human noroviruses ( novs ) are a primary cause of viral gastroenteritis throughout the world ( siebenga et al .
2009 ) , and the principle cause of foodborne illness in europe ( kroneman et al . 2008 ; phillips et al . 2010 ) and the united states ( mead et al . 1999 ; scallan et al . 2011 ) . the number of estimated cases in the united states was recently revised to 5.5  million annually ( scallan et al .
2011 ) , while england has an estimated 2  million cases per year ( phillips et al .
norovirus illness is contracted through contaminated food and water and direct person - to - person transmission .
contamination often arises during the handling and preparation of foods , although there are many instances where foods , particularly shellfish , are contaminated within their environment .
products requiring extensive handling are also prone to contamination . in spite of interventions to eliminate product contamination , like thorough cooking , some products , such as salads and molluscan shellfish
are typically eaten raw or only lightly cooked and such products represent the greatest risk to the consumer ( richards 2001 ; richards et al . 2010 ) .
the development of commercial processing strategies for foods has been largely based on reductions in bacterial pathogens , and only to a lesser extent on the inactivation of enteric viruses .
for some of the enteric viruses , like poliovirus and related picornaviridae , astroviruses , parvoviruses , rotaviruses , and adenoviruses 40 and 41 , the levels of infectious virus particles can be determined using cell or tissue culture , since these viruses are propagatable .
unfortunately , nov can not be routinely propagated in cell culture or animal models . human exposure and resulting illness
scientists and regulators have relied on virus detection in foods based on the physical presence of viral rna , as determined by reverse transcription - pcr , which presently can not distinguish between infectious and inactivated viruses ( richards 1999 ) , although methods are being developed toward that goal ( li et al . 2011 ;
the lack of a suitable assay for infectious nov and other unculturable viruses , like most wild - type strains of hepatitis a virus ( hav ) , has led the research community to focus on viral surrogates .
the selection of a surrogate , at least in regard to novs , has been based on the ability of the surrogate to be propagated in culture , and its genetic , physical , or chemical relatedness to the pathogen . over the years
, different surrogates have been selected for use in determining the uptake , persistence , distribution , and inactivation of viruses in foods , water , and environmental samples as well as in chemical disinfection studies on surfaces .
studies with surrogates have been used with the intent to develop a better understanding about the pathogens they represent as well as applied applications for pathogen elimination . a respiratory virus known as feline calicivirus ( fcv ) in the genus vesivirus
was first used as a nov surrogate in the late 1990s ( doultree et al .
2006 ; dsouza and su 2010 ; doultree et al . 1999 ; gehrke et al . 2004
; hudson et al . 2007 ; jimemez and chiang 2006 ; kampf et al . 2005 ; lages et al . 2008 ; malik et al . 2006 ; malik and goyal 2006 ; mori et al . 2007 ; morino et al . 2009 ; poschetto et al . 2007 ; sattar et al . 2011 ; steinmann 2004 ; urakami et al . 2007 ; whitehead and mccue 2010 ) ; and processing interventions , like heating ( buckow et al . 2008 ; butot et al . 2009 ; cannon et al .
; fino and kniel 2008 ; nuanualsuwan et al . 2002 ) , marinating / acidification ( cannon et al . 2006 ; hewitt and greening 2004 ) , and high pressure processing ( chen et al . 2005 ; kingsley et al .
( 2006 ) identified murine norovirus-1 ( mnv ) as a closer genetic relative of nov .
now , mnv has become the more commonly used surrogate for chemical disinfection studies ( belliot et al .
2006 ; dsouza and su 2010 ; lee et al . 2008 ; lim et al .
2010 ; magulski et al . 2009 ; park et al . 2010 ; sattar et al .
2011 ) ; and studies on processing interventions like heating ( baert et al . 2008a ; cannon et al .
; sow et al . 2011 ; tanner 2009 ) , freezing ( baert et al .
