Journal of General Virology (2012), 93, 2195–2203 DOI 10.1099/vir.0.044974-0

Global transmission of influenza viruses from humans to swine

Martha I. Nelson,1 Marie R. Gramer,2 Amy L. Vincent3 and Edward C. Holmes1,4

Correspondence 1Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA Martha I. Nelson 2University of Minnesota Veterinary Diagnostic Laboratory, St Paul, MN 55108, USA [email protected] 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 State University, 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 Received 6 June 2012 emphasize the need to improve biosecurity measures at the human–swine interface, including Accepted 6 July 2012 influenza vaccination of swine workers.

INTRODUCTION major continents: Africa (Njabo et al., 2012), Asia (Kim et al., 2011; Sreta et al., 2010), Australia (Holyoake et al., The 2009 pandemic H1N1 virus (H1N1pdm09) represents 2011), Europe (Hofshagen et al., 2009; Howard et al., 2011) the best-documented emergence of a swine pathogen in and South America (Pereda et al., 2010). Upon introduction humans, particularly as the virus was associated with into the swine population, H1N1pdm09 viruses co-circu- widespread global morbidity, mortality and years of life lost lated with endemic swine influenza viruses and exchanged in humans (Viboud et al., 2010). The H1N1pdm09 virus genome segments via reassortment (Ducatez et al., 2011; was generated by a reassortment event between Eurasian Lam et al., 2011; Vijaykrishna et al., 2010). In 2011, 12 swine H1N1 influenza viruses and North American triple- reassortant H3N2 (H3N2v) swine influenza viruses with reassortant H1 viruses, with the former contributing the N1 matrix (M) segments of pandemic H1N1pdm09 origin were and M segments and the latter donating the PB2, PB1, PA, isolated from humans in the USA (CDC, 2012). H1, NP and NS segments (Garten et al., 2009). Although similar influenza virus reassortants containing segments of Seasonal human influenza viruses have also been isolated both Eurasian swine virus and triple-reassortant swine virus periodically from swine, with a few major lineages becoming origins have been detected in Asia (Lam et al., 2011; Smith endemic in swine: most notably the human-origin H3N2 et al., 2009), no ‘smoking gun’ viruses that are closely related viruses that proliferated in European swine in the 1970s progenitors have been detected in swine in any locality. (Ottis et al., 1982), the H1d lineage of human origin that Following the identification of H1N1pdm09 in humans in emerged in North American swine in 2003 (Karasin et al., April 2009, the virus was transmitted rapidly from humans 2006), and the human H3, N2 and PB1 segments that were back into swine. The first isolation of a H1N1pdm09 virus associated with the triple-reassortant viruses that were in swine was from a pig farm in Alberta, Canada, in May identified in 1998 in North American swine (Zhou et al., 2009 (Howden et al., 2009), and the H1N1pdm09 virus was 1999). In addition, singletons and smaller clusters of swine subsequently isolated from outbreaks in swine in all other influenza viruses that contain one or more segments of human seasonal influenza virus origin have been identified Seven supplementary figures and two supplementary tables are in several countries, including Argentina (Cappuccio et al., available with the online version of this paper. 2011; Pereda et al., 2011) and Italy (Moreno et al., 2012).

044974 Printed in Great Britain 2195 2196 others and Nelson I. M. 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 of H1N1pdm09 influenza virus from humans into swine during 2009–2011

Introduction Country of State or province of Month/year of No. of isolates Bootstrap support (%) for viral introductionsD no. collection* collection (if available)* collection (swine) 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

ora fGnrlVirology General of Journal 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

93 38 USA IA, IL Nov–Dec 2010 3 98 97d Human-to-swine transmission of influenza virus

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 main aim of our study was therefore to estimate, using a simple D phylogenetic approach, the total number of introductions of the H1N1pdm09 virus from humans into swine using a dataset of H1N1pdm09 viruses collected globally in swine during 2009–2011. We then compared this estimate with the equivalent transmission frequency of human seasonal

; IL, Illinois; KY, Kentucky; MN, (non-pandemic) H1 and H3 into swine since 1990.

RESULTS

Frequent transmission of H1N1pdm09 virus from humans into swine Across the haemagglutinin (HA), neuraminidase (NA) and

Bootstrap support (%) for viral introductions concatenated internal gene phylogenies, we identified a total of 49 discrete introductions of H1N1pdm09 influenza 70 %. [ , virus from humans into swine during 2009–2011 Table 1; Figs

Internal gene phylogeny HA phylogeny NA phylogeny 1 and S1–S3 (available in JGV Online)]. These introductions were observed in 12 countries and semi-autonomous regions: Australia, Canada, China, Costa Rica, Cuba, Hong Kong (SAR), Italy, Singapore, South Korea, Taiwan, Thailand and the USA. The majority of viral introductions (swine) into swine were identified in the USA and Canada (19 and No. of isolates eight introductions, respectively; Table 1), reflecting the higher number of H1N1pdm09 influenza virus sequences available from North American swine. Given our strict criteria for defining viral introductions (bootstrap values ¢70 %) and limited global sampling, these numbers are likely to underestimate the extent of global spillover from

collection humans to swine of the H1N1pdm09 virus significantly.

Month/year of Our conservative methods are particularly prone to miss introductions involving single isolates. For example, single H1N1pdm09 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 phylogenies presented in Figs S1–S3. State or province of collection (if available)* Frequency of human-to-swine transmission of seasonal influenza viruses Across the H1, H3, N1 and N2 phylogenies, at least 23 separate

70 %, but only for a subset of the isolatesintroductions in a given cluster. of seasonal influenza virus from humans to ¢ swine were observed during the period 1990–2011 (Figs S4– collection* Country of S7). The vast majority of these introductions were identified after 1996, when surveillance of influenza virus in swine increased. Six human-to-swine transmission events involved

cont. the human seasonal H1 segment, eight involved the human H3 segment, six involved the human N1 segment and 16 involved the human N2 segment (Table 2). The higher Introduction no. 394041424344 USA45 USA46 USA47 USA48 USA49 USA USA IA USA IL USA MN, USA NE NC, USA PA IL MN IL MN Nov 2010 Nov 2010–May IL, 2011 KY, Nov OH 2010–Mar Nov 2011 MN 2010–Feb 2011 TX Nov 2010–Feb Dec 2011 2010–Jan 2011 3 Jan–Mar 2011 3 Dec 2010–Mar Dec 2 2011 2010–Jan 2011 2 Feb–Apr 2011 3 2 Jul 2011 2 2 9 2 2 99 100 100 100 100 92 100 100 95 100 100 100 94 100 100 98 92 99 100 Bootstrap support was Boxes that are empty indicate that no sequences were available for these genome segments or that bootstrap support was d Table 1. Minnesota; MO, Missouri; NE,D Nebraska; OH, Ohio; NC, North Carolina; PA, Pennsylvania; TX, Texas. *HK, Hong Kong. For Canadian provinces: ALB, Alberta; MB, Manitoba; QC, Quebec; SK, Saskatchewan; for Australia: VIC, Victoria; for thenumber USA: IA, Iowa of introductions of the human N2 segment is http://vir.sgmjournals.org 2197 M. I. Nelson and others

