Optimal Methods For Recovery Of Murine Norovirus Biology Essay


Human Norovirus continues to be a major public health concern, accounting for an estimated two-thirds of foodborne illness cases in the U.S. NoV is commonly transmitted by person-to-person spread, and many outbreaks of NoV may be linked to the preparation of food by infected food handlers. In particular, salads containing fresh produce are of particular concern because the product often does not undergo an intervention step prior to consumption. In this study, methods for inoculation and recovery of murine norovirus (MNV-1), a NoV surrogate, from food preparatory surfaces were optimized, and the effect of hand washing on removing viral contamination was examined. MNV-1 (approx. 7-log PFU) was inoculated onto the surface of poly, vinyl, and latex gloves; the fingers and palm of human hands; and the outer surface of fresh-cut romaine lettuce leaves. After air drying, inoculated virions were recovered from gloves and hands using the glove juice method. Several eluents were compared for their recovery efficiency, including Dulbecco's Modified Eagle's Media (DMEM), 3% beef extract and stripping solution (0.04% K2HPO4, 1.01% Na2HPO4, 0.1% Triton X-100). Stripping solution was an effective eluent for MNV-1 from gloves, recovering between 34.9 and 40.9% of inoculated MNV-1 from gloves and 23.7% from bare hands. Beef extract (3%) was an effective eluent for MNV-1 on lettuce, recovering between 24.5 and 27.0%, depending on whether the lettuce was vortexed, shaken or stomached. The efficacy of hand washing in removing virions from the hands of volunteers was also tested, and the results indicated only a marginally increased reduction could be achieved when washing hands for 20 seconds with liquid (2.96-log) or foam (2.90-log) soap, compared to a five second rinse with tap water (2.80-log).

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Food-borne diseases are a major cause of morbidity and hospitalization worldwide. Each year 76 million food-borne illnesses, more than 300,000 hospitalizations and 5,000 deaths are estimated to occur in the United States, indicating that one in four Americans develops food-borne illness each year and at least 1 in 1,000 is hospitalized (1). Even after major advances and improvements in the field of the food safety and related surveillance systems, food-borne diseases are still a global public health problem (2). Foodborne diseases occur at a very high rate in the elderly people, pregnant women, immuno-compromised people, and children. Only 20% of food-borne illnesses are caused by the known pathogens. Interestingly, of these illnesses caused by known pathogens, viral agents are estimated to account for more than two-thirds (3).

Human exposure to enteric viruses through contaminated food products has been a growing concern in the last decade (4). Viruses are the most common pathogens transmitted via food, for example they are estimated to cause 66.6% of food related illnesses in the United States, compared to 9.7% and 14.2% for Salmonella and Campylobacter, respectively (10). Research on viral food-borne diseases is just emerging and is very limited as compared to bacterial agents of food-borne illness (4). Hepatitis A virus (HAV), norovirus (NoV), rotavirus, and astrovirus are widely known human enteric viruses which are collectively estimated to cause more than 30 million illnesses per year and NoV alone cause approximately 23 million illnesses annually (10, 11). In Europe, viral agents were responsible for 10.2% of the foodborne outbreaks during 2006 and were pointed out as the second most common causative agent, after Salmonella. Moreover, NoV is the most common single foodborne agent in Sweden, causing estimated 135,000-220,000 cases of foodborne illness annually (3). This clearly indicates that of the known enteric viruses, the NoV is the virus of particular concern (5, 8).

The common route of transmission for human enteric viruses through foods is via consumption of minimally processed foods like Ready to Eat foods (11). Many types of fresh produce like Vegetables, including different types of salad vegetables and green onions, have also been associated with outbreaks of viral hepatitis and gastroenteritis as they are minimally processed. ( 4,6,7,8).

The noroviruses are classified as the genus Norovirus within the family Caliciviridae, and are now considered the most common cause of outbreaks of non-bacterial gastroenteritis (13,14,15). Caliciviridae contains four genera: Norovirus, Sapovirus, Lagovirus, and Vesivirus. The prototype strain of human norovirus is the Norwalk virus (NV/Human/US/1968), which was first discovered in an outbreak of gastroenteritis in an elementary school in Norwalk, OH, in 1968 (16).