; park et al . 2011 ) , gamma irradiation ( feng et al . 2011 ) , electron beam ( sanglay et al . 2011 ) , and high pressure processing ( kingsley et al . 2007 ; lou et al . 2011 ; tang et al . 2010 ) .
poliovirus and bacteriophage ms2 have also been used as potential surrogates for nov ( bae and schwab 2008 ; dsouza and su 2010 ; dawson et al .
another potential surrogate is the recently discovered rhesus monkey calicivirus , known as tulane virus , which can replicate in cell culture , but as indicated by tan and jiang ( 2010 ) , may not serve as a good model for human nov because it is not in the norovirus genus and has not been shown to cause gastroenteritis like the human novs .
unfortunately , there are fundamental differences in the inactivation rates of many closely related viruses  differences which may limit their role as surrogates .
surrogate viruses are generally expected to mimic the viruses they represent , although studies are proving this concept invalid . in direct comparisons between fcv and mnv , their susceptibility to temperatures , ph , and environmental conditions has been shown to be dramatically different ( cannon et al .
2006 ) . in these studies , mnv was significantly more resistant to both acidic and alkaline phs than fcv ; mnv was more resistant than fcv to chloroform , freon , and vertrel ; fcv was more stable than mnv at 56c ( p  <  0.05 ) , but differences were not significant at 63 and 72c ; and mnv was more stable in solution at room temperature ( cannon et al .
gibson and schwab ( 2011 ) reported that fcv was significantly less stable than mnv at 50c , but not significantly different at 60c . in these same studies ,
hav was significantly more resistant than fcv and mnv to heat treatment at 50 and 60c ( gibson and schwab 2011 ) .
bae and schwab ( 2008 ) evaluated the persistence of fcv , mnv , ms2 and poliovirus in surface water and groundwater and found that fcv was significantly less stable than mnv , ms2 , and poliovirus .
( 2011 ) compared the effects of ethanol - based hand rubs to eliminate fcv and mnv .
they showed that over a short contact time ( 20  s ) that fcv was 100 times more resistant to inactivation than mnv . in another study on hand sanitizers ,
fcv was more sensitive to some low ph sanitizers than mnv ; however , mnv was more readily inactivated by alcohols , thus the recommendation was made to include both surrogates when testing hand sanitizers ( park et al . 2010 ) .
a study on the inactivation of fcv and mnv by uv irradiation ( 254  nm ) showed fcv to be more sensitive than mnv ( park et al . 2011 ) .
dsouza and su ( 2010 ) evaluated the effects of chemical treatments to inactivate fcv , mnv , and ms2 and reported that a 2% trisodium phosphate treatment for 1  min decreased fcv and ms2 by over 6 logs , but mnv by only 1 log .
they also showed that 2% glutaraldehyde reduced fcv and mnv titers by 6 logs , but ms2 by <3 logs .
seventy percent ethanol was reported to cause no reduction in titers for any of the viruses ( dsouza and su 2010 ) .
( 2007 ) compared the persistence of nov and fcv in the digestive tissues of the oyster after depuration for 10  days and concluded that fcv was rapidly depleted , whereas nov persisted .
we now know that nov binds to histo - blood group antigens ( hbgas ) present on gastrointestinal cells of oysters , clams , and mussels ( le guyader et al .
2006 ; tian et al . 2006 , 2007 ) , which may account for differences in virus uptake by shellfish .
tulane virus also binds to hbgas ( farkas et al . 2010 ) , while mnv and fcv bind to sialic acid on the host cell surface ( stuart and brown 2007 ; taube et al .
these results should lead one to conclude that not all virus surrogates are equal ; some surrogates are more similar while others are quite different from the pathogens they represent .
a major limitation in these studies is a lack of correlation in the inactivation rates of the surrogates and of the pathogen . koopmans and duzier ( 2004 ) suggested the use of the most resistant enteric virus in developing food safety guidelines .
this may over regulate the industry if nov is substantially more susceptible than the surrogate . on the other hand
, even the most resilient surrogate may not be as resistant as the pathogen , and may lead to a false sense of security relative to the safety of food or water .
unfortunately , there is often no information available to directly correlate the pathogen with its ascribed surrogate , leaving regulators unable to promulgate new food regulations based on surrogate studies .
in addition , a surrogate may be representative of a pathogen s response to a particular processing scenario ( e.g. , pasteurization ) , but may not necessarily represent a pathogen s response under other processing conditions or in other food matrices .
data obtained must be carefully scrutinized and treated as presumptive evidence of how the pathogen may respond to a particular treatment . the use of fcv as a surrogate has diminished as more researchers are utilizing mnv , which is genetically more similar to nov than fcv .
consequently , past fcv research is being looked on as unreliable , while mnv is rapidly gaining popularity as a more suitable surrogate , in spite of the fact that fcv seems more resistant to chemical disinfectants than mnv .
the reason is that mnv may fail to respond in the same manner as the pathogen it represents .
this was demonstrated in a volunteer study involving the inactivation of nov in oysters using high pressure processing ( leon et al .