Fig. 1. Phylogenetic relationships of the six concatenated internal gene segment (PB2, PB1, PA, NP, M, NS) sequences from 100 H1N1pdm09 influenza virus isolates collected in swine globally during 2009–2011, and 1008 H1N1pdm09 influenza viruses collected globally in humans during the same time period (total dataset, n51108 sequences; colour-coded black). Each swine H1N1pdm09 isolate is colour-coded by the country of origin: USA, brown; Canada, light blue; Thailand, purple; South Korea, 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. An identical phylogeny with all viral introductions labelled in detail is provided in Fig. S1. probably related to several factors, including (a) the overall our final estimate, particularly those involving singletons. higher number of H3N2 virus introductions (n512) due to Six singleton swine isolates, including three from Argentina, the dominance of the H3N2 subtype in humans during the which were identified to be of human origin on both the HA period of this study, (b) the frequency of introductions (n53) and NA trees but were not supported by high bootstrap of the human H1N2 variant that circulated globally in humans values, are highlighted (letters a–g, Table 2; Figs S4–S7). from 2001 to 2003, and (c) the frequency of reassortment Additionally, eight swine H3N2 isolates collected in Hong involving N2 segments of human origin and HA segments of Kong during 2000–2002 are interspersed with human swine origin. For example, the 20 H1N2 reassortant viruses isolates collected from that same time period on both the from Italy that are associated with introduction #11 have N2 H3 and N2 phylogenies, suggesting additional introductions sequences that are related most closely to the N2 of human of human H3N2 viruses into swine in Hong Kong, although seasonal H3N2 isolates collected in 1997–1998, but H1 again lacking the phylogenetic resolution required to be sequences related to European swine influenza viruses. defined as discrete introductions. Twelve of the introductions of seasonal influenza virus were first identified in Asia, six were first identified in the USA, Estimated time periods of human-to-swine three in Canada and two in Europe (Table 2). As with the transmission H1N1pdm09 viruses, the methods that we used to define a human-to-swine transmission event are highly conservative, The long branch lengths associated with several introduc- and numerous probable introductions were excluded from tions of human-origin influenza viruses into swine indicate

2198 Journal of General Virology 93 http://vir.sgmjournals.org 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 bootstrap support ¢70 % for 23 introductions of seasonal influenza viruses (H1, H3, N1 and N2 segments) from humans into swine during 1990–2011

Introduction Country and year of collection Subtype Related human No. of isolates Bootstrap support (%) for viral introductions no. viruses (year) (swine) 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), H3N2 1995–1996 10 100 97 S. Korea 2005–2006, China 2007 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 H3N2 1998 126D 100 97, 70d (triple-reassortant II) 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 virus influenza of transmission Human-to-swine 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 2199 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. M. I. Nelson and others that numerous viral introductions circulated in swine for viruses has occurred in the USA, as six viral US introduc- many years before being detected. Given the high intensity tions include multiple states, representing viral dissemina- of sampling of influenza virus in humans, the duration of tion within the Midwestern USA (Table 1). Limited spread unsampled circulation in swine can be estimated simply within Canada between Alberta and Manitoba was also and directly by the differences in time between when the detected (introduction #2; Table 1). Within-country spread isolates were collected in swine and when the most closely was not observed in other countries where information on related human isolates were collected. For example, the the province or region of viral collection was also available, eight H3N2 viruses associated with introduction #13 were including China and Australia. collected in swine in Thailand in 2004–2009, but are related Similarly, 18 of the 23 introductions of human seasonal H1 most closely to A/Wuhan/359/1995(H3N2)-like viruses from 1995–1996 on the H3 and N2 phylogenies (Figs S4 and H3 influenza viruses into swine involve a single and S6, respectively), representing 8 years of unsampled country (Table 2). Six of these single-country introductions circulation in swine. The time difference between the 2008 involve singleton swine influenza viruses, with no evidence H3N2 and 2010 H1N2 isolates collected in swine in Argen- of onward transmission. In contrast, four viral introduc- tina and the most closely related human seasonal isolates is tions include isolates collected in different countries, approximately 6–8 years. Likewise, the isolate A/sw/Argentina/ indicating viral migration within North America and into CIP051-StaFeN2/2010(H1N2) is related most closely to the and within Asia. Aside from the major introduction of reassortant H1N2 viruses that circulated in humans during triple-reassortant H3N2 viruses that spread from North 2002–2003 (Figs S5 and S6), suggesting that this virus has America to Asia (introduction #4) and from the USA to circulated undetected in swine in Argentina or elsewhere Canada (introduction #10), possible limited viral migra- since at least 2003, when the H1N2 viruses disappeared tion occurred between Taiwan and Indonesia (introduc- globally in humans. Incorporating more viral sequence data tion #9) and the USA and South Korea (introduction #8). from swine by also including partial HA (HA1) sequences filled in some of these gaps in surveillance (H1–HA1 and H3–HA1 trees available from the authors upon request). For DISCUSSION example, the USA 2007–2011 (d-1) introduction (#18) is Despite evolving the capability to transmit efficiently in detected 2 years earlier in 2005 when HA1 data are included. humans, our results indicate that the H1N1pdm09 influenza Finally, 11 years separate the three H3N2 swine isolates virus has retained its ability to transmit back into swine and collected in South Korea in 2007 (introduction #17) from to co-circulate with other swine influenza viruses, increasing the most phylogenetically related human seasonal H3N2 genetic diversity and providing the opportunity for reassort- viruses collected in 1995–1996. However, the relatively short ment. The large number of H1N1pdm09 transmission events branch lengths associated with these 2007 South Korean observed in this study, even using conservative methods, isolates raises the question of whether the evolutionary rate highlights the frequency of human–swine contact rates was particularly low on this branch, or whether these se- globally and the continual exposure of swine to human quences could be erroneous. The short branch length that influenza viruses. Indeed, this transmission rate is consi- separates A/sw/Fujian/0325/2008(H1N1) (introduction #19) derably higher than would be inferred from the relatively from the most closely related human seasonal H1N1 viruses lower number (n523) of human-to-swine transmission collected in 2000–2001, relative to the approximately 7–8- events of seasonal H1 and H3 influenza viruses that year difference in collection date, also raises the question of were identified during the past two decades. Importantly, possible sequencing error. detection of seasonal human influenza viruses in swine did not increase during 2009–2011, despite widespread Spatial patterns of human-origin influenza viruses global circulation of seasonal H3N2 viruses in humans in swine during 2010–2011. Hence, the different transmission frequencies observed are not explained by the increase The number of swine H1N1pdm09 isolates associated with in swine influenza surveillance since 2009 and are unlikely each introduction (ranging from one to ten isolates; Table 1) to be an artefact of sampling. Furthermore, the low represents the extent of onward swine-to-swine transmission phylogenetic resolution of the H1N1pdm09 phylogeny, that can be inferred from our phylogenetic analysis. Although due to the virus’s recent emergence and relatively low the vast majority of introductions of human H1N1pdm09 genetic diversity, impeded the identification of what are viruses into swine exhibit local onward transmission in pigs, likely to be many more introductions of this virus into reflected in introductions that are associated with more than swine than could be defined here. one swine isolate, we infer that global viral movement has not yet been important in the dissemination of the H1N1pdm09 Although it is tempting to compare the frequencies of virus in swine. Specifically, each cluster of swine H1N1pdm09 human-to-swine transmission with the 27 swine-origin influenza viruses that are associated with a human-to-swine influenza viruses that were identified in humans in the USA transmission event is highly spatially structured, with no over a time period similar to that of our study (1990–2010) swine introduction including viruses collected in more than (Shu et al., 2012), such a comparison is biased greatly one country. However, within-country spread of H1N1pdm09 by the substantially higher levels of global surveillance of

