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Global transmission of influenza viruses fromhumans to swine
Martha I. Nelson,1 Marie R. Gramer,2 Amy L. Vincent3
and Edward C. Holmes1,4
Correspondence
Martha I. Nelson
Received 6 June 2012
Accepted 6 July 2012
1Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA
2University of Minnesota Veterinary Diagnostic Laboratory, St Paul, MN 55108, USA
3Virus and Prion Diseases Research Unit, National Animal Disease Center, USDA-ARS, Ames,IA 50010, USA
4Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania StateUniversity, University Park, PA 16802, USA
To determine the extent to which influenza viruses jump between human and swine hosts, we
undertook a large-scale phylogenetic analysis of pandemic A/H1N1/09 (H1N1pdm09) influenza
virus genome sequence data. From this, we identified at least 49 human-to-swine transmission
events that occurred globally during 2009–2011, thereby highlighting the ability of the
H1N1pdm09 virus to transmit repeatedly from humans to swine, even following adaptive evolution
in humans. Similarly, we identified at least 23 separate introductions of human seasonal (non-
pandemic) H1 and H3 influenza viruses into swine globally since 1990. Overall, these results
reveal the frequency with which swine are exposed to human influenza viruses, indicate that
humans make a substantial contribution to the genetic diversity of influenza viruses in swine, and
emphasize the need to improve biosecurity measures at the human–swine interface, including
influenza vaccination of swine workers.
INTRODUCTION
The 2009 pandemic H1N1 virus (H1N1pdm09) representsthe best-documented emergence of a swine pathogen inhumans, particularly as the virus was associated withwidespread global morbidity, mortality and years of life lostin humans (Viboud et al., 2010). The H1N1pdm09 viruswas generated by a reassortment event between Eurasianswine H1N1 influenza viruses and North American triple-reassortant H1 viruses, with the former contributing the N1and M segments and the latter donating the PB2, PB1, PA,H1, NP and NS segments (Garten et al., 2009). Althoughsimilar influenza virus reassortants containing segments ofboth Eurasian swine virus and triple-reassortant swine virusorigins have been detected in Asia (Lam et al., 2011; Smithet al., 2009), no ‘smoking gun’ viruses that are closely relatedprogenitors have been detected in swine in any locality.
Following the identification of H1N1pdm09 in humans inApril 2009, the virus was transmitted rapidly from humansback into swine. The first isolation of a H1N1pdm09 virusin swine was from a pig farm in Alberta, Canada, in May2009 (Howden et al., 2009), and the H1N1pdm09 virus wassubsequently isolated from outbreaks in swine in all other
major continents: Africa (Njabo et al., 2012), Asia (Kimet al., 2011; Sreta et al., 2010), Australia (Holyoake et al.,2011), Europe (Hofshagen et al., 2009; Howard et al., 2011)and South America (Pereda et al., 2010). Upon introductioninto the swine population, H1N1pdm09 viruses co-circu-lated with endemic swine influenza viruses and exchangedgenome segments via reassortment (Ducatez et al., 2011;Lam et al., 2011; Vijaykrishna et al., 2010). In 2011, 12reassortant H3N2 (H3N2v) swine influenza viruses withmatrix (M) segments of pandemic H1N1pdm09 origin wereisolated from humans in the USA (CDC, 2012).
Seasonal human influenza viruses have also been isolatedperiodically from swine, with a few major lineages becomingendemic in swine: most notably the human-origin H3N2viruses that proliferated in European swine in the 1970s(Ottis et al., 1982), the H1d lineage of human origin thatemerged in North American swine in 2003 (Karasin et al.,2006), and the human H3, N2 and PB1 segments that wereassociated with the triple-reassortant viruses that wereidentified in 1998 in North American swine (Zhou et al.,1999). In addition, singletons and smaller clusters of swineinfluenza viruses that contain one or more segments ofhuman seasonal influenza virus origin have been identifiedin several countries, including Argentina (Cappuccio et al.,2011; Pereda et al., 2011) and Italy (Moreno et al., 2012).
Seven supplementary figures and two supplementary tables areavailable with the online version of this paper.
Journal of General Virology (2012), 93, 2195–2203 DOI 10.1099/vir.0.044974-0
044974 Printed in Great Britain 2195
Table 1. Country and state/province of virus collection, month and year of collection, number of swine isolates, and bootstrap support ¢70 % for 49 introductions ofH1N1pdm09 influenza virus from humans into swine during 2009–2011
Introduction
no.
Country of
collection*
State or province of
collection (if available)*
Month/year of
collection
No. of isolates
(swine)
Bootstrap support (%) for viral introductionsD
Internal gene phylogeny HA phylogeny NA phylogeny
1 Canada ALB May 2009 2 85d 95
2 Canada ALB, MB Aug 2009 2 94
3 Australia VIC Aug 2009 6 95d 86
4 Singapore Aug 2009 1 72
5 USA MN Aug–Sep 2009 4 100
6 Canada MB Sep–Oct 2009 3 92 73
7 HK Oct 2009 2 100
8 Canada QC Nov 2009 1 85
9 Canada SK Nov 2009 1 95
10 Canada Nov–Dec 2009 3 84 72
11 Canada QC Nov–Dec 2009 4 97
12 Italy Nov 2009 1 92
13 S. Korea Nov–Dec 2009 3 100d 98 70d
14 Canada QC Nov–Dec 2009 5 90 84d
15 USA IL Nov 2009 1 71
16 Taiwan Nov 2009–Sep 2010 4 98 82d
17 Thailand Nov 2009–Jan 2010 6 100
18 China Guangdong Dec 2009 3 100 89
19 China Guangdong Dec 2009 3 100 85
20 HK Dec 2009 2 100 94
21 HK Dec 2009 5 100 96
22 Italy Dec 2009 2 100 98
23 S. Korea Dec 2009 8 100 84 93
24 S. Korea Dec 2009–Jan 2010 9 100 72 90
25 USA IL, MN Dec 2009–Feb 2010 8 87 72d
26 USA NC Dec 2009–Mar 2010 2 90
27 USA IL Dec 2009–Jan 2010 3 94d 76
28 China Nanchang Jan 2010 4 100 81
29 China Guangdong Jan 2010 5 100
30 Thailand Feb–Jun 2010 4 97
31 USA IL, MO Mar 2010 3 98
32 China May 2010 10 100
33 USA IL May 2010 3 92d 97
34 Thailand Sep 2010 2 100 80
35 Costa Rica Nov 2010 6 100 100
36 Cuba Nov 2010 3 98d 100
37 Thailand Nov 2010 2 99
38 USA IA, IL Nov–Dec 2010 3 98 97d
M.I.N
elsonand
others
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The increased global surveillance of influenza in pigs pro-vides the first opportunity to estimate the extent of human-to-swine transmission of the H1N1pdm09 virus. The mainaim of our study was therefore to estimate, using a simplephylogenetic approach, the total number of introductionsof the H1N1pdm09 virus from humans into swine using adataset of H1N1pdm09 viruses collected globally in swineduring 2009–2011. We then compared this estimate withthe equivalent transmission frequency of human seasonal(non-pandemic) H1 and H3 into swine since 1990.
RESULTS
Frequent transmission of H1N1pdm09 virus fromhumans into swine
Across the haemagglutinin (HA), neuraminidase (NA) andconcatenated internal gene phylogenies, we identified atotal of 49 discrete introductions of H1N1pdm09 influenzavirus from humans into swine during 2009–2011 [Table 1; Figs1 and S1–S3 (available in JGV Online)]. These introductionswere observed in 12 countries and semi-autonomousregions: Australia, Canada, China, Costa Rica, Cuba, HongKong (SAR), Italy, Singapore, South Korea, Taiwan,Thailand and the USA. The majority of viral introductionsinto swine were identified in the USA and Canada (19 andeight introductions, respectively; Table 1), reflecting thehigher number of H1N1pdm09 influenza virus sequencesavailable from North American swine. Given our strictcriteria for defining viral introductions (bootstrap values¢70 %) and limited global sampling, these numbers arelikely to underestimate the extent of global spillover fromhumans to swine of the H1N1pdm09 virus significantly.Our conservative methods are particularly prone to missintroductions involving single isolates. For example, singleH1N1pdm09 isolates from Mexico, Japan, England, Came-roon, Brazil and Colombia (A/sw/4/Mexico/2009, A/sw/Osaka/1/2009, A/sw/England/73690/2010, A/sw/Cameroon/11rs149-198/2010, A/sw/Brazil/12A/2010, A/sw/Colombia/1403/2010) are likely to represent additional introductions.These additional probable introductions are identified by #
on the detailed HA, NA and concatenated phylogeniespresented in Figs S1–S3.
Frequency of human-to-swine transmission ofseasonal influenza viruses
Across the H1, H3, N1 and N2 phylogenies, at least 23 separateintroductions of seasonal influenza virus from humans toswine were observed during the period 1990–2011 (Figs S4–S7). The vast majority of these introductions were identifiedafter 1996, when surveillance of influenza virus in swineincreased. Six human-to-swine transmission events involvedthe human seasonal H1 segment, eight involved the humanH3 segment, six involved the human N1 segment and 16involved the human N2 segment (Table 2). The highernumber of introductions of the human N2 segment isT
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Human-to-swine transmission of influenza virus
http://vir.sgmjournals.org 2197
probably related to several factors, including (a) the overallhigher number of H3N2 virus introductions (n512) due tothe dominance of the H3N2 subtype in humans during theperiod of this study, (b) the frequency of introductions (n53)of the human H1N2 variant that circulated globally in humansfrom 2001 to 2003, and (c) the frequency of reassortmentinvolving N2 segments of human origin and HA segments ofswine origin. For example, the 20 H1N2 reassortant virusesfrom Italy that are associated with introduction #11 have N2sequences that are related most closely to the N2 of humanseasonal H3N2 isolates collected in 1997–1998, but H1sequences related to European swine influenza viruses.
Twelve of the introductions of seasonal influenza virus werefirst identified in Asia, six were first identified in the USA,three in Canada and two in Europe (Table 2). As with theH1N1pdm09 viruses, the methods that we used to define ahuman-to-swine transmission event are highly conservative,and numerous probable introductions were excluded from
our final estimate, particularly those involving singletons.Six singleton swine isolates, including three from Argentina,which were identified to be of human origin on both the HAand NA trees but were not supported by high bootstrapvalues, are highlighted (letters a–g, Table 2; Figs S4–S7).Additionally, eight swine H3N2 isolates collected in HongKong during 2000–2002 are interspersed with humanisolates collected from that same time period on both theH3 and N2 phylogenies, suggesting additional introductionsof human H3N2 viruses into swine in Hong Kong, althoughagain lacking the phylogenetic resolution required to bedefined as discrete introductions.
Estimated time periods of human-to-swinetransmission
The long branch lengths associated with several introduc-tions of human-origin influenza viruses into swine indicate
Fig. 1. Phylogenetic relationships of the six concatenated internal gene segment (PB2, PB1, PA, NP, M, NS) sequences from100 H1N1pdm09 influenza virus isolates collected in swine globally during 2009–2011, and 1008 H1N1pdm09 influenzaviruses collected globally in humans during the same time period (total dataset, n51108 sequences; colour-coded black). Eachswine H1N1pdm09 isolate is colour-coded by the country of origin: USA, brown; Canada, light blue; Thailand, purple; SouthKorea, dark green; China, pink; Hong Kong, red; Taiwan, orange; Italy, olive; Mexico, light green; Singapore, yellow; Australia,dark blue. The first introduction of H1N1pdm09 identified in swine, in May 2009 in Canada, is denoted by an asterisk. Anidentical phylogeny with all viral introductions labelled in detail is provided in Fig. S1.
M. I. Nelson and others
2198 Journal of General Virology 93
Table 2. Country and year of collection, viral subtype, year of collection of the most phylogenetically related human influenza viruses, number of swine isolates, and bootstrapsupport ¢70 % for 23 introductions of seasonal influenza viruses (H1, H3, N1 and N2 segments) from humans into swine during 1990–2011
Introduction
no.
Country and year of collection Subtype Related human
viruses (year)
No. of isolates
(swine)
Bootstrap support (%) for viral introductions
H1 H3 N1 N2
1 Japan 1992 H1N1 1991 1 79
2 Canada 1997 H3N2 1997 1 § 98
3 Japan 1997 H3N2 1997 4 § 84
4 USA 1998–1999 (triple-reassortant I),
S. Korea 2005–2006, China 2007
H3N2 1995–1996 10 100 97
5 HK 1999–2000, China 2003–2008 H3N2 1999 26 93 94*
6 France 1999 H3N2 1998 1 § 99
7 HK 1999–2004, China 2004–2010 H1N2|| 1996 18 100
8 USA 1999, S. Korea 2004 H3N2 1999 2 § 95
9 Taiwan 2002, Indonesia 2004 H3N1 H3N2 1990 2 96
10 USA 2003–2011, Canada 2005–2011
(triple-reassortant II)
H3N2 1998 126D 100 97, 70d
11 Italy 2003–2010 H1N2|| 1997–1998 20 100
12 Canada 2004 H1N2 2002–2003 2 92
13 Thailand 2004–2009 H3N2 1995–1996 8 100 100
14 China 2004–2006 H1N1 1997 2 93 100
15 USA 2005–2011 (d-2) H1N1 H1N2 2002–2003 43D 97 100* 97, 70d
16 HK 2006 H1N1 2006–2007 1 100 88
17 S. Korea 2007 H3N2 1995–1996 3 100 97
18 USA 2007–2011 (d-1) H1N2 2002–2003 97 97 97, 70d
19 China 2008 H1N1 2000–2001 1 § 80
20 USA 2009 H1N1 2007 1 90
21 Canada 2009 H1N1 2008–2009 2 81 90
22 China 2010, HK 2011 H3N2 2005 3 100 100
23 Vietnam 2010 H3N2 2005 6 100 100
a HK 1999 H3N2 1999–2000 1 – –
b HK 2000–2002 H3N2 2000–2002 8 – –
c Canada 2003 H1N2 2002–2003 1 – –
d USA 2003 H3N2 2001–2002 1 – –
e Argentina 2008 H3N2 2001–2002 1 – –
f Argentina 2009 H1N1 2005–2007 1 – –
g Argentina 2010 H1N2 2002–2003 1 – –
*Bootstrap support was ¢70 %, but only for a subset of the isolates in a given cluster.
DIn cases of reassortment, the number of isolates on HA phylogeny is provided.
dIn cases of reassortment, bootstrap values for both NA clades that are associated with an introduction on the HA tree are provided.
§The HA associated with this introduction was identified to be of human seasonal influenza virus origin on the HA phylogeny (or the HA1 phylogeny; introduction no. 3), but was not associated
with a significant bootstrap value.
||These H1N2 viruses are not related to the seasonal H1N2 viruses that circulated globally in humans in 2001–2003, but rather are human–swine reassortants.
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that numerous viral introductions circulated in swine formany years before being detected. Given the high intensityof sampling of influenza virus in humans, the duration ofunsampled circulation in swine can be estimated simplyand directly by the differences in time between when theisolates were collected in swine and when the most closelyrelated human isolates were collected. For example, theeight H3N2 viruses associated with introduction #13 werecollected in swine in Thailand in 2004–2009, but are relatedmost closely to A/Wuhan/359/1995(H3N2)-like virusesfrom 1995–1996 on the H3 and N2 phylogenies (Figs S4and S6, respectively), representing 8 years of unsampledcirculation in swine. The time difference between the 2008H3N2 and 2010 H1N2 isolates collected in swine in Argen-tina and the most closely related human seasonal isolates isapproximately 6–8 years. Likewise, the isolate A/sw/Argentina/CIP051-StaFeN2/2010(H1N2) is related most closely to thereassortant H1N2 viruses that circulated in humans during2002–2003 (Figs S5 and S6), suggesting that this virus hascirculated undetected in swine in Argentina or elsewheresince at least 2003, when the H1N2 viruses disappearedglobally in humans. Incorporating more viral sequence datafrom swine by also including partial HA (HA1) sequencesfilled in some of these gaps in surveillance (H1–HA1 andH3–HA1 trees available from the authors upon request). Forexample, the USA 2007–2011 (d-1) introduction (#18) isdetected 2 years earlier in 2005 when HA1 data are included.Finally, 11 years separate the three H3N2 swine isolatescollected in South Korea in 2007 (introduction #17) fromthe most phylogenetically related human seasonal H3N2viruses collected in 1995–1996. However, the relatively shortbranch lengths associated with these 2007 South Koreanisolates raises the question of whether the evolutionary ratewas particularly low on this branch, or whether these se-quences could be erroneous. The short branch length thatseparates A/sw/Fujian/0325/2008(H1N1) (introduction #19)from the most closely related human seasonal H1N1 virusescollected in 2000–2001, relative to the approximately 7–8-year difference in collection date, also raises the question ofpossible sequencing error.
Spatial patterns of human-origin influenza virusesin swine
The number of swine H1N1pdm09 isolates associated witheach introduction (ranging from one to ten isolates; Table 1)represents the extent of onward swine-to-swine transmissionthat can be inferred from our phylogenetic analysis. Althoughthe vast majority of introductions of human H1N1pdm09viruses into swine exhibit local onward transmission in pigs,reflected in introductions that are associated with more thanone swine isolate, we infer that global viral movement has notyet been important in the dissemination of the H1N1pdm09virus in swine. Specifically, each cluster of swine H1N1pdm09influenza viruses that are associated with a human-to-swinetransmission event is highly spatially structured, with noswine introduction including viruses collected in more thanone country. However, within-country spread of H1N1pdm09
viruses has occurred in the USA, as six viral US introduc-tions include multiple states, representing viral dissemina-tion within the Midwestern USA (Table 1). Limited spreadwithin Canada between Alberta and Manitoba was alsodetected (introduction #2; Table 1). Within-country spreadwas not observed in other countries where information onthe province or region of viral collection was also available,including China and Australia.
Similarly, 18 of the 23 introductions of human seasonal H1and H3 influenza viruses into swine involve a singlecountry (Table 2). Six of these single-country introductionsinvolve singleton swine influenza viruses, with no evidenceof onward transmission. In contrast, four viral introduc-tions include isolates collected in different countries,indicating viral migration within North America and intoand within Asia. Aside from the major introduction oftriple-reassortant H3N2 viruses that spread from NorthAmerica to Asia (introduction #4) and from the USA toCanada (introduction #10), possible limited viral migra-tion occurred between Taiwan and Indonesia (introduc-tion #9) and the USA and South Korea (introduction #8).
DISCUSSION
Despite evolving the capability to transmit efficiently inhumans, our results indicate that the H1N1pdm09 influenzavirus has retained its ability to transmit back into swine andto co-circulate with other swine influenza viruses, increasinggenetic diversity and providing the opportunity for reassort-ment. The large number of H1N1pdm09 transmission eventsobserved in this study, even using conservative methods,highlights the frequency of human–swine contact ratesglobally and the continual exposure of swine to humaninfluenza viruses. Indeed, this transmission rate is consi-derably higher than would be inferred from the relativelylower number (n523) of human-to-swine transmissionevents of seasonal H1 and H3 influenza viruses thatwere identified during the past two decades. Importantly,detection of seasonal human influenza viruses in swinedid not increase during 2009–2011, despite widespreadglobal circulation of seasonal H3N2 viruses in humansduring 2010–2011. Hence, the different transmissionfrequencies observed are not explained by the increasein swine influenza surveillance since 2009 and are unlikelyto be an artefact of sampling. Furthermore, the lowphylogenetic resolution of the H1N1pdm09 phylogeny,due to the virus’s recent emergence and relatively lowgenetic diversity, impeded the identification of what arelikely to be many more introductions of this virus intoswine than could be defined here.
Although it is tempting to compare the frequencies ofhuman-to-swine transmission with the 27 swine-origininfluenza viruses that were identified in humans in the USAover a time period similar to that of our study (1990–2010)(Shu et al., 2012), such a comparison is biased greatlyby the substantially higher levels of global surveillance of
M. I. Nelson and others
2200 Journal of General Virology 93
influenza in humans than consistently occurs in swine.However, the intensity of influenza surveillance in humansdoes facilitate estimation of the time of emergence of the 23introductions of human seasonal influenza viruses into swine.For example, the introductions of H1N1pdm09 viruses weregenerally detected in swine within 1 year, whereas several ofthe human seasonal influenza viruses that were introducedinto swine were not detected for 5–10 years. Whilst the timeto detection in swine may relate to intrinsic viral charac-teristics (e.g. the virus must first adapt, then replicate com-petently in the new host, then spread to many hosts beforebeing detected), it is also possible that the difference in timebetween when a human-to-swine transmission event is de-tected in swine and the year of collection of the most closelyrelated human influenza viruses provides an indication ofintensity of swine surveillance in a given region. The smallestgaps in detection are in Canada, the USA and Hong Kong,where ongoing viral surveillance is conducted in swine,while larger time gaps occurred in countries such as SouthKorea, Italy, Thailand and Argentina, where samplingappears to be more sporadic, based on publicly availablesequence data.
The global frequency of introductions of H1N1pdm09viruses into swine also provides an opportunity to track, indetail, the onward transmission and spatial dissemination ofthese new viral clades and how they relate to swine move-ments (Nelson et al., 2011). It will be particularly importantto determine which of these H1N1pdm09 lineages sustainstransmission in swine over the long term, to observe theirpopulation dynamics in a new swine host and to identifywhich lineages spread globally. The multiple introductionsof human influenza viruses into swine provide a naturalexperiment for observing adaptive evolution of influenzafrom human to swine hosts, in identifying instances ofparallel adaptive evolution, and in the comparison of ratesof evolutionary change and selection pressures along thebranches that define human-to-swine transmission events.Clearly, the ability to trace such dynamics relies upon thecontinued surveillance of influenza in swine globally, evenafter the initial interest in the 2009 H1N1 pandemic hassubsided.
METHODS
Sequence data used in this study. To estimate the number ofhuman-to-swine transmission events of the H1N1pdm09 virus, wecompiled gene sequence data from 263 H1N1pdm09 influenza virusesthat were collected in swine during 2009–2011 in 18 countries:Argentina, Australia, Brazil, Cameroon, Canada, China, Colombia,Costa Rica, Cuba, Hong Kong, Japan, Mexico, Singapore, SouthKorea, Taiwan, Thailand, the UK and the USA (Table S1). Due to thefrequency of reassortment between H1N1pdm09 viruses and otherswine influenza viruses involving the HA and NA segments, con-catenated internal gene segments (PB2, PB1, PA, NP, M and NS) andthe HA and NA segments were studied separately. Separate phylo-genies were also inferred for each of the internal gene segments toidentify and remove any viruses with reassorted internal genes. Asbackground data, 1008 complete genome sequences from H1N1pdm09influenza viruses that were collected in humans during 2009–2011 were
downloaded from the Influenza Virus Resource on GenBank; due
to the size of the original dataset (n52718 genomes), 50 genome
sequences were selected randomly from highly sampled countries (i.e.
those with .50 genome sequences) (Bao et al., 2008).
To identify swine influenza viruses related to human seasonal H1 and
H3 influenza viruses, four phylogenetic trees were inferred using all
full-length H1 (n5991, excluding pandemic viruses), H3 (n5326),
N1 (n5858) and N2 (n5559) sequences collected from swine since
1990 that are available in GenBank (accession numbers are available
from the authors). Due to the frequency of reassortment and low
availability of whole-genome sequence data for these swine viral
isolates, alignments of the concatenated internal genome segments
were not included for this part of the study. These data came from
Argentina, Belgium, Canada, China, Denmark, France, Germany,
Hong Kong, Hungary, Italy, Japan, Spain, South Korea, Sweden,
Taiwan, Thailand, the UK and the USA. From these phylogenies, 152
human-origin swine H1 sequences, 221 human-origin swine H3
sequences, 18 human-origin swine N1 sequences and 430 human-
origin swine N2 sequences were identified globally and used in the
final analysis. This analysis also included, as background data, 1000
randomly selected human seasonal H1, H3, N1 and N2 segments
collected from 1990 to 2011. A large clade of European sequences (e.g.
A/sw/Scotland/410440/1994) that were of human seasonal H1 virus
origin were not included because they clearly were introduced from
humans into swine at least a decade prior to 1990, the beginning of
our study period.
To identify any additional human-to-swine transmission events that
could not be detected from trees inferred with full-length HA
sequence data, two additional phylogenies were inferred using all
partial HA1 sequence data (918 nt) from the swine H1 (n51200
sequences) and H3 (n51290 sequences) subtypes available since 1990,
including HA1 sequence data from the same 1000 human influenza
viruses that were selected as background in the full-length HA
phylogenies. Using HA1 data, we identified 50 additional swine H1
sequences of human origin and 70 additional swine H3 sequences
of human origin. However, all but two isolates [A/sw/Obihiro/3/
1993(H3N2) and A/sw/Obihiro/1/1994(H3N2)] were associated with
introductions that are already identified in the analysis of full-length
HA and NA sequences.
Phylogenetic analysis of H1N1pdm09. Alignments were con-
structed manually using the Se-Al program (Rambaut, 2002) for three
sets of swine H1N1pdm09 virus sequence data, with the 1008 human
H1N1pdm09 isolates included as background: (i) the HA segment[n5232 swine H1N1pdm09 isolates, 199 of which were downloaded
from GenBank, and 33 provided by the University of Minnesota
Veterinary Diagnostic Laboratory (UMVDL) from samples collected
from their routine veterinary diagnostic laboratory submission and/or
tested via the US Department of Agriculture (USDA) National
Animal Health Laboratory Network (NAHLN) Swine Influenza Sur-
veillance System]; (ii) the NA segment (n5229 swine H1N1pdm09
isolates, 197 from GenBank and 32 from UMVDL); and (iii)
concatenated internal gene segments (n5100 swine H1N1pdm09
isolates, all downloaded from GenBank, with all reassortants
identified from individual gene trees and excluded). For each
alignment, a maximum-likelihood (ML) tree was inferred using the
PhyML v3.0 program (Guindon et al., 2010), employing SPR (subtree
pruning and regrafting) branch-swapping and a general time-reversible
model (GTR) model of nucleotide substitution with gamma-distributed
rate variation among sites. Statistical support for individual nodes was
estimated using 1000 replicate neighbour-joining trees inferred using
PAUP* and assuming the GTR+gamma model of nucleotide substi-
tution (Swofford, 2003). Well-supported nodes (¢70 % bootstrap
support) defining human-to-swine viral introductions were identified
visually on each phylogeny. To mitigate possible effects of sequencing
Human-to-swine transmission of influenza virus
http://vir.sgmjournals.org 2201
error, the several isolates that were separated by extremely long branch
lengths were only categorized as discrete introductions when multiple
isolates from the same country exhibited the same phylogenetic pattern.
