REVIEW

The zoonotic potential of rotavirus

Nigel Cooka,*, Janice Bridgerb, Kevin Kendallc, Miren Iturriza Gomarad,Laila El-Attarb, Jim Grayd

aCentral Science Laboratory, Sand Hutton, York YO41 1LZ, UKbRoyal Veterinary College, London, UKcAskham Bryan College, Askham Bryan, York, UKdHealth Protection Agency, Colindale, London NW9 5HT, UK

Accepted 23 January 2004

KEYWORDSZoonotic; Rotavirus;

Reassortants

Summary Rotaviruses are generally species-specific, but cross-species transmission ispossible, as has been demonstrated experimentally. Several case studies haveindicated infection of humans by animal rotaviruses. Comparison of genetic sequencesof human and animal rotaviruses often reveals close identity. Surveillance ofcirculating rotaviruses in the human population has revealed the presence of severaluncommon genotypes. Many of these have been found in domestic animals, and it ispossible that they arose in the human population through zoonotic transmission. Thelow incidence of uncommon strains would suggest that such transmission, or at leastthe establishment of an animal rotavirus or a human/animal reassortant virus in thehuman population, does not happen with any great frequency. However, many millionsof people will be exposed year on year to animal rotaviruses. This happens withinfarming communities, and potentially to visitors to the countryside. There may besome measure of environmental contamination through livestock excrement. Thisexposure may not result in high levels of infection, but some infection could occur.There may be a continual input of rotavirus strains or sequences into the humanpopulation from the animal population albeit at a very low level.Crown Copyright Q 2004 Published by Elsevier Ltd on behalf of The British InfectionSociety. All rights reserved.

Introduction

Rotaviruses have a wide host range, infectingmany animal species as well as humans. As itwas found that certain animal rotavirus strainshad antigenic similarities to some humanstrains,1 speculation increased about whether

animals play a role as a source of rotavirusinfection in humans. But it was observed, usingRNA–RNA hybridization assays, that most of thecorresponding genes of animal and humanrotaviruses do not have a high degree ofhomology whereas those of rotaviruses fromthe same species do.2 –4 These observations ledto the view that rotaviruses have a restrictedhost range in nature due to lower fitness in non-host tissues in terms of replication efficiency,and under natural conditions animal rotaviruses

0163-4453/$30.00 Crown Copyright Q 2004 Published by Elsevier Ltd on behalf of The British Infection Society. All rights reserved.doi:10.1016/j.jinf.2004.01.018

Journal of Infection (2004) 48, 289–302

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*Corresponding author. Tel.: þ44-1904-462-623; fax: þ44-1904-462-111.

E-mail address: n.cook@csl.gov.uk

do not infect humans or vice versa.1 There ishowever an alternative view that animalrotaviruses can indeed infect humans and causedisease whenever the chance exists.5 This isbased on the identification of unusual rotavirustypes, with properties of strains more commonlyfound in animals, which were isolated fromvarious cases of human infection. These unusualhuman rotavirus types may have arisen either aswhole virions or as genetic reassortants betweenhuman and animal strains during coinfection of asingle cell. The segmented nature of the genomesuggests that, like other viruses with segmentedgenomes such as influenza virus,6,7 rotavirusesare able to form new strains by a mechanism ofreassortment. Reassortment can occur when tworotaviruses of two different strains infect thesame cell, and during replication and packagingthey exchange genome segments.8 The 11genome segments of the parental virus strainscan theoretically reassort into 2048 (211) differentpossible genome constellations, if reassortment israndom.9

Gouvea and Brandtly9 hypothesized that rota-viruses exist as mixed populations of reassortants,and that reassortment was the driving forcebehind diversity. A prerequisite of diversity isco-circulation of many different rotavirus typesin a population; and more diversity, and morefrequency of uncommon strains, is seen in yearswith the highest number of co-circulatingstrains.10,11 Gouvea and Brandtly9 consideredthat mixed populations of rotaviruses are beingcontinually propagated in human and animalhosts, resulting in new and diverse progeny popu-lations of rotavirus.

There is a question as to whether transmission ofrotaviruses from animals to humans are ‘deadends’, i.e. do the viruses involved have anycapacity for secondary transmission to otherhumans and for initiating an outbreak? Viruseswhich have arisen by reassortment betweenhuman and animal rotaviruses however, may havegreater fitness and become established in thehuman population.

With regard to new rotavirus strains arisingthrough reassortment, a concept of zoonoticgenes may be developed. These can be defined asgenes originating in animal rotaviruses which caninteract with genes of human rotaviruses, to forminfectious rotavirus particles which are seriallypropagated in the human population.

It is possible that reassortant rotaviruses canarise not only through simultaneous infection bytwo different strains, but also after asynchro-nous infection where one strain infects a host,

after (e.g. a few days) another has initiatedinfection.8,12

Experimental evidence for zoonotictransmission of rotavirus

It has been demonstrated many times that animalsof one species can be infected by rotaviruses whichhave been isolated from another species, includinghumans. The early studies of this type werereviewed by Theil.13 Two more recent examplesof experimental cross-species infectivity areworthy of note. The first is that the human G1P[8] rotavirus strain Wa is pathogenic for exper-imental pigs14 and is currently used as a pathogenicchallenge inoculum in pigs to assess efficacy ofpotential human rotavirus vaccines.14,15 The rota-virus NSP1 and NSP4 non-structural proteins showspecies specificity, but phylogenetic analyses of theWa NSP1 and NSP4 genes show that both genescluster with those of porcine rotaviruses.16,17 Inaddition, G1 rotaviruses are known to infect pigs.18

It is possible that these relationships may explainthe molecular basis for the observed infectivity ofhuman rotavirus Wa for pigs. A second example ofexperimental cross-species infectivity where thegenetic composition of the infecting rotavirus hasbeen examined is that of the bovine rotavirusisolate PP-1, which was highly pathogenic toexperimental pigs but replicated poorly in exper-imental cattle.18 Sequence analysis showed thatthis G3 rotavirus had porcine NSP4 and VP4 genesbut a bovine NSP1 gene. This strongly suggestedthat PP-1 was a reassortant between bovine andporcine rotaviruses but that a species-specific NSP1was not critical in cross-infection between cattleand pigs. These two examples unequivocallydemonstrate that cross-species rotavirus infectiondoes occur, at least experimentally, and that thisinfection can be pathogenic in the heterologousspecies.