2200 Journal of General Virology 93 Human-to-swine transmission of influenza virus influenza in humans than consistently occurs in swine. downloaded from the Influenza Virus Resource on GenBank; due However, the intensity of influenza surveillance in humans to the size of the original dataset (n52718 genomes), 50 genome does facilitate estimation of the time of emergence of the 23 sequences were selected randomly from highly sampled countries (i.e. those with .50 genome sequences) (Bao et al., 2008). introductions of human seasonal influenza viruses into swine. For example, the introductions of H1N1pdm09 viruses were To identify swine influenza viruses related to human seasonal H1 and generally detected in swine within 1 year, whereas several of H3 influenza viruses, four phylogenetic trees were inferred using all the human seasonal influenza viruses that were introduced full-length H1 (n5991, excluding pandemic viruses), H3 (n5326), 5 5 into swine were not detected for 5–10 years. Whilst the time N1 (n 858) and N2 (n 559) sequences collected from swine since 1990 that are available in GenBank (accession numbers are available to detection in swine may relate to intrinsic viral charac- from the authors). Due to the frequency of reassortment and low teristics (e.g. the virus must first adapt, then replicate com- availability of whole-genome sequence data for these swine viral petently in the new host, then spread to many hosts before isolates, alignments of the concatenated internal genome segments being detected), it is also possible that the difference in time were not included for this part of the study. These data came from between when a human-to-swine transmission event is de- Argentina, Belgium, Canada, China, Denmark, France, Germany, tected in swine and the year of collection of the most closely Hong Kong, Hungary, Italy, Japan, Spain, South Korea, Sweden, related human influenza viruses provides an indication of Taiwan, Thailand, the UK and the USA. From these phylogenies, 152 human-origin swine H1 sequences, 221 human-origin swine H3 intensity of swine surveillance in a given region. The smallest sequences, 18 human-origin swine N1 sequences and 430 human- gaps in detection are in Canada, the USA and Hong Kong, origin swine N2 sequences were identified globally and used in the where ongoing viral surveillance is conducted in swine, final analysis. This analysis also included, as background data, 1000 while larger time gaps occurred in countries such as South randomly selected human seasonal H1, H3, N1 and N2 segments Korea, Italy, Thailand and Argentina, where sampling collected from 1990 to 2011. A large clade of European sequences (e.g. appears to be more sporadic, based on publicly available A/sw/Scotland/410440/1994) that were of human seasonal H1 virus sequence data. origin were not included because they clearly were introduced from humans into swine at least a decade prior to 1990, the beginning of The global frequency of introductions of H1N1pdm09 our study period. viruses into swine also provides an opportunity to track, in To identify any additional human-to-swine transmission events that detail, the onward transmission and spatial dissemination of could not be detected from trees inferred with full-length HA these new viral clades and how they relate to swine move- sequence data, two additional phylogenies were inferred using all ments (Nelson et al., 2011). It will be particularly important partial HA1 sequence data (918 nt) from the swine H1 (n51200 to determine which of these H1N1pdm09 lineages sustains sequences) and H3 (n51290 sequences) subtypes available since 1990, transmission in swine over the long term, to observe their including HA1 sequence data from the same 1000 human influenza population dynamics in a new swine host and to identify viruses that were selected as background in the full-length HA phylogenies. Using HA1 data, we identified 50 additional swine H1 which lineages spread globally. The multiple introductions sequences of human origin and 70 additional swine H3 sequences of human influenza viruses into swine provide a natural of human origin. However, all but two isolates [A/sw/Obihiro/3/ experiment for observing adaptive evolution of influenza 1993(H3N2) and A/sw/Obihiro/1/1994(H3N2)] were associated with from human to swine hosts, in identifying instances of introductions that are already identified in the analysis of full-length parallel adaptive evolution, and in the comparison of rates HA and NA sequences. of evolutionary change and selection pressures along the branches that define human-to-swine transmission events. Phylogenetic analysis of H1N1pdm09. Alignments were con- structed manually using the Se-Al program (Rambaut, 2002) for three Clearly, the ability to trace such dynamics relies upon the sets of swine H1N1pdm09 virus sequence data, with the 1008 human continued surveillance of influenza in swine globally, even H1N1pdm09 isolates included as background: (i) the HA segment after the initial interest in the 2009 H1N1 pandemic has [n5232 swine H1N1pdm09 isolates, 199 of which were downloaded subsided. 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 METHODS Animal Health Laboratory Network (NAHLN) Swine Influenza Sur- Sequence data used in this study. To estimate the number of veillance System]; (ii) the NA segment (n5229 swine H1N1pdm09 human-to-swine transmission events of the H1N1pdm09 virus, we isolates, 197 from GenBank and 32 from UMVDL); and (iii) compiled gene sequence data from 263 H1N1pdm09 influenza viruses concatenated internal gene segments (n5100 swine H1N1pdm09 that were collected in swine during 2009–2011 in 18 countries: isolates, all downloaded from GenBank, with all reassortants Argentina, Australia, Brazil, Cameroon, Canada, China, Colombia, identified from individual gene trees and excluded). For each Costa Rica, Cuba, Hong Kong, Japan, Mexico, Singapore, South alignment, a maximum-likelihood (ML) tree was inferred using the Korea, Taiwan, Thailand, the UK and the USA (Table S1). Due to the PhyML v3.0 program (Guindon et al., 2010), employing SPR (subtree frequency of reassortment between H1N1pdm09 viruses and other pruning and regrafting) branch-swapping and a general time-reversible swine influenza viruses involving the HA and NA segments, con- model (GTR) model of nucleotide substitution with gamma-distributed catenated internal gene segments (PB2, PB1, PA, NP, M and NS) and rate variation among sites. Statistical support for individual nodes was the HA and NA segments were studied separately. Separate phylo- estimated using 1000 replicate neighbour-joining trees inferred using genies were also inferred for each of the internal gene segments to PAUP* and assuming the GTR+gamma model of nucleotide substi- identify and remove any viruses with reassorted internal genes. As tution (Swofford, 2003). Well-supported nodes (¢70 % bootstrap background data, 1008 complete genome sequences from H1N1pdm09 support) defining human-to-swine viral introductions were identified influenza viruses that were collected in humans during 2009–2011 were visually on each phylogeny. To mitigate possible effects of sequencing http://vir.sgmjournals.org 2201 M. I. Nelson and others error, the several isolates that were separated by extremely long branch Hofshagen, M., Gjerset, B., Er, C., Tarpai, A., Brun, E., Dannevig, B., lengths were only categorized as discrete introductions when multiple Bruheim, T., Fostad, I. G., Iversen, B. & other authors (2009). isolates from the same country exhibited the same phylogenetic pattern. Pandemic influenza A(H1N1)v: human to pig transmission in Norway? Euro Surveill 14, 19406. Phylogenetic analysis of seasonal H1 and H3. Alignments were Holyoake, P. K., Kirkland, P. D., Davis, R. J., Arzey, K. E., Watson, J., manually constructed using Se-Al for four sets of swine influenza Lunt, R. A., Wang, J., Wong, F., Moloney, B. J. & Dunn, S. E. (2011). virus sequences that were identified as of human seasonal (non- The first identified case of pandemic H1N1 influenza in pigs in pandemic) influenza virus origin: (i) 152 H1 sequences, (ii) 221 H3 Australia. Aust Vet J 89, 427–431. sequences, (iii) 18 N1 sequences and (iv) 430 N2 sequences. As Howard, W. A., Essen, S. C., Strugnell, B. W., Russell, C., Barass, L., background, 1000 human influenza viruses were selected randomly Reid, S. M. & Brown, I. H. (2011). Reassortant pandemic (H1N1) 2009 for each segment and downloaded from GenBank. For each virus in pigs, United Kingdom. Emerg Infect Dis 17, 1049–1052. alignment, an ML tree was inferred as described above to identify well-supported nodes (¢70 % bootstrap support) defining introduc- Howden, K. J., Brockhoff, E. J., Caya, F. D., McLeod, L. J., Lavoie, M., tions of influenza viruses from humans to swine. The estimated time Ing, J. D., Bystrom, J. M., Alexandersen, S., Pasick, J. M. & other periods of human-to-swine transmission were inferred directly from authors (2009). An investigation into human pandemic influenza the phylogeny by identifying the most closely related human viruses virus (H1N1) 2009 on an Alberta swine farm. Can Vet J 50, 1153– (the sampling date of which is known). This simple phylogenetic 1161. method produced results (i.e. times to common ancestry) similar Karasin, A. I., Carman, S. & Olsen, C. W. (2006). Identification of to those obtained using the Bayesian Markov chain Monte Carlo human H1N2 and human-swine reassortant H1N2 and H1N1 approach available in the BEAST program (Drummond & Rambaut, influenza A viruses among pigs in Ontario, Canada (2003 to 2005). 2007) and performed during a previous analysis of human influenza J Clin Microbiol 44, 1123–1126. viruses that were transmitted to swine (Nelson et al., 2011). Kim, S. H., Moon, O. K., Lee, K. K., Song, Y. K., Yeo, C. I., Bae, C. W., Yoon, H., Lee, O. S., Lee, J. H. & Park, C. K. (2011). Outbreak of pandemic influenza (H1N1) 2009 in pigs in Korea. Vet Rec 169, 155. ACKNOWLEDGEMENTS Lam, T. T., Zhu, H., Wang, J., Smith, D. K., Holmes, E. C., Webster, R. G., This research was conducted within the context of the Multinational Webby, R., Peiris, J. M. & Guan, Y. (2011). 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http://vir.sgmjournals.org 2203 State of the Field