Phylogenetic analysis of seasonal H1 and H3. Alignments were
manually constructed using Se-Al for four sets of swine influenza
virus sequences that were identified as of human seasonal (non-
pandemic) influenza virus origin: (i) 152 H1 sequences, (ii) 221 H3
sequences, (iii) 18 N1 sequences and (iv) 430 N2 sequences. As
background, 1000 human influenza viruses were selected randomly
for each segment and downloaded from GenBank. For each
alignment, an ML tree was inferred as described above to identify
well-supported nodes (¢70 % bootstrap support) defining introduc-
tions of influenza viruses from humans to swine. The estimated time
periods of human-to-swine transmission were inferred directly from
the phylogeny by identifying the most closely related human viruses
(the sampling date of which is known). This simple phylogenetic
method produced results (i.e. times to common ancestry) similar
to those obtained using the Bayesian Markov chain Monte Carlo
approach available in the BEAST program (Drummond & Rambaut,
2007) and performed during a previous analysis of human influenza
viruses that were transmitted to swine (Nelson et al., 2011).
ACKNOWLEDGEMENTS
This research was conducted within the context of the Multinational
Influenza Seasonal Mortality Study (MISMS), an ongoing inter-
national collaborative effort to understand influenza epidemiology
and evolution, led by the Fogarty International Center, NIH, with
funding from the Office of Global Affairs at the Department of Health
and Human Services (DHHS).
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State of the Field
Plant Viruses. Invaders of Cells and Pirates ofCellular Pathways1
Richard S. Nelson and Vitaly Citovsky*
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401 (R.S.N.);and Department of Biochemistry and Cell Biology, State University of New York, Stony Brook,New York 11794–5215
Plant viruses, discovered over a century ago whenthe science of virology was born (for review, seeCreager, 2002), are obligate parasites on their hosts.Through their life cycle, from virus accumulation tointracellular, local, and systemic movement, viruses uti-lize plant proteins, normally involved in host-specificactivities, for their own purposes. Although the firstidentification of a host protein interacting with plantviral RNA took place more than 25 years ago (forreview, see Buck, 1999; Waigmann et al., 2004), the truecomplexity of this interaction between plant virusesand their hosts to allow virus accumulation and spreadis just now becoming understood.
In addition, the ability of the host to defend itselfagainst virus replication and spread is now known to bemuch more complex than was thought not long ago.During the early 1990s, the first findings were pub-lished suggesting that a plant host defense system tar-geting viral RNA with extreme sequence specificityexisted (e.g. de Haan et al., 1992; Lindbo and Dougherty,1992). Initially, these observations were not fully un-derstood to represent an RNA-mediated host defensesystem now referred to as the RNA interference(RNAi), but with time were well differentiated fromthe better studied host and transgene defense systemsmediated through proteins (e.g. the hypersensitivereaction of Nicotiana tabacum cv Xanthi NN againsttobacco mosaic virus [TMV] and coat protein-mediatedresistance; Beachy, 1999; Marathe et al., 2002). In the lastfew years, plant molecular virologists and biologistshave moved with increasing speed to document theincredibly complex interactions between virus and hostfactors necessary to allow or defeat virus infections inthe presence of RNAi (e.g. Baulcombe, 2004). Thus,plant viruses, besides their traditional role as causativeagents of numerous plant diseases, represent moleculartools to examine and dissect diverse basic cellularprocesses in plants, ranging from intracellular trans-port and nucleocytoplasmic shuttling (Lazarowitz and
Beachy, 1999; Oparka, 2004) to intercellular transport(Waigmann et al., 2004) to gene silencing (Moissiardand Voinnet, 2004).
This focus issue reports new insights into howviruses may utilize host factors to accumulate andmove intracellularly to position for intercellular move-ment (Chen et al., 2005; Ju et al., 2005; Liu et al., 2005).Also, information further illuminating the ‘‘give andtake’’ between virus and host factors battling forcontrol during RNAi is presented (Chellappan et al.,2005; Liu et al., 2005; Schwach et al., 2005). Updatearticles on virus-host interactions during virus repli-cation and movement in this issue review recentinformation in these areas to provide clues for pro-ductive future research (Boevink and Oparka, 2005;Thivierge et al., 2005). In this State of the Field editorial,we introduce the research and Update articles in thisissue and review recent literature on virus-host inter-actions not addressed in the Updates.
VIRUS ACCUMULATION
For both DNA and RNA plant viruses, the accumu-lation of progeny virus involves translation and rep-lication of viral sequences (Buck, 1999; Ahlquist et al.,2003; Noueiry and Ahlquist, 2003; Hanley-Bowdoinet al., 2004; Ishikawa and Okada, 2004; Rajamaki et al.,2004, and refs. therein). These plant viruses rely on thehost to provide factors to aid their accumulation. TheUpdate article by Thivierge et al. (2005) presentsa summary of recent insights into the mechanisms bywhich positive-sense single-stranded RNA virusestake advantage of the host cell mRNA processingand translation machinery.
Research on virus-host interactions during DNAvirus accumulation has also moved forward. For ex-ample, an NAC domain protein, SINAC1, from tomato(Solanum lycopersicum) that interacts with a geminivirusreplication enhancer (REn) protein was identified andsuggested to participate in viral replication (Selth et al.,2005). NAC family members, which function in plantdevelopment and defense responses (e.g. Xie et al.,2000; Hegedus et al., 2003), are known to interact withother geminivirus proteins, such as RepA, but in thoseinstances they inhibited rather than promoted viralreplication (Xie et al., 1999). Furthermore, a NAC pro-tein interaction with an RNA virus coat protein is
1 The work in our labs is supported by grants from the NationalInstitutes of Health, National Science Foundation, U.S. Departmentof Agriculture, U.S.-Israel Binational Research and DevelopmentFund, and U.S.-Israel Binational Science Foundation to V.C., and bythe Samuel Roberts Noble Foundation to R.S.N.
* Corresponding author; e-mail [email protected];fax 631–632–8575.
www.plantphysiol.org/cgi/doi/10.1104/pp.104.900167.
Plant Physiology, August 2005, Vol. 138, pp. 1809–1814, www.plantphysiol.org � 2005 American Society of Plant Biologists 1809
necessary during a resistance response in Arabidopsis(Arabidopsis thaliana; Ren et al., 2000). That host proteinsfrom a single family display different functions duringDNA and RNA virus infection illustrates the complex-ity of the virus-host interaction process.
Large-scale screening for host factors that affectRNA virus accumulation has been undertaken usingyeast as an alternative host distinguished by a wealthof well-characterized mutants (e.g. Kushner et al.,2003; Panavas et al., 2005). These experiments showedthat host genes involved in viral accumulation maydiffer between viruses. For example, while bromemosaic virus and tomato bushy stunt virus each areaffected in their accumulation by approximately 100host genes, only 14 of these genes overlap betweenviruses. The overlapping genes encoded proteins be-longing mainly to three functional groups: proteinbiosynthesis, protein metabolism, and transcription/DNA remodeling (Panavas et al., 2005). Interestingly,no overlap existed between tomato bushy stunt virusand brome mosaic virus for genes involved in proteintargeting, membrane association, vesicle transport, orlipid metabolism (Panavas et al., 2005), suggesting thatthere are important differences between these virusesfor host membrane targeting and intracellular trans-port. Although analysis in yeast allows a high-throughput analysis of yeast host factors that affectplant virus accumulation, it is important to supplementthese data with information obtained in plant cells, forexample, using a recently developed technology tostudy virus replication in a cell-free system of mem-brane-containing extract from uninfected evacuolatedplant protoplasts (Komoda et al., 2004). The potential toutilize protoplasts from mutant plants silenced forexpression of specific plant genes identified throughthe yeast-based selection is very exciting.
INTRACELLULAR ANDINTERCELLULAR MOVEMENT
To spread between cells, viruses must first movefrom their replication sites to plasmodesmata at the cellperiphery and then traverse these intercellular chan-nels to enter the neighboring cell. Cell-to-cell transportof most plant viruses is mediated by specific virallyencoded factors termed movement proteins (MPs), thefunction of which may be augmented by other viralproteins (for review, see Morozov and Solovyev, 2003;Rajamaki et al., 2004; Waigmann et al., 2004). Themajority of the cell-to-cell transport machinery, how-ever, is presumed to be provided by the host cell. Onesuch host transport apparatus is the cytoskeleton.Although plant cytoskeletal elements were implicatedin viral cell-to-cell transport a decade ago (Heinleinet al., 1995; McLean et al., 1995), the relative roles ofmicrotubules and microfilaments in the transport pro-cess are just emerging. Recent data suggest that, forTMV, microfilaments participate in the cell-to-cellmovement of the virus, whereas microtubules andmicrotubule-associated proteins may be involved in
degradation of the viral MPs (Gillespie et al., 2002;Kragler et al., 2003). In this issue, Liu et al. (2005)demonstrate the role of microfilaments in cell-to-cellmovement of TMV. Disruption of microfilaments bypharmacological agents or by virus-induced gene si-lencing compromised TMV spread from cell to cell, butit did not significantly affect viral accumulation withinthe infected cells (Liu et al., 2005). Furthermore, thisstudy demonstrated the potential involvement of an-other TMV factor, the 126-kD protein, in viral transportalong microfilaments; the 126-kD protein was shown toassociate with viral replication complexes, modulatetheir size, and potentially mediate their interactionwith and movement along the microfilament network(Liu et al., 2005).
Increasing evidence suggests that the cytoskeletalnetwork does not function alone in viral transport toand through plasmodesmata. Instead, it may act to-gether with the endomembrane transport system of thehost cell. Specifically, many viral MPs may be deliveredto plasmodesmata via the endoplasmic reticulum (ER),while actin/myosin filaments may regulate the flow ofproteins in the ER membrane (Boevink and Oparka,2005). Two articles in this issue address the role of ER inviral cell-to-cell transport and plasmodesmal targeting.Ju et al. (2005) show that the potato potexvirus X (PVX)triple gene block (TGB) p2, one of the proteins requiredfor movement of this group 2 member of the TGB-containing viruses, associates with ER-derived vesi-cles, which in turn colocalize with actin filaments.Intriguingly, no association of the TGBp2 with Golgivesicles was detected (Ju et al., 2005), consistent withfindings in a recent report studying the movement ofTGBp2-containing structures in tissue infected withpotato mop-top virus, a group 1 member of the TGB-containing viruses (Haupt et al., 2005). Thus, theseviruses likely use an ER-dependent pathway forplasmodesmal targeting, which is different from theGolgi-dependent targeting to plasmodesmata recentlydemonstrated for some cellular proteins (Sagi et al.,2005).
The association of the potexviral TGBp2 MP withmicrofilaments and ER resembles similar associationsof the tobamoviral MP and the 126-kD protein (McLeanet al., 1995; Heinlein et al., 1998; Hagiwara et al., 2003;Liu et al., 2005). This resemblance indicates physicaland functional similarities between MPs and move-ment-associated proteins of potexviruses and tobamo-viruses, suggesting that both viral groups utilizea similar method of intracellular movement, at leastthrough a portion of this passage (Nelson, 2005).
For TMV, the role of the ER translocation and plas-modesmal targeting was explored by Chen et al. (2005)using calreticulin, a cellular protein that localizes toplasmodesmata (Baluska et al., 1999; Michalak et al.,1999; Chen et al., 2005). This study showed that theN-terminal signal peptide was critical for the ability ofcalreticulin to accumulate within plasmodesmata (Chenet al., 2005). Based on these observations, it is tempting tospeculate that plasmodesmal targeting involves two dis-
Nelson and Citovsky
1810 Plant Physiol. Vol. 138, 2005
tinct signals, a signal to enter the ER network and a puta-tive plasmodesmata localization signal. Consistent withthis idea, several types of viral MPs that ‘‘gate’’ plasmo-desmata (e.g. Waigmann et al., 1994; Tamai and Meshi,2001) have been shown also to associate with the ER (e.g.Heinlein et al., 1998; Haupt et al., 2005; Ju et al., 2005).
Chen et al. (2005) also showed that calreticulininteracts with TMV MP and that overexpression ofcalreticulin in transgenic plants redirects TMV fromplasmodesmata to microtubules and compromises cell-to-cell transport of the virus. A potential, albeit indirect,functional link between viral MPs and calreticulin alsomay be inferred from the observations that one of thetwo MPs of the turnip crinkle virus (TCV) interactswith an Arabidopsis protein containing two RGD cell-attachment sequences (Lin and Heaton, 2001) that arerecognized by integrins (Campbell et al., 2000), whichin turn interact with calreticulin (Dedhar, 1994).
Possible roles of the calreticulin-MP interaction inregulation of plasmodesmal permeability are dis-cussed in the Update article by Boevink and Oparka(2005) in this issue. These authors present a review ofthe latest trends and discoveries regarding the role ofthe ER/actin network in intracellular transport, rec-ognition of adhesion sites at the cell periphery, mod-ification of plasmodesmata by alteration of the cellwall structure, Hsp70 chaperones as potential trans-location factors, and regulation of viral cell-to-cellmovement (Boevink and Oparka, 2005).
Recently, a potential link between virus accumula-tion and cell-to-cell movement was identified when theeukaryotic translation factors eIF4E and eIF(iso)4E,which are required for accumulation of potyviruses(Duprat et al., 2002; Lellis et al., 2002; Ruffel et al., 2002;Nicaise et al., 2003), were also shown to aid in virus cell-to-cell movement (Gao et al., 2004). These observationssupported earlier findings where plant mutants withaltered eIF4E activity exhibit limited virus spread(Arroyo et al., 1996). It has been speculated that poty-virus intracellular movement may occur via an inter-action of eIF4E with eIF4G, which then bindsmicrotubules (Lellis et al., 2002). Regardless of themechanism of eIF4E-mediated virus movement, it isimportant to realize that host proteins may function inseveral steps of the virus infection process, e.g. in thecase of eIF4E, both in virus translation and/or replica-tion and in viral cell-to-cell movement.
Finally, in recent years, viral MPs have been shownto interact with numerous other cellular proteins,such as pectin methylesterases (Dorokhov et al.,1999; Chen et al., 2000; Chen and Citovsky, 2003),protein kinases (Yoshioka et al., 2004), homeodomainproteins (Desvoyes et al., 2002), DnaJ-like proteins(Soellick et al., 2000; von Bargen et al., 2001), rabacceptor-related proteins (Huang et al., 2001), b-1,3-glucanase-interacting proteins (Fridborg et al., 2003),and transcriptional coactivators (Matsushita et al.,2001, 2002). To date, only protein kinases have beenshown to play a role in viral intercellular move-ment (Citovsky et al., 1993; Kawakami et al., 1999;
Waigmann et al., 2000; Trutnyeva et al., 2005), whilethe functions of other MP-interacting proteins in thisprocess remain obscure, awaiting future studies.
VIRUSES VERSUS RNAi HOST DEFENSE
Virus-host interactions during RNAi in plants arecomplex and understood only at a rudimentary level.In general, plants have multiple RNA silencing path-ways with diverse biological roles (Baulcombe, 2004).These include the regulation of gene expression andimportantly, for this short review, the control of virusaccumulation. The analysis of the RNAi pathwaycontrolling virus accumulation is complicated becausesome of the host genes involved in this process alsofunction in regulating host gene expression. In addi-tion, viruses themselves modify the final outcome bytheir expression of proteins that defeat the system, i.e.suppressors of RNA silencing. For a more completeunderstanding of this rapidly evolving area, there aremany excellent recent reviews (Baulcombe, 2004; Dinget al., 2004a; Moissiard and Voinnet, 2004).
RNA silencing involves the recognition of a targetRNA and its subsequent destruction. This occurs viaa multistep enzymatic pathway including, in plants,an RNA-dependent RNA polymerase (RdRP; nowreferred to as RDR), an RNase-III-type dicer-likeendonuclease (DCL), putative members of the RNA-induced silencing complex such as Argonaute, whichlikely binds RNA, and other proteins that may supportRNA-induced silencing complex activity, such asDEAD box helicases (SDE3; for review, see Baulcombe,2004; Meister and Tuschl, 2004). The majority of theseproteins are members of gene families, and it is thismultiplicity of family members that allows the plant torespond to widely varying needs (e.g. plant develop-ment and defense against virus invasion) and compli-cates our ability to understand each system.
One way to simplify this issue is to identify naturalor created plant knockout mutants for each geneinvolved in RNA silencing and study their loss-of-function phenotype during virus challenge. Using thisapproach, Arabidopsis DCL2 was found to be re-quired for protection against TCV (Xie et al., 2004).Arabidopsis SDE3 was required for protection againstcucumber mosaic virus (CMV) but not tobacco rattlevirus (TRV; Dalmay et al., 2001). For the RDRs, thetobacco RDR1 was required for protection againstTMV and PVX, while its Arabidopsis homolog wasrequired for protection against TMV-cg, a tobamovirusvery closely related to turnip vein clearing virus(TVCV; Lartey et al., 1993), and TRV (Xie et al., 2001;Yu et al., 2003). Interestingly, Nicotiana benthamiana isa natural mutant for RDR1, and transgenic expressionof an RDR1 ortholog from Medicago truncatula en-hanced its susceptibility to TMV, TVCV, and sunnhemp mosaic virus (a tobamovirus, but only distantlyrelated to TMV and TVCV), but not CMVor PVX (Yanget al., 2004). Similarly, RDR6 was required for pro-tection against CMV, but not turnip mosaic virus,
State of the Field
Plant Physiol. Vol. 138, 2005 1811
TVCV, TCV, or TRV in Arabidopsis (Dalmay et al.,2000; Mourrain et al., 2000). Thus, specific RDRs likelyrecognize different viruses; RDR1 is required for pro-tection against tobamoviruses and TRV, while RDR6 isrequired for protection against CMV.
In this issue, Schwach et al. (2005) report that RDR6in N. benthamiana is required to inhibit infections byPVX, potato virus Y, and CMV, in the presence of its Ysatellite RNA, but has no effect on infections by TMV,TRV, TCV, and CMV, in the absence of the Y satelliteRNA. During infection with PVX, RDR6 prevented thesystemic (including meristems), but not local, infectionof plants (Schwach et al., 2005). Grafting experimentsshowed that RDR6 is required for cells to respond toa systemically moving silencing signal. The results ofthis study suggested that RDR6 produces double-stranded RNA precursors from the silencing signalthat are used to generate short-interfering RNAs(siRNAs), which in turn allow an immediate silencingresponse against the target virus on its arrival(Schwach et al., 2005). This information advances ourunderstanding of the mechanism of the host RNAi-mediated resistance pathway against virus infection.For example, as Schwach et al. (2005) suggest, exclu-sion of virus from the meristem is mediated by RNAi,and RDR6 is involved in this process. These resultsalso raise issues to consider for future work in thisarea. For example, what are the virus and satelliteRNA targets telling us about the substrate structuralrequirements for each RDR, or is it that factors otherthan substrate suitability control the ability of partic-ular RDRs to control accumulation by specific viruses?Also, why does the Arabidopsis RDR6, but not N.benthamiana RDR6, protect against CMV?
It was also interesting that Schwach et al. (2005)showed that RDR6 did not control cell-to-cell move-ment of PVX, indicating that the silencing pathway inwhich this enzyme functions does not target virusintracellular or intercellular movement. In this issue,Liu et al. (2005) reported that mutant TMVs expressing126-kD protein silencing suppressors of varyingstrengths were also not altered in cell-to-cell move-ment (for TMV suppressor characterization, see Ku-bota et al., 2003; Ding et al., 2004b). Earlier, such anunlinking of RNA silencing suppressor activity fromcell-to-cell movement was demonstrated for the P15suppressor from peanut clump peculovirus (Dunoyeret al., 2002). It will be interesting to determine whetheror not RNA silencing ever directly targets the intra-cellular or cell-to-cell movement forms of the viralRNA. It may be that these forms are always protectedfrom the host silencing machinery.
Another article in this issue reports the effect oftemperature on the production of siRNAs in plantschallenged with various geminiviruses, demonstrat-ing that RNA silencing increased as the temperaturewas raised from 25�C to 30�C (Chellappan et al., 2005).This finding extends to DNA viruses what was foundfor an RNA virus, Cymbidium ringspot virus, in N.benthamiana (Szittya et al., 2003). Importantly, the in-
crease in siRNA steady-state levels was most striking(3- to 6-fold) for geminiviruses not associated witha recovery phenomenon (i.e. producing fewer symp-toms over time) compared with those that wereassociated with a recovery phenomenon. This dra-matic increase in siRNAs also was correlated with thepresence of one of two viral suppressors in thesegeminiviruses (Vanitharani et al., 2004; Chellappanet al., 2005). The critical importance of controlling tem-perature when studying RNAi or applying it in agri-culture is also highlighted in this work (Chellappanet al., 2005).
Last, it is interesting that connections between theinduction of stress in cells, which could be considereda host defense response, and virus movement mayexist. For example, exposure of plants to abiotic stress,e.g. low levels of heavy metal cadmium, blocks viralsystemic movement (Citovsky et al., 1998; Ghoshroyet al., 1998; Ueki and Citovsky, 2002). At the otherextreme, stress may aid movement because host heatshock protein (HSP) 70 (Aoki et al., 2002) and virus-encoded HSP70-related proteins (Medina et al., 1999;Alzhanova et al., 2001; Prokhnevsky et al., 2002) likelyhelp viral and/or host macromolecular transportthrough plasmodesmata. Interestingly, the inductionof host HSP70s during infection by plant RNA virusesis driven by a general mechanism that senses the levelof misfolded proteins in the cell, regardless of proteinorigin, viral or host (Aparicio et al., 2005). The study byJu et al. (2005) in this issue touched on the role of stressin viral movement by showing that turnover of TGBp2was greater during virus infection than when it wasexpressed alone in plant cells. They also determinedthat, in plant cells, TGBp2:green fluorescent proteinhad a longer half-life than free green fluorescentprotein. Based on these observations, Ju et al. specu-lated that cell stress, represented by increased proteinturnover, could aid movement of PVX between cells bytranslocating TGBp2 or viral movement complex outof the ER into the cytosol and making it available notonly for degradation but also for transport throughplasmodesmata (Ju et al., 2005). It will be interesting tosee if viruses indeed have pirated the host stressresponse for their own purposes.
ACKNOWLEDGMENT
We thank Drs. Elison Blancaflor and Ping Xu for valuable comments on
the manuscript.
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1814 Plant Physiol. Vol. 138, 2005
Table 1 | Viruses from the PRD1–adenovirus lineageFamily* Host Morphology Genome
topologyGenome length (kb)
Tectiviridae Gram-negative and Gram-positive bacteria
Icosahedral Linear ~15
Corticoviridae Gram-negative bacteria Icosahedral Circular ~10
STIV Archaea (Crenarchaeota) Icosahedral Circular 17,663
MVV Archaea (Euryarchaeota) Unknown Circular 12,039
TKV4 Archaea (Euryarchaeota) Unknown Circular 18,818
Adenoviridae Eukarya (vertebrates) Icosahedral Linear 26–45
Iridoviridae Eukarya (vertebrates and invertebrates)
Icosahedral Linear 140–303
Asfarviridae Eukarya (vertebrates) Icosahedral Linear 170–190
Phycodnaviridae Eukarya (unicellular) Icosahedral Linear or circular
160–560
Mimivirus Eukarya (unicellular) Icosahedral Linear 1,181,404
Poxviridae Eukarya (vertebrates and invertebrates)
Oval Linear 130–230
Ascoviridae Eukarya (invertebrates) Oval Circular 120–180
*If a virus has not been ascribed to a family, its name is provided. STIV, Sulfolobus turreted icosahedral virus.
Viruses are the most abundant biological enti-ties on the Earth (there are 1031–1032 virions in the biosphere; see Glossary)1. Nevertheless, classification of living organisms on the basis of their 16S rRNA genes left no room for viruses on the universal tree of life. However, it is now appreciated that viruses are not only ancient, but might also have played a major part in the emergence and consequent struc-ture of modern cellular life forms2–5. It is thus clear that comprehension of the virosphere, including evolutionary relationships between individual viruses and viral families, is crucial to fill the gaps in our understanding of the biosphere. It has been generally considered that viruses which infect evolutionary distant hosts (for example, bacteria and humans) have different origins6. However, recent accumulation of structural informa-tion has led to the proposal of a more unified common-ancestral-based virus classification7.