NoV is a small round virion of 27-38 nm in diameter and possesses a single-stranded, positive-sense, polyadenylated, RNA genome of 7400-7700 nucleotides (17,18). NoV has a nonenveloped structure, which is similar to those of other human enteric viruses, such as poliovirus (PV), coxsackievirus, and echovirus. NoV is classified into five genogroups (GI-GV), which can be further classified into 25 or more different human NoV genotypes (17). Genogroup GIII has only been found in cattle and GV only in mice. Genogroups GI (8 genotypes) and GII (17 genotypes) contain most of the strains infecting humans. Genogroup GII.4 is the predominant genotype circulating worldwide over the last decade causing 80% of all norovirus outbreaks (12). 

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The main mode of the NoV transmission is fecal-oral route through person-to-person (26). The outbreaks occur generally in crowded and closed environments i.e. where the flow of the human traffic is very minimal like the nursing homes, health care institutions, and cruise ships and also in the schools and prisons (22). This is clearly evident from this study which was conducted in a children's hospital of 59% of all NoV infections diagnosed in that hospital were acquired from the hospital (25).The primary reason for this being the long term exposure of the people to the infected environmenet n case of an outbreak. Factors such as a very low infectious dose like 10 NoV particles are enough to cause the illness, the absence of long-lasting immunity, the stability of the virus in the environment, and its transmission by a variety of routes lead to the high impact of NoV outbreaks (20, 21). The Infections are not only limited to person to person, food-borne transmissions do sometimes occur, through an analytical epidemiological study food items were identified as likely transmission vehicles of norovirus for some of the norovirus outbreaks (23). NoV causes a relatively severe illness, characterized by acute onset of gastrointestinal symptoms including abdominal pain, vomiting and diarrhea. The illness is generally self-limiting, and symptoms usually resolve within 2 or 3 days. The incubation period for the illness ranges from 6 to 48 hours. Even after the infection subsides the shedding of the NoV continues for weeks, as demonstrated in a study which was conducted in young children diagnosed for NoV illness in a hospital. Shedding of NoV was observed for more than one month in 22.6% of these children during the regular follow-ups (25).Typically high concentrations of NoVs (~109 viral particles/ml) are present in the feces or vomit of infected patients and highly infectious and because of their low infectious dose they are considered very infectious. Noroviruses are classified as class B biological agents due to their high infectivity and stability and to the suddenness of outbreaks and the debilitating nature of the disease (27). Despite the fact that it has a significant economic impact and considerable morbidity, no drug or vaccine is currently available to treat or prevent human norovirus disease. The reason for this is aspects of norovirus biology are not well understood. This is due in large part to the absence of a cell culture system or small animal model for human noroviruses (28). Since there are no methods for cultivation of human NoV, appropriate surrogates of NoV are usually used instead. The most commonly used surrogate viruses are feline calicivirus (FCV) and murine norovirus (MNV), which are both culturable in the laboratory using tissue culture techniques, and neither are believed to be infectious to humans.

A commonly used surrogate for studying the virucidal activity of chemical disinfectants on the human NoVs has been FCV. Within the family Caliciviridae, FCV belongs to the genus Vesivirus and causes infection in cats (31, 32, 33). FCV is known to be a respiratory pathogen, therefore, survival and inactivation characteristics may differ from those of human NoVs, which are transmitted by the fecal-oral route. As a result, FCV may not be the most suitable surrogate for studying the characteristics of human NoV. Recently, cultivation of MNV was achieved in the dendritic cells and macrophages(27). Since like the human NoV, MNV is a member of the genus Norovirus and passes through the gastrointestinal tract. As a result, MNV seems to be a more suitable surrogate for human NoV than FCV (34).