Plant Viruses. Invaders of Cells and Pirates of Cellular 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 when Beachy, 1999; Oparka, 2004) to intercellular transport the science of virology was born (for review, see (Waigmann et al., 2004) to gene silencing (Moissiard Creager, 2002), are obligate parasites on their hosts. and Voinnet, 2004). Through their life cycle, from virus accumulation to This focus issue reports new insights into how intracellular,local, and systemic movement, viruses uti- viruses may utilize host factors to accumulate and lize plant proteins, normally involved in host-specific move intracellularly to position for intercellular move- activities, for their own purposes. Although the first ment (Chen et al., 2005; Ju et al., 2005; Liu et al., 2005). identification of a host protein interacting with plant Also, information further illuminating the ‘‘give and viral RNA took place more than 25 years ago (for take’’ between virus and host factors battling for review, see Buck, 1999; Waigmann et al., 2004), the true control during RNAi is presented (Chellappan et al., complexity of this interaction between plant viruses 2005; Liu et al., 2005; Schwach et al., 2005). Update and their hosts to allow virus accumulation and spread articles on virus-host interactions during virus repli- is just now becoming understood. cation and movement in this issue review recent In addition, the ability of the host to defend itself information in these areas to provide clues for pro- against virus replication and spread is now known to be ductive future research (Boevink and Oparka, 2005; much more complex than was thought not long ago. Thivierge et al., 2005). In this State of the Field editorial, During the early 1990s, the first findings were pub- we introduce the research and Update articles in this lished suggesting that a plant host defense system tar- issue and review recent literature on virus-host inter- geting viral RNA with extreme sequence specificity actions not addressed in the Updates. existed (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 defense VIRUS ACCUMULATION system now referred to as the RNA interference (RNAi), but with time were well differentiated from For both DNA and RNA plant viruses, the accumu- the better studied host and transgene defense systems lation of progeny virus involves translation and rep- mediated through proteins (e.g. the hypersensitive lication of viral sequences (Buck, 1999; Ahlquist et al., reaction of Nicotiana tabacum cv Xanthi NN against 2003; Noueiry and Ahlquist, 2003; Hanley-Bowdoin tobacco mosaic virus [TMV] and coat protein-mediated et al., 2004; Ishikawa and Okada, 2004; Ra¨jamaki et al., resistance; Beachy, 1999; Marathe et al., 2002). In the last 2004, and refs. therein). These plant viruses rely on the few years, plant molecular virologists and biologists host to provide factors to aid their accumulation. The have moved with increasing speed to document the Update article by Thivierge et al. (2005) presents incredibly complex interactions between virus and host a summary of recent insights into the mechanisms by factors necessary to allow or defeat virus infections in which positive-sense single-stranded RNA viruses the presence of RNAi (e.g. Baulcombe, 2004). Thus, take advantage of the host cell mRNA processing plant viruses, besides their traditional role as causative and translation machinery. agents of numerous plant diseases, represent molecular Research on virus-host interactions during DNA tools to examine and dissect diverse basic cellular virus accumulation has also moved forward. For ex- processes in plants, ranging from intracellular trans- ample, an NAC domain protein, SINAC1, from tomato port and nucleocytoplasmic shuttling (Lazarowitz and (Solanum lycopersicum) that interacts with a geminivirus replication enhancer (REn) protein was identified and

1 suggested to participate in viral replication (Selth et al., The work in our labs is supported by grants from the National 2005). NAC family members, which function in plant Institutes of Health, National Science Foundation, U.S. Department development and defense responses (e.g. Xie et al., of Agriculture, U.S.-Israel Binational Research and Development Fund, and U.S.-Israel Binational Science Foundation to V.C., and by 2000; Hegedus et al., 2003), are known to interact with the Samuel Roberts Noble Foundation to R.S.N. other geminivirus proteins, such as RepA, but in those * Corresponding author; e-mail [email protected]; instances they inhibited rather than promoted viral fax 631–632–8575. replication (Xie et al., 1999). Furthermore, a NAC pro- www.plantphysiol.org/cgi/doi/10.1104/pp.104.900167. tein interaction with an RNA virus coat protein is