Structural virology, already in its youth when human rhinoviruses were found to be structurally related to small plant RNA viruses, offered unexpected answers to the vogue questions regarding origin and evolution of viruses8–10. The realization that the bacterial tectivirus PRD1 (Tectiviridae family) and human adenovirus (Adenoviridae family), which infect hosts from two different domains of life, might be evolutionary related was even
more surprising11. Not only did PRD1 and adenovirus contain linear double-stranded (ds) DNA genomes that were replicated in a protein-primed manner, but the topology of their major capsid proteins (MCPs) and the organization of these subunits in the capsid surface lattice, as well as the struc-ture of their penton and spike proteins, were found to be similar12,13. In the same
way, parallels were drawn between other icosahedral viruses: tailed dsDNA bacteri-ophages (Caudovirales order) and herpes-viruses (Herpesviridae family)14–17, as well as dsRNA bacteriophages (Cystoviridae family) and reoviruses (Reoviridae fam-ily)18–20. These observations gave rise to the viral lineage hypothesis, which predicts a common origin for viruses that share the same capsid architecture, but infect hosts from different domains of life7,21–23. Membership of a lineage is not strictly dependent on genome-sequence similarities, mode of replication or genome organization (circular versus linear), as these traits vary between the members of the proposed lineages. Rather, it relies on common principles of virion architecture. The set of genes that encode a capsid is considered to be the hallmark of the virus, as only these determinants can define a replicon as belonging to a virus rather than some other entity; for example, a plasmid. This block of genes is therefore likely to be inherited vertically in every virus lineage21. Ascribing viruses into such structure-based lineages not only bypasses the problem of the mosaic nature of viral genomes24, but also limits the enormous virosphere to a comparatively small number of lineages.
o P i n i o n
Virus evolution: how far does the double β-barrel viral lineage extend?Mart Krupovic and Dennis H. Bamford
Abstract | During the past few years one of the most astonishing findings in the field of virology has been the realization that viruses that infect hosts from all three domains of life are often structurally similar. The recent burst of structural information points to a need to create a new way to organize the virosphere that, in addition to the current classification, would reflect relationships between virus families. Using the vertical β-barrel major capsid proteins and ATPases related to known viral genome-packaging ATPases as examples, we can now re-evaluate the classification of viruses and virus-like genetic elements from a structural standpoint.
NATuRE REVIEwS | MicrobioloGy VoluME 6 | DECEMbER 2008 | 941
PErSPECTIVES
Nature Reviews | Microbiology
PBCV-1 Mav-1_CI
Adenovirus PRD1
PM2
STIVa
b
Figure 1 | Structural features common to members of the PrD1–adenovirus lineage. a | A comparison of the available X-ray structures of the double β-barrel major capsid proteins (MCPs) and the pseudoatomic model of the putative MCP of Maverick 1 from sea squirt Ciona intestinalis (Mav-1_CI). STIV MCP (Sulfolobus turreted icosahedral virus MCP) is coloured cyan, human adenovirus MCP is coloured purple, bacteriophage PrD1 MCP is coloured green, bac-teriophage PM2 MCP is coloured blue and Paramecium bursaria Chlorella virus type 1 MCP (PBCV-1 MCP) is coloured red. The putative MCP of Mav-1_CI is coloured red, whereas the insertions with respect to the corresponding PBCV-1 sequence are white. Protein sequences of Mavericks were downloaded from the repbase Update database of repetitive elements79 (see Further information). BioInfoBank Meta Server60 was used to predict the tertiary structure of the putative MCP of Mav-1_CI. The structure of PBCV-1 MCP38 was determined to be the best template for structural modelling. The sequence of the PBCV-1 MCP was aligned with the cor-responding protein sequences of Mav-1_CI, and a three-dimensional model of the putative Mav-1_CI MCP was built. b | Architecture of the PrD1 virion (triangulation number of 25). Apices of the red triangle combine the fivefold vertices (light shading) outlining the triangular virus facet. Grey hexagons depict the morphology of the double β-barrel trimers. The inset shows a close-up X-ray model of one trimer (top view) in which individual monomers are shown in different colours11. FIG. 1b courtesy of N.G.A. Abrescia, J.J. Cockburn and D.I. Stuart, University of Oxford, UK.
Several structure-based viral lineages have been proposed to date7. one of these lineages, the PRD1–adenovirus lineage, unites dsDNA tailless viruses that use similar architectural principles and are present in the largest number of different viral families; this lineage will be used in
this opinion as an example of a structur-ally defined viral lineage7,22,23. we discuss the available virion and capsid protein structures to show how far the lineage hypothesis can be extended and what it tells us about virus origin and evolution in this particular lineage.
Viruses with double β‑barrel proteinsThe PRD1–adenovirus lineage comprises icosahedral tailless viruses with dsDNA genomes. The hallmark of these viruses is their characteristic trimeric MCP, which has a double β-barrel structure (FIG. 1a). After trimerization of the MCP, hexagonal capsomers are formed, which are then arrayed as triangular plates to form the icosahedral virus shell7 (FIG. 1b). Members of the PRD1–adenovirus lineage infect cells in all three domains of cellular life. General characteristics of the current members of this lineage are briefly described below and summarized in TABLES 1,2.
Bacterial viruses. The best characterized bac-terial member of the PRD1–adenovirus line-age is bacteriophage PRD1, the type member of the Tectiviridae family, which infects a range of Gram-negative bacteria25. The PRD1 virion consists of an icosahedrally organ-ized proteinaceous capsid that surrounds a protein-rich lipid membrane. This membrane encloses the linear dsDNA genome, which is replicated by the phage-encoded type b polymerase (Polb) in a protein-primed man-ner12,25–27. The structure of the PRD1 MCP11 (FIG. 1a), and subsequently the entire 66 MDa PRD1 virion12,26, was solved by X-ray crystallography, which revealed an inter-action between the amino-terminal α-helices of the MCP subunits and the internal phage membrane. This interaction forces the viral membrane to obey icosahedral sym-metry. Another tectivirus, bam35, that infects Gram-positive Bacillus spp. was found to be different from PRD1 genetically28, but nearly identical structurally29.
bacteriophage PM2, the type member of the Corticoviridae family, infects marine Pseudoalteromonas species30. The PM2 virion and its major structural proteins have been analysed by both X-ray crystallography and cryo-electron microscopy (EM)31,32. Interestingly, X-ray analysis revealed that the double β-barrel MCP of PM2 (FIG. 1a) lacks the bottom part, the typical N-terminal α-helix, which contacts the viral membrane in the PRD1 virion31 (discussed below).
Archaeal viruses. Viruses that infect the archaeal domain are represented by the crenarchaeal Sulfolobus turreted icosahedral virus (STIV). Cryo-EM analysis of the STIV virion revealed similarities to bacteriophage PRD1 (REF. 33). Subsequent X-ray analysis of the STIV MCP revealed that three-dimensional structures of PRD1 and STIV MCPs are nearly identical34 (FIG. 1a). Although STIV is the only archaeal virus isolated so far,
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Table 2 | Genome lengths and T numbers of the double β‑barrel viruses
Virus Family Genome (bp) T ref.
Eukaryotic host
Mimivirus Not assigned 1,181,404 ~1,179 45
PpV01 Phycodnaviridae? ~415,000 219 80
PBCV-1 Phycodnaviridae 330,743 169 39
CIV Iridoviridae 212,482 147 39
Adenovirus Adenoviridae ~35,000 25 36
Prokaryotic host
STIV Not assigned 17,663 31 33
Bam35 Tectiviridae 14,935 25 29
PrD1 Tectiviridae 14,927 25 12
PM2 Corticoviridae 10,079 21 32
CIV, Chilo iridescent virus; PBCV-1, Paramecium bursaria Chlorella virus type 1; STIV, Sulfolobus turreted icosahedral virus, T, triangulation.
two putative proviruses, TKV4 and MVV, have been recently identified in the genomes of archaeal species from the second major phylum of Archaea, the Euryarchaeota35.
Eukaryotic viruses. Adenoviruses infect higher eukaryotes and have linear dsDNA genomes that are replicated by a protein-primed mechanism. Various adenoviruses have been subjected to intensive EM and X-ray analyses36,37. unlike other members of the PRD1–adenovirus lineage, adenoviruses do not have an internal membrane (dis-cussed below). In addition, a high-resolu-tion X-ray structure of the MCP, as well as a cryo-EM-based reconstruction of the entire virion, is available for Paramecium bursaria Chlorella virus type 1 (PbCV-1; a member of the Phycodnaviridae family)38,39 (FIG. 1a). The Phycodnaviridae family unites geneti-cally diverse viruses that infect both marine and freshwater algae40. Some members of the Phycodnaviridae can lysogenize their host cells by integrating into the cellular chromosome41.
Comparative genome analyses of large eukaryotic dsDNA viruses exposed a conserved set of genes for four virus families — Phycodnaviridae, Iridoviridae, Asfarviridae, Poxviridae — and the recently isolated Mimivirus, which suggests that these groups share a monophyletic origin. It has been proposed that these group-ings should be collectively called the nucleo-cytoplasmic large DNA viruses (NClDVs)42,43. Phycodnaviruses, asfarviruses, iridoviruses and the Mimivirus all possess an internal membrane that is surrounded by a large icosahedral capsid39,44,45, whereas poxviruses are brick shaped46. However, vaccinia virus (Poxviridae family) encodes
a highly conserved scaffolding protein (D13) that is homologous to the MCPs of other NClDVs42,43, and is known to play an essential part during the formation of genome-containing, spherical immature virions47,48. Subsequent transition from imma-ture to mature brick-shaped virions seems to be associated with the loss of D13 (REF. 49). Intriguingly, D13 was shown to trimerize and form an external, honeycomb-like lattice that covers the lipid bilayer of the immature virion49. Similarly, a three-dimensional density map of the trimeric D13 homologue in orf virus (Poxviridae family) was found to accommodate the electron-density map of the PbCV-1 MCP50, indicating that D13 also adopts a double β-barrel fold. These observa-tions suggest that the immature virions of poxviruses resemble the mature icosahedral virions of the PRD1–adenovirus lineage49,50.
Similar morphological transitions might have given rise to viruses of the Ascoviridae family. Ascoviruses attack lepidopteran larvae and pupae, and vary in shape from ovoid to bacilliform depending on the species51. Comparative genomic and phylogenetic anal-yses unexpectedly revealed that ascoviruses are likely to have evolved from an iridovirus ancestor52,53.
Mavericks: the missing link?As discussed above, members of the Adenoviridae are outliers in the PRD1–adenovirus lineage (BOX 1). on the one hand, the adenoviral MCP is much larger than the MCPs of the other members of the PRD1–adenovirus lineage (FIG. 1a) and adenoviruses lack lipids as structural components. on the other hand, adenovi-ruses have the same genome organization and mode of replication as tectiviruses. we
assume that the large MCP of adenoviruses evolved from a smaller version that is still found in NClDVs and prokaryotic viruses. Identification of a virus with an NClDV-like capsid protein and the genome organization of the adenovirus would be the best proof of such a hypothesis.
A class of self-synthesizing DNA transposons was recently identified in a wide range of eukaryotic organisms (from nematodes to vertebrates)54,55. These trans-posable elements were named Mavericks54 or Polintons55. Interestingly, Mavericks possess several features that are common to tectiviruses and adenoviruses, including genome organization and gene content55,56
(FIG. 2). A typical Maverick is 15–20 kb long, is flanked with inverted terminal repeats and encodes approximately 10 protein spe-cies55,56. The most conserved set of encoded proteins includes a protein-primed Polb, an adenoviral cysteine protease, an ATPase (similar to the packaging ATPases of internal membrane-containing viruses) and an integrase55,56. It is important to note that the ATPase is the second protein that is common to all membrane-containing members of the PRD1–adenovirus lineage57. These ATPases form a distinct clade in the FtsK–HerA superfamily of cellular chromosome-pumping ATPases58 that is evolutionary distinct from the packaging ATPases of tailed bacteri-ophages and herpesviruses58,59. In addition
Glossary box
BiosphereThe global ecological system that integrates all living organisms.
CapsomerThe basic structural unit of the capsid of a virus.
Convergent evolutionThe process by which organisms that are not monophyletic independently evolve similar traits as a result of adaptation to ecological niches or similar environments.
Divergent evolutionThe accumulation of differences between groups that can lead to the formation of new species, usually as a result of the adaptation of different groups of the same species to different environments.
TransposonA genetic element that can move from one locus of a chromosome to another through a recombinase-mediated reaction.
Triangulation number(T ). Used to describe the number of subunits that exist in a capsid: an icosahedral capsid of triangulation number T will possess 60 T subunits.
VirosphereThe total sum of all viruses.
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Box 1 | Capsid‑size variation
An interesting characteristic of the trimeric double β‑barrel major capsid proteins (MCPs) is their ability to serve as building blocks to form shells of highly variable sizes (FIG. 1b). The X‑ray structure of bacteriophage PRD1 virions (~4 Å resolution) provided the first insights into the possible mechanisms of such a scalable assembly12. The linearly extended protein P30 was found to connect the icosahedral vertices and consequently define the size of the virion. Cryo‑electron microscopy analysis revealed a similar protein in bacteriophage Bam35 (REF. 29). Interestingly, a mutant of African swine fever virus (Asfarviridae family) that forms tubular, MCP‑coated membrane structures instead of icosahedral particles has been recently characterized72: a mutation in a single gene that encodes for a structural protein, pB438L, was responsible for this change. This is reminiscent of the phenotype observed during the infection with tape‑measure protein P30‑deficient mutants of bacteriophage PRD1 (REF. 73). Additional information is needed to determine whether pB438L of ASFV is an equivalent of the tape‑measure protein of PRD1.
The size of the capsid is proportional to the interior volume and consequently to the size of the genome, which must be fitted into the capsid (TABLE 2). Strategies used by members of the PRD1–adenovirus lineage to increase the interior volume of the capsid in response to environmental changes (for example, evolution of the immune system of eukaryotic hosts) are shown in the figure. The numbers denote the different strategies: alteration of the triangulation (T) number by viruses of the nucleo‑cytoplasmic large DNA virus (NCLDV) group (1); the use of larger building blocks (pseudohexamers) and removal of the internal membrane by adenoviruses (2) and conservation of the original capsid size by Mavericks at the expense of transition to a predominantly intracellular persistence as integrated transposable elements (3).
The beauty of the scalable system used by PRD1 is its predicted capacity to change the interior volume depending on the length of the genetic material to be packaged by altering the T number (see the figure). The ability to package larger genomes might be crucial for successful infection. Although some scientists favour the hypothesis of reductive evolution to explain the emergence of NCLDVs4,74, recent analyses of the 1.2 Mb Mimivirus genome revealed that although most of its genes were acquired by lateral gene transfer either from its amoebal host or from bacteria that were parasitizing the same hosts, the Mimivirus genome evolved through multiple gene and segmental genome‑duplication events75,76. Similar conclusions were drawn from a recent analysis of the entire NCLDV group77. These findings suggest that NCLDVs evolved from smaller viruses rather than from organisms that were even more complex.
The other possible way to increase the inner capsid volume is to alter the MCP so that it can form larger capsomers and remove virion components that reside inside the protein shell. This strategy seems to be successfully used by adenoviruses (see the figure). Adenoviruses form capsids of the same T number (T = 25) as the tectiviruses, whereas the genome size of adenoviruses is considerably larger (TABLE 2). The larger capacity of the capsid is due to two main reorganizations. First, compared with PRD1, the two β‑barrels in the adenovirus hexon are further apart, which creates a trimer with a width of 97 Å compared with 80 Å in the MCP of PRD1 (REF. 11). Second, the internal membrane of adenoviruses has been removed. However, it should be noted that once the larger capsid is formed, packaging of the original (smaller) genome would be unlikely to lead to the formation of infectious virions, as a lower genome‑packaging density would not be sufficient to drive the initial injection of the viral genome into the host cell78.
Nature Reviews | Microbiology
2
3
Mavericks?
1
to walker A and walker b motifs, ATPases of these internal membrane-containing viruses contain a conserved and essential P9/A32 motif, which was proposed to be specific to membrane-containing viruses57. Adenoviruses seem to use the ATP-binding cassette (AbC)-type ATPase for genome packaging, which might have been acquired
from the host together with loss of the inter-nal membrane59. Interestingly, the ATPases encoded by Mavericks possess a P9/A32 motif. In addition to the four proteins mentioned above — Polb, cysteine protease, ATPase and integrase — Mavericks encode four conserved hypothetical proteins, which have been named PX, PY, Pw and PZ55.
using Structure Prediction Meta Server60, the MCP of PbCV-1 was found to be the best template for structural modelling of PY proteins38. In a subsequent analysis, the high stereochemical quality of a three-dimensional model of a selected PY protein (FIG. 1a) indicated that the Maverick protein PY has the potential to adopt the same fold as the MCP of PbCV-1: the double β-barrel fold (M.K. and D.H.b., unpublished observations).
So what are these large transposable DNA elements of eukaryotes? It is possible that Mavericks represent the remnants of ancient viruses, as proposed by Pritham and colleagues56. based on the presence of the putative MCP and the packaging ATPase, the progenitor virus is proposed to be a member of the PRD1–adenovirus lineage. Retention and high conservation of these two genes as part of the Maverick genome, as well as the restricted size of the genome itself, are intriguing. one explanation might be that Mavericks are still capable of forming virions and can sometimes spread as genuine viruses. An alternative scenario is that the possible ancestor virus of Mavericks, in contrast to the two evolutionary schemes proposed above for NClDVs and adenoviruses (BOX 1), evolved a third tactic to avoid the immune system of the evolving eukaryotic host (see the figure in BOX 1). Instead of coding for a larger capsid that would accom-modate a larger genome, Mavericks changed their life style to persist inside the host cell without destroying it. However, unless this change in life style occurred recently, this scenario does not explain the conservation of the two putative genes that encode for struc-tural virion proteins or explain the apparent conservation of genome length, which is likely to be constrained by capsid size.
Divergent versus convergent evolutionThe viral lineage hypothesis assumes that the origin of viruses in each lineage is mono-phyletic and that diverse viral families in a lineage arose as a result of divergent evolution. unfortunately, a lack of significant sequence similarity between viral genomes of the PRD1-like viruses prevents us from verifying such an assumption. Although it is not pos-sible to reject with certainty that convergent evolution led to the appearance of the double β-barrel fold, it should be realized that the protein fold is not the only feature shared by viruses in the lineage. The most conserved feature of viruses of the PRD1–adenovirus lineage is the use of double β-barrel MCPs to build the protein shell: pseudohexameric trimers of the MCP are arrayed as triangular
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Nature Reviews | Microbiology
PolBATPase MCP Pro
PolBATPase MCPPro Int
PolB ATPase MCPINT
PolB ATPase MCP
RCR ATPase MCPInt
RCR ATPase MCP
PolB ATPase MCP
MCM ATPase MCP
RCR ATPase MCP Int
Int
5 kb
Virus or element
Frog adenovirus*
Maverick-2_XT
Maverick-1_CI
PRD1*
Bam35
TKV4
MVV
PM2*
Corticoelement
STIV*ATPase MCP
Pro
Figure 2 | Genome organization of selected members of the PrD1–adenovirus lineage from all domains of life. Presented are the genome organizations of members of the PrD1–adenovirus line-age from the Bacterial domain (PrD1, Bam35, PM2 and a corticoelement in the Vibrio splendidus 12B01 genome), the Archaeal domain (Sulfolobus turreted icosahedral virus (STIV), and proviruses TKV4 and MVV) and the Eukarya (Frog adenovirus). The genomic organizations of Maverick-1_CI and Maverick-2_XT are also provided, which reside in the genomes of Ciona intestinalis and Xenopus tropicalis, respectively79 (see Further information). Genomic sequences were aligned using major capsid protein (MCP)-coding genes. Arrows indicate the direction of transcription. MCP-coding genes are coloured blue, ATPase-coding genes are coloured yellow, replicase-coding genes are coloured red, recombinase-coding genes are coloured green and adenoviral protease-coding genes are coloured purple). The last five genomes are arbitrarily linearized for a more convenient alignment. Asterisks denote the viruses for which MCP X-ray structures are available. The genomes of Paramecium bursaria Chlorella virus type 1 (PBCV-1) and other nucleo-cytoplasmic large DNA viruses are much larger than those shown and were therefore omitted from the alignment. Int, integrase; PolB, polymerase B; Pro, protease; rCr, rolling circle replication.
plates to form the icosahedral virus capsid. Importantly, in all these viruses, the axes of the β-barrels are perpendicular to the capsid shell. Furthermore, the position of genes in the genome is often conserved35. This is most clearly observed for prokaryotic members of the PRD1–adenovirus lineage (FIG. 2). It should be noted that these viruses possess genomes of different topologies, and replication is carried out with the aid of non-homologous proteins, suggesting that genome replication and virion assembly are not coupled. This was confirmed by the finding that four different replication systems are used by PM2-like corticoviral elements61. Nevertheless, the genes that encode these pro-teins are often found at equivalent positions in the genome (FIG. 2).
Structurally similar viruses that can infect hosts from different domains of life have dif-ferent gene contents, as interactions with the host cell change the gene content of the virus. we therefore find it useful to divide viral genes into two groups. one category, referred to as the non-self genes21, includes viral genes that are involved in the interaction with the host and can be exchanged between unrelated viruses or even between the virus and the host chromosome through hori-zontal gene transfer24. Consequently, their evolutionary histories cannot be followed with certainty. This category includes genes for DNA and RNA metabolism (replication, recombination and transcriptional factors), host recognition, evasion of the immune system and release of the virus progeny35,61–64.
by contrast, the other category, the viral self genes21, includes genes that encode the key structural components of the virion. Such components eventually determine how the virion is constructed and are specific to a given viral lineage, which indicates that they are inherited vertically. The probability that so many similarities between viruses within a viral lineage would arise as a result of convergent evolution is low.
Evolutionary origin of the lineageA number of hypotheses have been put forward to explain the origin of viruses (see, for example, REFS 4,5,21,65). These hypoth-eses converge to one common feature: the viral capsid protein is the only component that is unique to the viral world. Extending this idea, Raoult and Forterre66 have even proposed to divide biological entities into ribosome-encoding organisms, which include eukaryotic, archaeal and bacterial organisms, and capsid-encoding organisms, which include viruses. Acquisition of the protein shell by a replicon was a cornerstone in the origin of viruses. This event seems to have occurred multiple times, giving rise to diverse viral lineages, some of which still exist today. The origin of capsid proteins is speculative. one possibility, as suggested by Koonin et al.5, is that viral hallmark genes (one of which is the MCP-coding gene) might have descended directly from a primordial gene pool, as they are missing from the cellular gene content.
Structural and genomic data allow us to extrapolate back in time and suggest an evolutionary trail of the double β-barrel pro-teins and consequently viruses that use these proteins for their assembly. FIGURE 3 combines information about a number of viral families that infect hosts from all three cellular domains. It should be noted, however, that the scenario presented is highly hypothetical, and is meant only to stimulate further research and discussions on virus evolution. The numbers represent the possible major turning points in the evolution of the members of the PRD1–adenovirus lineage and will be discussed in numerical order.
The double β-barrel fold seems to arise through gene duplication9,23,31,38. In line with this proposal was the recent identification of SH1 and P23-77, two membrane-containing viruses that infect archaeal and bacterial hosts, respectively67,68. The structure of the SH1 and P23-77 virions has been determined by cryo-EM and image reconstruction to 9.6 Å and 14 Å resolutions, respectively68,69. Analysis of the two virions revealed that hex-agonal capsomers are similarly arranged in a T = 28 (T represents the triangulation number)
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icosahedral lattice. The subnanometre reso-lution structure of the SH1 virion, together with biochemical data, suggested that hex-agonal capsomers are formed from six indi-vidual β-barrels that are oriented normal to the capsid surface: the central axis of the bar-rels was orientated perpendicular to the cap-sid surface and was stabilized by additional decorating proteins69. Importantly, SH1 and P23-77 both encode a putative packaging ATPase with the P9/A32-specific motif that is common to all membrane-containing members of the PRD1–adenovirus lineage57 (M. Jalasvuori and J. K. bamford, personal communication). on the basis of these com-mon virion assembly principles, we propose that such single β-barrel viruses should be grouped together with the double β-barrel viruses into a vertical β-barrel superlineage (FIG. 3). Such a superlineage would include all viruses that use similar virion architec-ture regardless of whether the hexagonal capsomer is composed of six monomers or three dimers.
Duplication and subsequent fusion of the gene that encodes for a vertical β-barrel capsid protein probably led to the emergence of viruses with the double β-barrel MCPs (FIG. 3, branching point 1). The use of double β-barrel trimers instead of single β-barrel hexamers reduces the number of protein subunits needed to build the hexagonal cap-somer (from six in SH1 to three in PRD1) and would account for a lower assembly
error probability, as proposed by Jäälinoja and colleagues69. Furthermore, double β-barrel trimers are likely to be more stable than hexamers, which could account for the absence of decorating or stabilizing proteins. The simplest double β-barrel is found in corticovirus PM2 and was designated the minimal double β-barrel31 (FIG. 1a). Structural comparison of the two PM2 β-barrels with those of other viruses from the same lineage revealed that the two barrels in PM2 are more similar to each other than to any other coat protein, which supports the hypothesis that the protein arose by gene duplica-tion23,38. Importantly, an interaction with the internal membrane in PM2 is achieved by separate viral integral-membrane proteins. All other membrane-containing viruses of the lineage possess larger and more elaborate MCPs that directly interact with the underly-ing viral membrane12,34,38, which suggests that they diversified from the minimal double β-barrel fold (FIG. 3, branching point 2).
PM2, and all other corticoviral elements that can frequently be found integrated into the genomes of aquatic bacteria, seem to possess circular genomes that are apparently replicated through four different mecha-nisms61. Circular dsDNA genomes are also characteristic of crenarchaeal virus STIV and the euryarchaeal proviruses, TKV4 and MVV33,35. Interestingly, the integrated genome of MVV is only slightly larger than that of PM2, and replication of both genomes seems
to be carried out through a rolling-circle mechanism35,70. Notably, none of these (pro)viruses encodes a DNA polymerase for independent genome replication, but rather they depend on the cellular replica-tion machinery. This also seems to be true for SH1, for which no replication protein could be identified71, and P23-77, which encodes a putative replication initiation protein (M. Jalasvuori and J. K. bamford, personal communication). by contrast, all other members of the PRD1–adenovirus lineage, including those that infect bacteria or eukaryotes, encode Polb (FIG. 3b). It is possible that the polB gene was acquired only once, and then diversified into the protein- and RNA or DNA-primed Polb (FIG. 3, branch-ing point 3). Tectiviruses, adenoviruses and Mavericks encode a protein-primed version of Polb, whereas all the other members of the PRD1–adenovirus lineage, the NClDVs and ascoviruses, encode an RNA or DNA-primed Polb43,52,55.