, pressures of up to 600 megapascals ( mpa ) for 5  min at room temperature were required to totally eliminate 4 logs of nov ( leon et al .
only 250  mpa was required to inactivate 7 logs of fcv in cell culture media under the same conditions , while 4 logs of mnv required 400  mpa for 5  min at 5c for inactivation ( kingsley et al .
one of the problems in comparing such studies is differences in processing conditions or matrix between assays . here , we see different matrices ( cell culture media and oysters ) and different processing conditions ( room temperature and 5c ) ; however , no other comparative studies exist for this processing technology .
another example of the differences in the inactivation of related viruses may be seen for poliovirus and hav .
poliovirus has been used as a surrogate for hav because of its similarity in size , shape , and structure to hav ; however , high pressure processing can inactivate hav at pressures around 400  mpa , but poliovirus persists at 600  mpa ( kingsley et al .
another study showed that different strains of cell culture - adapted hav have different sensitivities to heat and high pressure , where heating to 60c for 10  h and pressures of 420  mpa reduced virus infectivity by anywhere from 3 to 5 logs , depending on the strain ( shimasaki et al .
this represents a 100-fold difference in the inactivation of different strains of hav and highlights how different strains of the same virus do not accurately portray the inactivation dynamics of all hav strains .
much like the differences in inactivation noted among hav strains , different responses to inactivation should be anticipated among the nov strains .
different strains of fcv also showed widely differing susceptibilities to inactivation by three alcohol mixtures and a chlorine compound ( di martino et al .
if strains of the same virus give discordant inactivation results , then one might expect virus surrogates , which are only slightly related to the pathogens , to be poor indicators of the pathogen s inactivation kinetics .
novs include a variety of genetically similar strains ( also called genotypes or clusters ) within two genogroups ( i and ii ) .
the uptake of different nov strains by oysters varied with genogroup i , cluster 1 nov ( norwalk virus strain ) efficiently bioconcentrated , but genogroup ii , cluster 4 nov poorly bioconcentrated ( maalouf et al .
2011 ) . consequently , the use of a surrogate for viruses that differ widely in their genetic composition and likely in their response to various processing techniques , chemical disinfection , or environmental conditions seems counterproductive .
although the most resistant surrogates may be used to evaluate processing effectiveness , the pathogens could be several orders of magnitude more resistant to treatment than the surrogate .
predictive models have been proposed for nov and hav inactivation in shellfish , based on the use of surrogates , either fcv or a cell culture - adapted strain of hav ( buckow et al .
2008 ; grove et al . 2009 ) ; however , such models are not likely to accurately portray the inactivation of pathogenic viruses on or within foods . with the likelihood that mnv will not accurately reflect the conditions necessary to inactivate nov in foods , one questions the funding of surrogate studies
. will mnv become another fcv , with millions of dollars spent on research but little confidence in the results ?
considerable research funding is provided globally each year to conduct surrogate studies on nov ; however , there is little benefit derived from these studies in regard to the development of food processing strategies .
after years of research with fcv as the surrogate , investigators are considering those results highly presumptuous , particularly in light of mnv which is now generally perceived as a better surrogate .
the time , effort , and cost that were devoted to surrogate research with fcv may have been misdirected . at what cost was the fcv research conducted ?
no one can be sure , but certainly millions of dollars have been spent on what is now considered by most as an ineffectual surrogate .
a similar fate will befall mnv when researchers identify still better surrogates for nov and similar or greater dollar losses can be expected .
uncertainty concerning the reliability of surrogate studies has , to the best of this author s knowledge , prevented results from being implemented in regulatory actions or new food processing procedures .
if information were available on how much money was spent on surrogate studies and one were to conduct a cost  benefit analysis on the fcv or mnv studies performed to date , results would be startling , since there has been much money spent , but with little change in the way we process foods or sanitize the workplace . at the time of this writing ( september , 2011 ) , a number of papers had already been published during 2011 on the use of mnv as a nov surrogate , including the use of varying processing technologies , like electron beam ( sanglay et al .
