. Ultrafiltration concentrates viral particles based on molecular weight (Croci et al., 2008). Filters equipped with membrane pores permit the passage of liquids and low molecular mass particles (less than 50 to 100 kDa) and the viruses are captured by the filter (Le Guyader et al., 2004b). Similar to ultracentrifugation, the virus eluates must undergo additional purification to prevent clogging of the filters. One advantage of ultrafiltration is that RTPCR inhibitory components are not co-isolated with the viral particles (Table 6) (Rutjes et al., 2005). Recovery can be increased by treating the filters with bovine serum albumin (BSA) or by sonication of the purified virus eluate (Jones et al., 2009). Scherer et al. (2010) report that the recovery of NoV from lettuce, raspberries, and ham using ultrafiltration as concentration method was 9%, 3% and 7%, respectively, but PEG precipitation resulted in significantly higher recoveries of 23%, 7% and 24%, respectively. Using an ultrafiltration-based technique, recoveries from 1.7% to 19.6% were found in fresh strawberries, frozen raspberries, frozen blueberries, and fresh raspberries (Butot et al., 2007). In contrast to Scherer et al., these authors found a significantly higher recovery of NoV from raspberries and strawberries using ultrafiltration compared to PEG precipitation. Ultrafiltration has also resulted in a 0.1 to 1% NoV recovery for lettuce (Rutjes et al., 2006). In a RoV food-borne outbreak, ultrafiltration combined with a neutral elution was successfully used as virus extraction step to detect RoV in potato stew sample responsible for a food-borne outbreak (Mayr et al., 2009) (Table 2). 3.1.2.4. Other concentration methods. Other strategies for concentration of eluted viruses from foods include cationic separation and immunoconcentration. During NoV immunoconcentration, magnetic beads are covered either with histo-bloodgroup antigens (HBGA) types A, B, H(2) and H(3) or with type III porcine gastric mucin to bind NoV particles after eluting them from food samples. The bound NoV are subsequently eluted from the magnetic beads using an appropriate buffer (Tian and Mandrell, 2006; Park et al., 2008; Tian et al., 2008; Morton et al., 2009). For HAV detection, magnetic beads covered with monoclonal (K3-2F2) HAV antibodies have been used (Bidawid et al., 2000). This strategy has successfully been tested on lettuce, green onions, strawberries, and ham (Bidawid et al., 2000; Morton et al., 2009). GI and GII NoV were recovered from strawberries with efficiencies of 14.1% and 29.5%, respectively (Park et al., 2008) and this approach has been used to detect NoV in rolled cabbage and macaroni in a NoV food-borne outbreak (Kobayashi et al., 2004). While immunomagnetic capture allows very specific extraction of food-borne viruses in various food types (oysters, produce) and enables efficient removal of PCR inhibitors, questions could be

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raised about the long-term use of antibodies due to the immunogenetic drift of these food-borne viruses (Table 6). Cationic separation is based on the hypothesis that positively charged magnetic particles could be used in conjunction with a magnetic capture system to concentrate and purify viral particles from food matrices. The purification of the viral agent is believed to occur because the negatively charged proteins of the virus capsid bind to the positively charged magnetic particles. Fumian et al. (2009) showed recovery efficiencies of 5.2% to 56.3% using a filter-based cationic method for NoV in lettuce and Minas cheese. An automated cationic separation system named Pathatrix TM (Matrix MicroScience, Newmarket, UK) is commercially available and has been tested with varying success. Promising results have been reported when using Pathatrix TM to concentrate HAV from virus eluates of lettuce, green onions, strawberries, deli-turkey, oysters, and cake, with recovery efficiencies ranging between 17.0 and 81.7% (Papafragkou et al., 2008). However, less consistent results were obtained for NoV recovery from lettuce, strawberries, raspberries and green onions, both in combination with an immunoconcentration step and without that step (Morton et al., 2009; Morales-Rayas et al., 2010). Cationic separation has similar disadvantages to ultracentrifugation as it requires expensive equipment and specialized personnel (Table 6). 