Diversification of bacterial tectivi-ruses from adenoviruses, Mavericks and NClDVs was probably associated with the emergence of the bacteria and Eukarya domains (FIG. 3, branching point 4). This scenario is supported by a structure-based phylogenetic analysis7 of currently available double β-barrel MCPs, which revealed that MCPs of PbCV-1 and adenovirus cluster together31. It is hard to predict the order in which the radiation from the common
Nature Reviews | Microbiology
a b
Vertical β-barrelsuperlineage
Vertical β-barrelsuperlineage
Double β-barrellineage
Single β-barrellineage
or
PolBRNA or DNAprimed
Ascoviridae
AsfarviridaeIridoviridae
Phycodnaviridae
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NCLD
VsProtein primed
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Cellularreplicationmachinery 1
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Poxviridae
Mavericks Adenoviridae
PRD1Tectiviridae
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STIVTKV4
MVV
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34
Figure 3 | Possible origin and evolution of viruses that belong to the vertical β‑barrel superlineage. The schematic represents the hypotheti-cal evolutionary relationship between double-stranded DNA viruses that use vertical β-barrel pseudohexamers for icosahedral capsid shell assem-bly. It should be noted that the diagram is not a tree that is inferred from phylogenetic analysis but rather a summary of the available data on the depicted viral families. The same diagram is coloured with different shades to reflect the major capsid protein-based (part a) and genome replication
machinery-based (part b) relationship between different virus families that are unified into the vertical β-barrel superlineage. The numbers denote the key turning points in the evolution of this virus superlineage. The coloured circles represent the different domains of life from which the correspond-ing hosts reside. Bacteria are coloured red, Archaea are coloured green and Eukarya are coloured blue. The organization of the viral genomes (circular or linear) is indicated. NCLDV, nucleo-cytoplasmic large DNA virus; PolB, polymerase B; STIV, Sulfolobus turreted icosahedral virus.
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ancestor of adenoviruses, Mavericks and NClDVs might have proceeded. The monophyletic origin and relationship between individual virus families of the NClDV group, as well as the connection between iridoviruses and ascoviruses, was thoroughly discussed by Iyer et al.42,43 and Stasiak et al.53, respectively, and will not be repeated here. In contrast to the evolution of NClDVs, which advanced by increas-ing their capsid T number, adenoviruses modified their ancestral PbCV-1-like MCP and seem to have sacrificed their internal membrane to build larger shells and conse-quently obtain the ability to increase their gene content (BOX 1). This idea is supported by the identification of Mavericks, which pos-sess genome organization and gene content similar to adenoviruses and tectiviruses, but an MCP that is more similar to that of NClDVs (FIGS 1,2). It seems that increased genome size, and consequently capsid volume, is under positive selection, possibly owing to the benefit of incorporating addi-tional genes to ensure successful propagation in more complex eukaryotic cells.
ConclusionsMany technological advances have been achieved during the past decade, which has led to a rapid increase in available structural data on diverse viruses that infect hosts from all three domains of life. This led to the realization that the previ-ously unimaginably large virosphere can actually be divided into a small number of groups, giving rise to the viral superlineage hypothesis. If successfully implemented, this hypothesis could improve the cur-rent virus classification by reflecting evolutionary relationships between diverse virus families. For example, grouping the entire virosphere into structure-based superlineages would create the highest evolutionary-relevant taxonomic level and would therefore bring remote virus families together, despite differences in genome topology or gene content and even the fact that hosts might belong to different domains of life. Implementation of such an additional higher hierarchical level would not replace the use of current lower taxonomic levels, such as the family, subfamily and genus, which are certainly useful for classifying genetically closely related viruses.
The most widespread viral superlineage described to date seems to be the vertical β-barrel superlineage, which combines a large number of viral families that use hexagonal capsomers for capsid formation. Structural data on virion architecture,
complemented with genomic data analyses, have allowed us to propose a scenario of how these icosahedral dsDNA viruses might have evolved. These findings not only provide order to the entire domain of viruses but may also shed light on the origins and evolu-tion of early life, when viruses may have substantially contributed to the composition of the biosphere by facilitating genetic exchange.
Mart Krupovic and Dennis H. Bamford are at the Department of Biological and Environmental
Sciences and Institute of Biotechnology, Biocenter 2, PO BOX 56 (Viikinkaari 5),
00014 University of Helsinki, Finland. Correspondence to D.H.B.
e‑mail: [email protected]
doi:10.1038/nrmicro2033
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AcknowledgementsWe thank J. Ravantti for his invaluable comments, and M. Jalasvuori and J.K. Bamford for sharing their unpublished data. This work was supported by the Finnish Center of Excellence Program (2006-2011) of the Academy of Finland (grants 1213467 and 1213992 to D.H.B. and grant 1210253). M.K. is supported by the Viikki Graduate School in Biosciences.
DATABASESentrez Genome: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genomeBam35 | Orf virus | PBCV-1 | PM2 | PrD1 | STIVProtein Data Bank: http://www.rcsb.org/pdb/home/home.doadenovirus MCP | PrD1 MCP | PBCV-1 MCP | PM2 MCP | STIV MCP
FURTHER inFoRMATionDennis H. Bamford’s homepage: http://blogs.helsinki.fi/bamford-group/BioinfoBank Meta server: http://meta.bioinfo.pl/submit_wizard.plrepbase Update: http://www.girinst.org/repbase/update/index.html
All linkS Are ActiVe in tHe online PDF
by 30 September 2007, 708 bacterial genome sequences had been submitted to publicly accessible databases, such as those at the European Molecular biology laboratory’s European bioinformatics Institute (EMbl–EbI), the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the National Center for biotechnological Information (NCbI) (see Further information). Many other genomes are currently being sequenced and several sequences have been completed but have not yet been placed in the public domain. The number of publicly available genomes has doubled approximately every 16 months
o P i n i o n
e-Science: relieving bottlenecks in large-scale genome analysesTracy Craddock, Colin R. Harwood, Jennifer Hallinan and Anil Wipat
Abstract | The development of affordable, high-throughput sequencing technology has led to a flood of publicly available bacterial genome-sequence data. The availability of multiple genome sequences presents both an opportunity and a challenge for microbiologists, and new computational approaches are needed to extract the knowledge that is required to address specific biological problems and to analyse genomic data. The field of e-Science is maturing, and Grid-based technologies can help address this challenge.
since 2000. At this rate, more than 2,000 bacterial genomes will be available by the end of 2010 (FIG. 1).
In most cases, the genome of only a single representative of a species has been sequenced. Increasingly, however, replicate genome sequences are appearing in the public domain (FIG. 1). For example, there are now 14 publicly available Staphylococcus aureus genomes, with 2 additional strains at the ‘fin-ishing’ stage. using replicate genomes, direct comparisons can be made between closely related strains, providing unprecedented insights into the mechanisms involved in genome plasticity and rates of evolution. As
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Rev. sci. tech. Off. int. Epiz., 1991, 10 (3), 733-748
Virus survival in the environment E.C. PIRTLE and G.W. BERAN *
Summary: Viruses pass into the environment from clinically ill or carrier hosts; although they do not replicate outside living animals or people, they are maintained and transported to susceptible hosts. Population concentrations and movement, both animal and human, have been steadily increasing in this century, enhancing transmission of respiratory and enteric viruses and compounding the difficulty of preventing environmental transmission.
Studies on environmental survival factors of viruses have been most definitive for polioviruses, foot and mouth disease viruses and Aujeszky's disease virus. In addition, heat resistance studies have been reported on adenoviruses, African swine fever virus and the Norwalk virus. Resistance to disinfectants has been studied for many viruses, including picornaviruses, papovaviruses, reoviruses and retroviruses. Survival of viruses in and on a variety of fomites has been studied for influenza viruses, paramyxoviruses, poxviruses and retroviruses. The subacute spongiform encephalopathy agents, under extensive current studies, are being found to have incredible stability in the environment.
KEYWORDS: Environment - Fomites - Inactivation - Resistance - Stability -Survival - Virus.
I N T R O D U C T I O N
In the triad o f infect ious disease transmiss ion involv ing aet io logical agents , susceptible hosts and the env ironment , the role o f the env ironment is the most a m b i g u o u s . The env ironment receives, mainta ins or protects and transports aetiological agents to susceptible hosts . Viruses may enter the environment in enormous quantities from clinically ill or inapparent carrier hosts ; w h e n extant outs ide the hosts which support their repl icat ion, they are the least u n d e r s t o o d o f infect ious agents . The greatest prospects for disease control for the future, however , lie in environmental measures to halt or reduce transmiss ion . Converse ly , failure to break the chains o f transmiss ion will result f rom failure to protect the env ironment or to m o d i f y it beneficial ly .
The increase in respiratory disease transmission through populat ion concentrations, in cities in the case o f h u m a n s and in conf inement product ion units in the case o f animals , is the m o s t striking example o f disease prevalence . A greater contemporary awareness o f the role p layed by the conf ined env ironment in increasing the transmission of some diseases while reducing that o f others has prompted more serious study o f the env ironment .
* Department of Microbiology, Immunology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, United States of America.
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This review of the survival of viruses in the environment attempts to consolidate data from reported studies. Further investigation of how numerous viruses survive in the environment is necessary. Current knowledge is fragmented and the fragments differ widely according to the infectious agents; this fact highlights the need for more comprehensive studies in the future.
This article discusses viral entry, survival and transport as they relate to any nonliving substances or living organisms which do not support viral replication. Viruses are considered by families and are ordered alphabetically. When more than one virus is discussed in a chapter, they are considered alphabetically in relation to the first virus mentioned in the chapter.
A D E N O V I R I D A E
Thermal inactivation studies have been reported for adenovirus 12, reovirus 1 and herpes simplex virus in raw milk, sterilized homogenized milk, raw chocolate milk and raw ice cream mix, with minimum essential medium (MEM) as control suspending fluid (55, 57). From approximately 10,000 plaque-forming units (PFU) per ml of each suspending medium, inactivation curves at 40 C-60°C were asymptotic to the base line, indicating that small amounts of these viruses survived, even at the higher temperature. At 65°C, the inactivation curves approached first order reactions, indicating that temperatures near pasteurization standards were effective in inactivating these three viruses. In the same studies, influenza A and Newcastle disease viruses showed stability in raw and sterilized milk equivalent to that in MEM. Thermal inactivation of Maloney virus, Rauscher leukemia virus and Rous sarcoma virus assayed in mice showed Rous sarcoma to be the most resistant.
A R E N A V I R I D A E
The hallmark of all rodent-borne arenaviruses is persistent infection in the rodent host in the presence of immunological response (38). Persistence is established in the natural host if virus transmission occurs in utero or shortly after birth. Most persistently infected rodents have permanent viruria and viremia. Viral persistence is a highly efficient means of virus perpetuation in most rodent offspring; it is also the most important source of contamination of the external environment and leads to transmission of infection.
H E R P E S V I R I D A E
Extensive studies have been published on assays of potentially contaminated fomites for both human and animal herpesviruses. Additional studies of virus survival on and in experimentally-contaminated fomites have been reported. In studies on the alphaherpesvirus, HSV2, assays of spa water failed to yield the virus (45). To
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simulate the conditions of survival of HSV2 on plastic-coated benches and seats in spa facilities, HSV2 (10 4 - 2 CCID50/0.5 ml) was placed on plastic surfaces in a humid atmosphere at 37°C-40°C. The virus was found to survive for up to 4.5 h under these conditions. The results of a study with HSV1 and HSV2 viruses demonstrated that HSV obtained directly from ulcerative or vesicular genital lesions was able to survive for several hours on fomites and hard surfaces and for several days on dry cotton gauze (35). HSV2 has been shown to survive for short periods outside the host; it is the opinion of some workers, however, that while the virus can persist on certain surfaces and porous items, such as towels, for relatively long periods of time, fomites are not particularly significant in transmission (22).
There has been considerable interest in environmental transmission of the betaherpesvirus, cytomegalovirus (CMV), among infants and personnel in medical facilities. During a four-month study, CMV was found in the urine of eight infants (54). Three of the isolates were found to be identical by restriction endonuclease analysis, which suggests that the three infants in question were infected with the same CMV strain. The evidence indicates that CMV was transmitted from one infant to the other two infants through unidentified fomites within the nursery. Samples from the immediate environment of these eight CMV-infected infants were obtained and submitted for virus assay in cell cultures (23). CMV was isolated from those objects which had come in direct contact with infected secretions, i.e. from six of eight oronasal suction bulbs, one feeding tube, four dry diapers in contact with genitalia, and from a pair of gloves worn by a nurse. While the conclusion was reached that CMV could be isolated for several hours after natural contamination, it was not determined that fomites were an important source of nosocomial CMV transmission. That personnel should wash their hands during patient care, however, was considered as essential.
Nosocomial transmission of CMV was investigated in a chronic care unit (CMV excretion prevalence 16%) and a neonatal unit (CMV excretion prevalence 0.7%) (19). In the chronic care unit, two infants were infected with homologous strains of CMV. No infants acquired CMV in the neonatal unit of the hospital, though seroconversion did occur in two nurses. CMV was isolated from diapers and from the hands of patients and personnel, but not from environmental surfaces.
In an epidemiological study of bovine herpes mammillitis (BHM) virus (27), pseudocowpox was confirmed in dual infections in nine of eleven BHM-positive milking herds. The pattern of BHM spread was not related to the incidence of pseudocowpox or to the order in which cows were milked. Meteorological data suggested that BHM occurred more frequently in those years conducive to the increase of large populations of insect vectors, and that the direction in which BHM spread to herds within a locality was related to the predominant wind direction.
Fomites were experimentally identified as a risk in the transmission of CMV (53). Urine and saliva inoculated with 1 x 10 4 P F U / m l of either a wild or laboratory strain of CMV could be recovered, at 37°C, for 48 h and 2 h, respectively.
Survival studies on Aujeszky's disease virus (ADV) in this unit have focused on experimental contamination of fomites and vehicles present in and around swine operations. Virus suspended in saliva, nasal-washing or saline-glucose control fluids
7 3 6
was assayed for survival on solid fomites kept moist at 25°C. What follows are the maximum times needed to reach 99.99% inactivation:
Mean survival time of ADV in contact with fomites in saliva suspension was 4 5 % of virus in saline-glucose suspension; in nasal-washing suspension, the mean survival time was 30% of that for virus in saline-glucose suspension (52).
ADV suspended in liquid fomites at 25°C reached 99.99% inactivation at the following times (52):
- well water 7 days
- chlorinated water 2 days
- urine from swine on subtherapeutic medicated feed 14 days
- manure pit effluent <1 day
- anaerobic lagoon effluent 2 days.
ADV in aerosol decayed logarithmically with half-lives of 17.4-36.1 min at 22°C and 27.3-43.6 min at 4°C , with longer survival times at 5 5 % relative humidity than at 2 5 % or 85% relative humidity (51).
ADV fed to houseflies (Musca domestica) could be recovered for as long as seven days in the internal organs but for only a short time from the body surfaces. The longest survival time for virus was obtained when 3-day-old flies were exposed (compared to survival in 5- to 13-day-old flies), and when exposed flies were kept at 10°C as compared to 20°C or 30°C (61). Virus placed on the feet of pigeons and
- control fluid (no fomite) 58 days
- steel 18 days
- concrete 4 days
- plastic 8 days
- rubber 7 days
- denim cloth <1 day
- loam soil 7 days
- green grass 2 days
- shelled corn 36 days
- pelleted swine feed 3 days
- meat and bone meal 5 days
- alfalfa hay <1 day
- straw bedding 4 days
- sawdust bedding 2 days
- swine faeces 2 days.
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starlings could not be recovered, even when suspended in mucin, placed on washed surfaces of the toes and sampled promptly after exposure (M.A. Schoenbaum and G.W. Beran, unpublished data) .
ADV mixed into 80% lean ground pork sausage, p H 5.85, could still be recovered after 14 days at 4°C storage and after 40 days at -20°C storage (E.C. Pirtle and T.A. Proescholdt, unpublished data) .
I R I D O V I R I D A E
African swine fever virus (ASFV) is the only virus in the Iridoviridae family which is known to infect mammals . ASFV survives over a wide range of pH values and is particularly resistant to alkaline conditions. In the presence of protein, some infectious virus may survive for 7 days at pH 13.4 and for several hours below pH 4.0 (49).
N O R W A L K V I R U S
The Norwalk group of viruses is tentatively described as calici-like, but has not been definitely classified (32). In experiments, the Norwalk virus retained infectivity to volunteers producing gastroenteritis following exposure to pH 2.7 for 3 h at room temperature and after heating at 60°C for 3 min. Both the Norwalk and " W " (Wollan boarding school) agents were stable following treatment with 20% ether at 40°C for 18-24 h.
O R T H O M Y X O V I R I D A E
Orthomyxoviruses, influenza A and B (10 3 - 5 and 1 0 4 2 infectious doses/0.1 ml, respectively) were spread over stainless steel, plastic and absorbent material surfaces (30 to 50 mm) (4). These objects were subsequently sampled by rubbing with sterile swabs or " c l e a n " fingers. Both influenza A and B viruses survived on hard surfaces for 24 to 48 h, but for less than 8 to 12 h on porous surfaces. The conclusion was therefore reached that , under conditions of heavy environmental contamination, the transmission of influenza virus by fomites may be possible.
P A P O V A V I R I D A E
Objects used in the treatment of patients with human papillomavirus (HPV) infections were tested for recovery of H P V - D N A (25). H P V - D N A was identified by hybridization on 8 of 16 (50%) surgical gloves, on 23 of 62 (37%) and 1 of 62 (1.6%)
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biopsy forceps before and after sterilization in 30% tincture of Savlon for 30 min, and on 5 of 22 (23%) and 1 of 22 (4.5%) cryoprobe tips before and after cleaning with 90% ethanol for 1 min. Whether the H P V on fomites remained infectious was not evaluated; however, extreme caution on the part of patients and personnel was suggested.
P A R A M Y X O V I R I D A E
In studies on paramyxoviridae obtained from infants, a suspension of respiratory syncytial virus (RSV) containing approximately 10 5- 5 CCID50/ml was used to contaminate Formica counter tops, cloth gowns, rubber gloves, paper facial tissues and hands (28). The virus was recovered from counter tops for as long as 6 h, from rubber gloves for 1.5 h, from cloth gowns and paper tissues for 30 to 45 min, and from skin for up to 20 min. Self-inoculation by contact with contaminated infant secretions was therefore considered a potential method of nosocomial transmission of RSV.
Dilutions of parainfluenza viruses (PIV), types 1, 2 and 3, were experimentally studied on nonabsorptive and absorptive surfaces (9). Virus was recovered for up to 10 h from nonabsorptive surfaces and for up to 4 h from absorptive surfaces. Interpretation of the results suggests that fomites should be considered as sources of PIV transmission inside and outside hospitals.
Newcastle disease virus was isolated from the carcasses of frozen poultry for over 730 days and from buried carcasses for 121 days (31).
Rinderpest virus was reported to inactivate rapidly outside the body (48) and to be most stable at pH 4.0-10.2 at 4°C. Infected meat stored in a chilled state between 2°C and 7°C was still infective after seven days.
P I C O R N A V I R I D A E
Extensive investigations have been conducted on the survival, under natural and experimental conditions, of several viruses belonging to the Picornaviridae family (small RNA viruses). Results of these Picornavirus investigations are given herein.
The Mengo encephalomyocarditis virus, a Picornavirus of the Cardivirus genus, was examined for survival at various temperatures (21). The virus suspended in saline at 1 x 10 6- 6 infectious doses/ml was still detectable at titres of > 10 2 infectious doses/ml on the 25th day at 37°C, on the 102nd day at room temperature, and on the 117th day between 2°C and 10°C.
In studies in this unit, encephalomyocarditis virus (Florida strain) was mixed into 80% lean ground pork sausage, pH 5.85, held at 4°C and -20°C and assayed at regular intervals. By the 56th day, viral titres had decreased 99% in sausage stored at 4°C but not at all in sausage stored at -20°C (E.C. Pirtle and T .A. Proescholdt, unpublished data) .
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Foot and mouth disease (FMD) virus, a Picornavirus of the genus Aphthovirus, has been widely studied for its strong environmental stability. Virus shed from infected mammary glands was incorporated into milk micelles and fat droplets, thus affording thermal resistance (7). A port ion of the viral population was found viable in contaminated milk after pasteurization at 72°C for 15 seconds or after acidification to p H 4.6 (11). F M D virus has been recovered from cattle stalls 14 days after removal of infected cattle, from urine after 39 days, from soil after 28 days in au tumn and after 3 days in summer, and from dry hay at 22°C after 20 weeks storage. Salting, curing and drying of hides of infected cattle has not been effective in inactivating the virus (15). Carcasses of infected animals at 4°C have reached a tissue p H < 5.3 within a few days when chilled, thus inactivating FMD virus; however, virus has been recovered from bone marrow and lymph nodes after six months of refrigeration (1) and from swine blood after two months of refrigerated storage (15).
Numerous human pathogenic viruses exist among the enterovirus genus of the picornaviruses. Banker (3) has emphasized the detection of more than 100 different types of enteric viruses in drinking water, wastewater, sea water, as well as in soil, crops, foods and aerosols. Also emphasized was the possibility that viruses which are more resistant than indicator bacteria to conventional purification procedures, and which thus have greater potential for survival, may be contained in drinking water which meets bacteriological s tandards.
Three enteroviruses — polioviruses, echoviruses and coxsackieviruses — were used to contaminate soil and vegetables; their survival times, under various storage conditions, were then recorded (2). The concentration of the viruses employed varied from 1 x 10 4- 5 to 1 x 10 5- 5 C C I D 5 0 / m l . Depending on soil type, moisture content, p H and temperature, the viruses survived for 150 to 170 days in soil. When added to uncooked vegetables and stored under household conditions, the viruses survived for as long as 15 days.
Three of the numerous studies conducted on insects as possible survival hosts of enteroviruses are cited here. In one study (16), poliovirus types 1 and 3 were fed to two groups of blowflies (Phoenicia sericata) via sugar cubes contaminated with 1 0 7 . 5 P F U / c u b e . The flies were then alternately incubated between 40°C and 4°C . Dead flies were removed and titrated for the polioviruses. Viruses were recovered from flies for 13 days (type 3) to 15 days (type 1). Virus titres decreased during the study period; this indicated that the virus did not replicate in the flies, and that mechanical means accounted for survival and potential spread.
In the second study (17), cockroaches (Periplaneta americana) were fed poliovirus type 3 at approximately 10 6 . 5 P F U / r o a c h . Virus survived in roaches for as long as 50 days with no evidence of virus multiplication; as in the blowfly study, the cockroaches were considered to be potential mechanical spreaders of the surviving poliovirus.
In studies conducted on the M F strain of swine enterovirus on the initial isolation farm, houseflies which had been in contact with the swine environment were collected and then assayed in nine pools of 25 flies each. The virus was recovered from all pools at mean concentrations between 19 and 186 C C I D 5 0 per fly. Wash fluids from surfaces of the flies yielded a mean of 124 C C I D 5 0 per fly, while a mean of 66 CCID50 was obtained from ground tissues of the washed fly bodies. The authors concluded that contaminated houseflies could readily transport the virus between swine units. However, with regard to the widespread viral shedding in the environment by adult
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swine and the widespread early infection of young pigs, flies did not seem to play any essential role in the maintenance of the endemic state of infection (60).
Poliovirus type 1 and coxsackieviruses, types B1 and B6, were added to foods at total concentrations of 1 x 10 5 or 1 x 10 6 C C I D 5 0 , then held at room temperature, 10°C and -20°C (37). The viruses were still viable after one week, one month and five months , respectively. Decomposition did not affect virus viability at room temperature and antibiotics controlled bacterial contaminants during laboratory assay.
In a study of the survival of coxsackievirus B2 experimentally inoculated on ground beef, a 7 5 % recovery rate (7,000 PFU) was achieved by suspension, elution and inoculation of monkey kidney cell cultures (55). The same technique was used to recover naturally contaminating polioviruses and echoviruses from three of twelve purchases of market-purchased beef at levels between 1 and 195 P F U per 5 g. One loaf of beef yielded poliovirus type 1 and echovirus type 6, one yielded poliovirus type 2, and one yielded poliovirus types 1 and 3.
In a study on experimental contamination during the preparat ion of Thuringer sausage, approximately 85% of the coxsackievirus A9 (10 4 to 7.5 X l 0 5 CCTD50) which was added to the sausage was lost during 24 h fermentation at 30°C (30). Addit ional heating of the prepared sausage at 49°C resulted in further progressive loss of virus; after 6 h at 49°C, however, an average of 1.1 x 10 3 CCID50 of virus/g of sausage still remained of the initial 7.5 x 10 5 CCID50/g. Despite the counts of spoilage bacteria which rose progressively, coxsackievirus A9 suspended in ground beef was found to survive, at levels considered as infectious, for up to eight days at both 23°C and 4°C.
So that parameters for virus isolation from environmental samples might be better defined, the effects of inoculum size and cell culture density on cytopathic effect (CPE) or plaque assay were assessed with poliovirus type 1 and Buffalo green monkey (BGM) cells (47). A linear relationship was obtained with an inoculum volume of 1.0 ml /25 cm 2 . Depending on the sensitivity of the cell cultures, maximum titres were obtained when 25,000 to 75,000 cells/ml were incubated for six days before exposure to virus.
Survival of enterovirus 70 (EV70), the aetiologic virus of acute haemorrhagic conjunctivitis, was assayed at various temperatures (20°C to 35°C) and relative humidities (20, 50, 80 and 95%) (50). Ultrahigh relative humidity (95%) best protected EV70 from inactivation at 20°C, but the virus was least stable between 33°C and 35°C at this humidity level.