Epidemiological evidence for zoonotictransmission of rotavirus

Until recently, specific rotavirus types have beenassociated with specific animal species. For example,human rotaviruses most commonly belong to G types1–4 and P types [4] and [8],19 whereas bovinerotaviruses most commonly belong to G types 6, 8and 10 and P types [1], [5] or [11].20 Rotavirussubgroup II is strongly associated with human strains(L. Svensson: personal communication). As more

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rotaviruses have been characterized, the hostspecies specificity of P and G types has becomeless distinct. Human group A rotavirus strains thatposses genes commonly found in animal rotaviruseshave been isolated from infected children in bothdeveloped and developing countries. Strains such asG3 (found commonly in species such as cats, dogs,monkeys pigs, mice, rabbits and horses), G5 (pigsand horses), G6 and G8 (cattle), G9 (pigs andlambs), and G10 (cattle) have been isolated fromthe human population throughout the world.21 Gand P type combinations which are found in manhave also been found in animal species. Forexample, G10P[11] was found in American andCanadian cattle by Lucchelli et al.22 and in Indiancows and buffaloes by Gulati et al.23 G3P[6] andG4P[6] were found in pigs in Poland and the USA byWiniarczyk et al.24 and G1P[8] and G5P[8] werefound in pigs in Brazil by Santos et al.25 Theemerging G9 strains26 –28 may have arisen in humansthrough transfer from animals. They have beenfound in lambs and pigs.25,29 In humans, they appearto cause more severe symptoms than the commonrotavirus strains,26,30 which might be due to lessimmunity to these emerging strains, or to greatervirulence being conferred by their genetic makeup.

Several studies have indicated symptomaticinfection of humans by animal viruses. Nakagomiand Nakagomi31 reported that almost all genesegments of a rotavirus G3 strain (AU228) isolatedfrom a child with a pet cat were identical to thoseof a feline rotavirus strain (FRV-1). Strains verysimilar to this may have become established inhumans.5 A three week-old baby in an Israelihousehold which had a young dog (,6 months old)was infected with an animal rotavirus G3 strain.32

An outbreak of group B rotavirus gastroenteritisoccurred in Maryland, USA in 1985.33 Six out of 16people (adults and children) with gastroenteritisexcreted a virus similar to a group B rotavirus strainin rats.

Das et al.34 reported that a G8 rotavirus whichhad widely circulated in newborn infants in India,causing asymptomatic infection, had VP7 and VP4gene sequences which were identical to those of abovine rotavirus strain. It was not certain whetherthis strain was a pure bovine virus, or whether someof is genes were derived from human rotaviruses viareassortment. Its NSP1 gene was highly homologousto that of a G3 strain which was later isolated fromcattle, suggesting reassortment had occurred.35

Cooney et al.36 suggested that, since G8 strainspossess diverse P-types, the exchange of serotypeG8 VP7 genes between humans and cattle may haveoccurred on more than one occasion, or may becontinuing. Nakagomi and Nakagomi5 considered

that available evidence suggested that whereassome feline and canine rotavirus strains havespread into human populations as whole virions,bovine rotaviruses were involved in reassortmentwith human rotaviruses, leading to the emergenceof unusual strains in various parts of the world.

Apparent dual infection with human and animalrotaviruses has been observed. Nakagomi et al.37

recovered G1P[5] and G1P[8] strains from an infantwith severe diarrhoea. The G1P[5] rotavirus wasgenotypically similar to bovine strains. It was notisolated from the infant in high titre, and possiblyhad little if any effect on the child’s disease.Nonetheless it would have had the potential toreassort with the coinfecting strain.

It is possible that some human rotaviruses havemultiple origins. Holmes et al.38 speculated that anunusual G8 P[14] type was derived from reassort-ment between human, bovine and lapine strains.

This review will examine the potential forzoonotic transmission of rotaviruses from a UnitedKingdom perspective, describing the incidence ofuncommon rotavirus strains, detailing potentialroutes of transmission of animal rotaviruses tohumans, and discussing risks of exposure, whichshould be illustrative of the situation in a widergeographical context.

Evidence for zoonotic group Arotaviruses in the United Kingdom

During a survey of circulating rotavirus strainscarried out in the UK between 1995 and 1998,39

several uncommon genotypes were identified.Reassortment between common human strainscould explain the presence of some of theseuncommon strains, but not all. Some of the strainswith unusual G and/or P types, i.e. G1P[9], G3P[6],G3P[9], G8P[8], G9P[6] and G9P[8] could be theresult of zoonotic transmission or of gene transferby reassortment. Many of these virus types arefound circulating in domestic animals and pets.21

Iturriza-Gomara et al.40 considered that the G9strains resulted from the recent introduction intothe UK of G9P[6] rotavirus, followed by reassort-ment with the common G1P[8], G3P[8] or G4P[8]viruses. They observed that the G9P[8] strainsincreased in incidence between 1995 and 1998,whereas that of G9P[6] declined: this could suggestthat G9P[6] has a replicative disadvantage inhumans, which could be consistent with an animalorigin. Cubitt et al.26 observed that childreninfected with G9 strains were older, and had moresevere symptoms than those infected with other

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strains; this would support the recent introductionhypothesis, since this population would thus lackimmunity to these strains. Furthermore, G9 strainswere not detected in archived rotaviruses collectedbefore 1995 in the UK.