3.2. Direct RNA extraction Direct viral RNA extraction techniques involve treatment of the food product with a guanidinium isothiocyanate (GITC)/phenolbased reagent, followed by purification of the extracted RNA. Direct RNA extraction has been successfully applied on fat/protein-based foods, as 1 to 10 2 NoV RT-PCRU could be recovered in 10 to 30 g of hamburger, turkey, roast beef, penne, tagliatelle, and deli ham (Table 4) (Schwab et al., 2000; Boxman et al., 2007; Baert et al., 2008). In a variety of 10 g ready-to-eat foods, detection of 10 6 NoV GI/GII genomic copies was consistently possible, while 10 4 NoV GI/ GII genomic copies could only be occasionally detected (Stals et al., 2011b). Successful recovery of 10 RT-PCRU in 0.15 g of oyster digestive tissue has been observed from direct RNA extraction combined with shredding of the digestive tissue using zirconium beads (Table 2) (de Roda Husman et al., 2007). This approach has also been used to demonstrate NoV presence in oysters involved in a food-borne NoV outbreak (Table 2; (Boxman et al., 2006). However, Baert et al. (2007) demonstrated that an elution–concentration protocol was more sensitive than a direct RNA extraction protocol when detecting NoV GII in mussel digestive tissue. Nevertheless, Nenonen et al. (2009) have used the direct RNA extraction approach as virus extraction method of choice to confirm oysters as source of a NoV foodborne outbreak (Table 2). 3.3. Proteinase K treatment A recent method combining proteinase K and heat treatment at 65 °C (Jothikumar et al., 2005; Comelli et al., 2008) has been selected by the CEN/TC275/WG6/TAG4 working group for the extraction of the most common enteropathogenic viruses from shellfish digestive tissue (Lees, 2010). This enzymatic/thermal treatment damages the viral capsid, thereby releasing the nucleic acids (Nuanualsuwan and Cliver, 2002). Until now, this approach has only been tested on shellfish foods (Table 4). This approach has proven its reliability during various NoV food-borne oubreaks related to shellfish. (Table 2) 4. Purification virus eluate/concentrate or extracted RNA In most protocols, purification (removal of food debris and inhibitory substances) of the eluted or concentrated viruses is performed through either filtration or treatment with Freon 113, Vertrel® XF

or chloroform:butanol. This is particularly important when using a molecular detection method after purification. Due to its physical and chemical properties, Freon 113 (1,1,2trichloro-1,2,2-trifluoroethane) is able to extract lipids and lipid bilayers without extracting proteinaceous (polar) material such as non-enveloped viruses (Rieht et al., 1993; Liebermann and Mentel, 1994; Kobayashi et al., 2004). This reagent has therefore been used for the purification of eluted viruses in food, but use of Freon has been progressively eliminated because of its implications in ozone layer depletion and accompanying environmental concerns (Taylor, 1996). A less damaging Freon substitute developed by DuPont called Vertrel® XF (1,1,1,2,3,4,4,5,5,5-decafluoropentane) has provided similar results when purifying NoV RNA from fecal samples (Mendez et al., 2000). Its use has successfully been evaluated in several protocols for virus extraction from fat/protein based foods (Papafragkou et al., 2008; Fumian et al., 2009). The use of Vertrel® XF has not yet been investigated in protocols for virus extraction in shellfish. A second alternative for Freon is RNA purification by organic solvents such as a chloroform:butanol mixture, which produced better results than Freon when recovering HAV from oysters (Mullendore et al., 2001). This purification method has been used for direct RNA extraction protocols in shellfish and when detecting NoV from carbohydrate/water-based foods (Baert et al., 2007, 2008; Stals et al., 2011a). Finally, filter-purification of the eluent has been performed by filtering through cheesecloth (Leggitt and Jaykus, 2000) or through 0.45 μm and 0.20 μm filters (Scherer et al., 2010). 5. Quality control In general, quality control of methods for detection of viruses in food samples implies the use of adequate controls throughout the different steps that are considered critical for correct detection. For molecular detection, the use of negative and positive controls has been reviewed elsewhere (Rijpens and Herman, 2002; Hoorfar et al., 2003, 2004). As virus extraction is often performed by lengthy and complex (in-house) methods, process controls might be required to assure the reliability of obtained results. A process control should be used to indicate the effect of the food matrix on the virus extraction efficiency. This may be very useful due to the great variety of foods at risk for viral contamination. At the most basic level, process controls consist of adding a control virus to a (parallel) tested sample. Process controls can be added to the same sample as internal process controls (IPC) or in a parallel analyzed sample within the same setup as an external process control (EPC) (Croci et al., 2008). The use of an IPC is more labor-intensive as it requires the addition of an extra molecular detection assay, but it allows the determination of the extraction efficiency from every individual sample as this can differ with respect to viral recovery and inhibitor removal (Hoorfar et al., 2004). Ideally, process controls should be: 1) genetically related to the tested virus, 2) easy to cultivate, and 3) unlikely to naturally contaminate the tested food sample. The