Recently the first MNV was isolated and adapted to cell culture on dendritic cells and macrophages cells like RAW 264.7(ref 39). Four isotype MNV strains have reportedly been discovered so far, including MNV-1, MNV-2, MNV-3, and MNV-4 (35). MNV-1 is one of these strains isolated from research mouse facilities and has been used as a model of human norovirus infection (36). Even most of the noroviruses which are associated with infections in the humans, cattle, swine and mice have also been identified. Of these potential norovirus surrogates, the MNV is the only norovirus that can be cultured in the cell culture and also in a small animal. In addition, laboratory mice are versatile and inexpensive model for studying the viral pathogenesis, making MNV one of the surrogate for studying the characteristics of the NoV (36, 37). The MNV capsid structure, genomic organization, and replication cycle are very similar to those of human NoVs . Recently Cannon et al. showed that MNV and feline calicivirus (FCV) were resistant to organic solvents and their inactivation rates were similar at 63 and 72°C. However, MNV was more resistant than FCV to basic and acidic pHs. Long-term resistance was also higher for MNV than for FCV at room temperature (38).so like human NoV, MNV which is a member of the genus Norovirus and passes through the gastrointestinal tract. Because of this, MNV seems to be a more suitable surrogate for human NoV than FCV (40).

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Fresh produce is generally considered as an essential part of a healthy diet; In the US alone, the per capita consumption of fresh fruits and vegetables increased from 254 pounds in 1980 to 328 pounds in 2000. This growing trend in increased consumption of fruits and vegetables is expected to continue till 2020, with fruit consumption increasing by 24 to 27% and vegetable consumption increasing by 19 to 24% (54). Fresh produce grows in a natural habitat which is vulnerable to contamination by human pathogens, and an increase in food-borne outbreaks associated with consumption of berries, sprouts, and vegetables indicates that consuming fresh food products poses risk to the human health (41). While a substantial amount of research has been performed on the survival, transmission and inactivation of bacterial pathogens associated with fresh produce, published research regarding the stability of human pathogenic viruses on these food products is limited. Unlike bacteria, viruses cannot grow in or on foods but may be present in fresh produce as a result of fecal contamination. The source of contamination is not often clear. It may occur at the source, i.e. at the harvesting area, as a result of contact with water contaminated with untreated human sewage. Alternatively, handling of the produce by an infected person who can be shedding the viruses after harvesting may contaminate it with virus and transmit the infection (42).

The danger posed by enteric virus contamination of fresh produce was exposed during a (insert year of outbreak) outbreak of hepatitis A, where consumption of contaminated green onions caused 3 deaths and 601 cases of illness (9). The unique characteristics of these viruses include greater resistance to conditions such as low pH, low temperatures, and low water activity, which are conditions that bacteria may not be able to survive or grow ( 12).

Norovirus outbreaks in the fresh and frozen produce are very high, especially because of the attribute that the noroviruses can survive in the cold environment. Contamination of the produce with noroviruses often is a result of handling of the produce by infected food handlers at the source in the growing and harvesting areas (43). According to Baert et al, fresh produce can be considered as high-risk foods since at the post-harvest stage, handling of the produce by the infected food handlers ignoring the hygiene practices play a vital role. The produce handled manually which is not heated before consumption is at a very high risk compared to the other which are heat treated (44). Un-processed foods, such as vegetable or fruit salads, sandwiches, and bakery items, are most often associated with viral disease outbreaks. Butot et al.2009 reviewed outbreaks associated with salad vegetables and fruits in England and Wales between 1992 and 2000. Of 83 outbreaks, 13 (15.7%) were caused by noroviruses, and 23 (28%) were caused by unknown agents, but clinical and epidemiological features of these illnesses suggested that the majority may have been caused by noroviruses (45). A NoV outbreak occurred in 2000, where at least 333 persons became ill with gastroenteritis across 13 U.S. states, was due to the consumption of a catered meal that was prepared in Ohio and distributed to 52 car dealerships. An epidemiological survey conducted after the outbreak indicated contamination of the salads likely occurred during preparation by handling of the produce items by infected food handlers (46). In the U.K. an outbreak was reported where 55 of the 111 guests who attended a wedding reception became ill with NoV gastroenteritis. This outbreak was also linked to foodservice personnel, and resulted from a vomiting espisode of a food handler into a sink used for food preparation. Though the sink was cleaned and sanitized with a chlorine based disinfectant, the same sink was used for preparation of a potato salad the following day. This outbreak highlighted the ease of NoV cross contamination as well as the considerable resistance of NoV to chemical disinfectants (47)

A similar outbreak was reported from Michigan, involving three NoV outbreaks that affected at least 100 people The majority of those ill consumed sandwiches that were prepared by a single food handler who returned to work several hours after suffering NoV symptoms and was still excreting NoV in his stools. Lettuce, jalapeno peppers and onions were significantly linked to NoV illness in one of the three outbreaks. Epidemiologic investigation discovered that the food handler who tested positive for NoV sliced lettuce each morning by hand, and washed heads of lettuce in the same sink that employees washed their hands and no sanitizing step was performed before or after heads of lettuce were washed (48).