H u m a n enterovirus 72, the aetiologic virus of human hepatitis A , has shown a relative stability which is typical of the majority of enteroviruses. Rapidly developing epidemics of hepatitis A have often followed faecal contamination of a single source, such as drinking water, food, or milk, with human enterovirus 72 (40, 42, 43, 58, 59).
A swine enterovirus was tested for stability to dimethyl ether, arsenilic acid, penicillin, dihydrostreptomycin, butyl parasept and Oxytetracycline. It was also tested at p H values 5 to 9 and at incremental temperatures between 23°C and 70°C (5). No depression of infectivity titre was obtained with any of the chemicals or compounds tested. The virus was stable at p H values 5 to 9 at 4 °C for 28 days, at 23°C for two weeks, and at 37°C for four days. It was inactivated within 30 min at 50°C; inactivation was immediate at 56°C and higher.
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Swine enterovirus, which was excreted in large amounts in the faeces, was shown to survive in faeces at ambient temperatures for periods up to 27 days (20).
Swine vesicular disease virus (SVDV) has been isolated from infected faeces after 28 days (18). It was stable over a wide p H range (2 to 12) and survived at p H 5 to 10 at 4°C for long periods. SVDV. has been isolated from infected premises for as long as eleven weeks after swine herd depopulat ion and disinfection of the premises. Only processes which involved heat t reatment at or above 60° C for at least 30 min effectively inactivated SVDV. Contaminated meats, which had been imported and refrigerated, were found to remain dangerous almost indefinitely; for example, SVDV at -20°C yielded viable virus for 300 days.
Domestic sewage was found to contain about 500 enteric virus units/100 ml, while polluted surface water contained no more than one uni t /100 ml (12). The ratio of enteric viral density to coliform bacterial density in human faeces was approximately 15 virus units for every 106 coliforms. The activated sludge process removed 90 to 98% of enteric viruses in raw sewage. Hypochlorous acid (HC10) was shown to be effective in inactivating viruses in water, with the rate depending on the virus, p H , temperature and contact time. Polioviruses, coxsackieviruses and hepatitis A viruses were more resistant to HClO than coliform or enteric pathogenic bacteria.
Coxsackieviruses in human stools were thermally inactivated at 55°C for 15 min or at 71.1°C for 15 seconds in water. Dairy products offered these viruses a measure of protection against thermal inactivation, but minimum conditions recommended for pasteurization (61.7°C for 30 min or 71.1°C for 15 seconds) proved adequate for inactivation of the faecal strains (34).
Surface sea water from the Mediterranean inactivated 10 3 C C I D 5 0 / m l of poliovirus type 1 in six to nine days (39). The sea water lost part of this virucidal activity after boiling, but Seitz filtration was found to have no such activity. Artificial sea water was less deleterious to the virus. It was therefore concluded that the virucidal activity was partly due to heat-labile substance(s) in sea water.
A study was conducted in the Philippines on the survival of enteroviruses in sea water alone, or in sea water containing oysters. In sea water alone, 99.99% of the decay in virus titre occurred for poliovirus type 1 in fourteen days and for poliovirus types 2 and 3 in seven days. The same percentage occurred for coxsackievirus B4 in three days, for coxsackieviruses A9, Bl and B6 in seven days, for coxsackieviruses B3 and B5 in fourteen days, for echoviruses 5 and 7 in seven days and for echoviruses 1, 12, 13, 17 and 20 in fourteen days. The same survival periods were recorded for poliovirus types 1-3 in sea water with living oysters; however, at three days the oysters were found to have concentrated poliovirus type 1 at titres of 10 5- 5 CCID50/g of digestive tract and 10 6- 5 C C I D 5 0 / g of flesh compared to 10 3- 5 C C I D 5 0 / m l of sea water. At seven days, > 9 9 . 9 % of the poliovirus type 1 was gone from the digestive tracts and tissues of the oysters; at fourteen days, virus levels in the oysters were at nearly the same levels as those found in their ambient sea water. Poliovirus types 2 and 3 were not taken up from the contaminated sea water by the oysters (G.W. Beran and J .C . Nakao , unpublished data) .
In a flow-through sea water system, oysters were found to concentrate poliovirus type 1 at rates of 10 to 60 times the concentration of the virus in sea water. The oysters later cleansed themselves of virus in UV-treated fresh sea water. E. coli assayed as indicator showed that experimental conditions were optimal for the study (41).
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The results of another study indicated that poliovirus added to ground beef may be more resistant to the levels of heat commonly employed when cooking if the meat contains fat levels approaching 30% (26).
In studies on the survival of virus during the manufacture of Cheddar cheese, 9 8 % of poliovirus type 1, and nearly 100% of influenza A virus and vesicular stomatitis (VS) virus, were inactivated (13). Poliovirus persisted in cheese throughout the aging process, but was inactivated 10 6-fold when milk which was to be made into cheese was given minimal prior heat treatment.
When suspended in water, milk or ice cream, poliovirus in faecal material from poliomyelitis patients was inactivated at pasteurization temperatures, 61.7°C for 30 min or 71.1°C for 15 seconds (33). Poliovirus in the spinal cords of infected monkeys suspended in the same media was also destroyed at pasteurization temperatures.
When cattle were inoculated in tongue epithelium with 10 5 bovine ID50 of foot and mouth disease virus (FMDV) (15), thyroid, adrenal and rumen yielded 1.2, 3.2 and 1.2 log10 PFU, respectively, at eight days post inoculation. FMDV was recovered from bone marrow in titres of 1.2 to 1.5 log10 PFU after seven months storage at 1°C to 4°C.
P O X V I R I D A E
In studies involving one- to two-day-old chicks, avipox virus strain used as modified live virus (MLV) vaccine with 10 6 CCID50/ml of drinking water proved more efficacious than 10 4 CCID50/ml when administered either by drinking water or by aerosol (44). This was probably due to the better survival rate of avipox virus in the higher initial concentration of 10 6 C C I D 5 0 / m l .
The persistence of capripoxvirus in sheep flocks has been attributed in large part to the survival of infectious virions in skin scales which fall on pasture plants or the soil (24).
R E O V I R I D A E
Reovirus type 1, influenza A, and parainfluenza 3 viruses survived for three days or less in low moisture processed food. However, poliovirus and echovirus 6 survived in such food for more than two weeks at room temperature and more than two months at 5°C (14).
In studies on the survival of rotaviruses, approximately 10 7 PFU of human rotavirus suspended in faecal matter were placed on disks of stainless steel, glass and plastic (36). Twenty-seven disinfectants were tested for ability to inactivate these viruses. Only nine of the twenty-seven formulations proved to be effective. The results indicated a relative resistance of human rotaviruses to a wide range of chemical disinfectants; they also emphasized the need to evaluate thoroughly the virucidal potential of formulations employed so as to prevent and control outbreaks of rotavirus diarrhoea in human patients.
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It has been demonstrated that bluetongue is a noncontagious disease transmitted primarily by insects (46). The bluetongue virus has been shown to lose infectivity at pH 3.0, and lipid solvents to reduce infectivity approximately tenfold.
R E T R O V I R I D A E
The constantly increasing number of cases of human immunodeficiency virus (HIV) infections and the recognition of retroviral infections in animals has led to a search for effective methods to disinfect contaminated materials. Hypochlorite-releasing disinfectants have been evaluated against HIV (8). Sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) were tested by quantitative suspension. HIV suspensions of 10 4 to 105 syncytial forming units/ml were prepared in isotonic NaCl or NaCl plus 10% plasma. Results indicated that disinfection (10 3-10 4 reduction in 2 min) could be achieved using N a D C C and NaOCl at concentrations of 50 ppm and 2,500 ppm available chlorine (Av Cl2) for virus in NaCl and NaCl plus plasma, respectively. Spilled blood required disinfection with high Av Cl 2 concentrations of NaDCC and NaOCl solutions of 5,000 ppm of blood to effect complete inactivation within 2 min.
R H A B D O V I R I D A E
As rabies virus is transmitted by bites — unless accidentally injected under highly unusual conditions — and thus circumvents the need to survive in the environment, it is epidemiologically unimportant even when viable in the environment. The lipoprotein envelope and glycoprotein projections subject the virions to oxidative or ultraviolet light inactivation as well as to desiccation. Rare occurrences of aerosol transmission have involved high concentrations of virus through laboratory accidents or in caves where depleted oxygen levels, combined with a relative increase in virus-protecting nitrogen, allowed the accumulation of virus excreted by infected, cave-dwelling bats (6).
The vesiculovirus, vesicular stomatitis virus (VSV), is resistant to marked pH changes and is moderately resistant to heat and chemical inactivation (29). The virus remains infective for up to three weeks, depending on the medium in which it is suspended; frozen VSV has remained viable for several years.
S C R A P I E A G E N T
The scrapie agent remains unclassified. Scrapie agent-infected brains from infected hamsters were ground, made into suspensions and centrifuged, after which the supernatant fluids were suspended in soil (10). The soil suspensions were placed into petri dishes and buried for three years. Of the 10 5 hamster infectious units thus interred, between 10 2 and 10 3 of infectious prions were recovered after three years in the soil.
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P R I O R I T Y A R E A S F O R F U R T H E R S T U D Y
Further investigations of the viruses which enter the human and animal environments are needed, especially as soil and water receive increasing concentrations of these infectious agents. Determinations of the viability of viruses identified in the environment are important , as such viruses do not replicate. In the absence of applicable laboratory culture procedures, differentiating viable virions from degraded ones may be difficult. Nucleic acid hybridization is a promising method of detecting viral DNA.
Of specific importance are hepatitis A virus, the calici- and calici-like viruses (Norwalk, Ditchling or W, Cockle, Parramatta and Snow Mountain), astroviruses (Marin County), Sapporo and Otofuke agents, parvoviruses, coronaviruses and the enteric adenoviruses. Emerging in importance are the agents of subacute spongiform encephalopathies in humans and animals, the unique protein structures of which are being termed prions. These agents are highly resistant to any degradation or inactivation.
In the future, more emphasis needs to be placed on the survival of these viruses in specific environments where intimate human and animal contact exists. The movement of viruses over long distances in air, particularly in aerosols of different droplet sizes, in droplet nuclei and by adherence to dry particles, is a critical area which has yet to be adequately delineated. The survival of viruses in water remains a priority, while the survival of viruses in foods is emerging as a new priority. If properly developed, environmental virology will clearly be a significant discipline in the future.
* * *
SURVIE DES VIRUS DANS L'ENVIRONNEMENT. - E.C. Pirtle et G.W. Beran.
Résumé: Les virus se transmettent dans l'environnement à partir de porteurs sains ou malades. Bien que leur réplication ne se produise que dans l'organisme des animaux ou des hommes, ils peuvent être transmis à des hôtes sensibles. L'augmentation régulière, depuis un siècle, des concentrations et des déplacements des populations tant animales qu'humaines, facilite la transmission des virus à tropisme respiratoire et entérique, et en rend la prévention plus complexe.
L'étude des facteurs liés à la survie des virus dans l'environnement a donné des résultats concluants dans le cas des poliovirus, des virus de la fièvre aphteuse et du virus de la maladie d'Aujeszky. La résistance à la chaleur des adénovirus, du virus de la peste porcine africaine et du virus de Norwalk a fait l'objet d'études dont les résultats sont également connus. La résistance aux désinfectants de nombreux virus, dont les Picornavirus, les papovavirus, les réovirus et les rétrovirus, a été étudiée. La survie des virus sur ou dans divers vecteurs passifs a aussi été étudiée, en particulier pour les influenzavirus, les paramyxovirus, les poxvirus et les rétrovirus. Les agents responsables des encéphalopathies spongiformes subaiguës font preuve d'une stabilité extraordinaire dans l'environnement, comme le démontrent des recherches en cours.
MOTS-CLÉS : Environnement - Inactivation - Résistance - Stabilité - Survie -Vecteurs passifs - Virus.
* * *
745
SUPERVIVENCIA DE LOS VIRUS EN EL MEDIO AMBIENTE. - E.C. Pirtle y G.W. Beran.
Resumen: Los virus son transmitidos al medio ambiente por portadores sanos o enfermos. Aunque su replicación se produce únicamente en los organismos de los animales o del hombre, los virus pueden sobrevivir fuera de ellos y transmitirse a huéspedes sensibles. En el siglo presente, las concentraciones y los movimientos de poblaciones, tanto animales como humanas, han aumentado regularmente, favoreciendo así la transmisión de virus respiratorios y entéricos y haciendo más difícil su prevención.
Los estudios relativos a los factores de supervivencia de los virus en el medio ambiente han sido concluyentes para los poliovirus, los virus de la fiebre aftosa y el de la enfermedad de Aujeszky. También se tienen informes sobre estudios de la resistencia al calor de los adenovirus, del virus de la peste porcina africana y del virus de Norwalk. La resistencia a los desinfectantes se ha estudiado en numerosos virus, incluidos los Picornavirus, papovavirus, reovirus y retrovirus. Así mismo, se ha investigado acerca de la supervivencia de los virus dentro o sobre diversas fomites, sobre todo en lo relativo a los influenzavirus, paramixovirus, poxvirus y retrovirus. Se están llevando a cabo estudios que demuestran una increíble estabilidad en el medio ambiente de los agentes responsables de las encefalopatías espongiformes subagudas.
PALABRAS CLAVE: Estabilidad - Fomites - Inactivación - Medio ambiente -Resistencia - Supervivencia - Virus.
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46. O W E N N.C. (1964). - Investigations on the pH stability of bluetongue virus and its survival in mutton and beef. Onderstepoort J. vet. Res., 31 (2), 109-118.
47. PAYMENT P. & TRUDEL M. (1985). - Influence of inoculum size, incubation temperature, and cell culture density on virus detection in environmental samples. Can. J. Microbiol., 31 (11), 977-980.
48. PLOWRIGHT W. (1968). - Rinderpest virus. In Virology Monograph No. 3, Springer Verlag, Vienna & New York, 25-110.
49. PLOWRIGHT W. & PARKER J . (1967). - The stability of African swine fever virus with particular reference to heat and pH inactivation. Arch. ges. Virusforsch., 21, 383-402.
50. SATTAR S.A., DIMOCK K . D . , A N S A R S.A. & SPRINGTHORPE V.S. (1988). - Spread of acute haemorrhagic conjunctivitis due to Enterovirus-70: effect of air temperature and relative humidity on virus survival on fomites. J. med. Virol., 25 (3), 289-296.
51. SCHOENBAUM M.A., ZIMMERMAN J . J . & BERAN G . W . (1990). - Survival of Pseudorabies virus in aerosol. Am. J. vet. Res., 51 (3), 331-333.
748
5 2 . SCHOENBAUM M.A. , FREUND J . D . & BERAN G . W . ( 1 9 9 1 ) . - Survival of Pseudorabies virus in the presence of selected diluents and fomites. JAVMA, 198 ( 8 ) , 1 3 9 3 - 1 3 9 7 .
5 3 . SCHUPFER P.C. , M U R P H J . R . & B A L E J . F . ( 1 9 8 6 ) . - Survival of cytomegaloviruses in paper diapers and saliva. Pediat. infect. Dis., 5, 6 7 7 - 6 7 9 .
5 4 . SPECTOR S.A. ( 1 9 8 3 ) . - Transmission of CMV among infants in hospital documented by restriction endonuclease digestion analyses. Lancet, II, 3 7 8 - 3 8 1 .
5 5 . SULLIVAN R . & R E A D JR R . B . ( 1 9 6 8 ) . - Method of recovery of viruses from milk and milk products. J. Dairy Sci., 51 ( 1 1 ) , 1 7 4 8 - 1 7 5 1 .
5 6 . SULLIVAN R . , FASSOLITIS A . D . & R E A D JR R . B . ( 1 9 7 0 ) . - Method for isolating viruses from ground beef. J. Food Sci., 35 ( 5 ) , 6 2 4 - 6 2 6 .
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A REVIEW
Enterically infecting viruses: pathogenicity, transmission andsignificance for food and waterborne infection
M.J. CarterSchool of Biomedical and Molecular Sciences, University of Surrey, Guildford, UK
2004/0757: received 1 July 2004, revised 7 February 2005 and accepted 9 February 2005
1. SUMMARY
Enterically infecting viruses are ubiquitous agents, mostly
inducing silent infections. Several are however associated
with significant diseases in man from diarrhoea and vomiting
to hepatitis and meningitis. These viruses are drawn from a
variety of virus families and have different structures and
genetic material, yet all are suited to this means of
transmission: Normally they are shed in high numbers
(assisting environmental transit) and exhibit great particle
stability (permitting survival both outside the body and on
passage through the stomach). Human activities particularly
associated with food and water processing and distribution
have the capacity to influence the epidemiology of these
viruses. This review provides a description of viruses
spreading by these means, their significance as pathogens
and considers their behavior in these human-assisted
processes.
2. INTRODUCTION
The term virus stems from the Latin virus meaning �poison�,and in some ways virus contamination of food resembles
toxic contamination more than contamination with other
micro-organisms. Viruses are not free living; they are
dormant between hosts and have an absolute requirement
for living cells in which to replicate. Human viruses require
human cells in which to replicate, these are not present in
our food and thus such viruses cannot increase in number
during storage. The amount of any contaminating viruses
should actually decline during storage, and this can be
assisted by treatment with chemicals, heat or irradiation.
Human viruses do not cause food spoilage and contamin-
ation may provide no visible clues to its presence. These
features mean that measures to control bacteria will not
1. Summary, 1354
2. Introduction, 1354
3. Enterically infecting viruses and their targets, 1355
4. The viruses, 1355
4.1 Adenoviruses, 1357
4.2 Astroviruses, 1358
4.3 Caliciviruses, 1358
4.3.1 Noroviruses, 1358
4.3.2 Sapoviruses, 1359
4.4 Hepatitis A and E viruses, 1359
4.5 Parvoviruses, 1360
4.6 Picornaviruses, 1360
4.7 Rotavirus, 1360
5. Pathogenicity of food-borne viruses, 1360
5.1 Gastroenteritis, 1360
5.2 A novel virus toxin, 1361
5.3 Hepatitis, 1361
6. Virus stability, 1362
7. Waterborne virus infection, 1362
7.1 Wastewater treatment and virus survival, 1362
7.2 Potable water treatment and virus survival, 1365
8. Food-borne virus transmission, 1366
8.1 Shellfish, 1367
8.2 Soft fruit and salad vegetables, 1368
8.3 Other foods, 1369
8.4 Food handlers, 1369
9. Person-to-person transmission, 1370
9.1 Person-to-person or food-borne? 1371
10. Virus diagnosis and detection, 1371
10.1 Clinical samples, 1371
10.2 Food samples, 1372
11. Alternative indicators of virus contamination, 1373
12. Conclusions, 1373
13. Acknowledgements, 1374
14. References, 1374
Correspondence to: Michael J. Carter, School of Biomedical and Molecular Science,
University of Surrey, Guildford GU2 7XH, UK (e-mail: [email protected]).
ª 2005 The Society for Applied Microbiology
Journal of Applied Microbiology 2005, 98, 1354–1380 doi:10.1111/j.1365-2672.2005.02635.x
necessarily control viruses and could actually preserve them.
Food-borne viruses are infectious at very low doses and
could be introduced at any point in the food chain. Many are
difficult (or currently impossible) to culture and detection is
no simple task. Outbreaks and sporadic occurrences of food-
borne virus infection continue throughout the world. There
are simply too many to mention and there is no complete
data set. It seems likely that the documented outbreaks are
limited only by our ability to document them.
The cost of these events is likely to be phenomenal to the
community; a single food-borne outbreak of hepatitis A
virus (HAV) exposed up to 5000 persons in Colorado. In
this case the costs for medical treatment of those infected
amounted to approx. $50 000 whilst the cost of tracing and
controlling this single outbreak cost over half a million US$
(Dalton et al. 1996). The burden of infectious intestinal
disease (IID) in its broader sense is likewise huge, in the UK
the cost per case of norovirus (NoV)-induced gastroenteritis
involving a GP visit is estimated at £176 and two persons in
every 1000 will make such a visit each year (FSA 2000). The
interested reader is referred to several recent reviews (Lees
2000; Seymour and Appleton 2001; Sair et al. 2002;
Koopmans and Duizer 2004).
As enteric viruses cannot replicate outside their hosts, all
such virus transmission is in effect person-to-person.
Environmental transit time between hosts may be brief
or prolonged. Long-distance travel may take place, e.g.
through water systems or even the air. Long-distance
travel is accompanied by exposure to the environment and
dilution; thus viruses having prolonged environmental
transit times must be very stable to survive and are
(usually) shed in very large numbers. Enteric viruses meet
both requirements; they are acid stable and replicate to
prodigious titres in the gut before being shed in concen-
trated doses directly into the sewage system. All potentially
food-borne viruses can also be transmitted directly from
person to person via faecal contamination of the environ-
ment and viewed in this way food is simply another kind
of fomite in environmental transmission, it occupies a
special niche simply because of its privileged position in
terms of its introduction to the body and the potential it
may offer for widespread distribution through trade and
commerce.
The relative importance of food-borne vs more direct
person-to-person transmission is unclear; enteric infections
are ubiquitous, single occurrences are far too numerous to
mention and statistics usually record only outbreaks (when
several people are infected in one location or through one
common vehicle). However any one outbreak may involve
different types of spread; these viruses have a high
secondary attack rate and person-to-person transmission
will probably follow even if the virus was actually introduced
to that setting by food. This can potentially mask food-borne
introductions and it is likely therefore that food-borne
transmission is underestimated.
3. ENTERICALLY INFECTING VIRUSESAND THEIR TARGETS
There are two types of enterically infecting virus – the first
are capable of spreading elsewhere in the body. Infection by
these viruses is often subclinical but they may induce signs
and symptoms of disease in nonintestinal tissues. These
viruses include enteroviruses (e.g. polio or Coxsackie, which
may spread to the meninges, central nervous system;
skeletal/heart muscle or pancreas) and hepatitis viruses A
and E spreading to the liver. The second type of virus are
true gut inhabitants. These replicate in the enteric tract,
specific symptoms when they occur, are those of a gastro-
intestinal infection; usually diarrhoea and vomiting but the
extent of each component is variable.
4. THE VIRUSES
Table 1 lists the main viruses associated with enteric
infection and summarizes their key properties. The most
important are illustrated in Fig. 1. Enteric viruses are drawn
from a variety of virus families, they range approximately
10-fold in diameter and 20-fold in terms of genome size and
complexity. The major enterically transmitted and thus
potentially food/waterborne agents comprise (alphabetic-
ally) the human adenoviruses (AdV), astroviruses (HAstV),
caliciviruses, hepatitis E virus (HEV), parvoviruses, picor-
naviruses [including enteroviruses, kobuviruses and hepati-
tis A (HAV)], and the rotaviruses (RV). Most enteric
viruses are childhood infections; spreading largely person-
to-person and assisted by the lower hygiene levels in this
group. Food-borne transmission may be negligible (AdV) or
insignificant (RV) in the developed world. However in the
undeveloped world spread of these agents by these routes is
poorly characterized. Childhood infection leaves residual
immunity that may prevent (or mollify) infection over the
rest of an individual’s lifetime. Although this is not
universally true, in general viruses causing childhood illness
are not significant pathogens in healthy adults previously
exposed as children. The IID survey in England (FSA
2000) estimated the incidence of GP consultations for
intestinal disease by patient age and causative organism.
Data from this survey have been reanalysed (Fig. 2) to show
the proportion of consultations made for each virus in the
age groups <5 and >5 years. As expected GP consultations
induced by these viruses were biased towards children
<5 years; GP visits by older persons comprised only a small
proportion of the total consultations. There were two
exceptions to this observation; some 25–50% of GP
consultations for NoV infection were made by older
FOOD-BORNE VIRUS 1355
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
Table
1Properties
ofthemajor
entericallyinfectingviruses
Nam
eVirusfamily(genus)
Food-borne
Size(genom
e)Features
Associated
illness
Polio,Coxsackie,
echo,
enterovirus
Picornaviridae
(enterovirus)
Yes,mainly
water,
presentin
shellfish
28nm
(ssR
NA)
Littlesurfacedetail
Manycultivable
inVeroor
Helacells
Mainly
asymptomatic,caninduce
muscle
pains(Bornholm
disease),
cardiomyopathy,meningitis,CNS
motorparalysis
Aichivirus
Picornaviridae
(kobuvirus)
Yes,shellfish
28nm
(ssR
NA)
Knob-likeprojections
Cultivable
Verocells
Gastroenteritis
HepatitisA
virus
Picornaviridae
(hepatovirus)
Yes
28nm
(ssRNA)
Littlesurfacedetail
Fhrk-1
cells
Hepatitis,mildin
theyoung
HepatitisEvirus
Unclassified
Mainly
water
34nm
(ssR
NA)
Calicivirus-like
structure
Uniquegenetic
organization
Not
cultivable
Hepatitis,severein
pregnancy
Rotavirus
Reoviridae
Rareoftenwater
70nm
(dsR
NA)
Multilayered
Segmentedgenom
e
(11pieces)
Cultivable
Ma104
cells
Diarrhoea
–commonin
theyoung,
incidence
decreasingwithage,
but
increasesin
theelderly
AdenovirusgroupF,
types
40and41
Adenoviridae
Not
reported
100nm
(dsD
NA)
Distinctiveicosahedral
Cultivable
Graham
293cells
Milddiarrhoea,sheddingmay
be
prolonged,mainly
affectschildren
Saporovirus
Caliciviridae
(saporovirus)
Yes
(rare),mainly
shellfish
34nm
(ssR
NA)
Cup-likedepressionson
surface;
nonearecultivable
Gastroenteritis–commonin
children
believed
tobemilder
ineffect
Norovirus
Calicivirus(norovirus)
Yes
34nm
(ssR
NA)
Fuzzysurfacestructure;
not
cultivable
Explosiveprojectilevomitingin
older
children/youngadults
Human
astrovirus
Astroviridae
(mam
astrovirus)
Occasionally,water
andshellfish
28nm
(ssR
NA)
Eightserotypes
5or
6-pointstar
motif
Appearance
variable
Cultivable
CaC
O2cells
Mostly
infect
children,higher
serotypes
seen
inadults.Relatively
mildgastroenteritis,butprobably
underestimated
Wollan,ditchling,
Param
atta
andcockle
agents
Parvoviridae?
