Available online at www.sciencedirect.com
ScienceDirect Environmental persistence and transfer of enteric viruses Grishma Kotwal and Jennifer L Cannon Non-enveloped enteric viruses, such as Human Norovirus and Hepatitis A Virus, are readily transmitted via the fecal–oral route. Outbreaks are often prolonged due to the ability of these viruses to survive on environmental surfaces, on foods, and in water. Delineation of properties impacting enteric virus transfer and persistence in the environment has been the focus of several recent publications and is the topic of this review. Such information is important for modeling transmission scenarios, identifying risks of food-borne and water-borne virus contamination, and targeting prevention and control efforts for risk mitigation. Addresses Center for Food Safety, Department of Food Science and Technology, The University of Georgia, Griffin, GA 30223, USA Corresponding author: Cannon, Jennifer L ([email protected])
Current Opinion in Virology 2014, 4:37–43 This review comes from a themed issue on Environmental virology Edited by Lee-Ann Jaykus and John Scott Meschke For a complete overview see the Issue and the Editorial Available online 14th January 2014 1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2013.12.003
as through the extremely low pH of the stomach. A person infected with HuNoV can shed up to 1011 viruses per g of feces or 107 viruses per 30 ml of vomitus [5,6]. Similarly, high levels of shedding (up to 109 viruses per g of feces) have been reported for HAV [7,8]. Survival is also facilitated by organic debris of the clinical matrix in which the virus is shed (feces or vomit) and virus aggregate formation, offering protection from environmental insults encountered in route to new human hosts. In this review, transfer and environmental survival of food-borne and water-borne viruses outside of the human host will be discussed with primary focus on HuNoV and HAV. Studying HuNoV and HAV transfer and environmental survival is complicated by the fact that neither virus can be cultured from clinical or environmental samples [3,9]. For this reason, animal caliciviruses such as Feline Calicivirus (FCV) and Murine norovirus (MNV1) and a cell culture-adapted strain of HAV (HM-175) are commonly used to estimate transfer, survival and infectivity. Understanding the environmental survival and transfer of HuNoV and HAV is important for identifying risks of food-borne and water-borne virus contamination and targeting prevention and control efforts for risk mitigation.
Enteric virus transfer between hands, foods, and environmental surfaces Introduction Direct person to person contact is the most common transmission route for human noroviruses (HuNoV) which cause up to 21 million cases of acute gastroenteritis each year in the United States [1]. HuNoV and Hepatitis A Virus (HAV) are the most frequent causes of foodborne diseases of viral etiology in the US, causing over 5.4 million illnesses from HuNoV and over 1500 illnesses from HAV each year [2]. Indirect transmission following ingestion of contaminated food or water, or contact with contaminated surfaces (fomites) is also a frequent cause of HuNoV and HAV illnesses. While similar morphologically and in terms of genome size, HuNoV and HAV belong to distinct families of viruses; Caliciviridae and Picornaviridae, respectively. Both are non-enveloped, icosahedral viruses about 27-–35 nm in diameter containing a genome that is 7–7.5 kilobases of poly-adenylated, positive sense, single-stranded RNA [3,4]. Non-enveloped viruses are generally more environmentally persistent than their enveloped counterparts. The fecal-oral transmission route of HuNoV and HAV, and HuNoV’s transmission through aerosolized vomitus necessitates a robust viral capsid capable of surviving intermittent transfer outside of the human host, as well www.sciencedirect.com