1.8 Cross Contamination Studies

Though there is lot of epidemiological evidence for foods as vehicles for norovirus transmission as described above. Research on NoV transmission in food service settings is very limited. Such a study was performed by Bidawid et al. (2004) using feline calicivirus (FCV) as a surrogate virus for NoV, on the virus transfer from the finger pads of hands to selected foods like ham and lettuce, and a representative food service surface, stainless steel. Ten microliters of the FCV inoculum was dried on volunteer finger pads and were asked to press onto each surface for 10 s at a pressure of 0.2 to 0.4 kg/cm2. Nearly 46 ± 20.3, 18 ± 5.7, and 13 ± 3.6% of the FCV inoculum was transferred to ham, lettuce and stainless steel, respectively and 6 ±1.8, 14 ± 3.5, and 7 ± 1.9% transferred from ham lettuce and stainless steel respectively onto the hands. A significant reduction of FCV was observed when the finger pads were washed with 75% and 62% alcohol-based based sanitizers. This study just represents the food service setting but not the actual service setting as the viruses were only inoculated on the finger tips where as in a food service setting whole hand is used for handling the produce. FCV is not recommended as a surrogate to NoV due to its sensitivities to pH extremes and inability to persist on environmental surfaces for extended periods of time (49).

A study investigating bacterial transfer rates between hands and food (chicken breast and lettuce leaves) and food preparation surfaces (cutting boards and spigots) was performed with Enterobacter aerogenes B199A as a surrogate microorganism.Although the transfer rates of the bacterium were highly variable, ranging between 0.0005% and 100%, a normal distribution curve to describe the cross-contamination risk associated with various steps in the food preparation process. (50).

A number of studies have investigated factors related to improvements in hand hygiene compliance. One such study reported that the knowledge of the person in charge of a facility was related to hand hygiene practices and that training which included demonstration increased the likelihood that employees would wash their hands properly (51). A similar work was carried out on the efficiency of hand washing, and it estimated that a food handler on average has 8.6 work activities per hour that involve hand washing opportunities which clearly indicates the level of the risk associated with cross contamination during handling(52). Food preparers and food servers should wash their hands 6 and 11 times per hour to reduce the chances of cross contamination (53).

Chapter 2

materials and methods

Media and Chemicals Used

Growth medium- To 500 ml of Dulbecco's Modification of Eagle's Medium (DMEM; GIBCO-Invitrogen), 50 ml fetal bovine serum (FBS; GIBCO-Invitrogen) was added, along with 10 ml sodium bicarbonate, 10 ml L-glutamine and 5 ml HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). All components were purchased from GIBCO-Invitrogen, and all components stored at -18°C prior to use, except for DMEM (4°C), HEPES (4°C), and sodium bicarbonate room temperature). Growth medium was stored at 4°C prior to use.

Agarose- Low melting Sea plaque agarose (Lonza) was dissolved in D.I. water to 3% W/V in sterile glass bottles and autoclaved at 121°C for 45 min then stored in the refrigerator. Prior to use, the bottle is micro waved till the entire contents in the bottle are uniformly melted and then held at 45°C in a water bath.

2x MEM - Minimum Essential Medium (MEM) is added to the D.I. water at double strength and then sterilized and then stored in the refrigerator till further use

Complete 2XMEM - 2XMEM is supplemented with Newborn calf serum, L-glutamine, HEPES and sodium bicarbonate and then stored in the refrigerator till further use. Prior to use, the bottle is transferred into the incubator maintained at 37°C.

Antibiotic - Penicillin anti biotic is used to eliminate the bacterial growth in the sample added at a ratio of 1:100 to the sample.