Plant Physiology, August 2005, Vol. 138, pp. 1809–1814, www.plantphysiol.org Ó 2005 American Society of Plant Biologists 1809 Nelson and Citovsky necessary during a resistance response in Arabidopsis degradation of the viral MPs (Gillespie et al., 2002; (Arabidopsis thaliana; Ren et al., 2000). That host proteins Kragler et al., 2003). In this issue, Liu et al. (2005) from a single family display different functions during demonstrate the role of microfilaments in cell-to-cell DNA and RNA virus infection illustrates the complex- movement of TMV. Disruption of microfilaments by ity of the virus-host interaction process. pharmacological agents or by virus-induced gene si- Large-scale screening for host factors that affect lencing compromised TMV spread from cell to cell, but RNA virus accumulation has been undertaken using it did not significantly affect viral accumulation within yeast as an alternative host distinguished by a wealth the infected cells (Liu et al., 2005). Furthermore, this of well-characterized mutants (e.g. Kushner et al., study demonstrated the potential involvement of an- 2003; Panavas et al., 2005). These experiments showed other TMV factor, the 126-kD protein, in viral transport that host genes involved in viral accumulation may along microfilaments; the 126-kD protein was shown to differ between viruses. For example, while brome associate with viral replication complexes, modulate mosaic virus and tomato bushy stunt virus each are their size, and potentially mediate their interaction affected in their accumulation by approximately 100 with and movement along the microfilament network host genes, only 14 of these genes overlap between (Liu et al., 2005). viruses. The overlapping genes encoded proteins be- Increasing evidence suggests that the cytoskeletal longing mainly to three functional groups: protein network does not function alone in viral transport to biosynthesis, protein metabolism, and transcription/ and through plasmodesmata. Instead, it may act to- DNA remodeling (Panavas et al., 2005). Interestingly, gether with the endomembrane transport system of the no overlap existed between tomato bushy stunt virus host cell. Specifically, many viral MPs may be delivered and brome mosaic virus for genes involved in protein to plasmodesmata via the endoplasmic reticulum (ER), targeting, membrane association, vesicle transport, or while actin/myosin filaments may regulate the flow of lipid metabolism (Panavas et al., 2005), suggesting that proteins in the ER membrane (Boevink and Oparka, there are important differences between these viruses 2005). Two articles in this issue address the role of ER in for host membrane targeting and intracellular trans- viral cell-to-cell transport and plasmodesmal targeting. port. Although analysis in yeast allows a high- Ju et al. (2005) show that the potato potexvirus X (PVX) throughput analysis of yeast host factors that affect triple gene block (TGB) p2, one of the proteins required plant virus accumulation, it is important to supplement for movement of this group 2 member of the TGB- these data with information obtained in plant cells, for containing viruses, associates with ER-derived vesi- example, using a recently developed technology to cles, which in turn colocalize with actin filaments. study virus replication in a cell-free system of mem- Intriguingly, no association of the TGBp2 with Golgi brane-containing extract from uninfected evacuolated vesicles was detected (Ju et al., 2005), consistent with plant protoplasts (Komoda et al., 2004). The potential to findings in a recent report studying the movement of utilize protoplasts from mutant plants silenced for TGBp2-containing structures in tissue infected with expression of specific plant genes identified through potato mop-top virus, a group 1 member of the TGB- the yeast-based selection is very exciting. containing viruses (Haupt et al., 2005). Thus, these viruses likely use an ER-dependent pathway for plasmodesmal targeting, which is different from the INTRACELLULAR AND INTERCELLULAR MOVEMENT Golgi-dependent targeting to plasmodesmata recently demonstrated for some cellular proteins (Sagi et al., To spread between cells, viruses must first move 2005). from their replication sites to plasmodesmata at the cell The association of the potexviral TGBp2 MP with periphery and then traverse these intercellular chan- microfilaments and ER resembles similar associations nels to enter the neighboring cell. Cell-to-cell transport of the tobamoviral MP and the 126-kD protein (McLean of most plant viruses is mediated by specific virally et al., 1995; Heinlein et al., 1998; Hagiwara et al., 2003; encoded factors termed movement proteins (MPs), the Liu et al., 2005). This resemblance indicates physical function of which may be augmented by other viral and functional similarities between MPs and move- proteins (for review, see Morozov and Solovyev, 2003; ment-associated proteins of potexviruses and tobamo- Ra¨jamaki et al., 2004; Waigmann et al., 2004). The viruses, suggesting that both viral groups utilize majority of the cell-to-cell transport machinery, how- a similar method of intracellular movement, at least ever, is presumed to be provided by the host cell. One through a portion of this passage (Nelson, 2005). such host transport apparatus is the cytoskeleton. For TMV, the role of the ER translocation and plas- Although plant cytoskeletal elements were implicated modesmal targeting was explored by Chen et al. (2005) in viral cell-to-cell transport a decade ago (Heinlein using calreticulin, a cellular protein that localizes to et al., 1995; McLean et al., 1995), the relative roles of plasmodesmata (Baluska et al., 1999; Michalak et al., microtubules and microfilaments in the transport pro- 1999; Chen et al., 2005). This study showed that the cess are just emerging. Recent data suggest that, for N-terminal signal peptide was critical for the ability of TMV, microfilaments participate in the cell-to-cell calreticulin to accumulate within plasmodesmata (Chen movement of the virus, whereas microtubules and et al., 2005). Based on these observations, it is tempting to microtubule-associated proteins may be involved in speculate that plasmodesmal targeting involves two dis-

1810 Plant Physiol. Vol. 138, 2005 State of the Field tinct signals, a signal to enter the ER network and a puta- Waigmann et al., 2000; Trutnyeva et al., 2005), while tive plasmodesmata localization signal. Consistent with the functions of other MP-interacting proteins in this this idea, several types of viral MPs that ‘‘gate’’ plasmo- process remain obscure, awaiting future studies. desmata (e.g. Waigmann et al., 1994; Tamai and Meshi, 2001) have been shown also to associate with the ER (e.g. VIRUSES VERSUS RNAi HOST DEFENSE Heinlein et al., 1998; Haupt et al., 2005; Ju et al., 2005). Chen et al. (2005) also showed that calreticulin Virus-host interactions during RNAi in plants are interacts with TMV MP and that overexpression of complex and understood only at a rudimentary level. calreticulin in transgenic plants redirects TMV from In general, plants have multiple RNA silencing path- plasmodesmata to microtubules and compromises cell- ways with diverse biological roles (Baulcombe, 2004). to-cell transport of the virus. A potential, albeit indirect, These include the regulation of gene expression and functional link between viral MPs and calreticulin also importantly, for this short review, the control of virus may be inferred from the observations that one of the accumulation. The analysis of the RNAi pathway two MPs of the turnip crinkle virus (TCV) interacts controlling virus accumulation is complicated because with an Arabidopsis protein containing two RGD cell- some of the host genes involved in this process also attachment sequences (Lin and Heaton, 2001) that are function in regulating host gene expression. In addi- recognized by integrins (Campbell et al., 2000), which tion, viruses themselves modify the final outcome by in turn interact with calreticulin (Dedhar, 1994). their expression of proteins that defeat the system, i.e. Possible roles of the calreticulin-MP interaction in suppressors of RNA silencing. For a more complete regulation of plasmodesmal permeability are dis- understanding of this rapidly evolving area, there are cussed in the Update article by Boevink and Oparka many excellent recent reviews (Baulcombe, 2004; Ding (2005) in this issue. These authors present a review of et al., 2004a; Moissiard and Voinnet, 2004). the latest trends and discoveries regarding the role of RNA silencing involves the recognition of a target the ER/actin network in intracellular transport, rec- RNA and its subsequent destruction. This occurs via ognition of adhesion sites at the cell periphery, mod- a multistep enzymatic pathway including, in plants, ification of plasmodesmata by alteration of the cell an RNA-dependent RNA polymerase (RdRP; now wall structure, Hsp70 chaperones as potential trans- referred to as RDR), an RNase-III-type dicer-like location factors, and regulation of viral cell-to-cell endonuclease (DCL), putative members of the RNA- movement (Boevink and Oparka, 2005). induced silencing complex such as Argonaute, which Recently, a potential link between virus accumula- likely binds RNA, and other proteins that may support tion and cell-to-cell movement was identified when the RNA-induced silencing complex activity, such as eukaryotic translation factors eIF4E and eIF(iso)4E, DEAD box helicases (SDE3; for review, see Baulcombe, which are required for accumulation of potyviruses 2004; Meister and Tuschl, 2004). The majority of these (Duprat et al., 2002; Lellis et al., 2002; Ruffel et al., 2002; proteins are members of gene families, and it is this Nicaise et al., 2003), were also shown to aid in virus cell- multiplicity of family members that allows the plant to to-cell movement (Gao et al., 2004). These observations respond to widely varying needs (e.g. plant develop- supported earlier findings where plant mutants with ment and defense against virus invasion) and compli- altered eIF4E activity exhibit limited virus spread cates our ability to understand each system. (Arroyo et al., 1996). It has been speculated that poty- One way to simplify this issue is to identify natural virus intracellular movement may occur via an inter- or created plant knockout mutants for each gene action of eIF4E with eIF4G, which then binds involved in RNA silencing and study their loss-of- microtubules (Lellis et al., 2002). Regardless of the function phenotype during virus challenge. Using this mechanism of eIF4E-mediated virus movement, it is approach, Arabidopsis DCL2 was found to be re- important to realize that host proteins may function in quired for protection against TCV (Xie et al., 2004). several steps of the virus infection process, e.g. in the Arabidopsis SDE3 was required for protection against case of eIF4E, both in virus translation and/or replica- cucumber mosaic virus (CMV) but not tobacco rattle tion and in viral cell-to-cell movement. virus (TRV; Dalmay et al., 2001). For the RDRs, the Finally, in recent years, viral MPs have been shown tobacco RDR1 was required for protection against to interact with numerous other cellular proteins, TMV and PVX, while its Arabidopsis homolog was such as pectin methylesterases (Dorokhov et al., required for protection against TMV-cg, a tobamovirus 1999; Chen et al., 2000; Chen and Citovsky, 2003), very closely related to turnip vein clearing virus protein kinases (Yoshioka et al., 2004), homeodomain (TVCV; Lartey et al., 1993), and TRV (Xie et al., 2001; proteins (Desvoyes et al., 2002), DnaJ-like proteins Yu et al., 2003). Interestingly, Nicotiana benthamiana is (Soellick et al., 2000; von Bargen et al., 2001), rab a natural mutant for RDR1, and transgenic expression acceptor-related proteins (Huang et al., 2001), b-1,3- of an RDR1 ortholog from Medicago truncatula en- glucanase-interacting proteins (Fridborg et al., 2003), hanced its susceptibility to TMV, TVCV, and sunn and transcriptional coactivators (Matsushita et al., hemp mosaic virus (a tobamovirus, but only distantly 2001, 2002). To date, only protein kinases have been related to TMV and TVCV), but not CMVor PVX (Yang shown to play a role in viral intercellular move- et al., 2004). Similarly, RDR6 was required for pro- ment (Citovsky et al., 1993; Kawakami et al., 1999; tection against CMV, but not turnip mosaic virus,