2011 ) ; gamma irradiation ( feng et al . 2011 ) , and high pressure processing ( lou et al . 2011 ) to inactivate mnv in produce ; heat inactivation of mnv in clams ( sow et al . 2011 ) ; chemical disinfectants to eliminate mnv on produce and food contact surfaces ( predmore and li 2011 ) and hands ( park et al .
2011 ) ; and pulsed uv light inactivation of mnv on food contact surfaces ( jean et al .
such studies fail to provide reasonable expectations that the surrogates respond in a similar manner to nov or to different genogroups or strains of nov . in spite of the well intentioned and competent research that has been performed on nov surrogates , results derived from surrogate studies
have not answered the important questions regarding nov inactivation in foods , water , or on food contact surfaces .
we need to ask ourselves if fcv or mnv could ever be used in establishing food processing regulations when they only provide a glimmer of how the pathogen might respond to certain conditions . with all this uncertainty about surrogates ,
current practices to evaluate surrogate viruses and to employ molecular assays should be limited , particularly for nov inactivation studies .
it is hereby recommended that presumptive information derived from the use of surrogates be subjected to proof - of - principle testing and validated in volunteer studies using nov .
human challenge studies are essential to determine which processing techniques are effective in reducing nov in foods .
nov is considered the primary cause of gastrointestinal illness worldwide . with such high morbidity rates , strategically designed volunteer studies performed under controlled conditions
would seem prudent in order to assess the effects of cooking , freezing , irradiation , disinfectants , and other processing technologies on virus inactivation .
the cost for challenge studies would be high ; however , the results would be definitive .
a case in point was the recent volunteer study to assess the effectiveness of high pressure processing to inactivate nov in oysters , where it was definitively shown that pressure of up to 600  mpa for 5  min at room temperature would be required to inactivate 4 logs of the genogroup i.1 ( norwalk virus ) strain of nov ( leon et al .
does this mean that the same processing conditions would be required to inactivate the same level of other nov strains ?
no , not at all , but is seems likely that differences between nov strains would be less than differences between novs and their so called surrogates .
another example of the benefit of clinical trials involved a study performed in australia where shellfish containing nov were  purified  by depuration ( grohmann et al .
depurated oysters were subsequently fed to volunteers who became ill , thus demonstrating that commercial depuration was not effective in eliminating nov in contaminated oysters ( grohmann et al .
1981 ) . in this case , volunteer studies provided definitive answers about the infectiousness of the shellfish .
volunteer studies can provide a firm basis for developing food and water processing strategies and for making regulatory decisions . until such time that researchers develop a cell culture propagation system for nov , or other means to discriminate infectious from inactive nov , human challenge studies are our best hope for determining true virus inactivation parameters .
this recommendation to limit surrogate studies will be highly controversial , since funding for surrogate research has been plentiful and many researchers ( including those in my laboratory ) are accustomed to conducting nov research with virus surrogates .
however , a shift in research directions is essential if we are to identify realistic and practical processing strategies to improve food safety .
former and present research conducted with nov surrogates will likely contribute little to improving the world s food supply , while the costs of such studies could be pooled and redirected to more definitive clinical trials on nov inactivation .
future surrogate studies should be considered only when the research is well justified with a clear delineation of why a study with surrogates is appropriate and what definitive information is anticipated .
surrogate studies should be linked , to the extent possible , with nov volunteer studies to determine if the inactivation rates of surrogates and novs correlate after various processing interventions .
clinical trials will again be necessary to validate some of the new and upcoming assays that are designed to differentiate infectious from inactivated viruses , such as pretreatment of viruses with proteinase k and ribonuclease before rt - pcr ( nuanualsuwan and cliver 2002 ) , use of ethidium and propidium monoazide in conjunction with rt - pcr assays ( kim et al .