Transfer of viruses from infected persons to foods or environmental surfaces has been the source of several large outbreaks caused by infected food handlers. One outbreak investigation revealed 182 ill persons after eating salad prepared by a symptomatic food handler [10]. Over a weekend of weddings, 332 wedding guests became ill with norovirus traced back to two ill bakery employees that prepared the wedding cakes [11]. Environmental contamination can also lead to prolonged outbreak duration as demonstrated in a hotel where 372 guests and 72 employees became ill with norovirus after a guest vomited in the corridor of two floors [12]. In other studies, environmental swabs tested positive for HuNoV 14 days and even 9 weeks after outbreak initiation [13,14]. Outbreaks such as these have led to laboratory studies demonstrating the role of hands and environmental surfaces in virus transmission and generation of quantitative data for risk assessment (see Table 1). In 2006, D’Souza et al. studied the transfer of HuNoV genogroup 1 (GI) from stainless steel to lettuce surfaces and found 8 out of 9 lettuce samples to be positive after transfer [15]. Similarly, Bidawid et al. (2004) studied FCV transfer from fingers to/from food and food contact Current Opinion in Virology 2014, 4:37–43
38 Environmental virology
surfaces (ham, lettuce, brushed stainless steel), finding transfer percentages as high as 46 20.3% when contaminated fingers touched these surfaces, but only up to 14 3.5% when clean fingers touched contaminated surfaces [16]. The type of food coming in contact with the fomite surface was also found to be important. For example, Escudero et al. (2012) measured the transfer of a HuNoV genogroup II, genotype 2 (GII.2) strain and MNV-1 to foods from fomite surfaces, finding transfer percentages of viruses to lettuce (0–26%) to be lower than their transfer to deli meats (55–95%) [17]. Similarly, Stals et al. (2013) mimicked the preparation of deli sandwiches in the laboratory, measuring norovirus transfer from contaminated gloves and stainless steel to sandwich components (meat, lettuce, bun) and found different rates of transfer to each food type [18]. In addition, Sharps et al. (2012) found that the transfer rates of norovirus to raspberries, blueberries and grapes after handling with contaminated hands differed after direct contact [19]. This group and others have shown the extent of virus transfer (cross-contamination) occurring after norovirus-contaminated hands, foods, surfaces or kitchen items are sequentially used to contact other surfaces. Barker et al. (2004) demonstrated that fingers contaminated with HuNoV after contact with soiled toilet paper were capable of contaminating seven clean surfaces touched sequentially [20]. In addition, virus was detected on door handles, faucets and telephone receivers touched with contaminated fingers [20]. Sequential transfer of HAV and MNV-1 to produce items was demonstrated by Wang et al. (2013). In this study, contaminated produce was first prepared with a kitchen utensil (knife, grater, peeler, scrubbing pad), then the newly contaminated utensils were used to sequentially prepare seven produce items. In nearly all cases, virus was detected on the seventh item prepared with the contaminated utensil [21,22]. Two variables of transfer studies that have had a major influence on the results (transfer rates) are dry time of inoculum and the pressure applied during transfer. Increasing the dry time of the virus inoculum negatively correlates with virus transfer rates. For example, transfer of HAV from disks to fingerpads, fingerpads to disks, and between fingerpads after drying inoculum for 20 min resulted in roughly 3 log PFU virus transferred, but was reduced to 1 log PFU or less after the inoculum was dried for 4 hours [23]. Increasing the applied pressure of contact, on the other hand, increases virus transfer rate between surfaces. Mbithi et al. (1992) revealed that with a pressure increase from 0.2 to 1.0 kg/cm2 a 3-fold increase in virus transfer could be observed [23]. Similarly, Escudero et al. (2012) showed that a pressure increase from 100 to 1000 g/cm2 during MNV-1 transfer from formica, stainless steel or ceramic to lettuce surfaces, transfer rates increased from 0–4% to 8–20%, respectively [17]. Current Opinion in Virology 2014, 4:37–43