Plant Physiol. Vol. 138, 2005 1811 Nelson and Citovsky

TVCV, TCV, or TRV in Arabidopsis (Dalmay et al., crease in siRNA steady-state levels was most striking 2000; Mourrain et al., 2000). Thus, specific RDRs likely (3- to 6-fold) for geminiviruses not associated with recognize different viruses; RDR1 is required for pro- a recovery phenomenon (i.e. producing fewer symp- tection against tobamoviruses and TRV, while RDR6 is toms over time) compared with those that were required for protection against CMV. associated with a recovery phenomenon. This dra- In this issue, Schwach et al. (2005) report that RDR6 matic increase in siRNAs also was correlated with the in N. benthamiana is required to inhibit infections by presence of one of two viral suppressors in these PVX, potato virus Y, and CMV, in the presence of its Y geminiviruses (Vanitharani et al., 2004; Chellappan satellite RNA, but has no effect on infections by TMV, et al., 2005). The critical importance of controlling tem- TRV, TCV, and CMV, in the absence of the Y satellite perature when studying RNAi or applying it in agri- RNA. During infection with PVX, RDR6 prevented the culture is also highlighted in this work (Chellappan systemic (including meristems), but not local, infection et al., 2005). of plants (Schwach et al., 2005). Grafting experiments Last, it is interesting that connections between the showed that RDR6 is required for cells to respond to induction of stress in cells, which could be considered a systemically moving silencing signal. The results of a host defense response, and virus movement may this study suggested that RDR6 produces double- exist. For example, exposure of plants to abiotic stress, stranded RNA precursors from the silencing signal e.g. low levels of heavy metal cadmium, blocks viral that are used to generate short-interfering RNAs systemic movement (Citovsky et al., 1998; Ghoshroy (siRNAs), which in turn allow an immediate silencing et al., 1998; Ueki and Citovsky, 2002). At the other response against the target virus on its arrival extreme, stress may aid movement because host heat (Schwach et al., 2005). This information advances our shock protein (HSP) 70 (Aoki et al., 2002) and virus- understanding of the mechanism of the host RNAi- encoded HSP70-related proteins (Medina et al., 1999; mediated resistance pathway against virus infection. Alzhanova et al., 2001; Prokhnevsky et al., 2002) likely For example, as Schwach et al. (2005) suggest, exclu- help viral and/or host macromolecular transport sion of virus from the meristem is mediated by RNAi, through plasmodesmata. Interestingly, the induction and RDR6 is involved in this process. These results of host HSP70s during infection by plant RNA viruses also raise issues to consider for future work in this is driven by a general mechanism that senses the level area. For example, what are the virus and satellite of misfolded proteins in the cell, regardless of protein RNA targets telling us about the substrate structural origin, viral or host (Aparicio et al., 2005). The study by requirements for each RDR, or is it that factors other Ju et al. (2005) in this issue touched on the role of stress than substrate suitability control the ability of partic- in viral movement by showing that turnover of TGBp2 ular RDRs to control accumulation by specific viruses? was greater during virus infection than when it was Also, why does the Arabidopsis RDR6, but not N. expressed alone in plant cells. They also determined benthamiana RDR6, protect against CMV? that, in plant cells, TGBp2:green fluorescent protein It was also interesting that Schwach et al. (2005) had a longer half-life than free green fluorescent showed that RDR6 did not control cell-to-cell move- protein. Based on these observations, Ju et al. specu- ment of PVX, indicating that the silencing pathway in lated that cell stress, represented by increased protein which this enzyme functions does not target virus turnover, could aid movement of PVX between cells by intracellular or intercellular movement. In this issue, translocating TGBp2 or viral movement complex out Liu et al. (2005) reported that mutant TMVs expressing of the ER into the cytosol and making it available not 126-kD protein silencing suppressors of varying only for degradation but also for transport through strengths were also not altered in cell-to-cell move- plasmodesmata (Ju et al., 2005). It will be interesting to ment (for TMV suppressor characterization, see Ku- see if viruses indeed have pirated the host stress bota et al., 2003; Ding et al., 2004b). Earlier, such an response for their own purposes. unlinking of RNA silencing suppressor activity from cell-to-cell movement was demonstrated for the P15 suppressor from peanut clump peculovirus (Dunoyer ACKNOWLEDGMENT et al., 2002). It will be interesting to determine whether We thank Drs. Elison Blancaflor and Ping Xu for valuable comments on or not RNA silencing ever directly targets the intra- the manuscript. cellular or cell-to-cell movement forms of the viral RNA. It may be that these forms are always protected LITERATURE CITED from the host silencing machinery. Another article in this issue reports the effect of Ahlquist P, Noueiry AO, Lee WM, Kushner DB, Dye BT (2003) Host factors in positive-strand RNA virus genome replication. J Virol 77: temperature on the production of siRNAs in plants 8181–8186 challenged with various geminiviruses, demonstrat- Alzhanova DV, Napuli AJ, Creamer R, Dolja VV (2001) Cell-to-cell ing that RNA silencing increased as the temperature movement and assembly of a plant closterovirus: roles for the capsid was raised from 25°Cto30°C (Chellappan et al., 2005). proteins and Hsp70 homolog. EMBO J 20: 6997–7007 Aoki K, Kragler F, Xoconostle-Cazares B, Lucas WJ (2002) A subclass of This finding extends to DNA viruses what was found plant heat shock cognate 70 chaperones carries a motif that facilitates for an RNA virus, Cymbidium ringspot virus, in N. trafficking through plasmodesmata. Proc Natl Acad Sci USA 99: 16342– benthamiana (Szittya et al., 2003). Importantly, the in- 16347