The uncommon rotavirus strains in the UK studymade up 2.3% of the total identified.39 From thisdata, it can be estimated that a minimum of 850 ofthe reported 17,000 cases of rotavirus infection inEngland and Wales annually were caused by strainsof unusual types. If this extrapolated to the wholepopulation, a total of 25,000 infections withrotaviruses of unusual types might be expectedannually.39

The following sections will focus on the routeswhereby animal rotaviruses may be transmitted tohumans, to demonstrate the risk of exposure tothese agents in the UK population.

Direct contact with animals

In many developing countries, there is close contactbetween humans and domestic livestock. In areasprone to flooding, or with a monsoon climate, thiscan increase the chances of contact with animalfaeces. In the UK and other developed countries,contact with farm animals is relatively limited, interms of the proportion of the population workingon farms, but this contact could be sufficient toallow ingress of animal rotavirus strains or genesinto the human population. Exposure of humans toanimal rotaviruses may also be promoted throughcontact with domestic pets, particularly cats anddogs.

Contact with farm animals

All farm workers handling livestock, especially theyoung animals that are frequently handled, getcontaminated continuously with livestock faeces. Itis common for piglets, lambs and calves to behandled frequently during the first few weeks oflife. Not many farm workers wear gloves, and theymay eat using soiled hands. Smoking also may beanother way to acquire the viruses. Clothing andfootwear can be heavily contaminated, then takeninto residential accommodation. Because farmworkers are predominantly engaged in manualwork, their hands can get quite rough and providecracks where viruses can become located, evadingremoval by light washing. Aerosolised virusesproduced from cleaning practices, e.g. hosing ofpens, could also contaminate hair and skin. Often,it is not farm workers’ practice to shower after

work, but they can leave the farm and straightawayparticipate in a recreational activity, for instancemeeting friends in a public house, where others canget exposed to any pathogenic microorganismswhich they may harbour on their clothing or hands.

As well as faeces, dust and effluent may bepotential vehicles of transmission of rotavirusesbetween animals and farm workers, and also toothers with access to farms. Within piggeries, thereis a continuous cycle of rotavirus infection, withtransmission of rotavirus from piglet to piglet, andfrom litter to litter.41,42 Rotaviruses have beendetected in samples of dust, faeces and effluentcollected from a piggery.43 They were detected insamples taken from farrowing and weaner roomsbut not from fattener and sow houses. Thefollowing house had previously been cleaned anddisinfected, and the weaner house had actuallybeen free of piglets for 3 months. Porcine rota-viruses can survive in faeces for at least 4 monthswith only a 2 log10 reduction in the titre ofinfectious virus.43

In the UK, there are large numbers of farmanimals. The latest DEFRA statistics show that in2000 there was a total agricultural workforce of550,000, of which approximately 50% will come intodirect contact with farm animals. This is just under0.5% of the UK population.

As an illustrative example of a potential con-sequence of farm workers’ exposure to rotavirus, ahypothetical estimate of the number of infectionswith bovine rotaviruses through contact with calveson dairy farms can be made. In England, there areapproximately 18,000 dairy farms. It can beassumed that, since young cattle need extensivecare, a minimum of 1 person per day per farm is indirect contact with them. On dairy farms, thecalving season lasts all year. Therefore the numberof calf contact days per person per year in Englandis 6,570,000. The average number of dairy cattleper herd in approximately 80, each of which willcalve once per year. All the calves become infectedwith rotavirus, for a 5-day period in their second orthird week of life, and shed virus in their faeces at aconcentration of at least 1 £ 106 infectious rota-virus particles per ml. If it is assumed that onaverage each worker ingests 1 ml faecal materialper day, e.g. when eating lunch with soiled hands,them they will ingest at least 1 £ 103 infectiousrotavirus particles each day of contact withinfected calves. From the results of the study ofWard et al.,44 ingestion of 1000 infectious humanrotavirus particles will cause infection in approxi-mately 85% of people. Therefore, approximately5.5 million infections per year could occur throughfarm workers’ exposure to animal rotaviruses. Of

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course, this exposure would not in every instanceresult in infection, as animal rotaviruses will havelimited specificity for humans, and also farmworkers would have a degree of immunity againstthe animal rotaviruses through previous contact.But if there is, say, a one in a million chance of afarm worker becoming infected through ingestionof 1000 bovine rotavirus particles, then each yearon average at least five human infections will arisethis way in the UK. If the chance is one in athousand, then the number of human infections willbe 5000 per year. Many of these infections will beasymptomatic. But secondary spread to family orsocial contacts could occur. The opportunity forreassortment between the bovine rotavirus strainsand any human rotavirus strains which may beinfecting the farm workers or their secondarycontacts also exists.

Exposure to animal rotaviruses will also occurwith farm workers handling suckler (beef) calves,piglets and lambs, and similar calculations couldalso be performed for these situations.

Non-farm workers can also have direct contactwith rotavirus-infected livestock. Opportunities areafforded by access of children to farms, forexample (and perhaps significantly) during thelambing season, and the proliferation of openfarms rearing exotic species of domesticatedanimals, which may also harbour and excreterotaviruses. The protozoan parasite Cryptospori-dium parvum may provide an analogous example. Itis zoonotic, being found in a range of animalsincluding cattle.45 The transmissive stage of theparasite, the oocyst, cannot multiply outside ahost, but like a rotavirus particle, it can persist in aninfectious state in various environments.46 Also likerotaviruses, C. parvum oocysts are shed in largenumbers in the faeces of infected animals.45

Cryptosporidiosis can be acquired through directcontact with farm animals.47 –50 There have beenoutbreaks in non-farm workers after visits to openfarms, where faecally contaminated calves werehandled, or raw milk or animal feed consumed.51,52

There are approximately 300 farms in Englandwhich actively allow school visits (data from www.farmsforteachers.org.uk). Guidelines do, however,exist to help avoid infection during such visits.50

They recommend amongst other things fresh bed-ding for livestock to minimize the risk of childrencoming into contact with faeces, and the removalof animals with scour (diarrhoea).