Gloves - Food Service polyethylene gloves, vinyl gloves are supplied by the Ecolab and the latex gloves used is the regular laboratory gloves. Prior to use, gloves are placed under UV light in a biosafety cabinet for 10 min on each side to eliminate the bacteria and other microflora present on them which may interfere with the results of the experiment

Romaine Lettuce- Romaine lettuce is purchased from Jewel-Osco and stored in stored in the refrigerator till further use. Prior to use, lettuce leafs free from any damages are selected and rinsed under D.I. water thoroughly to remove the dirt and dried with the paper towel. Then the leaves are cut into 5-5 cm pieces, and placed under UV light in a biosafety cabinet for 10 min on each side to reduce levels of microflora present on the leafs which may interfere with the results of the experiment.

Neutral Red - Neutral red is obtained from the Sigma Aldrich and stored in the refrigerator.

Stripping Solution - 0.04% K2HPO4, 1% Na2HPO4, 0.1% Triton X-100 is added to the D.I. water and autoclaved at 121°C for 45 min in the autoclave and then stored in the refrigerator till further use.

3% Beef Extract - 3 % of desiccated Beef Extract is added to the D.I water and autoclaved at 121°C for 45 min in the autoclave and then stored in the refrigerator till further use.

Cell culture and Plaque Assay

Cell line. A mouse macrophage cell line RAW 264.7(ATCC® TIB-71TM) was used for murine norovirus infectivity assays. Cells were stored in cryogenic vials in liquid nitrogen at a concentration of 107 cells/ml. Cells were resuscitated from storage by thawing quickly under warm water then added to 25 ml of growth media in a tissue culture flask. Flasks were incubated at 37°C, with 5% CO2 for 2-3 days to obtain 85-90% confluence

Murine norovirus (MNV-1). MNV-1 was cultivated and quantified using RAW 264.7 cell line propagated inside the cell culture media DMEM. To cultivate the MNV-1, RAW 264.7 cells are grown upto 90% confluence in 75cm2 tissue culture flasks and then infected with thawed MNV-1 at a 0.05 multiplicity of infection .After 48-72 h cytopathic effects takes on the cells and the cells are seen floating in the cell culture media DMEM and this is very clearly evident when seen under microscope at 1000x, the flasks are then transferred into the -80°C freezer and to harvest the viruses the flasks has to undergo freeze thaw cycles at least 3 times. After the last thaw cycle the contents from the flask are transferred into 50 ml centrifuge tubes and centrifuged (3000 rpm, 20 min, 4°C) to remove the cell debris. Then the viruses are concentrated to 10 fold using centrifugal filter units which are decontaminated with laboratory grade ethanol (Amicon Ultra-15, Millipore, Billerica, MA) in the centrifuge operated at 2700 rpm, 4°C for 15 min till the desired 10 fold is achieved on the volume basis. Then the viruses concentrate on top of amicon unit are carefully pipetted out by using 1000µL pipette very carefully so as not to rupture the filter membrane into the 15 ml centrifuge tube and the it is filtered again through 0.2 µm filter to ensure that the viruses are dispersed properly and free from bacteria, and then from 15ml centrifuge tube it is aliquoted into 1ml sterilized 2 ml vials and stored in -80°C freezer for future use. The filtrate, the concentrate and the culture media before concentration are plaque assayed to enumerate the titre and compare the concentration by volume basis. On average the final titer of concentrated MNV-1 was approximately 8 log10 PFU/mL. Before the start of the experiment the virus titre is removed from the freezer and then thawed on the ice.

6 well Plates. For the Plaque assay, The cells grown to 90% confluence were brought down to 107 cells/ml and then 320µL of this was added to 12ml of the complete DMEM and 2ml of the cell suspension in the complete DMEM is dispensed into each of the well of the plate using a pipette this is called seeding of the cells. Then the Plates are incubated at 37°C, with 5% CO2 for 24 h to obtain 85 to 90% confluence.

Plaque assay. The Enumeration of the MNV1 viruses is done by this plaque assay which is also called the infectivity assay,10 fold serial dilutions of samples were performed in complete DMEM, following the addition of a 1:100 dilution of antibiotic-antimycotic solution. RAW 264.7 cells were seeded into 6-well plates as mentioned above are taken out on the day of the experiment and the complete DMEM was taken out from all the wells of each plate before inoculation of the viruses from the dilution tube. 400 µL of the virus suspension from each dilution was plated in duplicates on the 6-well plates. After 30 minutes of incubation the plates are shaken well and after 1h the inoculum is removed from the wells and 1st overlay is performed which is overlaying with 2ml 1:1 mixture of agarose and 2xMEM and then incubated at 37°C, with 5% CO2. After 48 hours incubation a second overlay was done with 2ml 1:1 mixture of agarose and 2xMEM containing 0.01% neutral red and incubated for 24 hours. Plates with 10 to 100 plaques were counted, and the virus titer was expressed as PFU/ml.