2010 ) , and various receptor and binding assays , like one developed by li et al .
virus surrogates may play a role in the development of assays for nov infectivity ; however , confirmation that the surrogates are truly representative of nov must be accomplished by volunteer studies .
for instance , receptor binding assays which show that mnv only binds when it is infectious , should be linked with volunteer studies designed to evaluate the assay using nov of demonstrated infectivity , as determined by clinical trials .
many in vitro surrogate studies suggest that nov can be inactivated by one treatment or another , but fail to carry the work forward to validate the results using similar treatments of nov in volunteer studies . over the years
my laboratory just completed a 5-year collaborative study that involved clinical trials at emory university in atlanta ( leon et al .
, there were seven separate trials involving 190 volunteers at emory university and the university of north carolina at chapel hill ( lindesmith et al .
2003 , 2005 , 2010 ) and there were no adverse reactions among any of the volunteers ( personal communication , christine moe , emory university ) .
other clinical trials involving novs have been performed at baylor college of medicine ( graham et al .
2002 , 2005 ) and elsewhere ( wyatt et al . 1974 ; dolin et al . 1982 ) .
nov illness is usually a temporary imposition where adverse effects outside of the usual nausea , diarrhea , and vomiting are seldom encountered ; therefore , there is reasonable expectation that trials may be conducted with minimal risk of complications .
oversight of clinical trials must be provided by institutional review boards for human subjects or similar entities whose mission is to evaluate potential patient risks , to ensure they are minimized to the extent possible , and to weigh the risks versus benefits of the proposed research .
costs for studies vary greatly , but an average volunteer may expect compensation of approximately $ 1000 ( us ) for their participation and incarceration in the hospital for 3 or 4  days .
hospital costs , labor , and overhead for researchers add to the overall expense , thus the cost of a volunteer study may approach $ 500,000 ( us ) in today s market , depending on the number of volunteers required to provide statistically significant results .
if monies that were normally used for surrogate studies were pooled , then multiple volunteer studies could likely be funded each year .
the threat of lawsuits , in the event of unforeseen circumstances , prevents some governments , universities , and hospitals from conducting or considering the funding of such studies . in reality
, there is far more risk in allowing millions of unsuspecting consumers to contract nov each year , when practical interventions are only a few volunteer studies away .
the price to pay for inactivity is great , with lost wages , medical costs , and regulatory and epidemiological expenses involved in tracking and managing outbreaks .
costs of supporting clinical trials would be a small price to pay for the considerable savings that would be brought about by even a modest reduction in nov outbreaks .
a change in attitudes and a shift in research priorities are essential if we are to win the battle against nov illness .
 the inability to propagate human norovirus ( nov ) or to clearly differentiate infectious from noninfectious virus particles has led to the use of surrogate viruses , like feline calicivirus ( fcv ) and murine norovirus-1 ( mnv ) , which are propagatable in cell culture . 
 the use of surrogates is predicated on the assumption that they generally mimic the viruses they represent ; however , studies are proving this concept invalid . in direct comparisons between fcv and mnv , 
 their susceptibility to temperatures , environmental and food processing conditions , and disinfectants are dramatically different . 
 differences have also been noted between the inactivation of nov and its surrogates , thus questioning the validity of surrogates . 
 considerable research funding is provided globally each year to conduct surrogate studies on novs ; however , there is little demonstrated benefit derived from these studies in regard to the development of virus inactivation techniques or food processing strategies . 
 human challenge studies are needed to determine which processing techniques are effective in reducing novs in foods . a major obstacle to clinical trials on novs 
 is the perception that such trials are too costly and risky , but in reality , there is far more cost and risk in allowing millions of unsuspecting consumers to contract nov illness each year , when practical interventions are only a few volunteer studies away . 
 a number of clinical trials have been conducted , providing important insights into nov inactivation . 
 a shift in research priorities from surrogate research to volunteer studies is essential if we are to identify realistic , practical , and scientifically valid processing approaches to improve food safety . 
Introduction
Limitations of Viral Surrogates
Costs and Benefits
Need for Clinical Trials