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Aparicio F, Thomas CL, Lederer C, Niu Y, Wang D, Maule AJ (2005) Virus Fridborg I, Grainger J, Page A, Coleman M, Findlay K, Angell S (2003) induction of heat shock protein 70 reflects a general response to protein TIP, a novel host factor linking callose degradation with the cell-to-cell accumulation in the plant cytosol. Plant Physiol 138: 529–536 movement of Potato virus X. Mol Plant Microbe Interact 16: 132–140 Arroyo R, Soto MJ, Martinez-Zapater JM, Ponz F (1996) Impaired cell-to- Gao Z, Johansen E, Eyers S, Thomas CL, Ellis THN, Maule AJ (2004) The cell movement of potato virus Y in pepper plants carrying the ya(pr21) potyvirus recessive resistance gene, sbm1, identifies a novel role for trans- resistance gene. Mol Plant Microbe Interact 9: 314–318 lation initiation factor eIF4E in cell-to-cell trafficking. Plant J 40: 376–385 Baluska F, Samaj J, Napier R, Volkmann D (1999) Maize calreticulin Ghoshroy S, Freedman K, Lartey R, Citovsky V (1998) Inhibition of plant localizes preferentially to plasmodesmata in root apex. Plant J 19: viral systemic infection by non-toxic concentrations of cadmium. Plant J 481–488 13: 591–602 Baulcombe D (2004) RNA silencing in plants. Nature 431: 356–363 Gillespie T, Boevink P, Haupt S, Roberts AG, Toth R, Valentine T, Beachy RN (1999) Coat-protein-mediated resistance to tobacco mosaic Chapman S, Oparka KJ (2002) Movement protein reveals that micro- virus: discovery mechanisms and exploitation. Philos Trans R Soc Lond tubules are dispensable for the cell-to-cell movement of Tobacco mosaic BBiolSci354: 659–664 virus. Plant Cell 14: 1207–1222 Boevink P, Oparka KJ (2005) Virus-host interactions during movement Hagiwara Y, Komoda K, Yamanaka T, Tamai A, Meshi T, Funada R, process. 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Lindbo JA, Dougherty WG (1992) Untranslatable transcripts of the tobacco Schwach F, Vaistij FE, Jones L, Baulcombe DC (2005) An RNA-dependent etch virus coat protein gene sequence can interfere with tobacco RNA polymerase prevents meristem invasion by potato virus X and is etch virus replication in transgenic plants and protoplasts. Virology required for the activity but not the production of a systemic silencing 189: 725–733 signal. Plant Physiol 138: 1842–1852 Liu J-Z, Blancaflor EB, Nelson RS (2005) The tobacco mosaic virus 126- Selth LA, Dogra SC, Rasheed MS, Healy H, Randles JW, Rezaian MA kilodalton protein, a constituent of the virus replication complex, alone (2005) A NAC domain protein interacts with Tomato leaf curl virus or within the complex aligns with and traffics along microfilaments. replication accessory protein and enhances viral replication. 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Mol Plant Pathol 5: 71–82 of AC2 and AC4 of cassava geminiviruses in mediating synergism and Morozov SY, Solovyev AG (2003) Triple gene block: modular design of posttranscriptional gene silencing suppression. J Virol 78: 9487–9498 a multifunctional machine for plant virus movement. J Gen Virol 84: von Bargen S, Salchert K, Paape M, Piechulla B, Kellmann J (2001) 1351–1366 Interactions between the tomato spotted wilt virus movement pro- Mourrain P, Be´clin C, Elmayan T, Feuerbach F, Godon C, Morel JB, tein and plant proteins showing homologies to myosin, kinesin, and Jouette D, Lacombe AM, Nikic S, Picault N, et al (2000) Arabidopsis DnaJ-like chaperons. Plant Physiol Biochem 39: 1083–1093 SGS2 and SGS3 genes are required for posttranscriptional gene silencing Waigmann E, Chen MH, Bachmaier R, Ghoshroy S, Citovsky V (2000) and natural virus resistance. Cell 101: 533–542 Phosphorylation of tobacco mosaic virus cell-to-cell movement Nelson RS (2005) Movement of viruses to and through plasmodesmata. protein regulates viral movement in a host-specific fashion. EMBO J In K Oparka, ed, Plasmodesmata. Blackwell Publishing, Oxford, pp 19: 4875–4884 188–211 Waigmann E, Lucas W, Citovsky V, Zambryski PC (1994) Direct functional Nicaise V, German-Retana S, Sanjua´n R, Dubrana MP, Mazier M, assay for tobacco mosaic virus cell-to-cell movement protein and Maisonneuve B, Candresse T, Caranta C, LeGall O (2003) The eukary- otic translation initiation factor 4E controls lettuce susceptibility to the identification of a domain involved in increasing plasmodesmal per- Potyvirus Lettuce mosaic virus. Plant Physiol 132: 1272–1282 meability. 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Proc Natl Acad Sci USA 102: 7326–7331 proteins, novel members of the NAC domain family, isolated by their Prokhnevsky AI, Peremyslov VV, Napuli AJ, Dolja VV (2002) Interaction interaction with a geminivirus protein. Plant Mol Biol 39: 647–656 between long-distance transport factor and HSP70-related movement Xie Z, Fan B, Chen C, Chen Z (2001) An important role of an inducible protein of beet yellows virus. J Virol 76: 11003–11011 RNA-dependent RNA polymerase in plant antiviral defense. Proc Natl Ren T, Qu F, Morris TJ (2000) HRT gene function requires interaction Acad Sci USA 98: 6516–6521 between a NAC protein and viral capsid protein to confer resistance to Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman turnip crinkle virus. Plant Cell 12: 1917–1925 D, Jacobsen SE, Carrington JC (2004) Genetic and functional diversi- Ruffel S, Dussault MH, Palloix A, Moury B, Bendahmane A, Robaglia C, fication of small RNA pathways in plants. PLoS Biol 2: 642–652 Caranta C (2002) A natural recessive resistance gene against potato Yang SJ, Carter SA, Cole AB, Cheng NH, Nelson RS (2004) A natural virus Y in pepper corresponds to the eukaryotic initiation factor 4E variant of a host RNA-dependent RNA polymerase is associated with (eIF4E). Plant J 32: 1067–1075 increased susceptibility to viruses by Nicotiana benthamiana. Proc Natl Ra¨jamaki ML, Ma¨ki-Valkama T, Ma¨kinen K, Valkonen JP (2004) Infection Acad Sci USA 101: 6297–6302 with potyviruses. In N Talbot, ed, Plant-Pathogen Interactions. Black- Yoshioka K, Matsushita Y, Kasahara M, Konagaya KI, Nyunoya H (2004) well Publishing, Oxford, pp 68–91 Interaction of Tomato mosaic virus movement protein with tobacco RIO Sagi G, Katz A, Guenoune-Gelbart D, Epel BL (2005) Class 1 reversibly kinase. 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way, parallels were drawn between other oPinion icosahedral viruses: tailed dsDNA bacteri- ophages (Caudovirales order) and herpes- Virus evolution: how far does the viruses (Herpesviridae family)14–17, as well as dsRNA bacteriophages (Cystoviridae double β-barrel viral lineage extend? family) and reoviruses (Reoviridae fam- ily)18–20. These observations gave rise to the viral lineage hypothesis, which Mart Krupoviˇc and Dennis H. Bamford predicts a common origin for viruses that Abstract | During the past few years one of the most astonishing findings in the share the same capsid architecture, but infect hosts from different domains of field of virology has been the realization that viruses that infect hosts from all life7,21–23. Membership of a lineage is not three domains of life are often structurally similar. The recent burst of structural strictly dependent on genome-sequence information points to a need to create a new way to organize the virosphere similarities, mode of replication or genome that, in addition to the current classification, would reflect relationships organization (circular versus linear), as between virus families. Using the vertical β-barrel major capsid proteins and these traits vary between the members of the proposed lineages. Rather, it relies on ATPases related to known viral genome-packaging ATPases as examples, we common principles of virion architecture. can now re-evaluate the classification of viruses and virus-like genetic elements The set of genes that encode a capsid is from a structural standpoint. considered to be the hallmark of the virus, as only these determinants can define a Viruses are the most abundant biological enti- more surprising11. Not only did PRD1 and replicon as belonging to a virus rather than ties on the Earth (there are 1031–1032 virions adenovirus contain linear double-stranded some other entity; for example, a plasmid. in the biosphere; see Glossary)1. Nevertheless, (ds) DNA genomes that were replicated in This block of genes is therefore likely to be classification of living organisms on the basis a protein-primed manner, but the topology inherited vertically in every virus lineage21. of their 16S rRNA genes left no room for of their major capsid proteins (MCPs) and Ascribing viruses into such structure-based viruses on the universal tree of life. However, the organization of these subunits in the lineages not only bypasses the problem of it is now appreciated that viruses are not only capsid surface lattice, as well as the struc- the mosaic nature of viral genomes24, but ancient, but might also have played a major ture of their penton and spike proteins, also limits the enormous virosphere to a part in the emergence and consequent struc- were found to be similar12,13. In the same comparatively small number of lineages. ture of modern cellular life forms2–5. It is thus clear that comprehension of the virosphere, Table 1 | Viruses from the PRD1–adenovirus lineage including evolutionary relationships between individual viruses and viral families, is crucial Family* Host Morphology Genome Genome to fill the gaps in our understanding of the topology length (kb) biosphere. It has been generally considered Tectiviridae Gram-negative and Gram-positive Icosahedral Linear ~15 that viruses which infect evolutionary bacteria distant hosts (for example, bacteria and Corticoviridae Gram-negative bacteria Icosahedral Circular ~10 humans) have different origins6. However, STIV Archaea (Crenarchaeota) Icosahedral Circular 17,663 recent accumulation of structural informa- MVV Archaea (Euryarchaeota) Unknown Circular 12,039 tion has led to the proposal of a more unified TKV4 Archaea (Euryarchaeota) Unknown Circular 18,818 common-ancestral-based virus classification7. Adenoviridae Eukarya (vertebrates) Icosahedral Linear 26–45 Structural virology, already in its Iridoviridae Eukarya (vertebrates and Icosahedral Linear 140–303 youth when human rhinoviruses were invertebrates) found to be structurally related to small Asfarviridae Eukarya (vertebrates) Icosahedral Linear 170–190 plant RNA viruses, offered unexpected answers to the vogue questions regarding Phycodnaviridae Eukarya (unicellular) Icosahedral Linear or 160–560 circular origin and evolution of viruses8–10. The realization that the bacterial tectivirus Mimivirus Eukarya (unicellular) Icosahedral Linear 1,181,404 PRD1 (Tectiviridae family) and human Poxviridae Eukarya (vertebrates and Oval Linear 130–230 adenovirus (Adenoviridae family), which invertebrates) infect hosts from two different domains of Ascoviridae Eukarya (invertebrates) Oval Circular 120–180 life, might be evolutionary related was even *If a virus has not been ascribed to a family, its name is provided. STIV, Sulfolobus turreted icosahedral virus.