Evidence against transmission of rotavirusfrom livestock

In a study undertaken in Panama between August

1979 and March 1980, Ryder et al.53 surveyed theprevalence of antibodies to rotavirus in, and theincidence of rotavirus diarrhoea among cattleranchers, their families, and their livestock. Onehundred and eighty farm workers, and 161 familycontacts were studies. The animals comprised 512cattle, 35 pigs and 19 sheep. At the start of thestudy, less than 30% of the people and animals on allthe farms had detectable antibody (IgG) to rota-virus, and there was no difference between theworkers who had daily exposure to the animals andthe women and children who rarely had contactwith the animals. No antibodies to rotavirus weredetected in pigs and sheep. By March, approxi-mately 70% of the humans and nearly 40% of thecattle had developed detectable antibodies torotavirus. No correlation could be observedbetween the rates of rotavirus infection in humansand animals, either overall or on individual farms.Rates of rotavirus infections among humans werethe same on farms with cattle as on those with pigsand sheep. Also, infection rates were similar in allworkers, regardless of how long they had beenworking with animals. Looking within the workers’families, however, it was observed that there was ahigh correlation between infection of children andinfection of adults. Infection rates were higheramongst parents with four or more children thanamongst those with fewer numbers or no children.The authors of the study concluded that forrotavirus infections in humans, children are thereservoir.

While the antibody prevalences were low in thisstudy, its results suggested that repeated episodesof rotavirus infection occurred in individuals,perhaps at intervals as short as 1 year. No typingof rotavirus isolated was performed. So it cannot beruled out that some of the human infections wererelated to the animal infections. As more of thefarms studied had animal containment structurescontiguous with the workers’ housing, close contactbetween children and animals may have occurredwith some regularity and transmission from childrento adults may have involved animal rotavirusstrains.

Contact with pets

Several case studies have indicated infection ofhumans by direct contact with household pets.Nakagomi and Nakagomi31 reported that a rotavirusgroup A G3 strain isolated from a child with a petcat was identical to a feline rotavirus strain. A 3-week old baby in an Israeli household which had ayoung dog (,6 months old) was infected with acanine rotavirus group A G3 strain.32

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Rotavirus infections in dogs are commonly sub-clinical.54 Human rotavirus has been shown, exper-imentally, to infect dogs.55 Dogs are alsosusceptible to infection with porcine56 and bovinerotaviruses.57 Sequence analysis of VP7 genes fromsome canine and feline rotavirus strains revealed ahigh degree of homology between them and mayreflect interspecies transmission.58

In a study of outpatients with diarrhoea on aNative American reservation, Engleberg et al.59

found that dog ownership was significantly ðP ¼

0:05Þ associated with rotavirus infection. Ownershipof cats, cattle, horses, or any other animal was notassociated with illness or seropositivity to rotavirus.The most significant factor was actually contactwith children under 2 years. Dogs were not thoughtto be the reservoir of infection, but to act astransmitters of the virus by rooting around on therefuse piles within the reservation (which containedsoiled nappies) and carrying viruses on their paws orsnouts.

In a survey of healthy cats in an animal shelter inMelbourne, Australia between March and October1984, Birch et al.60 found that 5% shed G3 typegroup A rotavirus. They noted that the most recenthuman infections (March to May 1984) were also dueto G3 rotavirus, and would not rule out thepossibility of interspecies transfer.

Of the 24.5 million UK households, just underaround 45% own a pet. In 2001 the number ofhouseholds owning pets was: dogs: 4.8 million;cats: 4.8 million. The number of pet dogs was 6.1million and pet cats 7.5 million (data from the PetFood Manufacturers’ Association; www.pfma.com).Also in the UK, there are around 1 million horses and3 million horse riders (data from the British HorseSociety www.bhs.org.uk). Handling and care ofinfected pets could directly expose humans torotaviruses. Also, household contamination ofobjects and surfaces with faeces from infectedanimals may also allow transmission of rotavirusesto humans.

Contamination of the environment andfood

Rotaviruses in livestock excreta

The excreta from infected cattle, pigs and sheepcontains large numbers of infectious rotavirusparticles. This is a potential source of contami-nation in various ways. Viruses in excreta depositedin fields could pass via run-off water into freshwaters such as rivers or lakes. Aerosolised virus

could be produced through disturbance of excreta,e.g. during cleaning practices. Also, excreta fromcattle and pigs can be stored, then spread ontoland. All the excreta are stored, including that fromdiarrhoeic animals. It is likely that rotavirusescould, via manure or slurry, contaminate arableland, water sources, and possibly also crops. As anexample, the potential extent of rotavirus con-tamination of land via cattle slurry from an averagedairy farm in the UK can be considered.