Yes,shellfish
25nm
(ssD
NA)
Smooth
featureless,poorly
characterizednoncultivable
Gastroenteritis–widespread
shellfish-associated
outbreaks,
largelycontrolled
throughcooking
1356 M.J. CARTER
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
children/adults; the highest of any virus, with astroviruses
close behind.
4.1 Adenoviruses
There are 51 serotypes of AdV; all are large icosahedral
DNA-containing viruses. About 30% of the serotypes are
pathogenic in man; most being upper respiratory tract
pathogens spreading primarily via droplets. However, even
the respiratory strains grow well in the gut and are present
in the faeces. Only types 40 and 41induce gastroenteritis and
these are shed in larger numbers. AdV are frequently found
in faecally polluted waters and have been identified in
shellfish (Girones et al. 1995; Pina et al. 1998a; Vantarakisand Papapetroupoulou 1998; Chapron et al. 2000), but havenot been appreciably associated with food-borne illness,
1 2 3
54
Fig. 1 Food-borne viruses. Electron micrographs of the most import enterically infecting and food-borne viruses found in clinical samples (human
faeces). All panels are reproduced at the same magnification; bar represents 100 nm. Panels show: human rotavirus; 2, enteric adenovirus; 3,
astrovirus; 4, norovirus; 5, sapovirus
Age group%<4 %>4
Per
cent
age
of to
tal
cons
ulta
tions
120·0
100·080·0
60·0
40·0
20·0
0·0
Fig. 2 Virus identifications in GP consultations for infectious
intestinal disease by age of patient. Viruses identified following GP
consultations have been segregated into the percentage of cases that
involving children below 4 years and those involving older children
and adults. Bias for infection of the young emerges clearly, even in the
case of noroviruses. Data are re-analysed from FSA (2000) IID survey
in England. j, NoV; , HastV; , AdV; , RV; (, SaV
FOOD-BORNE VIRUS 1357
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
presumably because most adults are immune and children
do not commonly eat shellfish. However outbreaks of other
strains associated with conjunctivitis (shipyard eye) and
pharyngitis are commonly associated with exposure to
polluted water, normally through recreational use (Crabtree
et al. 1997).Adenoviruses 40 and 41 account for 5–20% of US
hospital admissions for diarrhoea, mainly in children below
age 2 years (Uhnoo et al. 1984; Kotloff et al. 1989).
Incubation lasts 3–10 d, and illness (usually a watery
diarrhoea) may last a week. As children age, experience
with AdV infection gradually increases the levels of
population immunity. Only 20% of children below
6 months have antibody to these viruses, but by age 3 for
this has risen to 50%. In the IID survey in England AdV
infections were confined largely to children under 5 years
and accounted for approx. 12% of all viruses identified
(FSA 2000). Incidence was determined as 400 and 800 per
100 000 person years in the age groups 0–1 and 1–4 years.
Infection is not significant in healthy adults although it may
increase in significance again in the elderly (Dupuis et al.1995); fewer than 4% of enteric AdV GP consultations
involved persons over 5 years (Fig. 2). AdV are associated
with tumours in mice but no such association has ever been
made in man.
4.2 Astroviruses
Astroviruses are usually described as 28 nm rounded particles
with a smooth margin. In their centres they may bear a 5 or 6-
pointed �star�motif fromwhich they are named (Astron, a star)
(see Fig. 1). However, the appearance of these agents is
certainly variable, sometimes showing surface projections
(Appleton and Higgins 1975) and at other times resembling
caliciviruses (Willcocks et al. 1990). Morphology was subse-
quently used to present a classification scheme for many of
these small viruses associated with enteric infection and
including the caliciviruses (below) (Caul and Appleton 1982).
Cryo electron microscopy studies have now confirmed the
presence of surface projections (Matsui et al. 2001). Human
astroviruses comprise eight serotypes (HAst1–8); types 1 and
2 are rapidly acquired in childhood; by age 7 years 50% of
children are seropositive for type 1 and 75% by age 10 years
(Lee and Kurtz 1982; Kurtz and Lee 1984). Exposure to the
higher serotypes (4 and above) may not occur until adulthood.
Illness is generally mild, lasting 2–3 d after an incubation
period of similar length. This has led many to dismiss these
viruses as causative agents of significant disease in humans.
However astroviruses are the second most commonly
identified virus in symptomatic children (Herrmann et al.1991) and account for 5% of US hospital admissions for
diarrhoea – almost entirely of children (Ellis et al. 1984).Adults may be infected by higher serotypes and childhood
antibody may not prevent clinical disease: in Japan (1995),
1500 older children and teachers were affected in a
widespread food-borne outbreak of HAstV type 4 (Oishi
et al. 1994). Finally, astrovirus identification often relies on
electron microscopy but virus appearance is not always clear.
Astroviruses may be frequently mistaken for small round
(parvovirus)-like agents (Willcocks et al. 1991) and even for
NoVs (Madore et al. 1986). In England the IID survey
conducted between 1992 and 1995 (FSA 2000) found
astroviruses comprised 12% of all virus identifications with
incidences of 125 and 550 per 100 000 person years in the
age groups 0–1 and 2–4 years respectively. However, 16%
of GP consultations for astrovirus infection were made by
older children and adults (Fig. 2). As this survey identified
HAstV only by EM it remains possible that astrovirus
infections in the adult population were underestimated.
Culture conditions have been described (Willcocks et al.1990) and recently an ELISA-based detection kit has been
produced.
4.3 Caliciviruses
Caliciviruses appear under the electron microscope as if
covered in cup-like depressions, from which the virus takes
its name (calix ¼ a cup). The family includes two genera
that infect humans, the NoV and the sapoviruses (SaV). To
date neither of these can be cultured in the laboratory. The
nomenclature of these viruses has changed several times
recently. Formerly they were known by names derived from
their morphology (see Fig. 1): the small round structured
viruses, e.g. Norwalk virus appeared fuzzy and indistinct
whilst the human caliciviruses, e.g. sapporo virus, had a
more obvious calicivirus-like appearance (Caul and Apple-
ton 1982). Classification then moved to genomic organiza-
tion and the groups were renamed the Norwalk-like viruses
and the sapporo-like viruses. The nomenclature is now
hopefully settled with the refinement of these names to the
NoV and SaV respectively.
4.3.1 Noroviruses. Analysis suggests that the NoV are
the single most significant cause of IID in the developed
world. NoVs were first identified following an outbreak of
enteric illness amongst children and adults in the town of
Norwalk, OH (Alder and Zickl 1969). Although samples
were first collected in 1968, viruses were not clearly
identified until 1972 when antibody was used to clump the
particles (Kapikian et al. 1972). NoVs are now routinely
detected by PCR amplification of the RNA-polymerase
gene and by commercial ELISA kits, electron microscopy
is used as a back up. Sequence analysis of the PCR
products divides the NoV into two genogroups; group 1
exemplified by Norwalk virus itself and group 2 by Hawaii
virus (Lambden and Clarke 1994; reviewed in Clarke and
1358 M.J. CARTER
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
Lambden 2001). Recently genogroup 2 has been more
common in the UK.
Infections occur around the globe and throughout the year
but may be more common in winter giving rise to its former
name �winter vomiting disease�. Incubation lasts up to 48 h
and is followed by a self-limiting illness lasting 24–48 h.
NoV infection is not regarded as severe in otherwise healthy
adults, but it is debilitating and very unpleasant. In
vulnerable groups, the malnourished or elderly it can be
serious and may even precipitate death. It was thought that
subclinical and childhood infection was rare but recent
studies have shown this does occur in very young children
(Carter and Cubitt 1995). The IID study in England
estimated that 1% of children < 1 years would contract
NoV (FSA 2000).
Norovirus differs from other agents of gastroenteritis in
three ways: first, it causes disease in adults (teenagers and
above), thus NoVs are the most significant diarrhoeal virus
in terms of working/education days lost. Secondly, it
induces a high level of explosive projectile vomiting that
may be the first obvious sign of infection. Many cases are
identified at work with serious implications if a food handler
should be infected. Thirdly, although there are probably
multiple serotypes of NoV, immunity to all seems to be
short-lived. Thus individuals may be protected for only a
few months following an infection before they become
infectable once more by the same virus (Parrino et al. 1977).Some people appear to have an inherent resistance to
infection; community outbreaks that stemmed from com-
munal exposure by swimming pool contamination showed
familial clustering of symptomatic illness, and even in
middle age population antibody levels are only 50%. Many
of these seronegative individuals remain symptom-free and
it is now thought likely that they lack the cell-surface
receptor (a carbohydrate antigen) to which the virus must
bind to initiate infection (Hutson et al. 2003). Susceptiblepersons require several bouts of infection by the same virus
before antibody levels are boosted sufficiently to afford some
protection. In the recent IID survey in England NoV
accounted for 30–40% of all viruses identified, they were the
most commonly identified agent in the community study,
and the third most common agent that caused persons to
seek consultation with their GP (FSA 2000). Several reports
across the world have indicated a rise in NoV detection
during 2002–03. These included shipboard outbreaks,
multistate occurrences in the US, a sudden rise in outbreaks
in Canada and numerous hospital outbreaks throughout the
UK that forced many to close wards or cease new
admissions. These have been attributed in part to the
emergence of a new strain of NoV across the world
characterized by mutations in the polymerase gene (Lopman
et al. 2003, 2004). This might be more infectious than
previous strains and if such an event has occurred then the
mechanism underlying this process requires investigation:
food-borne transmission, perhaps via international trade
should be considered.
4.3.2 Sapoviruses. Sapoviruses induce symptomatic
infections mainly in children. They account for some 3%
of hospital admissions for diarrhoea in both the UK and US.
Most children are sero-positive by age 12 and seem to
become infected between 3 months and 6 years of age. SaV
were found most frequently in children below age 4 in the
England IID with an incidence of 460 per 100 000 person
years in those aged <1 years that fell to 150 in those between
2 and 4 years (FSA 2000), <2% of GP consultations for SaV
infection took place in those >5 years (Fig. 2). Infection is
particularly common in institutional settings such as schools
and day care centres. Incubation is between 24–48 h and
illness is usually mild and short-lived with diarrhoea
predominating. However in those cases when SaV have
been seen to infect adults then symptoms are very similar to
those of NoV (Cubitt 1989).
4.4 Hepatitis A and E viruses
Epidemic hepatitis has been recognized since ancient times,
and its infectious nature was appreciated in the middle ages.
However the causative agent was identified only in 1972
when the newly developed technique of immune electron
microscopy permitted the particles to be identified (Kapik-
ian et al. 1972). Subsequently named HAV to distinguish it
from serum hepatitis (hepatitis B), this agent was found to
be responsible for the bulk of infectious hepatitis. Symptoms
were seen mainly in older children and adults but infection
was common in younger children although in these it tended
to be symptom free. HAV was found to be a member of the
Picornavirus family (see below) and was initially placed in
the genus Enterovirus. However it possessed some unique
properties in relation to its genetic structure and replication
procedure and it was subsequently removed to a new genus
(Hepatovirus) of which it is the only member. HAV can be
cultured in FRhK-4 cells but this is slow and difficult,
especially for primary isolates.
Although responsible for most enterically transmitted
hepatitis in the developed world, it was clear that, HAV alone
could never account for all enterically transmitted hepatitis in
the undeveloped world. Thus the concept of enterically
transmitted non-A, non-B hepatitis grew up. This gap in
knowledge was filled in 1990 when a new agent, HEV was
identified by molecular means (Reyes et al. 1990; reviewedBradley 1990, 1992). The genetic organization and particle
structure of HEV resembled the Caliciviruses and HEV was
initially classified in this family. However the detail of the
genomic organization and the enzymic capacities encoded are
such that it could not remain in this family. Consequently it
FOOD-BORNE VIRUS 1359
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
now forms the only member of a group called the �hepatitisE-like viruses� (Green et al. 2000). HEV is rare in the
developed world with cases generally limited to travellers.
4.5 Parvoviruses
Parvoviruses are poorly characterized as agents of enteric
infection. Diagnosis is based solely on electron microscopy.
Although associated clearly with gut infection in animals
(e.g. canine parvovirus), the only infectious human parvo-
virus characterized to date is a nonenteric agent B19, causing
a maculopapular rash in children. Parvoviruses have been
associated with gastroenteritis in primary and secondary
schools in the UK (Wollan and Ditchling agents) and
Australia (Paramatta agent) (Paver et al. 1973; Christopheret al. 1978; Appleton 1987, 2001). The cockle agent was
identified following a large outbreak in England (Appleton
and Pereira 1977), and was associated with consumption of
contaminated seafood. There is a strong argument that
some/many of these viruses may actually be misidentified
astroviruses, caliciviruses (Willcocks et al. 1991).
4.6 Picornaviruses
Picornaviruses infecting the gut were formerly all contained
in the Enterovirus genus. Enteroviruses have a rather
featureless appearance under the electron microscope and
include the ECHO Coxsackie and polioviruses. Most grow
well in laboratory cell cultures such as HeLa or Vero.
Formerly much feared, the success of vaccination has
controlled polio and allowed the WHO to target this virus
for global elimination early this century. Picornaviruses were
thought not to be usually associated with diarrhoeal
symptoms in humans but this changed in 1993 when
Aichivirus was discovered as the agent responsible for an
outbreak of shellfish-associated gastroenteritis (Yamashita
et al. 1993). Aichivirus could be grown in Vero cells and
study showed that it had a genome organization typical of
the picornaviruses (Yamashita et al. 1998). However the
particle shows differences in structure from other picorna-
viruses and bears surface projections similar to those of
astroviruses. These viruses have now been recognized as a
new genus in the picornavirus family termed the kobu-
viruses.
4.7 Rotavirus
Rotaviruses are large RNA-containing viruses belonging to
the family Reoviridae. Their particles are multilayered and
complex, replicative functions may be built into the shell.
Particles are readily visible and distinctive in the EM where
the outer layer of the capsid can appear like the spokes of a
wheel from which the virus is named (rota, a wheel).
Illness develops after an incubation period of 4–7 d and
usually presents as diarrhoea and vomiting lasting approx.
7 d. Viruses are shed in extremely high numbers (perhaps
over 109 per gram of stool) and diagnosis is a relatively
simple matter. Virus is readily detected by direct examina-
tion by EM or PAGE (Moosai et al. 1984), antibody-basedbead-agglutination or ELISA systems. RV account for some
3Æ5 million cases of diarrhoea p.a. in the US equating to 35%
of hospital admissions for diarrhoea (Ho et al. 1988).
Approximately 120 children die each year in the US from
this virus and fatalities in the undeveloped world may
amount to millions (Parashar et al. 2003).Rotaviruses occur in five groups (A–E) but only groups
A–C infect humans. Group A is by far the most common
with sporadic episodes due to group C, group B is limited
largely to China. Only group A viruses can be cultured,
these are grown in Ma104 cells. Within each group RV are
divided into serotypes based on their surface-exposed
proteins. Within group A there are 14 types of VP7 (termed
G types) and approx. 20 of VP4 (termed P types). This
generates great antigenic diversity permitting serial infec-
tions which may be symptom free. The peak age for illness is
between 6 months and 2 years, by 4 years most persons
have been infected. Immunity to rotavirus is long-lasting,
thus sequential exposure leads to accumulated immunity
and frequency of illness decreases with age. Silent secondary
re-infections can occur (as in parents caring for infants) and
this provides another means for the virus to spread in the
community. RV were the most commonly identified enteric
pathogen in children <4 years in England and Wales and
comprised 25–35% of the virus identification made in this
study (FSA 2000). Only 11% of GP consultations for
rotavirus were made by persons >5 years. However this
raises two points, first not all adult infections are mild and
secondly this small percentage may actually reflect a greater
number of adult GP consultations for rotavirus infection
than for NoV (FSA 2000).
5. PATHOGENICITY OF FOOD-BORNEVIRUSES
5.1 Gastroenteritis
Gastroenteritic viruses replicate and destroy the mature
enterocyte covering the upper third of the intestinal villi.
Undifferentiated, immature cells do not support virus
replication. Destruction of functional mature cells disrupts
the reabsorption of water from the gut and diarrhoea ensues.
The villi retract in response to damage and decrease the
surface area available for absorption. At the same time the
crypt cells undergo rapid division and soon repopulate
the villi with young, as yet undifferentiated cells. These
immature cells are resistant to virus infection but cannot
1360 M.J. CARTER
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
replace the function of those that have been lost; they require
time to mature. Thus malabsorption continues until the cells
can develop the necessary ion uptake capabilities. This
exuberant cell division in the crypt is central to recovery.
Fortunately in humans viruses do not attack the crypt cells
themselves. When such infections do occur (e.g. canine
parvovirus) they result in bloody diarrhoea from which
recovery may not be possible (especially in a young animal).
5.2 A novel virus toxin
Although most viruses do not manufacture toxins some RV
are exceptional and induce the synthesis of a toxin-protein
termed NSp4 that can induce diarrhoea if administered
alone (Ball et al. 1996). NSP4 has no similarity to bacterial
toxins (Tian et al. 1995). It stimulates transepithelial
chloride secretion via a calcium ion-dependent path; it has
no effect on cAMP formation and is independent of the
cystic fibrosis channel. The mechanism involves stimulation
of inositol production (Dong et al. 1997). The protein also
has direct effects on brush border transport mechanisms
(Halaihel et al. 2000). It is proposed that at least one form of
soluble NSp4 is released from infected cells and binds to
neighbouring cells (Zhang et al. 2000).
5.3 Hepatitis
Hepatitis A and E viruses enter via the gut and may replicate
there, however both move rapidly to the liver and invade the
hepatocytes. Clinical features of both viruses are similar
although HEV is more severe and may have a fatality rate of
20–40% in late pregnancy. HEV has a longer incubation
period than HAV (60 d vs 48 d) and a more prolonged
viraemia (Clayson et al. 1995b; Scharschmidt 1995; Reid and
Dienstag 1997). The long incubations make identification of
the source of infection problematic as contaminated food will
usually have been eaten or disposed of before illness arises.
Haemoglobin breakdown by the liver is impaired and a
bilirubin (normally shed in the bile and thence in the faeces)
overflows into the blood. The skin and whites of the eye turn
yellow (jaundice) and faeces become pale. Bilirubin is filtered
from the blood by the kidneys, and urine becomes dark.
Virus particles are shed into the bile and thence in the faeces,
but in contrast to the cell destruction caused by gastroen-
teritis viruses there is little virus-induced liver cell damage.
HAV interferes only weakly with host cell activities and new
viruses are released inside membrane-bound packets without
necessity to lyse the cell. An immune response develops
2–3 weeks after infection and leads to immune attack on
infected liver cells. It is this host response rather than the
virus itself that causes the signs of liver damage. Eventually
the immune response eliminates all infected cells (and thus
the virus) from the body. Convalescence may be prolonged
(8–10 weeks) and some 15% of HAV cases may follow a
relapsing course over 12 months or more.
Hepatitis viruses A and E have been affected by human
activities. In former times, infection occurred early in life,
often whilst still protected by maternal antibody. Such
endemic infections tended to be mild or subclinical. Both
viruses are rare where sanitation is improved. This has
reduced exposure and so increased the age at which first
infection occurs; in Hong Kong, 30% of those under 30
were seropositive in 1979; by 1989 this had fallen to only
9%, although seropositivity in the elderly remained high. In
contrast, in France where sanitation has been good for many
years, 80% of persons over 30 have no antibody to HAV (i.e.
have never been infected).
This shift in age at infection increases the severity of
infection: below 3 years, HAV infection is virtually always
subclinical; but symptomatic infections predominate by
5 years and severity worsens with age (Hadler et al. 1980).Persons over 50 years of age account for only 12% of the
cases of HAV but have a case-fatality rate sixfold higher than
average (CDC 1994; Fiore 2004). This delay in infection
allows a pool of susceptible individuals to accumulate in the
community and establishing conditions for epidemic spread.
Analysis of annual incidence figures in the US reveals
evidence of epidemic behaviour (Fiore 2004). The Centers
for Disease Control, USA estimates 267 000 cases occurred
on average per year between 1987 and 2001, most were mild
or symptom free but 10–30 000 acute cases were registered
annually. Mead et al. (1999) estimate that 5% of cases are
food-borne. The situation in the UK has been summarized
(Crowcroft et al. 2001). The existence of susceptible adults
in some parts of the world is significant in the context of
food-borne infection since trade that could bring virus-
contaminated food grown/produced in areas of high ende-
micity to areas of low prevalence could pose a threat for
adults in those areas (see below).
Hepatitis E virus is not significant in the UK or US; most
infections are limited to returning travellers. Epidemics of
HEV are known, often spread by contaminated water; the
worst cases involved 30 000 people in New Delhi (1955);
100 000 in Xinjiang Uighar, China (1986), and 79 000 in
Kanpur India (1991) (Grabow et al. 1994; Scharschmidt
1995). More limited shellfish-associated outbreaks occur
sporadically around the Mediterranean. HEV replicates in
pigs (Balayan et al. 1990) and has been found in both wild anddomestic cows, goats and pigs (Clayson et al. 1995a). Repli-cation also occurs in laboratory rats (Maneerat et al. 1996;Meng et al. 1996). These findingsmean that animalsmight act
as reservoirs for infection (Kabrane-Lazizi et al. 1999; Wu
et al. 2000). Seropositivity has been estimated at 2–10% even
in areas free from human disease; 15% of homeless persons in
Los Angeles revealed antibody to HEV, possibly through
contact with infected urban rats (Smith et al. 2002). Similarly
FOOD-BORNE VIRUS 1361
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
HEV has been detected in sewage from areas in which clinical
disease is absent (Pina et al. 1998b).
6. VIRUS STABILITY
The stability of an enteric virus is of fundamental signifi-
cance to its transmission. First, it may limit the period for
which virus contamination remains a threat in the environ-
ment whether in sea or fresh water or dried onto a surface,
and secondly, it governs the efficiency of attempts to
deliberately destroy a virus, e.g. in food or water processing
(mainly sensitivity to temperature and chlorination). Many
enteric viruses are nonenveloped and contain RNA as their
genetic material (exceptions AdV and parvovirus that
contain DNA). RNA is labile, hydrolysed at both acid and
alkaline pH and destroyed by radiation or enzymic proces-
ses. DNA is more stable but is still sensitive to UV
irradiation. This emphasizes the crucial role of the virus coat
proteins that must protect the nucleic acid. The relative
stabilities of some of the enteric viruses are assembled from a
variety of sources and presented in Table 2. There is no
central data set for all viruses and their frequently used
surrogates; most studies use only a few viruses under
restricted conditions and have not considered the effects of
combined treatments. The environment in which inactiva-
tion proceeds will certainly affect the values determined and
this has central significance when considering survival in
different foodstuffs (e.g. inside shellfish or in milk) or in the
environment (marine vs fresh water etc.). As each study has
used slightly different conditions, this table should be
regarded as only indicative.
Although all these viruses are destroyed by boiling, the
thermal stability of some is remarkable. Temperatures above
90�C are required to inactivate HAV (Millard et al. 1987)and others suggest 100�C may be necessary (Croci et al.1999). Inactivation is often biphasic and a residue of
resistant virus may survive. This probably represents virus
in a protected microenvironment, e.g. aggregated or com-
bined with materials exerting a protective/stabilizing effect
(Tierney and Larkin 1978; Larkin and Fassolitis 1979;
Bidawid et al. 2000a).Virus persistence in dried matter depends on the surface
onto which it is dried, the presence of extra (faecal)
material and the temperature/humidity of storage. Many
enteric viruses survive for long periods on common surface
types requiring up to 60 d for a 2 log reduction in titre
(Abad et al. 1994a). In general reducing the temperature
and adding faecal contamination promotes virus survival
but viruses may respond differently to relative humidity;
HAV and rotavirus are stabilized at low relative humidity
whilst enteroviruses are stabilized at higher values (Mbithi
et al. 1991). This probably depends on the type of surface
onto which the viruses are dried (Abad et al. 1994a).
Values for inactivation times/persistence in water vary
widely with reported T90 values (i.e. time for 1 log titre
reduction or 1 TLR) for enteroviruses of 14–288 h. A 4 log
reduction times in sea water are likewise variable but are
probably measured in weeks (Chung and Sobsey 1993;
Callahan et al. 1995). Persistence in artificially contaminated
water has been demonstrated for >1 year (rotavirus and
poliovirus) (Biziagos et al. 1988) and 300 d for AdV 41
(Enriquez et al. 1995). These variations probably reflect
differences in the conditions used particularly, type of water,
illumination, turbidity and pH, and emphasize the need for
standardized studies of all relevant viruses (and common
surrogates) under directly comparable conditions. Despite
this variation all these viruses are probably capable of
survival for weeks/months at environmental temperatures
and at low temperatures, sheltered from UV irradiation
some may persist for years.
7. WATERBORNE VIRUS INFECTION
All these viruses enter the sewerage system and may survive
wastewater treatment to contaminate receiving waters. Here
they could pose a threat to recreational users or to
consumers of shellfish or other produce eventually destined
for the food chain. In addition, where such waters are later
abstracted and treated for use as drinking water, contam-
inating viruses might survive this treatment too. Drinking
water can also be contaminated after treatment if supplies
are not adequately separated from untreated sources.