Enteric virus persistence in water, on foods, and on environmental surfaces Consumption of contaminated water has caused several outbreaks [24], most commonly due to septic system malfunction [25,26]. There is abundant evidence indicating that non-enveloped enteric viruses survive for long periods of time (Table 2), be it on food or food contact surfaces or in waters (ground, surface, and drinking waters). For instance, Fallahi and Mattison (2011) revealed MNV-1 survival in bottled water over a period of 42 days at room temperature, with only a 1.6 log reduction in infectivity [27]. Cooler temperatures can further increase virus survival times in water as demonstrated by Bae and Schwab (2008), revealing MS2, FCV and Poliovirus (PV) survival in surface and ground waters to be greater at 48C than at 258C [28]. This was generally the case for bacteriophage (MS2, GA) and Human Adenovirus-2 (HAdV2) survival in ground water; however, HAdV2 demonstrated remarkable persistence in 208C water, decreasing in infectivity by 2.4 log at the end of the 120 day study [29]. HAdV2 DNA was also detected after 400 days in 208C water by Ogorzally and colleagues (2010), but the duration of infectious virus persistence could not be determined since detecting viral nucleic acids in water typically exceeds the duration of infectious virus survival. In a recent study by Seitz et al. (2011), longterm persistence of infectious HuNoV (GI.1 Norwalk virus) in water was demonstrated when human subjects became ill following consumption of ground water contaminated with the virus 61 days prior [30]. This was the maximum time point for the human challenge portion of the study, but noroviral RNA was still detected after 1266 days (3 years) of storage in the ground water. As previously described, hands are important vehicles for direct and indirect transmission of enteric viruses. Once transferred, enteric viruses can survive for long periods of time on contaminated hands, fomites, or foods coming in contact with these hands. A recent example of human norovirus persistence on fingerpads was demonstrated by Liu et al. (2009) where an approximate 0.2 log reduction in detected RNA occurred after 15 min, but then remained fairly stable up to the 120 min time point marking the end of data collection [31]. Enteric virus survival on stainless steel has been extensively studied with variable storage temperatures (4–378C) and relative humidity. These factors in addition to surface type influence virus survival. The inoculum matrix (stool vs cell culture lysate) also appears to influence virus survival. For example, Lamhoujeb et al. (2009) studied the persistence of HuNoV on stainless steel and plastic (PVC) disks for up to 56 days [32]. Persistence was highest (49–56 days) under low temperature (78C) and high humidity (86%) conditions and lowest (7 days) at room temperature (208C) with a low humidity (30%) environment. Similarly, Escudero et al. (2012), revealed a maximum average viral RNA reduction of 2 logs for GI.1 HuNoV and 2.3 logs for GII.2 HuNoV www.sciencedirect.com
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Table 1 Enteric virus transfer between hands, foods, and environmental surfaces Virus
Inoculum matrixa, titerb and volume
Surfaces involved c
HAV
SS 104 PFU in 10 ml
Fingers and kitchen surfaces
HAV
CL + 5% FBS 105 PFU in 10 ml Mucin, BSA and tryptone 105 PFU in 10 ml
Fingers and lettuce
GII
Results summary
200–1000 g/cm2 f or 10 s
Plaque assay
Plaque assay
Fingers, ham, l ettuce and SST
0.2 or 0.4 kg/cm2 for 10 s 0.2 or 0.4 kg/cm2 for 10 s
50–3484 PFU transferred depending on wet vs dry inoculum, pressure and surface type Virus transfer 9.2 0.9%
SS NP in 150 ml
Fingers and melamine
NP for 10 s
RT-PCR
GI or FCV
SS: 104 RT-PCRU in 10 ml CL: 106 PFU in 10 ml
SST and lettuce
0.01, 0.1, or 1 kg/9 cm2 for 15 s
RT-PCR or Plaque assay
MNV-1
CL in PBS 105 PFU in 1 or 3.5 ml
Wash water, onions and spinach
NP for 25 s or 2 min
Plaque assay
GII.2 or MNV-1
SS: 105 GC in 25 ml CL: 106 PFU in 50 ml
Ceramic, SST, Formica, lettuce and turkey meat
0.1 or 1 kg/9 cm2 for 15 s
RT-PCR and Plaque assay
HuNoV (GI/GII)and MNV-1
SS: 109 GC in 10 ml SS: 106 GC in 10 ml
Gloves, SST and small fruits
0.05 kg/cm2 for 5 s
RT-PCR
HAV or MNV-1
CL 104 PFU in 50 or 150 ml CL: 6.7 or 6.5 log PFU/ml in 25 ml, 50 ml, or 150 ml
Produce, knives and graters Produce and kitchen utensils
NP for NP
Plaque assay
NP for NP
Plaque assay
SS: 107 GC in 20 ml CL: 104 PFU in 20 ml
Gloves, SST, boiled ham, bun and lettuce
0.2 or 0.4 kg/cm2, twisted 908 for 10 s
RT-PCR or Plaque assay
Current Opinion in Virology 2014, 4:37–43
HAV or MNV-1
GII.4 or MNV-1
a b c
SS: stool suspension; CL: cell culture lysate; FBS: fetal bovine serum; BSA: bovine serum albumin NP: Not provided; GC: genome copies. SST: stainless steel.