The average number of dairy cattle per herd isapproximately 80, each of which will calve once peryear. One dairy cow produces 53 l excreta per day(data from Anonymous61); assuming it is all stored,the cattle slurry produced will be 1.5 £ 106 l. Thereare 80 calves produced per year, all of whichbecome infected with rotavirus for a 5-day period intheir second or third week of life. Assuming eachcalf produces 2 l of rotavirus-contaminated faecesper day during infection, the total volume ofexcreta the calves produce over the year is 800 l,containing at least 106 infectious rotavirus particlesper ml. After mixing with the slurry produced by theolder cattle, and assuming homogeneity, theconcentration will be at least 5 £ 105 infectiousrotavirus per litre of slurry. A recent ADASrecommendation62 is that slurries should be pre-ferentially stored for 3 months prior to landapplication. Bovine rotaviruses have a decay ratein stored slurry of less than 2 logs in 12 weeks;63

therefore, in the current example, the concen-tration of rotavirus after 3 months would be around5 £ 103 per litre. If the slurry is spread onto land,the contamination per hectare can be estimated.The area of land required for spreading waste fromone dairy cow housed for 6 months is 0.19 ha.61 So53 £ 182.5 ¼ 9.6 £ 103 l of cattle slurry is spreadonto 0.19 ha. Therefore 1 ha will be spread withapproximately 5 £ 104 l of cattle slurry. This areacould then be contaminated with 2.5 £ 108 infec-tious rotavirus particles. In other words, 1 m2 couldbe contaminated with 2.5 £ 104 infectious rotavirusparticles.

There is a round-the-year application of cattleslurry onto land in the UK.64 This will mediate acontinuous introduction of animal rotaviruses intothe environment. Besides cattle slurry, cattlemanure, pig waste, and sheep excrement will alsoprovide vehicles for environmental contaminationwith animal rotaviruses. Consequent humanexposure to animal rotaviruses through agriculturalwork is likely. With regard to farm visits, orrecreational activities such as rambling, the Cryp-tosporidium analogy may again be illustrative. Thenumber of cases of cryptosporidiosis in England andWales65 and Southern Scotland66 declined in 2001

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after the outbreak of foot and mouth disease.Hunter et al.67 considered that this was probablydue to the removal of access to the countryside,one of the control measures imposed, whichprevented people from coming into contact withfarm and wild animals and their excrement. It canbe reasonably expected that exposure to animalrotaviruses also occurs through similar contact, andit would be interesting to examine rotavirusincidence likewise. Furthermore, the potentialalso exists for contamination of watrcourses andfood crops with animal rotaviruses, via excrement.

Contaminated water

There are several enteric viruses which have beenproven to be transmissible by water,68 and waterhas been the vehicle in a number of outbreaks ofrotavirus gastroenteritis worldwide.

A risk of contamination of watercourses withanimal rotaviruses exists through animal excreta.As is known to occur with Cryptosporidium,46,69

rotaviruses shed from infected animals on pasture-land could be transported via run-off waters intowatercourses.

It has been shown that enteric virus excretedfrom cattle can be present in river water. Leyet al.70 demonstrated that bovine enterovirus, apicornavirus which can cause diarrhoea and abor-tions but which is generally asymptomatic, and canbe detected in the faeces of cattle could also befound in pasture pools, run-off streams and anadjacent river. In a study conducted in Switzerland,Metzler et al.71 detected rotavirus by RT-PCR inapproximately 40% of samples (23/58), whichincluded sewage effluent, river water, lake water,spring water and well water. Five isolates ofinfectious rotaviruses were isolated from thesamples by cell culture. When typed, three isolateswere G8P[1], one was G8 but unidentified P type,and another was unidentified. Rotaviruses of theG8P[1] type have been exclusively detected incattle, but when Metzler et al.71 examined 18faecal samples from diarrhoeic calves in the area oftheir study, G8P[1] rotaviruses were not detected.They did not state whether any human clinicalsamples were also screened, and the origin of theunusual rotaviruses was therefore not clear. Grata-cap-Cavalier et al.72 detected bovine and porcine(group A but type not reported) rotaviruses indrinking water from homes in Isere, France, wherethere were children with rotaviral gastroenteritis.The rotavirus isolates were not the same as thosewhich were infecting the children, but the authorsconsidered that consumption of animal rotavirus-

contaminated water could facilitate the occurrenceof human-animal virus reassortants.

The MAFF Water Code61 recommends that farm-yard manure or slurry should not be spread within10 m of any ditches, streams, rivers or ponds. But arecent survey of farms in England62 revealed thatthis precaution against contamination of water-courses was not being observed in all cases, with22% of respondents with manure/slurry and water-courses spreading less than the recommendeddistance. There has been much experimental workdone to show that enteric viruses can be trans-ported through soils,73 and a recent field study74

demonstrated that poliovirus seeded onto soil couldbe transported 21.5 m to contaminate a well.

Rotaviruses may be able to persist in freshwaterfor several days. In a study by Raphael et al.,75 itwas demonstrated that a 100-fold decline ininfectious particles in river water took approxi-mately 10 days at 20 8C, and 32 days at 4 8C. Thus,rotaviruses have the potential for wide dissemina-tion through watercourses after a contaminationevent. Humans could consequently becomeexposed to animal viruses through drinkinguntreated water, either directly or after failure ofa potable water treatment or distribution system.Bathing or recreational water would be anotherlikely source of exposure.

In the UK, there have been no reported water-borne outbreaks of rotavirus. But sporadic cases ofwaterborne viral gastroenteritis are difficult torecognise and are likely to go unnoticed.68 Thiswill be even more apposite of any animal rota-viruses, given the probable low risk of symptomaticinfection. But the size of the recorded outbreaks inother countries demonstrates the magnitude ofexposure which waterborne human rotavirus trans-mission can cause, and gives some indication of thepotential exposure of a human population to animalrotaviruses, which could be continually present insome water sources. It would be informative toverify, and ascertain the extent of, this presence bya survey of freshwater sources for animalrotaviruses.

Contaminated food

Animal rotaviruses could potentially be transmittedby foods directly, i.e. through consumption of meatfrom infected animals, or indirectly, through con-sumption of foods contaminated with organicwaste. Another indirect route of transmissionmight be handling of food, such as fresh produce,sold on farms, by farm workers exposed to infectedanimals.

The foods which are normally associated with

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transmission of enteric viral disease are those whichare eaten raw, such as soft fruit and saladvegetables, or only lightly cooked, such as shell-fish.76,77 However, foods which have been contami-nated by infected handlers post-cooking can alsocause outbreaks of viral disease.