Recovery Methods from Hands and Produce

2.3.1 Recovery from Gloves and Hand. Glove juice method was used to recover virus from surface of gloves and hands (58).Top side of the glove was spot inoculated with 25µL of MNV 1 and allowed to air-dry for 30 min in the biosafety cabinet. Mean while the same kind of glove was filled with 25 ml of the eluent. The inoculated gloves were worn over the hands and the glove with the eluent filled was worn on top of the inoculated glove and then the hand is massaged for 2 minutes (1 min on the palm and 1 min on the fingers). For the hand the inoculation was done on the bare hand after it is cleaned with rinsing with distilled water, after the massage the eluent from the glove is transferred into 50 ml centrifuge tube and stored on the ice till it is plaque assayed.

2.3.2 Recovery from Lettuce. The front side of the 5x5 cm romaine lettuce leaf pieces was spot-inoculated with 50µL MNV1 and spread evenly on the leaf with the pipette tip.

Then they were dried in biohazard cabinet for 45 minutes for the inoculum to dry. Three modes of recovery were compared namely pulsification, stomaching and vortexing

Stomacher bag was filled with 25 ml of the eluent (3% B.E /Stripping Solution) and one leaf with the dried inoculum was transferred aseptically using the sterile tweezers into the bag and sealed at the top. Then is the stomacher bag was transferred into the Pulsifier and Pulsifier is operated for 1 minute. This method is pulsification method and for stomaching method the bag is transferred into the Stomacher and Stomacher is operated at 230 RPM for 1 minute. For vortexing method unlike the other two methods the inoculated leaf was directly transferred to the centrifuge tube and vortexed for 1 minute at ten speed. After the treatments the eluent was stored on the ice till it is plaque assayed

Hand washing Trials

Volunteers were asked to rinse the hands with D.I water and wipe it down completely with paper towels. Left hand of the participant's hands was inoculated with 100 µl MNV-1 stock of 8-log PFU/ml and distributed evenly from each fingertip toward the palm. Volunteer was asked to air dry inoculated hands in a biohazard cabinet for 10 min. Three cleaning methods were studied a) Washing with tap water which was rising of the hands with regular tap water b) Washing with Liquid Soap was cleaning of the hands with application of soft soap and c) Washing with Foam Soap. Volunteers were escorted to the sink and running water and were asked to wash at least for 20 seconds as per the FDA code for hand washing in food service areas with one of the cleaning method until they feel clean and then asked to wipe off the hands with paper towels. The temperature of water was measured. Paper towels were discarded in the biohazard waste. Virions remaining on the hand after washing were recovered using the glove juice method as described in 2.3.1 and after the recovery the volunteer's hands were disinfected to remove the viruses completely from the hands.

Paper towel was soaked with 10% bleach and given to the volunteer and was asked to wipe their hands for alteast 30 seconds and same is done with 70% ethanol and then was asked to wash the hands in the running waster with Bacdown soap with lather up past wrists for at least 60 sec and same is repeated one more time with bacdown soap and then the hands are rinsed with waster and dried with paper towel. Then volunteers were asked to sanitize their hands with ethanol hand sanitizer.

Chapter 3

Results and discussion

Recovery from Gloves and Hands

The glove juice method (58) was investigated for its ability to recover MNV-1 from whole hands (palm and fingers), as opposed to other methods discussed in the literature, such as the finger pad method (56) and swabbing (57). Stripping Solution, growth medium, and 3% beef extract were compared to recover MNV-1 from inoculated bare and gloved hands. Initially, stripping solution was compared to 3% B.E. for MNV-1 recovery from latex gloves.