Plaque assay
Fingers to ham, lettuce, metal disks: 46 20.3, 18 5.7 and 13 3.6%; vice versa: 6 1.8, 14 3.5, and 7 1.9% Virus transferred to seven sequentially touched clean melamine surfaces SST to lettuce: GI up to 8/9 surfaces positive, the drier the inoculum, the less virus transfer; similar results with FCV Greater than 3 logs PFU MNV-1 transfer to onion bulbs and spinach leaves Transfer efficiency ranged from 0 to 26% for lettuce and from 55 to 95% for turkey Transfer % greater with wet inoculum; Indirect (clean glove to contaminated SST to fruits) was lower than direct transfer between two surfaces Contaminated produce to knives and graters: 0.9–5.1 log PFU Produce to utensils: >2 log PFU virus transfer; Transfer to up to 7 sequentially touched surfaces GII.4 (gloves to SST: 0.2%, vice versa: 0.9%), MNV-1 (Gloves to SST-0.1%, vice versa: 3.6%)
Reference [23]
[39] [16]
[20]
[15]
[34]
[17]
[19]
[22] [21]
[18]
Virus transfer and persistence Kotwal and Cannon 39
Test
FCV
Pressure and contact time
Enteric virus persistence in water, on foods, and on environmental surfaces Virus
HuNoV GI.1 MNV-1, PV or FCV
MS2
Inoculum matrixa, titerb and volume c
Surface or water type d
SS: 105 RT-PCR units per ml in NP CL: 107 or 104 PFU per ml in NP
Surface and ground water Surface and ground water
3–5 wk at 258C or 48C in the dark 3–5 wk at 258C or 48C in the dark
Plaque assay and qRT-PCR
SUP from E. coli cells: 107 PFU per ml in NP SS: 106 NPU in 50 ml
Surface and ground water
3–5 wk at 258C or 48C in the dark
Plaque assay and qRT-PCR
Nucleic acid reduction rate at 258C: 0.03 log10/day; at 48C: 0.02 log10/day Infectivity and nucleic acid reduction rate at 258C (log/ day): 0.09 and 0.05(MNV), 0.13 and 0.10 (PV),0.18 and 0.09 (FCV) Infectivity and nucleic acid reduction rate at 258C: 0.12 and 0.06 log10/day
10 days at 78C
Real-time NASBA
Virus detected on the 10th day on both surfaces
[37]
7 days at RT
Plaque assay
No decline in potable water for 1 week. No decline in wash water till day 6; 0.79 log reduction observed on 7th day No significant reduction in PFUs seen over 6 months
[34]
GI was more resistant than GII and the highest reduction of GII was observed in blueberries (2.3 log in 3 months) Little effect on HAV; FCV infectivity reduction most pronounced in raspberries and strawberries Virus detection: 56 days on PVC and up to 49 days on SST (78C); 7 and 28 days (208C for low and high RH)
[35]
Duration, temperature and environ. conditions
MNV-1
CL: 7.0 log PFU in 1 ml
Lettuce and turkey slices Potable or produce wash water
MNV-1
CL: 6.3 log PFU per ml in 1 ml or 2.5 ml SS: 105 or 106 RTPCRU in 50 ml
Shredded onions or spinach Berries, parsley, basil
6 months at
CL: 106 TCID50 in 50 ml SS: 102 NASBA positive units in 20 ml
Berries, parsley, basil SST or plastic (PVC)
90 days at 208C
HuNoV (GI.1 or GII.2)
SS: 107 or 109 GEP in 10 ml
Ceramic, Formica, SST or fingers
HuNoV (GII RNA)
Nuclease-free water: NP in 10 ml SUP or CL: 106 MPNCU or PFU per ml in NP
Formica or SST
HuNoV GII
HuNoV GI or GII
HAV or FCV HuNoV GII
Phage (MS2 or GA) or HAdV
MNV-1
www.sciencedirect.com
HuNoV (GI.1) or HuNoV RNA FCV, MNV-1, HAV, or PV HuNoV (GI.1 or GII.2) or MNV-1
HuNoV (GI or GII RNA) or MNV-1 RNA
SL: 105 or 106 PFU in 10 ml or 100 ml NP: 108 GEC per ml in 150 ml CL: 106.5 RT-PCRU in NP SS or CL: 105 RTqPCRU or 106 PFU in 25 ml or 50 ml Nuclease-free water NP in 10 ml
Ground water
Lettuce, SST, soil, bottled water Ground-, tap-and reagent-grade water Oysters Ceramic, Formica, SST, lettuce
Ceramic, Formica, SST, Lettuce
90 days at
218C 208C
56 days at 78C or 208C with high or low RH 42 days or 120 min at ambient RT and RH
7 days at ambient RT and RH 400 days at 48C or 208C in the dark
15 day or 42 days at RT 1266 days or 14 days at RT in the dark 21 days in a depuration tank 42 days or 14 days at RT or 48C at ambient RH and light Until undetected
Test
qRT-PCR
Plaque assay RT-PCR
RT-PCR and plaque assay Real-time NASBA
RT-qPCR
RT-qPCR RT-PCR and plaque assay
RT-PCR and plaque assay RT-qPCR RT-PCR RT-qPCR and Plaque assay
RT-qPCR
Results summary
HuNoV (GI): 1.5–2.9 log GEP average reduction after 21–28 days; (GII): 0.4–1.2 log GEP reduction in 42 days; Fingers: GI and GII: 0.15–0.20 log reduction in 15 min 3 log reduction in 8 hours; by day 7: RNA practically undetectable Infectivity reductions for HAdV: 0.008 (48C) to 0.03 (208C) log/day; phage: significantly higher; genome degradation for HAdV: None (48C), 0.004 log/day (208C) 1 log infectivity reduction after: 4 days (lettuce), 15 days (SST), 12 days (soil), 29 days (water) Virus RNA within intact capsid detected on 1266th day; Purified RNA detected on 14th day in all waters tested Persistence: HAV (>21 days, most acid resistant); MNV-1 (12 days); PV (1 day); FCV (