Although rotaviruses should be as easily trans-mitted by foods as most other enteric viralpathogens, foodborne disease outbreaks are seldomreported. In England and Wales, the most commonidentified cause of foodborne viral disease arenoroviruses,78 which between 1992 and 1995,accounted for 6% of reported outbreaks of food-borne gastroenteritis, and 95% of foodborne gastro-enteritis outbreaks where a viral agent waspositively identified.78 The reason for this dispro-portionate figure may be that most of the casesoccurred in large outbreaks, and the food vehiclewas relatively easily identified. It is unlikely thatthis would occur with animal rotaviruses, againgiven the probable low risk of symptomatic infec-tion. However, the possibility of sporadic food-borne infection cannot be ruled out. Also, thecorrelates of protection from rotaviruses andnoroviruses are poorly understood although symp-tomatic reinfection with noroviruses is not uncom-mon. Repeated rotavirus infections are usuallyasymptomatic or very mild.

Group C rotaviruses were the cause of a largefoodborne outbreak affecting schoolchildren andteachers in seven elementary schools in Fukui City,Japan:79 this affected more than 3000 individuals.One particular preparation center supplied lunch toall the affected schools, and this was the basis forattributing the outbreak to infected food, althoughno particular food item was incriminated throughquestionnaire responses and the virus could not bedetected in food samples. The rotavirus isolatedfrom the patients was immunologically similar toporcine group c rotaviruses, and Matsumoto et al.79

speculated whether it was actually of porcineorigin. As pork is usually thoroughly cooked it isunlikely that it was the vehicle in this outbreak;post-cooking contamination of foods by improperhygienic practice (e.g. contact with raw pork onpreparation surfaces) cannot however be dismissedas a possibility.

There is a potential risk of transmission of animalrotaviruses if crops, particularly those which areeaten raw, are exposed to farmyard waste. There isno published information on detection or survival ofrotaviruses in crops or soil amended with farmyardwaste, although information accruing from aEuropean research project on Cryptosporidium infood and water may be useful with regard toanalogous situations involving protozoan para-

sites.80 Group A rotaviruses have been detectedby EM and ELISA in lettuces sold in Costa Ricanmarkets.81 The detection by EM implied that theviruses were present on the lettuces in highnumbers (at least 105–106 particles per gram oflettuce), but their source was undetermined.

The Advisory Committee for the MicrobiologicalSafety of Foods78 considered that there was apossible role of sludge derived from human sewageas a potential source of viral contamination ofcrops. This concern could also be appropriate withregard to animal rotaviruses in manure or slurry. Ina report to the Department for the Environment,Transport and the Regions, Carrington et al.82,83

reviewed various experiments on virus survival insewage sludge-amended soils. They produced aquantitative interpretation of viral decay ratesfrom the data produced by these studies, andcalculated decimal reduction times for the virusesinvolved (enteroviruses including poliovirus). Thesestudies under review were mostly performed withNorth American soil types and indigenous climaticconditions, and information is lacking on persist-ence of viruses in soils and conditions pertaining toother countries. Carrington et al.83 pointed out thatin countries like the United Kingdom, mean soiltemperatures seldom exceed 15 8C at 10 cm depthis summer, and are about 5 8C in winter. Theysuggested that viral decay rates would be slowunder such conditions, with decimal reductiontimes from 24 days to over 100 days. This estimateis supported by the work of Damgaard-Larsenet al.,84 who studied the survival of enterovirusesin sludge-amended soil under Danish winter con-ditions. In December 1975 they added Coxsack-ievirus B3 to municipal sludges, which were thenplaced on soil lysimeters, and dug in by spade.Samples were taken each month until May 1976.The viral titre fell by 0.5–1 log10 per month, butsome viruses could still be detected at the end ofthe experiment. Carrington et al.83 considered thatcultivation of soil after sludge application wouldencourage viral decay by encouraging evaporation,but this practice could also result in aerosolisationof virus particles. Irrigation of crops with contami-nated water or organic waste is a potential meansof contaminating foofdstuffs with enteric viruses,and studies have demonstrated that rotaviruses canbe transferred to the surfaces of vegetables andpersist there for several days, following theapplication of sewage sludge to effluent. Once onfoodstuffs such as vegetables, viruses may persistunder normal storage conditions over the timesusual between purchase and consumption.85 Usingsimian rotavirus SA-11 as a model, Badawy et al.86

showed that rotavirus could survive on lettuce,

N. Cook et al.296

radishes and carrots for up to 30, 30, and 25 days,respectively, at refrigeration temperature; at roomtemperature, however, the survival periods were25, 4, and 15 days, respectively.

It is well known that zoonotic agents can betransmitted via meat contaminated at slaughter.Petersen et al.87 devised a rating scheme wherebythe public health significance of the potentialtransmission of zoonotic agents by beef could beprioritized. The list of 25 pathogens includedrotavirus, which was ranked 15th (the first threewere E. coli O157:H7, Salmonella, and Listeria, inthat order). Young animals, which are more likely tobe infected with rotavirus and display symptoms,are not likely to be slaughtered, but older animalscould be infected subclinically, and rotaviruses infaecal material could contaminate their meat atslaughter. Abattoirs in the UK grade livestock ontheir cleanliness, using a 1–5 scale (1 ¼ clean).Only clean animals get killed. This reduces thechances that carcasses are contaminated withzoonotic agents from faecal material from the skin.