murine norovirus 1 (MNV-1), the feline calicivirus (FCV), a genetically modified mengovirus (vMC0), and the MS2 bacteriophage have most frequently been used as process control for detection of enteric viruses in food samples, while CaCV and poliovirus (PV) have been less common. The use of MNV-1, a genogroup V NoV infectious to mice, as process control when detecting human infective NoV has been proposed due to its genetic similarities to the NoV genome (Wobus et al., 2006). MNV-1 has been successfully tested as process control in shellfish, soft red fruits and ready-to-eat foods (Kingsley, 2007; Stals et al., 2011a,b). FCV, a virus belonging to the Vesivirus genus and infectious to cats, has been applied as process control when detecting NoV in shellfish, bottled water and fresh produce (Kingsley, 2007; Mattison et al., 2009; Schultz et al., 2010; Uhrbrand et al., 2010). vMC0 is a genetically modified mengovirus strain not

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pathogenic to humans which can be cultivated in HeLa-cells (Hahn and Palmenberg, 1995). It has been widely used as process control for the detection of enteric viruses for NoV and HAV in shellfish (Comelli et al., 2008; Uhrbrand et al., 2010), and in bottled water (da Silva et al., 2007) as well. For this reason, the European Committee for Standardization/Technical Committee 275/Working Group 6/ Task Group 4 on virus detection in foods (CEN/TC275/WG6/TAG4) working group suggested it as a process control for the detection of enteric viruses in produce, shellfish and bottled water. Finally, the bacteriophage MS2, a nonpathogenic levivirus of the Leviviridae family, has also been proposed as a process control for detection of HAV in soft red fruits and water (Blaise-Boisseau et al., 2010). 6. Conclusions Detection of viral agents in foods is useful to complement epidemiological investigations of food-borne outbreaks. It is generally believed that the number of viral food-borne outbreaks far exceeds the number currently being reported and it is estimated that at least half of the viral food-borne disease outbreaks are not recognized because of inadequate sampling and detection methods (Svensson, 2000; Baert et al., 2009b). Ideally, methods for detection of viruses in foods should allow a quick and reliable confirmation of foods as sources for food-borne outbreaks (Goyal, 2006). Moreover, virus detection methodology could be used to preventively screen food products for virus presence. The first step in the detection methodology is virus extraction from the food, which remains a stumbling block for several reasons. A first reason involves the wide variety of described methods and protocols, which has complicated the standardization of virus extraction methodologies. As a result, laboratories investigating food-borne outbreaks and screening food products for virus presence use methods either published or developed and evaluated in-house, making comparison of results difficult. A first step towards harmonization could be the selection of a consensus sample size. However, only a limited number of publications have addressed the strategies for taking food samples. Bosch et al. (2011) discussed the sampling of foods for virus detection recently and mentioned that the first question to ask is whether a specific weight can be representative for the tested food. Studies that have developed and optimized methods for extraction of viruses in food have used a wide range of sample weights. While the sample weight varied between 0.15 and 50 g for shellfish, most studies have used 10 to 25 g samples for non-shellfish foods. The European Centre for Normalization (CEN) has proposed methods for detection of NoV and HAV using a sample weight of 2 g for shellfish (Lees, 2010). The second question is the number of samples that should be taken from a suspected batch, field, or truckload to obtain a statistically relevant number. However, no information on the homogenization of viral particles on contaminated foods is available in scientific literature. A second step towards harmonization could be the selection of (a set of) specific methods for virus extraction from specific food categories. For shellfish, the proteinase K approach seems like the most suitable candidate, as it has proven to work for shellfish samples naturally contaminated as well as inoculated with NoV. For carbohydrate/water based foods such as soft red fruits and lettuce at risk for contamination with NoV and HAV, an elution– concentration approach (whether or not in combination with an acid adsorption step) might be the most appropriate approach. However, the great variety of concentration methods makes selection of a single protocol not straightforward. For fat/protein based foods, the large number of different food products makes it unlikely that a single method will be able to detect food-borne viruses in this food category. Nevertheless, several studies have shown that food-borne viruses can be detected at low concentrations in naturally contaminated and both inoculated fat/protein-based foods using methodologies now available (Rutjes et al., 2006; Stals et al., 2011b). Recent efforts have