ScienceDirect Environmental persistence and transfer of enteric viruses Grishma Kotwal and Jennifer L Cannon Non-enveloped enteric viruses, such as Human Norovirus and Hepatitis A Virus, are readily transmitted via the fecal–oral route. Outbreaks are often prolonged due to the ability of these viruses to survive on environmental surfaces, on foods, and in water. Delineation of properties impacting enteric virus transfer and persistence in the environment has been the focus of several recent publications and is the topic of this review. Such information is important for modeling transmission scenarios, identifying risks of food-borne and water-borne virus contamination, and targeting prevention and control efforts for risk mitigation. Addresses Center for Food Safety, Department of Food Science and Technology, The University of Georgia, Griffin, GA 30223, USA Corresponding author: Cannon, Jennifer L ([email protected])
Current Opinion in Virology 2014, 4:37–43 This review comes from a themed issue on Environmental virology Edited by Lee-Ann Jaykus and John Scott Meschke For a complete overview see the Issue and the Editorial Available online 14th January 2014 1879-6257/$ – see front matter, # 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.coviro.2013.12.003
as through the extremely low pH of the stomach. A person infected with HuNoV can shed up to 1011 viruses per g of feces or 107 viruses per 30 ml of vomitus [5,6]. Similarly, high levels of shedding (up to 109 viruses per g of feces) have been reported for HAV [7,8]. Survival is also facilitated by organic debris of the clinical matrix in which the virus is shed (feces or vomit) and virus aggregate formation, offering protection from environmental insults encountered in route to new human hosts. In this review, transfer and environmental survival of food-borne and water-borne viruses outside of the human host will be discussed with primary focus on HuNoV and HAV. Studying HuNoV and HAV transfer and environmental survival is complicated by the fact that neither virus can be cultured from clinical or environmental samples [3,9]. For this reason, animal caliciviruses such as Feline Calicivirus (FCV) and Murine norovirus (MNV1) and a cell culture-adapted strain of HAV (HM-175) are commonly used to estimate transfer, survival and infectivity. Understanding the environmental survival and transfer of HuNoV and HAV is important for identifying risks of food-borne and water-borne virus contamination and targeting prevention and control efforts for risk mitigation.
Enteric virus transfer between hands, foods, and environmental surfaces Introduction Direct person to person contact is the most common transmission route for human noroviruses (HuNoV) which cause up to 21 million cases of acute gastroenteritis each year in the United States [1]. HuNoV and Hepatitis A Virus (HAV) are the most frequent causes of foodborne diseases of viral etiology in the US, causing over 5.4 million illnesses from HuNoV and over 1500 illnesses from HAV each year [2]. Indirect transmission following ingestion of contaminated food or water, or contact with contaminated surfaces (fomites) is also a frequent cause of HuNoV and HAV illnesses. While similar morphologically and in terms of genome size, HuNoV and HAV belong to distinct families of viruses; Caliciviridae and Picornaviridae, respectively. Both are non-enveloped, icosahedral viruses about 27-–35 nm in diameter containing a genome that is 7–7.5 kilobases of poly-adenylated, positive sense, single-stranded RNA [3,4]. Non-enveloped viruses are generally more environmentally persistent than their enveloped counterparts. The fecal-oral transmission route of HuNoV and HAV, and HuNoV’s transmission through aerosolized vomitus necessitates a robust viral capsid capable of surviving intermittent transfer outside of the human host, as well www.sciencedirect.com