Although rotaviruses have been detected inshellfish,88 shellfish have not been associated withtransmission of rotaviral disease. Metcalf et al.89

speculated that this may reflect some property ofthe stability of rotaviruses, e.g. loss of infectivityupon uptake by shellfish through possible removalof surface spikes. However, Ansari et al.90 believedthat shellfish harvested from sewage-polluted areasmay be a likely vehicle for rotavirus, and, aschildren seldom eat raw shellfish, and adultsseldom display symptoms of infection, shellfish-associated rotavirus gastroenteritis may go unno-ticed. Ley et al.70 found bovine enterovirus inoysters in a river which received run-off frompasture where cattle grazed. The virus could alsobe detected in the cattle faeces, and the run-offstreams.

Contaminated air

Many viral infections can be spread through air91

and it has been suggested that rotaviruses may beairborne.92 An airborne route of rotavirus trans-mission has been demonstrated in experimentalanimals responsible for outbreaks of epizooticdiarrhoea in infant mice.93,94 Although possiblyborne by aerosol droplets into the respiratorytract, rotaviruses may not replicate there, but betranslocated by mucocilliary activity into thegastrointestinal tract and ingested.95 Airbornedroplet spread of rotavirus has been suggested toexplain the universal occurrence of rotavirusdisease in children during the first 4 years of life,regardless of the level of hygiene that prevails or of

the quality of food and water available to them.Cook et al.92 drew some similarities with theepidemiology of measles, such as the markedseasonality of disease, the ubiquity of infection,and the high attack rate, to support the airborneroute hypothesis for spread of rotavirus.

In a study of rotavirus in pigs in Venezuela,Utrera et al.96 noted an increase in the number ofinfections during periods of low rainfall andhumidity. They suggested that the low relativehumidity promoted an increase in aerosol for-mation, particularly in relation to virus-laden dustfrom faecally contaminated surfaces, which couldresult in an increased spread of infection. A dryatmosphere, especially in combination with lowerair temperature, appears to be conducive to thestart of outbreaks of rotavirus gastroenteritis.97,98

The dry air indoors when it is heated during coldweather has been postulated99 as one of the causesof the seasonality of rotavirus infection, as it couldencourage aerosol transmission of rotavirus. Thehandling of rotavirus-contaminated material cancreate aerosols of various sizes. The larger of thesesettle out rapidly and can contaminate the immedi-ate surroundings, while the smaller (usually ,5 mmdiameter) become droplet nuclei which may remainairborne for prolonged intervals.99,100

Ansari et al.90 reviewed information derivedfrom experimental study of the survival of rotavirusin air. Although there are differences in resultswhich may be due to differences in the rotavirusstrains and the experimental protocols which havebeen employed, overall the information indicatesthat rotavirus could survive in air long enough topose a risk of infection to persons or animals in thesame environment.

Potential routes of airborne exposure to animalrotaviruses exist. For instance, intensive pig unitsare force-ventilated, as a form of temperaturecontrol and also to prevent airborne disease such aspneumonia in the pigs. The air is sucked out eitherthrough the side or through the roof. It isconceivable that this could result in disseminationof aerosolised rotavirus, to which not only workersbut also people living in the vicinity could beexposed. Also, spreading of animal wastes ontoland, in particular by slurry spreading mechanisms,which generate aerosol-sized droplets, could resultin such exposure. In the UK, over 90% of slurry isapplied to land by broadcast spreading,101 and it hasbeen shown that the technique can disseminatebacteria over distances of up to 650 m, especiallyunder windy conditions.102 A current DEFRA project(WA0804) is conducting extensive studies on bac-terial pathogen dissemination by land spreading ofsolid manure or slurry, and has identified the

Zoonotic rotavirus 297

highest risks to occur for instance from the use ofsplashplate tankers, spreading of fresh slurry (nopathogen die-off), and during high windspeeds (N.Nicholson: personal communiation).

Contaminated surfaces

Viral contamination of various objects and surfacescan occur either directly by contact with faeces orindirectly through virus-contaminated aerosols.91,

103,104 Hands frequently contact environmentalsurfaces, and the potential for transfer of rotavirusbetween surfaces and hands was demonstrated byAnsari et al.:105 their findings showed that rotaviruscould retain infectivity for several hours on skin,and could transfer in an infectious state to othersurfaces. Abad et al.106 examined the survival ofrotavirus on surfaces composed of severalmaterials, aluminum, china, glazed, tile, latexand polystyrene, commonly found in households,and found that infectious virus could persist for atleast 60 days. Moe and Shirley107 found thatrotavirus infectivity could persist for up to a weekunder conditions similar to those found in the homein temperate countries, namely 30% relativehumidity and 20 8C. This could permit the survivalof infectious virus on household objects longenough to infect persons living in the sameenvironment with an individual or animal which isshedding rotavirus. There is some inferentialevidence to indicate that virus-contaminated car-pets may be a vehicle for transmission of gastroin-testinal infection.108

Occupational exposure to animal pathogens maybe a source of household contamination. Riceet al.109 showed that Salmonella enterica couldbe cultured from the contents of vacuum cleanersused in houses where one or more occupant was incontact with infected cattle, or involved in aveterinary clinic outbreak of feline salmonellosis,or engaged in research on the bacterium. Thirtypercent (8/26) of the samples taken from thehouseholds whose occupant(s) had contact withinfected cattle were S. enterica positive, whereasno positive samples ðn ¼ 12Þ were identified fromhouses with no known occupational exposure. It isquite possible that occupational exposure to rota-virus-infected animals could similarly be a source ofhousehold contamination.

Conclusions

There is evidence that zoonotic transmission ofrotaviruses, or at least rotavirus genes, can occur.

The low incidence of uncommon strains in the UK,however, would suggest that such transmission, orat least the establishment of an animal rotavirus ora human/animal reassortant virus in the humanpopulation does not happen with any great fre-quency. However, the example of the G9 strainsmay be instructive. These viruses appearedrecently, possibly from animals: even if suchtransmission took place through a single event,they are now found worldwide. It cannot becompletely dismissed that zoonotic transmissionof rotaviruses could occur in the UK, nor can it bepredicted when such an occurrence could takeplace. The rapid movement of people of diversepopulations throughout the world allows the intro-duction and spread of rotaviruses generated indeveloping countries through zoonotic transmissionand reassortment. This could have implications fora vaccine strategy: it may be that if symptomaticinfections arising from the common human rota-virus types were reduced or eliminated, then otherinfectious strains may still exist within the animalpopulation.