Recovery with beef extract (4%) was significantly lower than that with stripping solution - 24% (P<0.001) (Figure 3.1). B.E has commonly been used as an eluent for recovering enteric viruses from fresh produce, whereas stripping solution is the eluent for hands recommended by the CDC (58). Stripping solution contains detergent (Triton X -100), lowering the surface tension of the eluent and in turn may assist washing more viruses from surfaces into the eluent. The promising result achieved with stripping solution encouraged further investigation of its effectiveness on other surfaces.

Figure 3.1. Comparison of 3% Beef extract and Stripping Solution for recovery of

MNV-1 from latex gloves

The Stripping solution appears to be more effective at eluting MNV-1 from the range of gloves tested compared to growth medium (Figure 3.2). There is not a statistically significant difference between the recovery of MNV-1 from latex gloves with stripping solution compared to recovery from growth medium (P > 0.05).

Figure 3.2. Comparison of DMEM and Stripping Solution for recovery of

MNV 1 from Gloves and Hands

The stripping solution was toxic to the RAW 264.7 cells, lifting of all the cells when inoculated on them in a 6 well plate at dilution 100 may be because of the detergent in the stripping solution. This significantly reduces the detectability of the viruses from the gloves or the hands during the transfer studies. Since there was no statistical difference between recoveries of stripping solution and growth medium. Though the Growth medium was recovering lower than the stripping solution it is found to be a better eluent with better detectability then the Stripping Solution. So the growth medium is recommended as an eluent for recovering viruses from hands and gloves for the further transfer scenario studies.

Recovery from Lettuce

In an event of cross contamination in the food service area, the produce items may be the carriers of viruses on the fecal- oral route. Since the produce items in the food service setting come in to contact with various food service surfaces like the gloves, cutting boards ,knives and utensils the chances of these produce items picking up the viruses during the contact may be high. It is important to quantify the virus transfer from these food surfaces to the produce. Three kinds of mechanical treatments (Pulsification, Stomaching and Vortexing) and two eluents (3% B.E and Stripping Solution) as shown in figure 3.3 were compared for recovery of MNV 1 from the lettuce surfaces. Vortexing with stripping solution yielded a maximum recovery of 38.35% and 3% B.E was more consistent with all the three recovery procedures.

Figure 3.3. Comparison of DMEM and Stripping Solution for recovery of

MNV 1 from Lettuce

Many methods have been developed for the recovery of MNV 1 from the produce items including lettuce, Methods like shaking, vortexing and stomaching are used with variation in the inoculation techniques like spot inoculation, inoculation by immeserion type of the inoculation methods, eluent and the elution methods. In most of the methods Vortexing with Beef Extract worked as a better combination giving a maximum recovery of the inoculated viruses from the surface of the produce [33,10]. Previous work in this laboratory indicated that vortexing romaine lettuce leaves in 3% beef extract at pH 8.5 recovered MNV-1 more effectively than other levels of pH tested (Liu et al., 2009, unpublished). Since stripping solution was better in recovering the viruses from the surface of the glove, it was interesting to study the same with the lettuce so the recovery methods were also studied with the stripping solution. interestingly except the pulsification treatment the recovery of the viruses were higher in the stomaching treatment and the vortexing with the stripping solution then the 3% beef extract and once again the vortexing treatment proved as a better method for recovering viruses from the lettuce with 38.35% with the stripping solution and 27.02% with the 3% beef extract. Even though the vortexing is giving a higher recovery, during the transfer studies recovery of the viruses needs to be done from more quantity of the produce 50 ml centrifuge tubes used in this methods are not viable, so stomaching with a second highest recovery rates of 26.55 % and 24.49% with the stripping solution and the 3% beef extract can be considered as the recovery methods during the transfer studies. There is not a statistically significant difference between the recoveries by beef extract in lettuce compared to stripping solution (P>0.05). Considering the toxicity of the stripping solution on the RAW 264.7 cells, 3% beef extract with stomaching technique can be used as the recovery tool for recovering viruses from the lettuce.

Hand Washing

3.3.1 Hand washing trials. The PFU recovered from one volunteer's inoculated hand each day of the trial was considered the initial titer, to which all calculations for washing efficacy were performed against. An average of 5.71±0.74 log PFU was recovered from inoculated hands, and this initial titer was consistent throughout the trials .Washing hands with soft soap for at least 20 s removed an average 2.96-log PFU/ml MNV-1 from hands, compared with 2.90-log and 2.80-log after washing with the foam soap and rinsing for at least 5 s with running tap water, respectively (Figure 3.4).