Transfer of viruses from infected persons to foods or environmental surfaces has been the source of several large outbreaks caused by infected food handlers. One outbreak investigation revealed 182 ill persons after eating salad prepared by a symptomatic food handler [10]. Over a weekend of weddings, 332 wedding guests became ill with norovirus traced back to two ill bakery employees that prepared the wedding cakes [11]. Environmental contamination can also lead to prolonged outbreak duration as demonstrated in a hotel where 372 guests and 72 employees became ill with norovirus after a guest vomited in the corridor of two floors [12]. In other studies, environmental swabs tested positive for HuNoV 14 days and even 9 weeks after outbreak initiation [13,14]. Outbreaks such as these have led to laboratory studies demonstrating the role of hands and environmental surfaces in virus transmission and generation of quantitative data for risk assessment (see Table 1). In 2006, D’Souza et al. studied the transfer of HuNoV genogroup 1 (GI) from stainless steel to lettuce surfaces and found 8 out of 9 lettuce samples to be positive after transfer [15]. Similarly, Bidawid et al. (2004) studied FCV transfer from fingers to/from food and food contact Current Opinion in Virology 2014, 4:37–43
38 Environmental virology
surfaces (ham, lettuce, brushed stainless steel), finding transfer percentages as high as 46 20.3% when contaminated fingers touched these surfaces, but only up to 14 3.5% when clean fingers touched contaminated surfaces [16]. The type of food coming in contact with the fomite surface was also found to be important. For example, Escudero et al. (2012) measured the transfer of a HuNoV genogroup II, genotype 2 (GII.2) strain and MNV-1 to foods from fomite surfaces, finding transfer percentages of viruses to lettuce (0–26%) to be lower than their transfer to deli meats (55–95%) [17]. Similarly, Stals et al. (2013) mimicked the preparation of deli sandwiches in the laboratory, measuring norovirus transfer from contaminated gloves and stainless steel to sandwich components (meat, lettuce, bun) and found different rates of transfer to each food type [18]. In addition, Sharps et al. (2012) found that the transfer rates of norovirus to raspberries, blueberries and grapes after handling with contaminated hands differed after direct contact [19]. This group and others have shown the extent of virus transfer (cross-contamination) occurring after norovirus-contaminated hands, foods, surfaces or kitchen items are sequentially used to contact other surfaces. Barker et al. (2004) demonstrated that fingers contaminated with HuNoV after contact with soiled toilet paper were capable of contaminating seven clean surfaces touched sequentially [20]. In addition, virus was detected on door handles, faucets and telephone receivers touched with contaminated fingers [20]. Sequential transfer of HAV and MNV-1 to produce items was demonstrated by Wang et al. (2013). In this study, contaminated produce was first prepared with a kitchen utensil (knife, grater, peeler, scrubbing pad), then the newly contaminated utensils were used to sequentially prepare seven produce items. In nearly all cases, virus was detected on the seventh item prepared with the contaminated utensil [21,22]. Two variables of transfer studies that have had a major influence on the results (transfer rates) are dry time of inoculum and the pressure applied during transfer. Increasing the dry time of the virus inoculum negatively correlates with virus transfer rates. For example, transfer of HAV from disks to fingerpads, fingerpads to disks, and between fingerpads after drying inoculum for 20 min resulted in roughly 3 log PFU virus transferred, but was reduced to 1 log PFU or less after the inoculum was dried for 4 hours [23]. Increasing the applied pressure of contact, on the other hand, increases virus transfer rate between surfaces. Mbithi et al. (1992) revealed that with a pressure increase from 0.2 to 1.0 kg/cm2 a 3-fold increase in virus transfer could be observed [23]. Similarly, Escudero et al. (2012) showed that a pressure increase from 100 to 1000 g/cm2 during MNV-1 transfer from formica, stainless steel or ceramic to lettuce surfaces, transfer rates increased from 0–4% to 8–20%, respectively [17]. Current Opinion in Virology 2014, 4:37–43