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been made within various EU projects, between reference laboratories, and within the CEN/TC275/WG6/TAG4 working group to stimulate the acceptance of standardized methods for detection of most common enteric viruses in carbohydrate/water based foods such as produce and hard fruits, as well as in shellfish and bottled water (Rodriguez-Lazaro et al., 2007; Croci et al., 2008; Lees, 2010). A second reason why extraction of viruses from foods remains a stumbling block involves the high variation in the recovery of viruses from food products, both quantitative and qualitative. This is often the result of a limited extraction efficiency. Although this problem might be due to the high number of steps involved in the methods used, the inclusion of adequate process controls is needed to picture these limitations. The correct use and interpretation of process controls has recently been reviewed by D'Agostino et al. (2011), especially for detection of viruses in foods. Regarding selection of a suitable process control, literature suggests the use of MNV-1 and FCV when detecting human NoV in foods. On the other hand, vMC0 is considered a more suitable process control in case of detection of HAV in foods. Finally, protocols for extraction of food-borne viruses from food samples are often time-consuming and laborious. Alternative timesaving methods for virus extraction requiring less effort are not yet available, however. Automatizing the techniques would save labor, but first the protocols and approaches should be harmonized and standardized. In conclusion, many challenges await researchers working on detection of food-borne viruses in food products, particularly relating to harmonization of the current methodologies. Acknowledgements This work was supported by the Belgian Science Policy AreaScience for Sustainable Development (SSD–NORISK–SD/AF/01) and by the Federal Public Service for Health, Food Chain and the Environment, grant RT 10/6 (TRAVIFOOD). The authors would like to thank Miriam Levenson for the careful reading of the manuscript. References Adams, A., 1973. Concentration of Epstein–Barr virus from cell culture fluids with polyethylene glycol. The Journal of General Virology 20, 391–394. Atmar, R.L., Neill, F.H., Romalde, J.L., Le Guyader, F., Woodley, C.M., Metcalf, T.G., Estes, M.K., 1995. Detection of Norwalk virus and hepatitis A virus in shellfish tissues with the PCR. Applied and Environmental Microbiology 61, 3014–3018. Baert, L., Uyttendaele, M., Debevere, J., 2007. Evaluation of two viral extraction methods for the detection of human noroviruses in shellfish with conventional and real-time reverse transcriptase PCR. Letters in Applied Microbiology 44, 106–111. Baert, L., Uyttendaele, M., Debevere, J., 2008. Evaluation of viral extraction methods on a broad range of ready-to-eat foods with conventional and real-time RT-PCR for Norovirus GII detection. International Journal of Food Microbiology 123, 101–108. Baert, L., Debevere, J., Uyttendaele, M., 2009a. The efficacy of preservation methods to inactivate foodborne viruses. International Journal of Food Microbiology 131, 83–94. Baert, L., Uyttendaele, M., Stals, A., Van Coillie, E., Dierick, K., Debevere, J., Botteldoorn, N., 2009b. Reported foodborne outbreaks due to noroviruses in Belgium: the link between food and patient investigations in an international context. Epidemiology and Infection 137, 316–325. Beuret, C., Baumgartner, A., Schluep, J., 2003. Virus-contaminated oysters: a threemonth monitoring of oysters imported to Switzerland. Applied and Environmental Microbiology 69, 2292–2297. Bidawid, S., Farber, J.M., Sattar, S.A., 2000. Rapid concentration and detection of hepatitis A virus from lettuce and strawberries. Journal of Virological Methods 88, 175–185. Bidawid, S., Farber, J.M., Sattar, S.A., 2001. Survival of hepatitis A virus on modified atmosphere-packaged (MAP) lettuce. Food Microbiology 18, 95–102. Blaise-Boisseau, S., Collette, C.H., Guillier, L., Perelle, S., 2010. Duplex real-time qRT-PCR for the detection of hepatitis A virus in water and raspberries using the MS2 bacteriophage as a process control. Journal of Virological Methods 166, 48–53. Bosch, A., Sanchez, G., Abbaszadegan, M., Carducci, A., Guix, S., Le Guyader, F.S., Netshikweta, R., Pintó, R.M., van der Poel, W.H.M., Rutjes, S., 2011. Analytical methods for virus detection in water and food. Food Analytical Methods 4, 4–12. Boxman, I.L.A., Tilburg, J.J.H.C., Te Loeke, N.A.J.M., Vennema, H., Jonker, K., de Boer, E., Koopmans, M., 2006. Detection of noroviruses in shellfish in the Netherlands. International Journal of Food Microbiology 108, 391–396.

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