Figure 3.4. Comparison of MNV 1 Removal from hand with different wash procedures

Variability was relatively high in these experiments, as was expected, due to the use of both male and female volunteers from a wide range of ages, and this variability is observed in both the scatter plot in Figure 3.4, as well as in the distribution plots in Figure 3.5 to 3.7. These distribution plots indicate the level of removal due to washing followed relatively normal distributions, ensuring further statistical analysis and modeling can be performed with this data.

Figure 3.5. Normal Distribution of MNV 1 Removal from hand with Water rinse

The above figure clearly indicates the variability of the reduction of the viruses from the hands during waster rinse the reduction was as low as 1 log and was as higher as the 6 log and this may be because of the reason that the hand washing style is very different between the volunteers and on a average there was 3 log reduction which clearly indicates that water removal alone is not effective in removal of viruses from the hand surfaces.

Figure 3.6 clearly indicates that the soap wash was more effective in removal of viruses from the hands with the least removal of 2 log against 1 log of the water rinse and the average was comparable with the water rinse, but interestingly the maximum removal was 4.5 log which was little lesser than the water rinse which has a maximum of 6 log and this again can be accounted for the volunteer style of hand washing.

Figure 3.6. Normal Distribution of MNV 1 Removal from hand with Soap Wash

Foam was very much comparable with the soap wash and the normal distribution clearly indicates that there is no much variability between both of them.

Figure 3.7. Normal Distribution of MNV 1 Removal from hand with Foam Wash

Only the left hand was inoculated in these hand washing trials. After washing, both the left and right hand were examined for MNV-1, and the results showed that there was a considerable transfer of viruses from the inoculated left hand to the non inoculated right hand during washing and this was again very variable between the volunteers and the normal distribution plot again of this data enables a sound risk assessment study.

Figure 3.8. Normal Distribution of MNV 1 transfer from left hand to right hand with Water rinse

This is really interesting to observe the transfer of viruses from inoculated left hand to the right hand during hand washing with water rinse, the log of the transfer % is depicted as shown in the normal distribution curve figure 3.8 and there is considerable amount of transfer and this clearly indicates the viral transfer during the hand contact even when rubbed vigorously under running water. This is also evidenced while doing a soap wash and foam wash

Figure 3.9. Normal Distribution of MNV 1 transfer from left hand to right hand with Soap Wash

Figure 3.10. Normal Distribution of MNV 1 transfer from left hand to right hand with Foam Wash

3.3.2 Hand Disinfection. After washing hands with laboratory soap, approximately 0.5-log was still detectable on the hands. Since the aim was to achieve no detectable MNV-1 on hands, the method was modified until no MNV-1 was detectable on hands. Few different combinations of hand wash was tried, Wiping the hands with bleach for 30 sec and 60 sec's, Reylon for 30 sec and 60 sec's, bleach with ethanol for 30 sec each, bleach with reylon for 30 sec each and non of them provided a satisfactory result, But finally bleach wipe and ethanol wipe 30 seconds each followed with consecutive hand washing's with bacdown soap for 1 minute each time provided a complete removal of viruses from hand and this hand disinfection procedure was used for all the volunteers participated in the studies for complete disinfection of their hands from viruses.

Chapter 3


Stripping solution worked better for the recovery of MNV 1 from hands, gloves and because of the reasons like the limitation in detectability and no statistical difference between the recoveries with Stripping solution and growth medium, Growth medium can be used as a eluent for recovery of MNV 1 from the gloves and the hand, As mentioned by the previous authors the 3% Beef extract was a better recovery eluent for the lettuce and stomaching technique is a better technique for the recovery of MV 1 from lettuce because of its consistency and no statistical variance from the other two methods. Since the recovery methods have been optimized for the various food service areas this helps in standardizing the methods for the transfer scenarios and conducting the volunteer studies.

The hand washing study also indicates the importance of the hand washing in removal of viruses from the hands and it also indicates the efficacy of the hand washing as per the FDA code for hand washing. This work also highlights the importance of the usage of the virucidal effect soap in the food industry.

Future work can be on the formulation of the soap with the ingredients having virucidal effect and its efficacy in removal of the viruses from the hands.