Enteric virus persistence in water, on foods, and on environmental surfaces Consumption of contaminated water has caused several outbreaks [24], most commonly due to septic system malfunction [25,26]. There is abundant evidence indicating that non-enveloped enteric viruses survive for long periods of time (Table 2), be it on food or food contact surfaces or in waters (ground, surface, and drinking waters). For instance, Fallahi and Mattison (2011) revealed MNV-1 survival in bottled water over a period of 42 days at room temperature, with only a 1.6 log reduction in infectivity [27]. Cooler temperatures can further increase virus survival times in water as demonstrated by Bae and Schwab (2008), revealing MS2, FCV and Poliovirus (PV) survival in surface and ground waters to be greater at 48C than at 258C [28]. This was generally the case for bacteriophage (MS2, GA) and Human Adenovirus-2 (HAdV2) survival in ground water; however, HAdV2 demonstrated remarkable persistence in 208C water, decreasing in infectivity by 2.4 log at the end of the 120 day study [29]. HAdV2 DNA was also detected after 400 days in 208C water by Ogorzally and colleagues (2010), but the duration of infectious virus persistence could not be determined since detecting viral nucleic acids in water typically exceeds the duration of infectious virus survival. In a recent study by Seitz et al. (2011), longterm persistence of infectious HuNoV (GI.1 Norwalk virus) in water was demonstrated when human subjects became ill following consumption of ground water contaminated with the virus 61 days prior [30]. This was the maximum time point for the human challenge portion of the study, but noroviral RNA was still detected after 1266 days (3 years) of storage in the ground water. As previously described, hands are important vehicles for direct and indirect transmission of enteric viruses. Once transferred, enteric viruses can survive for long periods of time on contaminated hands, fomites, or foods coming in contact with these hands. A recent example of human norovirus persistence on fingerpads was demonstrated by Liu et al. (2009) where an approximate 0.2 log reduction in detected RNA occurred after 15 min, but then remained fairly stable up to the 120 min time point marking the end of data collection [31]. Enteric virus survival on stainless steel has been extensively studied with variable storage temperatures (4–378C) and relative humidity. These factors in addition to surface type influence virus survival. The inoculum matrix (stool vs cell culture lysate) also appears to influence virus survival. For example, Lamhoujeb et al. (2009) studied the persistence of HuNoV on stainless steel and plastic (PVC) disks for up to 56 days [32]. Persistence was highest (49–56 days) under low temperature (78C) and high humidity (86%) conditions and lowest (7 days) at room temperature (208C) with a low humidity (30%) environment. Similarly, Escudero et al. (2012), revealed a maximum average viral RNA reduction of 2 logs for GI.1 HuNoV and 2.3 logs for GII.2 HuNoV www.sciencedirect.com
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Table 1 Enteric virus transfer between hands, foods, and environmental surfaces Virus
Inoculum matrixa, titerb and volume
Surfaces involved c
HAV
SS 104 PFU in 10 ml
Fingers and kitchen surfaces
HAV
CL + 5% FBS 105 PFU in 10 ml Mucin, BSA and tryptone 105 PFU in 10 ml
Fingers and lettuce
GII
Results summary
200–1000 g/cm2 f or 10 s
Plaque assay
Plaque assay
Fingers, ham, l ettuce and SST
0.2 or 0.4 kg/cm2 for 10 s 0.2 or 0.4 kg/cm2 for 10 s
50–3484 PFU transferred depending on wet vs dry inoculum, pressure and surface type Virus transfer 9.2 0.9%
SS NP in 150 ml
Fingers and melamine
NP for 10 s
RT-PCR
GI or FCV
SS: 104 RT-PCRU in 10 ml CL: 106 PFU in 10 ml
SST and lettuce
0.01, 0.1, or 1 kg/9 cm2 for 15 s
RT-PCR or Plaque assay
MNV-1
CL in PBS 105 PFU in 1 or 3.5 ml
Wash water, onions and spinach
NP for 25 s or 2 min
Plaque assay
GII.2 or MNV-1
SS: 105 GC in 25 ml CL: 106 PFU in 50 ml
Ceramic, SST, Formica, lettuce and turkey meat
0.1 or 1 kg/9 cm2 for 15 s
RT-PCR and Plaque assay
HuNoV (GI/GII)and MNV-1
SS: 109 GC in 10 ml SS: 106 GC in 10 ml
Gloves, SST and small fruits
0.05 kg/cm2 for 5 s
RT-PCR
HAV or MNV-1
CL 104 PFU in 50 or 150 ml CL: 6.7 or 6.5 log PFU/ml in 25 ml, 50 ml, or 150 ml
Produce, knives and graters Produce and kitchen utensils
NP for NP
Plaque assay
NP for NP
Plaque assay
SS: 107 GC in 20 ml CL: 104 PFU in 20 ml
Gloves, SST, boiled ham, bun and lettuce
0.2 or 0.4 kg/cm2, twisted 908 for 10 s
RT-PCR or Plaque assay
Current Opinion in Virology 2014, 4:37–43
HAV or MNV-1
GII.4 or MNV-1
a b c
SS: stool suspension; CL: cell culture lysate; FBS: fetal bovine serum; BSA: bovine serum albumin NP: Not provided; GC: genome copies. SST: stainless steel.