7.1 Wastewater treatment and virus survival
Enteric viruses are usually shed in large numbers, rotavirus,
titres can exceed 109 particles per ml and could comprise up
to 2 mg in every gram of stool. A value between 106–108 per
ml is common for astrovirus and AdV. Caliciviruses, entero-
and hepatitis viruses are often shed in lower (but still
appreciable) numbers; cultivable enteric viruses are ubiquit-
ous in human populations and levels in sewage can exceed
104 PFU per litre. Water treatments lacking a tertiary step
(e.g. UV treatment) reduce this load only poorly. Sewage
sludge production reduces numbers by 95% but many
viruses survive giving levels in receiving waters of up to
100 PFU per litre if contamination is serious and 1–10 PFU
per 100 l where it is less so (Gerba et al. 1985; Bloch et al.1990; Jothikumar et al. 2000; Scipioni et al. 2000; Pina et al.2001). Most enteric viruses have been detected in waste-
water, treated water and receiving waters over time, usually
by PCR techniques even if viruses are cultivable, e.g. HAV
(Dubrou et al. 1991; Goswami et al. 1993; Graff et al. 1993;Tsai et al. 1993; Jaykus et al. 1996; Schwab et al. 1996) andastrovirus (Le Cann et al. 2004). AdV have consistently been
detected in raw sewage and approx. 80% may be enteric
1362 M.J. CARTER
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
Table
2Virusstabilityvalues;assembledfrom
referencesbelow
.Nostudyhas
included
allthesevirusesunder
identicalconditionsandthusdataarenotalwayscomparable;values
presentedhereshould
betakenas
indicativeof
stabilityonly.Log
titrereduction(LTR),measuredbyinfectivityunless
otherwisestated.BacteriophageMS2,acommonsurrogatefor
entericvirusesisincluded
forcomparison
Virus
Persistence/inactivation
conditions
pH
stability
Thermal
inactivation
Freechlorine
(unless
stated)
UV
irradiation
(mJcm
)2)
Persistence
(dry)
Persistence
food
matrices
Persistence
seawater
Persistence
freshwater
Adenovirus
Stable
pH
6–9Æ5*
Inactivated
56deg,
10min*(6)
2LTRat
0Æ5
mgml)1
for10
min
(20)
3LTRat
1Æ0
mgml)1
for10
min
(20)
4LTRat
dose
216(23)
2Æ5
LTRon
drying,
further
3LTR
10d(19)
Not
significantly
food-borne
1LTRin
40d
15deg
(27)
3Æ2
LTRin
60d
at20deg
1LTRin
40d
at15deg
(27)
Astrovirus
Resists
pH
3(4)
Resists
50deg
1h,
60deg
5min
(4)
2LTRat
1mgl)1for
10min,
2Æ0
LTRat
0Æ5
mgl)1for
20min
(18)
Nodata
1LTRon
drying
then
stable
for
60dat
4deg
(19)
Nodata
Reportedly
lower
stabilitythan
freshwater
Drinkingwater:
2LTRat
4deg,
3Æ2
LTRat
20deg
both
60d
HepatitisA
virus
Resists
pH
1Æ0
for
2h(9)
Resists
60deg
for10
min;
cationsstabilize
to81
deg
Inactivated
98–100deg
(7)
Inactivated
by2–2Æ5
mgl)1
for15
min
(13)
CT
for99%
reduction¼
8(17)
1LTRat
0Æ5
mgl)1for
10min;2LTR
at1mgl)1
for10
min
(20);
3LTRat
4ppm
ClO
2for
10min
(16)
4LTRat
dose
16–39
(23);
1LTRat
dose
36Æ5
(30);1LTR
in1Æ3
min
at
42mW
cm)2in
seawater
(25)
Survives
>1month
at25
deg
in42%
humidity(10,
11);
3LTRin
8dand
then
stable
to
32d(16)
Resists
60deg
for19
min
in
oysters(12)
2LTRat
63deg
inwater
ormilk
for60
min
(16)
2LTRin
7din
creamywafer
cookies21deg
2LTRin
28dat
25deg
(11)
Estuarinewater:
2LTRin
28d
at25deg
(11)
PBS:1LTRin
56dat
25deg
Norovirus
Resists
pH
2Æ7
3h(14)
Resist60
deg
for30
min
(14)
Resists
exposure
to6Æ25mgl)1;
30min
(CT
187Æ5)
inactivated
at10
mgl)1
(CT
300)
(15);
otherssuggest
CTs30–60
required
(see
text)
1LTR(PCR)
at2mgl)1
chloramine
for3h(25)
Nodata–FCV
surrogate
T90
value
47Æ85(30)
NoV
Recoverable
from
disinfected
hospital
surfaces;
FCV
2LTR
in15
dat
4deg;or
in
30dat
20deg,
inactivatedin
1dat
37deg
(31)
Infectivityin
shellfish
not
reducedafter
1month
storageat
4
deg
orover
4monthsfrozen.
Stable
inice
1LTRin
24hat
10deg
(illuminated)
FCV
suspension–1
LTRper
day
at
37deg,4LTRin
60dat
4deg
(31)
FOOD-BORNE VIRUS 1363
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
Table
2(Continued)
Virus
Persistence/inactivation
conditions
pH
stability
Thermal
inactivation
Freechlorine
(unless
stated)
UV
irradiation
(mJcm
)2)
Persistence
(dry)
Persistence
food
matrices
Persistence
seawater
Persistence
freshwater
Picornavirus/
enterovirus
Resists
pH
3Æ0
(7)
Destroyed
42deg,
cations
stabilizeto
50deg(8)
2LTRat
free
chlorine
1Æ1–2Æ5mgl)1
(28)
1LTRby
chloramine,
2mgl)1for
3h(25)
1LTRin
1Æ3
min
atintensity
42mWscm
)2
inseawater
(25)
1LTRat
dose
24Æ1
(30)
2LTRon
drying,
further
3LTRin
60dat
4deg
>3LTRin
water
ormilkat
63deg,30
min
1LTRin
96hat
12deg;1LTRin
22hat
22deg
(22);
1LTR>670dat
4deg
(26)
1LTR24–31h
inriver
water
insitu
12–20
deg
(21)
Picornavirus/
kobuvirus
Resists
pH
3Æ5
(7)
Rotavirus
groupA
Stable
pH
3–9(5)
Resists
50deg
(3)
CT
values
for
2log
inactivation
0Æ01–0Æ05
(17)
GroupBvirus:
1Æ8
LTR10
min
1mgml)1;
1Æ5
LTR10
min
0Æ5
mgl)1(20)
4LTRat
56(23)
1LTRon
drying;
1further
LTRin
30d4deg
(19)
BovineRotavirus–
decay
0Æ5
LTR
per
day
natural
seawater
(29)
GroupA
virus:
2LTR>64d
tapwater
or10d
river
water
20deg
(27);GroupB
virus:3Æ2
LTR
in60d20
deg
(1,2)
MS2
Tolerates
low
pH
>1LTRin
15sat
72deg
inwater
ormilk(16)
1LTRby
monochloramine
2mgl)1,3h(25)
>3LTRby
10min
in2ppm
CLO2(16)
4LTRat
dose
750253nm
(24)
1LTRdose
23Æ04(30)
App3LTRin
4d(16)
Datasources:1.
Vondefechtetal.(1986);2.
Terrettetal.(1987);3.
Estes
(1991);4.
Monroeetal.(2000);5.
Mertensetal.(2000);6.
Russeletal.(1967);7.Kingetal.(2000);8.Dorval
etal.(1989);9.Scholzetal.(1989);10.McC
austlandetal.(1982);11.Sobseyetal.(1988);12.Petersonetal.(1978);13.Coulepsis(1987);14.Dolinetal.(1972);15.Keswicketal.(1985);
16.Miriam
andCliver(2000);17.Hoff(1986);18.Abad
etal.(1997);19.Abad
etal.(1994a);20.Abad
etal.(1994b);21.O’Brien
andNew
man
(1977);22.Bitton(1978);23.Cottonetal.
(2001);24.Som
eeretal.(2003);25.Shin
andSobsey(1998);26.Gantzer
etal.(1998);27.Enriquez
etal.(1995);28.Clark
etal.(1993);29.Loisyetal.(2004);30.Nuanualsuwan
etal.
(2002);31.Doultreeetal.(1999).
*Nonentericvirusstrains.
1364 M.J. CARTER
ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x
forms. These appear to survive sewage treatment well and
are frequently detected even in waters that lack enterovirus
contamination. This has led many to suggest that AdV may
be appropriate sentinels for the indication of faecal pollution
in water (Enriquez et al. 1995; Pina et al. 1998a; Wyn-Jones
and Sellwood 2001).
Noncultivable viruses have also been detected. NoV have
been found in sewage influent and effluent waters using PCR
techniques (Lodder et al. 1999), serial dilution of influent
waters suggested that levels may exceed 107 detectable virus
units although this cannot be readily correlated with particle
numbers or infectivity (Wyn-Jones et al. 2000). Estimates
using quantitative PCR obtained similar values (Laverick
et al. 2004). A study of treated water suggested that NoV
levels were virtually undiminished in primary treatment and
were reduced by only a little over 1 log in the final
(secondary) effluent (Cross 2004). Similarly astroviruses
have been detected in sewage treatment plant inlet and
effluent waters, indicating a reduction of approx. 2 log during
processing; effluent waters still contained 105 detectable
astrovirus genomes per litre (Le Cann et al. 2004). HEV was
found in sewage by molecular means (Pina et al. 1998b), andsurvives at least some wastewater treatments although its
removal has not been quantitated (Jothikumar et al. 1993).Viruses surviving water treatment and entering receiving
waters could persist and pose a risk for recreational users of
this water and studies have inferred potential exposure to
both NoV (Gray et al. 1997) and HAstV (Myint et al. 1994)from this source. Studies have indicated that the risks of
disease resulting from exposure to recreational water may be
as high as 1/1000 for AdV (Crabtree et al. 1997) and
perhaps higher for rotavirus (Gerba et al. 1996).The mechanisms of virus inactivation/removal during
wastewater treatment are not clearly understood and the
environment itself probably alters the effectiveness of each
process. Physical removal seems to be significant and much
virus (perhaps 95%) appears to be removed via activated
sludge or complexing with mineral particles. Active entero-
virus has been recovered from sludge (Albert and Schwa-
rzbrod 1991) and HAV was detected by both antigen capture
and PCR suggesting that particles were intact (Graff et al.1993). EU requirements for heat-treatment before sludge is
spread onto land should control virus contamination from
this source if followed [EU directive on sewage sludge in
agriculture (86/278/EEC) implemented under the sludge
(use in agriculture) regulations 1989] but there may be fewer
controls in the less developed world and these might
contaminate crops destined for export.
7.2 Potable water treatment and virus survival
Tap water may account for some 14–40% of gastrointestinal
illness and thus efforts to ensure its safety are vital (Payment
1988; Payment et al. 1997). The assurance of drinking waterquality begins with the source water, which should be
obtained from sources as far removed from potential
contamination as possible. In the US subsequent treatment
aims to reduce levels of contaminating virus by 99Æ99%.
Filtration can achieve an initial 10-fold reduction with a
further 1000-fold achieved by active disinfection (e.g.
chlorine, chlorine dioxide, ozone or UV irradiation). Of
these, chlorination is the most widespread; peak levels are
usually around 1 mg l)1 for 60–240 min (Grabow 1990;
Thurston-Enriquez et al. 2003) and water entering the US
distribution system should have a residual level of
0Æ2 mg l)1 to comply with requirements to control coliform
bacteria. Exposure to disinfectant is expressed by the contact
time (CT) parameter, concentration of agent (mg l)1)
multiplied by time (min). CT values for a 4 log inactivation
of enteric viruses fall in the range of 4–400 mgÆmin l)1 at a
contact concentration of 0Æ4 mg l)1 at 5�C. However the
rates of virus destruction and thus CT values required are
temperature dependent, doubling for every 10�C rise and
potentially defective around 0�C. Increasing the pH from 6
to 9 reduces the efficiency of free chlorine threefold, ozone
or chlorine dioxide are unaffected. Turbidity has the
greatest effect, shielding viruses from UV radiation, pro-
moting aggregation and also �mopping up� virucide. Increas-ing the turbidity from 1 to 10 Nephelometric Turbidity
Units decreases free chlorine effectiveness eight times
(LeChavallier et al. 1981; Hoff 1986; HECS 2003). Thur-
ston-Enriquez et al. (2003) re-evaluated the efficiency of
chlorine and found viruses to be 30 times more resistant
when aggregated. If this is so then procedures commonly in
use in the US should destroy most but possibly not all
aggregated viruses (Thurston-Enriquez et al. 2003).Norovirus was found relatively insensitive to chlorine; in
volunteer studies the virus survived exposure to 6Æ25 mg l)1
for 30 min (CT 187Æ5) and required 10 mg l)1 for destruc-
tion (CT 300 mgÆmin l)1) (Keswick et al. 1985). These
values imply that NoV could survive some water chlorin-
ation procedures. However, these experiments are difficult
to do and rely on clinical samples as NoV cannot be
cultivated. Keswick et al. (1985) used a crude sample of very
high titre that may well have protected the virus. A
protective effect due to such factors was later noted
(Meschke and Sobsey 2002). When purified, NoV has the
same sensitivity to chlorine as poliovirus (Shin and Sobsey
1998; Meschke and Sobsey 2002). A PCR-based study found
a CT value of 30–60 to be sufficient to inactivate the virus
(Shin and Sobsey 1998). However it is difficult to determine
which of these findings is the more relevant as viruses are
not found �pure� in the environment, or even in the same
form in all locations; exogenous material and aggregates are
always likely to be present. Rotavirus is inactivated
efficiently by chlorine (CT 112Æ5 at 3Æ75 mg l)1) and for
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this virus waterborne infections in the developed world are
usually linked to contamination of �clean� water post-
treatment (e.g. Hopkins et al. 1984).Enteric viruses have been found in drinking water
(1–20 PFU per 1000 l) (Payment 1988; Gerba and Rose
1990; Payment et al. 1997). Studies in France showed that
the presence of astrovirus RNA in tap water was correlated
with an increased risk of intestinal disease (Gofti-Laroche
et al. 2003). However it is not at all clear what an
acceptable level of virus contamination might be; a risk of
infection of 1 · 10)4 per person per year has been accepted
in both the US and the Netherlands (USA EPA 1991;
Staatscourant 2001). Using a model based on rotavirus
dose–response data, Regli et al. (1991) calculated that
2Æ22 · 10)7 viruses l)1 would be consistent with this risk
factor. A 4 log reduction achieved from even highly
rotavirus-contaminated source water (Raphael et al. 1985)would meet this level (Gerba et al. 1996). Estimates based
on enterovirus levels suggest that higher reductions might
be required (Regli et al. 1991). Such assessments are
variable, poor culture efficiency would underestimate the
levels present and increase risk to the consumer; uneven
distribution of contamination would cause an uneven risk
across the country and differences in levels of consumption
(and the proportion that is boiled before drinking) also
affect risk. Finally models based on infection may be less
relevant than models based on symptomatic illness and
could thus exaggerate the risk (Gerba et al. 1985; Gale
1996; Payment et al. 1997; Haas et al. 1999; Hurst et al.2001).
Most documented instances of drinking water contamin-
ation by viruses have been attributed to contamination of
previously �clean� water. This can result simply from
contamination of drinking water supplies, e.g. through
heavy rainfall or flood overwhelming treatment works and
permitting untreated or partially treated sewage to contam-
inate wells (Cannon et al. 1991; Kukkula et al. 1997, 1999).Failures in treatment processes themselves can also allow
contamination, e.g. pressure failure (Kaplan et al. 1982),
insufficient disinfection, or exceptionally high levels of virus
overwhelming a correctly applied procedure (Payment 1988;
Gerba and Rose 1990; Payment et al. 1997; Bitton 1999;
Gofti-Laroche et al. 2003). Virus leakage from sewers or
septic tanks can contaminate clean water (Hedberg and
Osterholm 1993). Waterborne outbreaks of enteric virus
infection are common; between 1980 and 1994 the US
recognized 28 outbreaks and 11 195 cases of waterborne
illness, of these 9000 cases were NoV and nearly 400 HAV
(HECS 2003). None of these viruses are appreciably affected
by freezing (which may actually preserve infectivity for long
periods) and thus infection is also spread when ice is
manufactured and distributed using contaminated water
(Cannon et al. 1991).
8. FOOD-BORNE VIRUS TRANSMISSION
Food-borne transmission may be divided into two areas:
in primary contamination food materials are already
contaminated before they are harvested, e.g. shellfish
grown in contaminated waters, or soft fruits irrigated/
sprayed with contaminated water. Secondary contamin-
ation occurs at harvest or during processing and empha-
sizes the role of the food handler preparing foods for
others with whom he/she does not come into direct
contact. Food handlers in this context include field
harvesters, production plant workers right through to
professional chefs and caterers. Contamination from these
persons involves not only transfer of virus from infected
persons but also their use of polluted water or materials in
processing. It is worth noting that transfer of virus from
an infected individual to food would be counted as a food-
borne infection if it takes place in the commercial/
industrial setting, but as person-to-person infection if it
occurs in the home.
It is difficult to assess the extent of food-borne transmis-
sion in the community, first because person-to-person and
food-borne routes may overlap (above), but secondly
because it is technically difficult to detect viruses in foods,
apart from shellfish most are not routinely tested. This has
led to two assumptions, first most food-borne outbreaks in
which a vehicle is not demonstrated are likely to have arisen
by contamination from food-handlers – at or close to the
point of serve – and the second is that most (if not all) cases
occurring in the community (i.e. outside outbreak situations)
probably arise from person-to-person transmission. These
assumptions should be reconsidered because both disguise
or negate potential instances of primary or distant secondary
contamination.
Estimates of the incidence of food-borne disease have
relied on statistics obtained from outbreaks. In recent years,
reports of viral gastroenteritis, particularly NoV outbreaks
have shown a dramatic increase in England and Wales; 418
cases were identified in 1992 and 2387 in 1996. In 1994 UK
reports of NoV outbreaks outnumbered those of Salmonellafor the first time. This increase may be partially explained
by increased awareness and improvements in virus identi-
fication techniques, thus more recently attempts have been
made to estimate the extent of community illness. Mead
et al. (1999) estimated that 67% of food-borne illness in the
US was caused by viruses and that 40% of NoV infections;
1% astrovirus, 1% rotavirus and 5% of HAV infections,
respectively, were food-borne. NoVs were responsible for
23 million cases per year with over 9 million food-borne
events. NoV then emerged as the most significant
food-borne infection in the US. Annually NoV was
estimated to be responsible for 20 000 hospitalizations
and 120 deaths.
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8.1 Shellfish
Enterically transmitted viruses are shed into sewage and
become rapidly diluted as they travel through the water
system. Filter-feeding molluscan shellfish (particularly
cockles, mussels and oysters) partially reverse this dilution
because they are filter-feeders. They are harvested from
close-to-shore locations where water will contain virus
inputs from sewage effluents; viruses become concentrated
within the shellfish and may be retained for some time.
Levels of virus in shellfish may be 100–1000-fold higher
than concentrations of viruses in their surrounding water.
European regulation divides shellfish harvesting waters
into different categories depending on the levels of faecal
pollution (Table 3). Very badly contaminated waters may not
be used to harvest shellfish for human consumption at all,
whilst intermediate levels may be used provided that the
shellfish are relayed into cleaner waters, heat-treated or
depurated before harvest. Depuration is a process in which
the animals are kept in a recirculation tank; water is passed
over the shellfish, disinfected by UV treatment and recycled.
The shellfish gradually purge their bodies of faecal indicator
bacteria and may be sold when these reach a target level. It is
now clear that bacteria are not an adequate indicator of the
presence of enteric viruses, and numerous virus infections
have been documented from shellfish compliant with these
regulations (Chung et al. 1996; Griffin et al. 1999; reviewedby Lees 2000). Bacteria are purged more rapidly than viruses.
Schwab et al. (1998) found that a 95% reduction in
Escherichia coli was achieved by 48 h depuration but NoV
was reduced by only 7% in the same period. The efficiency of
virus removal is influenced by a number of factors chiefly
temperature (Dore et al. 2000). Most gastroenteritis viruses
are more common in winter (even if not shellfish transmitted)
and increased community illness coupled with less efficient
purging by the shellfish could increase the risks associated
with shellfish consumption at these times. Viruses retain
infectivity well in shellfish and no loss of infectivity was
observed over 1 month in refrigerated storage (Tierney et al.1982) or 4 months when frozen (Di Girolamo et al. 1970).Trade in shellfish permits long-distance transmission: in
2002 oysters from Cork Bay spread NoV infection around the
world to Hong Kong (ProMED-mail 2002a; 20020331.3850),
and in 2004 Chinese frozen oysters were implicated in a
(presumed NoV) outbreak in Singapore (ProMED-mail
2004; 20040107.0075). In 1993 a multistate outbreak of
NoV illness occurred in the US affecting up to 186 000
people (Berg et al. 2000). This was traced to contaminated
oysters harvested in the Gulf of Mexico. Oysters in Europe
accumulate a mixture of all viruses present in their environ-
ment, but in these Gulf oysters only one strain was present.
This indicated that input virus must have originated from a
restricted number of infected persons and was possibly
attributable to incorrect disposal of faeces or vomitus at sea.
This remarkable occurrence indicates firstly just how
efficiently the shellfish can accumulate virus from their
environment and secondly just how significant commercial
trade and distribution can be in the dissemination of
infection. It further demonstrates the power of molecular
epidemiology in linking occurrences that would otherwise
not necessarily have been connected. Sporadic outbreaks
occur throughout the developed world via consumption of
virus-contaminated shellfish (reviewed by Lees 2000).
Many shellfish are subject to minimal cooking (if any),
linking this to their known ability to concentrate environ-
mental viruses the fact that they are the most commonly
identified source of food-borne virus infection should not be
surprising. The most significant viruses in this context are
NoV first reported as an Australia-wide outbreak (Murphy
et al. 1979), and hepatitis viruses. Recently Aichivirus has
been associated with infection from this source in Japan but
reported cases are few. However the viruses most commonly
detected in shellfish are enteroviruses (reviewed byGerba and
Goyal 1978). These seldom give rise to symptomatic disease
although some cases have been reported (Cliver 1997).
Virus concentration by shellfish is nonspecific and thus
their consumption can expose the consumer to a cocktail of
viruses that might have different effects when present
together. Limitations on current technology mean that
during investigations is it usual to seek only a few defined
virus types (those that succeed in inducing clinical disease).
However the possibility of multiple simultaneous infection
brought about by the consumption of shellfish should not be
overlooked, viruses might act synergistically to increase the
Table 3 Microbiological classification of
shellfish harvesting waters and requirements
for marketing for human consumption (taken
from EC directive 91/492). Virus contamin-
ation is not assessed
Class Standard Requirements for marketing for consumption
Class A <230 E. coli or 300 faecal coliforms May be consumed directly
Class B 90% compliance <4600 E. coli and
600 faecal coliforms
Depuration or relaying until
class A standard is met, or apply
heat-treatment
Class C <60 000 faecal coliforms Relay for 2 months to meet class A
or heat-treat when class B standards
met. Or heat-treat
Prohibited >60 000 faecal coliforms Prohibited
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severity of symptoms or to present confusing clinical
pictures.
Shellfish transmission of HAV is well documented and
includes the largest ever outbreak that of Shanghai (1988);
300 000 persons were believed to have been infected
through consumption of contaminated clams (Halliday et al.1991). Studies around the world have consistently identified
shellfish consumption as a major risk factor for the
contraction of hepatitis. In some studies the risk was
equivalent to that of contact with an infected person (Koff
et al. 1967; Kiyosawa et al. 1987). HAV is rarely detected in
food itself, largely because of the long incubation period for
this illness which means that no samples of suspect food are
likely to be available for testing. Even so shellfish are
believed to be the major vehicle for HAV infection and
Salamina and D’Argenio (1998) estimated that up to 70% of
all hepatitis A in Italy was contracted from shellfish. In the
UK it is recommended that shellfish flesh be raised to 90�Cfor 1Æ5 min (Millard et al. 1987) and continuous flow
methods now ensure that all shellfish are subjected equally
to this treatment. Since the implementation of these
recommendations, there have been no reports of outbreaks
in the UK of either viral gastroenteritis or hepatitis A
associated with shellfish heat-treated in this way (Appleton
2000). Work by Croci et al. (1999) suggested that even these
standards may not be sufficient to completely eliminate the
virus and they recommended treatment at 100�C for 2 min.
Hepatitis E is not commonly transmitted by shellfish
(Chan 1995; Stolle and Sperner 1997) but has been
identified in sewage even originating from nonendemic
areas (Pina et al. 1998b). This finding may be related to the
seroconversions also identified in such nonendemic regions
(see above). Thus the possibility of shellfish transmission
must be considered.
There are few reports of astrovirus transmission via
shellfish consumption (Caul 1987) although recently shell-
fish were implicated in a large outbreak involving teachers
and young adults at a school in Japan (Oishi et al. 1994).This was unusual in that it affected older persons and was
attributed to a higher serotype virus. Astroviruses are
frequently detected when sought in oysters and the apparent
lack of resultant illness is presumably attributable to prior
immunity remaining from childhood infection. Similar
considerations apply to rotavirus contamination.
8.2 Soft fruit and salad vegetables
After shellfish, food-borne virus illness is most commonly
linked to salads and soft fruits. These items are almost
always subject to handling immediately before serving and
this presents an opportunity for contamination. In virtually
all cases where this type of food is implicated an infected
food handler is suggested as the origin but in only a small
proportion of such cases is this link actually proved. Soft
fruits and salad vegetables, like shellfish are eaten raw but
also share other features; first, they all have a high water
content – absorbed from groundwater during growth;
secondly, many are eaten without peeling which would
remove external contamination. Consequently, primary
contamination is possible externally by spraying or internally
by uptake of viruses from contaminated irrigation water or
fertilizer. Surface splash may be significant for fruits close to
the ground (e.g. strawberries) and multi-state outbreaks of
HAV may have arisen in this way (Niu et al. 1992; Hutin
et al. 1999). Wastewater may be used for irrigation,
especially in dry areas where water is more precious, and
these communities show elevated incidence of hepatitis virus
infection although reasons have not been identified (Katze-
nelson et al. 1976).Viruses can enter plants through root damage (Katzenel-
son and Mills 1984), and this occurs universally through
root abrasion with soil particles. Other workers found virus
uptake from roots to the leaves (but not the fruits) of tomato
plants even when virus was introduced below the soil surface
eliminating the possibilities of aerial contamination (Oron
et al. 1995). Our own data also show that internal contam-
ination can occur but is probably of low level compared with
external contamination (M.J. Carter, unpublished observa-
tions). However, a lower amount of virus inside a crop plant
could potentially have an effect equivalent to a much higher
external dose because it cannot be removed by either peeling
or washing and may persist for longer because it is shielded
from UV inactivation. Washing is not always efficient as a
means of removing contamination, for instance HAV was
found to adhere well to produce, especially lettuce (Croci
et al. 2002). It is difficult to detect viruses in plant material
and in only a few cases has contamination been shown
directly, e.g. NoV in raspberries (Gaulin et al. 1999). Most
associations are made by epidemiological connection and
identification of the infectious agent in clinical samples from
those affected (reviewed by Seymour and Appleton 2001).