In the UK, contact with animals, and the inputinto the environment of animal wastes, mustfrequently expose many thousands of people toanimal rotaviruses. This may not result in highlevels of infection, but some infection could occur.There may be a continual, albeit very low level, ofinput of rotavirus strains or sequences into thehuman population from the animal population.Because of the nature of the exposure, this inputwill occur continually, year on year.

It is clear that there is much information lacking,which would allow a full assessment of any riskwhich animal rotaviruses pose to the UK populationthrough zoonotic transmission. In particular, thereis no data on rotaviruses circulating in animals inthe UK. Whilst detailed surveys of the G and P typeshave been undertaken recently in man in the UK, nocomparable survey has been conducted in animalsand there are no published surveys of the G and Ptypes circulating in cattle/pigs/lambs/cats/dogs/rodents in the UK. There is a lack of sequence datafrom animal rotaviruses, which prevents closecomparison between human and animal strains,and little is known about the long-term molecularepidemiology of rotavirus infections in animals. Thereplicative or pathogenic potential of animalrotaviruses in human hosts is not optimallyassessed. There is no information on survival ofrotaviruses in slurry- or manure-amended soil, andthere is no information on whether rotaviruses maybe detected in UK watercourses, or on foodstuffs inthe UK.

Several questions remain to be answered. For

N. Cook et al.298

example: is genotyping correct? Some publishedG10 primers cross-react with G3 types.20 Do‘common’ human strains have a zoonotic back-ground, i.e. are gene segments other than thoseencoding VP7 and VP4 derived from animal strains?Examining only G and P sequences from clinicalisolated does not reveal whether other segments,such as segment 11 (NSP4) may have a zoonoticbackground. Are there species-specific clusters ofgenotypes, e.g. G types? Species-specific geneticclusters of NSP4 have been described.17

There are several recommendations which canbe made. In order of priority, these are:

1. To establish whether there is any relationshipbetween rotavirus strains infecting humans andanimals, a survey should be performed ofconcomitantly circulating strains. The surveyshould compare rural and urban areas, withperhaps two examples of each. The study shouldinvolve GP practices for collection of humansamples, and veterinary practices for collectionof animal samples (livestock and pets). The ruralareas should be chosen for high prevalence ofdifferent livestock types, e.g. North Humbersidefor pigs, Cheshire for cattle. The study shouldfocus on group A rotaviruses, as the typing andclassification scheme for these is more fullydeveloped than for other groups (althoughinformation on group B and C rotaviruses wouldalso be useful), and examine the G and P types ofthe isolates. Genotyping should be performedwith fully validated primer sets. To provide afuller picture, the NSP1 and NSP4 genes of eachisolate should also be typed. Sequencing of RT-PCR amplicons should be performed as far aspossible, to facilitate identification of lineages.

2. A database of animal rotavirus sequences shouldbe established. Attributes such as (1) Species oforigin (2) Strain name, (3) Isolation date, (4)Geographical origin of isolate and (5) Samplingmethod, i.e. whether it was sub-cultured orsampled directly, should be included. Thisinformation could be used to order the compli-cated rotavirus classification system as well asdetermine phylogenetic relationships more effi-ciently. These attributes would be equally asuseful for human rotavirus isolates, and thisinformation should be included in futureaccessions.

3. In order to further develop the phylogeneticanalysis it would be advantageous to findhistorical rotaviral sequences to include in anyphylogenetic analysis. If a human sequence wassegregating into a human clade in the past butwas segregating into an animal clade at a later

date, then this would be a further proof thatsegments were being transferred from animal tohuman rotaviruses.

4. The potential for infection of humans by animalrotaviruses through direct contact with animalscould be studied by monitoring a group, e.g.incoming veterinary students, likely to handleinfected livestock or pets.

5. It would be beneficial to acquire dose–responseinformation to directly determine the infectivityof animal rotaviruses to humans. Examination ofthe data from vaccine trails should give reason-able information in this regard.

6. The evidence for cross-species infection raisesinteresting questions about the genetic andcellular basis of species specificity, and thisshould be established. This would provide anelement of prediction of replication in heter-ologous species.

7. Studies to determine how long animal rotaviruscan survive in soil amended with manure or slurrywould provide useful exposure assessment data.Such studies should model UK agricultural prac-tice, and involve in situ fields settings usinglysimeters over several seasons, as well aslaboratory microcosm experimentation, tothoroughly determine survival potential andexamine the factors which may influence it.The potential for transfer of animal rotavirusesto crops in soil which has been amended withmanure or sewage sludge could also be studied inthese tests.

8. Examination of watercourses for animal rota-viruses would facilitate assessment of the extentof exposure to viruses by this means.

If these recommendations are followed, theinformation obtained will allow a fuller under-standing of the zoonotic potential of rotavirus.

Acknowledgements

We acknowledge the support of the United KingdomDepartment for the Environment, Food and RuralAffairs (Defra). The views expressed in this paperare the authors’ and not necessarily those held byDefra. We would like to thank the following forconsultation and advice: Dr Ulrich Desselberger; DrDavid Cubitt, Great Ormond Street Hospital,London; Mr Andrew Schofield, Minster VeterinaryPractice, York; Professor Lennart Svensson, Uni-versity of Linkoping, Sweden. Many thanks also to DrNick Nicholson, of ADAS, Boxworth, Cambridge andto Professor Huw Smith of the Scottish ParasiteDiagnostic Laboratory, Glasgow.

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