Plaque assay
Fingers to ham, lettuce, metal disks: 46 20.3, 18 5.7 and 13 3.6%; vice versa: 6 1.8, 14 3.5, and 7 1.9% Virus transferred to seven sequentially touched clean melamine surfaces SST to lettuce: GI up to 8/9 surfaces positive, the drier the inoculum, the less virus transfer; similar results with FCV Greater than 3 logs PFU MNV-1 transfer to onion bulbs and spinach leaves Transfer efficiency ranged from 0 to 26% for lettuce and from 55 to 95% for turkey Transfer % greater with wet inoculum; Indirect (clean glove to contaminated SST to fruits) was lower than direct transfer between two surfaces Contaminated produce to knives and graters: 0.9–5.1 log PFU Produce to utensils: >2 log PFU virus transfer; Transfer to up to 7 sequentially touched surfaces GII.4 (gloves to SST: 0.2%, vice versa: 0.9%), MNV-1 (Gloves to SST-0.1%, vice versa: 3.6%)
Reference [23]
[39] [16]
[20]
[15]
[34]
[17]
[19]
[22] [21]
[18]
Virus transfer and persistence Kotwal and Cannon 39
Test
FCV
Pressure and contact time
Enteric virus persistence in water, on foods, and on environmental surfaces Virus
HuNoV GI.1 MNV-1, PV or FCV
MS2
Inoculum matrixa, titerb and volume c
Surface or water type d
SS: 105 RT-PCR units per ml in NP CL: 107 or 104 PFU per ml in NP
Surface and ground water Surface and ground water
3–5 wk at 258C or 48C in the dark 3–5 wk at 258C or 48C in the dark
Plaque assay and qRT-PCR
SUP from E. coli cells: 107 PFU per ml in NP SS: 106 NPU in 50 ml
Surface and ground water
3–5 wk at 258C or 48C in the dark
Plaque assay and qRT-PCR
Nucleic acid reduction rate at 258C: 0.03 log10/day; at 48C: 0.02 log10/day Infectivity and nucleic acid reduction rate at 258C (log/ day): 0.09 and 0.05(MNV), 0.13 and 0.10 (PV),0.18 and 0.09 (FCV) Infectivity and nucleic acid reduction rate at 258C: 0.12 and 0.06 log10/day
10 days at 78C
Real-time NASBA
Virus detected on the 10th day on both surfaces
[37]
7 days at RT
Plaque assay
No decline in potable water for 1 week. No decline in wash water till day 6; 0.79 log reduction observed on 7th day No significant reduction in PFUs seen over 6 months
[34]
GI was more resistant than GII and the highest reduction of GII was observed in blueberries (2.3 log in 3 months) Little effect on HAV; FCV infectivity reduction most pronounced in raspberries and strawberries Virus detection: 56 days on PVC and up to 49 days on SST (78C); 7 and 28 days (208C for low and high RH)
[35]
Duration, temperature and environ. conditions
MNV-1
CL: 7.0 log PFU in 1 ml
Lettuce and turkey slices Potable or produce wash water
MNV-1
CL: 6.3 log PFU per ml in 1 ml or 2.5 ml SS: 105 or 106 RTPCRU in 50 ml
Shredded onions or spinach Berries, parsley, basil
6 months at
CL: 106 TCID50 in 50 ml SS: 102 NASBA positive units in 20 ml
Berries, parsley, basil SST or plastic (PVC)
90 days at 208C
HuNoV (GI.1 or GII.2)
SS: 107 or 109 GEP in 10 ml
Ceramic, Formica, SST or fingers
HuNoV (GII RNA)
Nuclease-free water: NP in 10 ml SUP or CL: 106 MPNCU or PFU per ml in NP
Formica or SST
HuNoV GII
HuNoV GI or GII
HAV or FCV HuNoV GII
Phage (MS2 or GA) or HAdV
MNV-1
www.sciencedirect.com
HuNoV (GI.1) or HuNoV RNA FCV, MNV-1, HAV, or PV HuNoV (GI.1 or GII.2) or MNV-1
HuNoV (GI or GII RNA) or MNV-1 RNA
SL: 105 or 106 PFU in 10 ml or 100 ml NP: 108 GEC per ml in 150 ml CL: 106.5 RT-PCRU in NP SS or CL: 105 RTqPCRU or 106 PFU in 25 ml or 50 ml Nuclease-free water NP in 10 ml
Ground water
Lettuce, SST, soil, bottled water Ground-, tap-and reagent-grade water Oysters Ceramic, Formica, SST, lettuce
Ceramic, Formica, SST, Lettuce
90 days at
218C 208C
56 days at 78C or 208C with high or low RH 42 days or 120 min at ambient RT and RH
7 days at ambient RT and RH 400 days at 48C or 208C in the dark
15 day or 42 days at RT 1266 days or 14 days at RT in the dark 21 days in a depuration tank 42 days or 14 days at RT or 48C at ambient RH and light Until undetected
Test
qRT-PCR
Plaque assay RT-PCR
RT-PCR and plaque assay Real-time NASBA
RT-qPCR
RT-qPCR RT-PCR and plaque assay
RT-PCR and plaque assay RT-qPCR RT-PCR RT-qPCR and Plaque assay
RT-qPCR
Results summary
HuNoV (GI): 1.5–2.9 log GEP average reduction after 21–28 days; (GII): 0.4–1.2 log GEP reduction in 42 days; Fingers: GI and GII: 0.15–0.20 log reduction in 15 min 3 log reduction in 8 hours; by day 7: RNA practically undetectable Infectivity reductions for HAdV: 0.008 (48C) to 0.03 (208C) log/day; phage: significantly higher; genome degradation for HAdV: None (48C), 0.004 log/day (208C) 1 log infectivity reduction after: 4 days (lettuce), 15 days (SST), 12 days (soil), 29 days (water) Virus RNA within intact capsid detected on 1266th day; Purified RNA detected on 14th day in all waters tested Persistence: HAV (>21 days, most acid resistant); MNV-1 (12 days); PV (1 day); FCV (