Fruit and salad vegetables are traded around the world,
originating from areas where sunlight permits growth but
where water quality is not always assured. This trade either
as raw materials or as ready-to-eat products can aid
widespread virus dissemination. In 2003 an outbreak of
hepatitis A occurred in the US, this affected several hundred
persons in four states and resulted in four deaths. The
source was eventually traced green onions imported from
Mexico although virus was not identified in the food;
identification was possible only by analysis of the chain
of importation and transport (ProMED-mail 2003;
20031128.2946). In 1997 contaminated strawberries were
used in a school lunch preparation that was distributed to 17
states in the US and may have exposed up to 9000 children
(ProMED-mail 1997; 19970402.0689). A total of 200 cases
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of HAV were reported in Michigan. Similarly, pre-prepared
salads distributed across the US were responsible for a
multi-state outbreak of NoV affecting over 300 persons
(ProMED-mail 2000; 20000318.0377). Imported raspberries
were responsible for an outbreak in Canada (Gaulin et al.1999). Viruses are not destroyed by freezing or freeze-
drying, although there are few studies of the latter process in
contaminated food, in the laboratory freeze-drying is an
efficient means of preserving virus infectivity and should be
expected to preserve viruses in produce. Both hepatitis A
(Reid and Robinson 1987) and NoV (Ponka et al. 1999) havebeen transmitted by frozen fruit. The growth in the
worldwide trade in freeze-dried soft fruits (particularly
raspberries and strawberries) offers another potential vehicle
for transmission.
There are no data concerning levels or persistence of
potential internal virus contamination of fruits and vegeta-
bles, although Sadovski et al. (1978) recovered poliovirus
from cucumbers that had been irrigated with contaminated
water 8 d earlier an upper limit to virus persistence was not
established. In areas of high UV illumination surface
contamination may be short-lived in the field, Badawy et al.(1990) studied virus survival on sewage irrigated grass in
southern USA and found 2 log reductions in titre were
achieved in just 8–10 h in the summer, slightly longer in the
winter (16–24 h). However viruses can survive well on the
surface of produce once harvested, a number of studies have
indicated survival times of longer than a week and some-
times up to 30 d for a range of enteric viruses under typical
storage conditions (Konowalchuk and Speirs 1974, 1975a,b;
Badawy et al. 1985; Pirtle and Beran 1991; Kurdziel et al.2001; Croci et al. 2002). Increasing temperature decreased
virus survival times but conditions usually associated with
produce storage were conducive to virus survival. It is not
clear what effect microenvironments on a plant surface
might have, waxy cuticles, hairy projections and curled,
crinkled or convoluted leaves can all modify the way that
virus contamination might behave, the amount of wetting
will affect the way that a droplet dries whilst convoluted leaf
surfaces could shield a virus from UV or increase local
humidity. These effects are difficult to replicate experi-
mentally. Data from Kurdziel et al. (2001) and Ward and
Irving (1987) show that there is variability in the times that
poliovirus may survive on different artificially contaminated
plants stored at 4�C. Similarly these potentially sheltered
environments suggest that washing processes will likewise
vary in efficiency even over sections of the same plant.
These data suggest that contaminated fruit and vegetables
might account for a large number of food-borne infections.
A study in the UK indicated that these might present a
significant opportunity for virus infections (O’Brien et al.2000). However, the criticism frequently levelled at this
implication is that these infections are simply not observed.
To some extent this may arise from the assumption that
community illness is entirely or mainly spread by the direct
person–person route even though supporting data for this
assumption is weak. When investigations have attempted to
distinguish between person-to-person and food-borne ill-
nesses in the community the latter can be identified by
statistical means and suggest that there are potentially many
thousands of such instances per year (see below).
8.3 Other foods
Few other foods are associated with primary contamination
but a recent example is the consumption of raw liver from
wild boar or venison. Both have resulted in the transmission
of HEV to humans (Matsuda et al. 2003; Tei et al. 2003;Tamada et al. 2004). Consumption of raw liver is rare but
virus contamination of utensils used in handling it should be
borne in mind. Supermarket pork liver has also tested
positive for HEV (Yazaki et al. 2003) – even in countries
with no significant HEV infection. This suggests that pork
liver is not a significant source of infection for humans.
8.4 Food handlers
Food handlers may contaminate foods at any point from
harvest to serving. Toilet arrangements (including hand
washing) should be provided for harvesters in the field to
prevent contamination of produce as it is picked. Infected
food handlers may or may not be symptomatic at the time of
contamination. HAV infections may be mild or asympto-
matic but large numbers of particles are nonetheless shed in
the faeces. PCR technology has shown that even in
symptomatic illness, NoV shedding may precede symptoms
by 10–30 h and may continue (albeit it at a much lower
level) for 1–2 weeks afterwards (Graham et al. 1994;
Okhuysen et al. 1995; Estes and Leparc-Goffart 1999).
NoV transmission by pre-, post- or asymptomatic food
handlers has been demonstrated (Patterson et al. 1993;
Parashar et al. 1998; Gaulin et al. 1999). Cowden et al.(1995) recommend a 48-h period of exclusion following the
cessation of symptoms but as virus shedding is likely to
continue for some weeks returnees must follow strict hand
washing procedures. Inadequate hand washing/personal
hygiene has been implicated in outbreaks of both NoV and
HAV (Bean et al. 1990; Bidawid et al. 2004) and recently its
effectiveness has been demonstrated. Bidawid et al. (2000b)considered the likely levels of hand contamination by HAV,
and then estimated the resultant transfer of viruses to foods
by handling. They concluded that up to 10% (or appprox.
1000 particles) could easily be transmitted via the fingers to
food items (lettuce). If proper washing procedures were
followed this transfer fell to 0Æ3–0Æ6%. Other experiments
have shown that virus transfer rate is high immediately after
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hand contamination but reduces as the virus dries (Larson
1985; Ansari et al. 1988; Springthorpe and Sattar 1998).
Whilst this might imply that the window for contamination
closes rapidly, it should be borne in mind that virus does
survive drying onto the skin and reapplication of moisture
(as in handling washed and wet foods) could then lead to
increased transfers once more. Findings such as these
emphasize the importance of proper hygiene in controlling
food-borne infection from this source. However, given the
likelihood that this will not be achieved in all cases,
Koopmans and Duizer (2004) attempted to estimate the
risks associated with food contaminated in this way. Using
the value of 1000 particles for transfer (above), they reasoned
that a 3 log reduction in virus viability would be required to
ensure safety after such a contamination event. They then
estimated the effects of various common food-processing
procedures that might follow handling (e.g. freezing,
fermentation, acidification, pasteurization, etc.). With the
exception of boiling (and other high temperature treatments)
no procedure achieved a 3 log reduction suggesting that
viable virus would remain in almost all cases to present a
threat to the consumer. Mariam and Cliver (2000) also
showed that HAV would survive common pasteurization
procedures for milk (30 min at 63�C and 15 s at 72�C).The sudden onset of projectile vomiting and associated
aerosolization of virus particles is particularly problematic
when this occurs in a food handler. As vomiting may be the
first overt sign of infection such a situation is virtually
impossible to prevent. Given the survival characteristics of
these viruses all uncovered food should be destroyed or
cooked even if at some distance from the event. Decontam-
ination of catering premises presents similar problems to
that of hospitals. A sink into which an infected food handler
had vomited was thoroughly cleansed with chlorine bleach.
The following week the same sink was used to prepare salad
and resulted in an outbreak of NoV amongst the consumers
(Patterson et al. 1997). Food handlers certainly can con-
taminate foods but this is usually proved in only a minority
of cases. Attempts were made to assign transmission as
person-to-person or food-borne in an analysis of 2149
outbreaks occurring between 1992 and 1995 (ACMSF
1998). About 33% were attributed to NoV, 2% to RV and
0Æ5% each to astroviruses and small round (parvovirus)-like
agents. Although not robust, this analysis suggested that
many of the NoV outbreaks were food-borne and a food
vehicle was suggested in 35 cases. Virus was detected in only
two of the suspect foods (both oysters), in the remaining
cases contamination was attributed to a food handler but
subsequent investigation confirmed food handler infection in
only 20%. Food vehicles implicated were diverse but
included a variety of fresh fruits and salads as well as other
items that would commonly either contain or be served with
a salad or fresh fruit garnish. These included items such as
sandwiches, pies, vegetable salads, gateaux, fish, lobsters and
prawns. Consequently there was potential for primary (or at
least remote secondary) contamination in 80% of these
cases.
9. PERSON-TO-PERSON TRANSMISSION
Most enteric viruses are highly contagious, for most the
infectious dose is believed to lie between 10 and 100 particles
and for NoV possibly lower. Given rotavirus shedding at a
level of 109 per ml, as little as 0Æ1 ll of stool contamination
could contain an infectious dose. Such tiny quantities are
invisible and easily retained on skin. Although hand washing
is effective as a disinfectant (particularly alcohol-containing
washes) in the case of rotavirus (Estes et al. 1979) infectionsare easily transmitted from person to person either directly
or via contamination of objects and surfaces.
Vomit is also significant; 30 million particles may be shed
by this route (Reid et al. 1988; Caul 1994) and RV may
survive for up to 9 d in the air (Sattar et al. 1984). Proximity
to vomit at the time of vomiting may be more significant
than exposure later on; in a hotel NoV outbreak infection
was related to distance between the secondary cases and the
primary case at the time of the event (Marks et al. 2000) andin a hospital setting, persons in the proximity of a patient
who vomited were at greater risk of acquiring NoV than
those who actually cleaned up the vomit (Chadwick and
McCann 1993). This implies that aerosolization is a potent
means of infection of those in the immediate vicinity.
Airborne transmission is distinct from respiratory transmis-
sion as the route of infection remains via the gut rather than
the respiratory tract. Infection could result from the
trapping of aerosolized droplets containing virus in the
nasal passages and subsequent swallowing of virus. Alter-
natively aerosolized virus may travel some distance before
settling and contaminating surfaces or uncovered food.
Spread may be assisted by artificial ventilation (Chadwick
et al. 1994).Person-to-person transmission is clearly favoured by
closed settings, persons cannot leave cruise-ships, hospitals
or care homes and once a virus has been introduced by
whatever route efficient spread within that setting is to be
expected. In 2002 the CDC, Atlanta, recorded 21 outbreaks
of NoV on 17 ships docking in the US (ProMED-mail
2002b; 20021212Æ6049); this included one vessel on which
more than 200 cases were reported. Once a virus is present it
can be very hard to eliminate even by thorough disinfection
(Chadwick et al. 2000; Barker et al. 2004). Some ships
showed serial outbreaks amongst each new set of passengers
coming aboard. This is not surprising as even cleaning in a
hospital context may fail – serial astrovirus infections were
experienced in a bone marrow transplant unit despite
vigorous attempts to disinfect the unit between patients
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(Cubitt et al. 1999). Conventional cleaning of surfaces can
fail to remove detectable NoV virus (Green et al. 1998;
Barry-Murphy et al. 2000).
9.1 Person-to-person or food-borne?
Unsurprisingly, contact with an infected person is a major
risk factor for contraction of these viruses: secondary attack
rates of up to 95% have been reported for HAV within the
home (Villarejos et al. 1982). HEV appears to infect less well
in the domestic setting and secondary attack rates are lower
(Purcell 1997) even though virus shedding may be more
prolonged (Clayson et al. 1995b). Contact with an infected
person was found to constitute the greatest single risk for
infection by NoV in studies in the UK, US and the
Netherlands (Mead et al. 1999; FSA 2000; De Wit et al.2003). However this does not mean that all community
spread must be via the person-to-person route, because
unlike other viruses (which may be maintained entirely by
this route), enteric viruses can also spread through food or
water. It is difficult to estimate the contribution of each
route to the total burden of infection in the community.
Most figures consider only outbreaks, but the IID survey in
the UK revealed the true extent of these �missed� commu-
nity infections for the first time (FSA 2000). This survey
estimated that for every case of NoV that is laboratory
confirmed, over 1500 occur in the community. The report
attributes community cases (almost) entirely to person-to-
person transmission but presents no data on this point.
Recently De Wit et al. (2003) have made a thorough attempt
to determine the extent of food-borne transmission in the
community. They found that person-to-person transmission
was indeed common, much of it occurring in the home and
in many cases actually involving contamination of food
prepared for others. This is not counted as �food-borne�infection (see above). The analysis also showed for the first
time that person-to-person transmission alone could not
account for all community illness and the authors concluded
that 12–16% of infections could be attributed to contam-
inated food/water entering the household. In the UK there
are some 9Æ5 million cases of IID annually and has been
estimated that some 16% (1Æ52 · 106) are caused by NoV.
Assuming that all of these took place in a household of four
persons and that all persons in each household were
infected, this would correspond to a minimum of 380 000
infected households. Lastly, if 12–16% of these resulted
from incoming contaminated food as above, then contam-
inated food could instigate directly some 45–60 000 food-
borne cases in the UK per year as a minimum estimate.
Estimates in the Netherlands using less conservative
assumptions suggest 80 000 food-borne NoV infections
could occur annually even in this smaller country (De Wit
et al. 2003).
10. VIRUS DIAGNOSIS AND DETECTION
10.1 Clinical samples
An assessment of the impact of these viruses in human
disease requires the provision of a sensitive and reliable
detection method for application to clinical samples. Rout-
ine reagents for the detection of rotavirus, HAV and
poliovirus have been available for some time but technology
for the other viruses of interest has only recently moved
from the specialist laboratory into widespread use. As a
result the burden of disease contributed by other gut-
infecting viruses has not been so clearly established.
Diagnostic reagents have formerly been hard to provide
and in their absence perhaps the simplest method has been
to examine the faeces using electron microscopy. This is a
catch-all method and requires no prior knowledge of the
virus in order to detect it but it is expensive, makes high
operator demands and its efficiency depends on a virus
being readily recognizable; Viruses that are large, of
distinctive appearance, and present in significant numbers
(>106 particles per ml) are relatively easily observed.
However, small, diffuse or fuzzy viruses can often be
overlooked. Identification makes high demands on the
operator and this is even worse when a virus may have
several distinct appearances (see astroviruses). Historically it
is thought that this method has identified AdV and rotavirus
infections quite well but has been less successful in the cases
of caliciviruses, astroviruses and parvoviruses.
Most gut-dwelling bacteria are routinely identified by
culture; however, this route has not been open to the
detection of viruses. All viruses require living cells as hosts
and are totally dependent on the processes that their host
cells are able to provide, e.g. for protein synthesis and
protein processing, but the gut is a very specialized habitat
in virological terms. It contains a multiplicity of different
cell types, each having a different enzyme content and
surface protein composition; they may even vary in these
properties at different stages of their differentiation. Fur-
thermore, they are all bathed in a solution of the various
secreted products produced by other specialized cells,
notably of course proteases and bile salts. These features
make the gut a very difficult cellular environment to mimic
in culture. A virus may replicate in one type of cell, at one
stage of its differentiation and may require soluble products
released from quite different cells entirely. It is probably for
this reason that gut-replicating viruses have been very
difficult to culture in the laboratory. Those that can be
cultivated often require that the culture be supplemented
with proteases (usually trypsin) and in one case (porcine
enteric calicivirus) with duodenal juice, bile salts being the
active ingredient (Chang et al. 2004). Specialized cells are
often needed, e.g. differentiating colonic carcinoma cells for
astroviruses. Viruses that penetrate beyond the gut, invading
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other tissues are often (but by no means always) simpler to
cultivate. The significance of this point cannot be overstated,
most extracellular parasites will grow reasonably efficiently
in a broth which mimics the gut contents, but this is not true
of viruses and there are very good reasons why the
characterization and detection of the viruses of gastroenter-
itis have been problematic. Culture for viruses is not
generally used even to identify agents present in a faecal
sample. The chances of using such a procedure for the
detection of the lower quantities of virus present in food are
still more remote.
In view of the above difficulties in EM detection, reagent
availability in the past and difficulties in culture most
workers have acknowledged the existence of a �diagnosticgap� represented by the large proportion of infections for
which no obvious cause could be found. Even in the IID
survey in England and Wales, when causative agents were
vigorously pursued, target organisms were not identified in
63% of community samples and 45% of General Practice
samples (FSA 2000). Given the difficulties in virus diagno-
sis, it was always likely that a large part of this diagnostic gap
would probably be caused by viruses, but probably by the
smaller, less well-characterized and less-readily observed
viruses. The extent of any such bias in detection has recently
become clearer with the development of objective and
sensitive methods for the detection of many viruses: the
development of recombinant antigens for astroviruses and
especially NoV, and the development of ELISA systems for
both have at last provided a reproducible supply of antigen
from which to prepare and validate diagnostic serological
reagents as well as to conduct serosurveys of the incidence
and age-at-first-infection profiles of these viruses. This type
of detection method has been used in the IID survey in
England and Wales – although not all tests in all cases (FSA
2000), and in similar studies in Europe (Koopmans et al.2000) and the US (Mead et al. 1999). These studies all
support the conclusion that virus-associated enteric illness
has been greatly underestimated and currently suggest (in
the absence of concrete data) that most, if not this entire
diagnostic void is made up of NoV infections. Similarly data
from the FSA survey in England (2000) suggested that for
every case of NoV actually detected in the diagnostic
laboratory, on average 1562 have probably occurred in the
community.
10.2 Food samples
Virus detection in food and water (i.e. preinfection) is even
more problematic than detection postinfection, in clinical
samples. Levels of virus are orders of magnitude lower and
the challenge is to detect a potentially infectious dose
(perhaps as low as 10 particles) in a quantity of a size likely
to be consumed as a single portion. As these viruses can
either not be cultivated or cultivated only with difficulty,
most detection methods have concentrated on PCR tech-
nology. This has proved extremely successful when applied
to NoV detection in water and shellfish and can even
provide quantitative information (Laverick et al. 2004; LeCann et al. 2004). However PCR-based detection has two
problems, the first is that most of these viruses are RNA
viruses, PCR cannot be used unless the RNA has been
reverse transcribed (RT) to manufacture a DNA copy and
the efficiency of this RT step is notoriously variable.
Secondly PCR targets only a short section of the virus
genome (usually 200–300 bases) and reveals nothing at all
about the presence or integrity of the rest of the genome.
Strictly then, even quantitative PCR for such a virus detects
only the levels of cDNA to a specific region of the virus, it
cannot tell us how the numbers of DNA copies detected
relate to the number of RNA copies actually present in each
case and nor can it tell us whether the target detected was
present as an intact genome inside a viable particle or
present as a short section of free RNA released by virus
degradation. The first of these difficulties can be tackled by
careful incorporation of internal controls, although these
may of themselves interfere with target amplification. The
second is harder to address. It is often assumed that RNA
degradation occurs in two phases; virtually none at all whilst
the particle is intact followed by rapid and total degradation
once the particle is breached. Consequently levels of free
RNA should be insignificant and most detectable RNA
would be contained inside an intact particle. However it is
not clear that this assumption is valid; whilst Slomka and
Appleton (1998) used FCV as a model for NoV and found
that RNA was indeed rapidly degraded after inactivation of
the virus in shellfish they also confirmed that some PCR-
positive samples were not cultivable. Similar findings were
reported for poliovirus and bacteriophage MS2 (Shin and
Sobsey 1998; Sobsey et al. 1999). Recently a study by
Nuanualsuwan and Cliver (2003) suggests that the situation
could be yet more complex. These workers inactivated
calicivirus and picornaviruses (including hepatitis A) by
exposure to high temperature (72�C), UV radiation and
hypochlorite disinfection. They found that infectivity of all
viruses could be vastly reduced by these means. However,
the RNA inside the particle appeared to survive, the
reduction in infectivity being attributed to a loss of capsid
function. Inactivated particles could no longer attach to host
cells and were no longer recognized by antibodies. Despite
this failure the RNA remained intact within them and
continued to be detected by PCR; furthermore it was still
protected from degradation by hydrolytic enzymes suggest-
ing that the disjunction between PCR detection and virus
viability would become worse with time. These findings are
of great significance as they suggest that PCR-based
detection and quantitation would also detect virus particles
1372 M.J. CARTER
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that are inactive and pose no threat to a consumer. This
would overestimate the extent of virus contamination.
Antibody-based detection methods (or PCR detection
following an antibody-mediated concentration step) should
overcome this effect and this has also been observed (Sobsey
et al. 1999). A technical account of the primers and
conditions required for virus detection is beyond the scope
of this review but have been summarized elsewhere (Sair
et al. 2002).One drawback of PCR-based detection is that the test
consists of the observation of amplification or failure to
amplify. Specificity relies on the choice of primers which
should amplify only the selected target viruses. This approach
is intrinsically unsuitable for the identification of multiple
viruses simultaneously. It yields no information on the
presence of other virus types not specifically sought by the
procedure, or more distantly related to the �known� virusesfromwhich primers were designed. PCR for NoV for instance
can identify only 90–95% of EM-positive samples, presum-
ably because of inherent sequence variability within the virus
family. However microarray technology now offers the ability
to use multiple and redundant PCR primers to target and
amplify a range of viruses present in a sample. Specificity is
then achieved at the array hybridization stage when hybrid-
ization to a microarray plate containing several hundred
probes for individual viruses or groups of viruses detects each
simultaneously. Such detection technologies offer the chance
of defining at last the true extent of virus contamination of
both food and the environment.
11. ALTERNATIVE INDICATORS OF VIRUSCONTAMINATION
In view of the difficulties in detecting viruses above, much
use has been made of indicator organisms to provide clues to
the likelihood of virus contamination (reviewed by Kator and
Rhodes 1987). Cultivable enteroviruses such as poliovirus
have been extensively used. This agent has been ubiquitous
in sewage-polluted environments because of widespread
vaccination in the community and the resultant faecal
shedding from vaccinees. However it is difficult and expen-
sive to routinely extract and assay poliovirus from contam-
inated food and water, further the WHO aim to eliminate this
virus from the world and work with it is already subject to
control. As a substitute many workers now suggest AdV (see
above). However an alternative approach has been to use
nonhuman viruses such as the bacteriophages (Power and
Collins 1989, 1990) and chief amongst these are the male-
specific bacteriophages of the Levivirus family such as MS2.
These bacteriophages contain an RNA genome of similar size
to the picorna-, calici- and astroviruses and have particle sizes
in a similar range. Bacteriophage are frequently found in
faecally contaminated materials (and shellfish associated with
gastroenteritis outbreaks) and are thought to show similar
stabilities to the enteric viruses. These data suggest that they
may be good models for virus contamination in these systems
(Dore et al. 1998, 2000). However it should be borne in mind
that indicator organisms such as these can only indicate the
possibility of human virus contamination; they cannot prove
either its presence or its absence. F+-specific bacteriophage
can be specifically enumerated using an engineered pilus-
bearing Salmonella typhimurium host that cannot be infected
by non-RNA coliphage (Havelaar and Hogeboom 1983). Use
of this host yields data relatively free of contaminating
somatic coliphage but does not easily separate contamination
of animal origin from that of human. Further work would be
necessary to make such a distinction using oligonucleotide
hybridization (Beekwilder et al. 1995; Hsu et al. 1995). Atpresent there are no data concerning the use of these
organisms as potential indicators of faecal contamination for
soft fruits and vegetables. Mariam and Cliver (2000)
attempted to use bacteriophage to determine the extent of
virus inactivation likely in response to certain treatments in
various food processes and Dawson et al. (2005) used phage
to monitor the survival of viruses on soft fruits. Slomka and
Appleton (1998) used feline calicivirus in a similar manner.
However these experiments require careful analysis as
neither are exactly equivalent to the viruses of interest.
Mariam and Cliver (2000) found that both MS2 and UX174were more labile than HAV in their analysis. However they
nevertheless concluded that these organisms could be useful
if appropriate relationships could be determined between
phage inactivation rates and those of viruses of concern. Such
work demands parallel experiments using the actual viruses
of concern to �calibrate� the response of the indicators in a
variety of circumstances and such data is urgently required.
12. CONCLUSIONS
Food-borne viruses are ubiquitous; it is likely that their
incidence has been underestimated in the past not only in
terms of the number of infections but also in terms of the
frequency with which they are food-borne. Sewage treat-
ment without disinfection should be recognized as inad-
equate and UV treatments of final effluents should be
encouraged to protect both recreational waters and espe-
cially shellfish harvesting areas. Intrinsic contamination of
foods other than shellfish needs to be considered and more
work is needed to characterize the uptake, survival and
removal properties of viruses in plant tissues. Given the
difficulty of excluding (or even identifying) food handlers
likely to be shedding virus at any one time and the growth
in national/international trade in foods requiring minimal
cooking we may expect food-borne viruses to increase in
significance in the future. This effect may be of most
significance in the case of HAV where the divergence in
FOOD-BORNE VIRUS 1373
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infection patterns between producer and consumer coun-
tries is greatest.
13. ACKNOWLEDGEMENTS
I thank Dr Martin Adams and Dr Peter Wyn-Jones for
constructive criticism of the manuscript and provision of the
results from their �data-mining�.
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