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a Viruses with double β‑barrel proteins STIV Adenovirus PRD1 The 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 PM2 PBCV-1 Mav-1_CI 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 b 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 Figure 1 | Structural features common to members of the PrNaD1–adenovirusture Reviews | Micr lineage.obiology a | A comparison of the available X-ray structures of the double β-barrel major capsid proteins Gram-positive Bacillus spp. was found to be 28 (MCPs) and the pseudoatomic model of the putative MCP of Maverick 1 from sea squirt Ciona different from PRD1 genetically , but nearly intestinalis (Mav-1_CI). STIV MCP (Sulfolobus turreted icosahedral virus MCP) is coloured cyan, identical structurally29. human adenovirus MCP is coloured purple, bacteriophage PrD1 MCP is coloured green, bac- bacteriophage PM2, the type member teriophage PM2 MCP is coloured blue and Paramecium bursaria Chlorella virus type 1 MCP of the Corticoviridae family, infects marine (PBCV-1 MCP) is coloured red. The putative MCP of Mav-1_CI is coloured red, whereas the Pseudoalteromonas species30. The PM2 virion insertions with respect to the corresponding PBCV-1 sequence are white. Protein sequences and its major structural proteins have been 79 of Mavericks were downloaded from the repbase Update database of repetitive elements (see analysed by both X-ray crystallography Further information). BioInfoBank Meta Server60 was used to predict the tertiary structure of and cryo-electron microscopy (EM)31,32. 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- Interestingly, X-ray analysis revealed that responding protein sequences of Mav-1_CI, and a three-dimensional model of the putative the double β-barrel MCP of PM2 (FIG. 1a) Mav-1_CI MCP was built. b | Architecture of the PrD1 virion (triangulation number of 25). lacks the bottom part, the typical N-terminal Apices of the red triangle combine the fivefold vertices (light shading) outlining the triangular α-helix, which contacts the viral membrane virus facet. Grey hexagons depict the morphology of the double β-barrel trimers. The inset in the PRD1 virion31 (discussed below). 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 Archaeal viruses. Viruses that infect of Oxford, UK. the archaeal domain are represented by the crenarchaeal Sulfolobus turreted icosahedral Several structure-based viral lineages this opinion as an example of a structur- virus (STIV). Cryo-EM analysis of the STIV have been proposed to date7. one of these ally defined viral lineage7,22,23. we discuss virion revealed similarities to bacteriophage lineages, the PRD1–adenovirus lineage, the available virion and capsid protein PRD1 (REF. 33). Subsequent X-ray analysis unites dsDNA tailless viruses that use structures to show how far the lineage of the STIV MCP revealed that three- similar architectural principles and are hypothesis can be extended and what it dimensional structures of PRD1 and STIV present in the largest number of different tells us about virus origin and evolution in MCPs are nearly identical34 (FIG. 1a). Although viral families; this lineage will be used in this particular lineage. 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 assume that the large MCP of adenoviruses evolved from a smaller version that is still Virus Family Genome (bp) T ref. found in NClDVs and prokaryotic viruses. Eukaryotic host Identification of a virus with an NClDV-like Mimivirus Not assigned 1,181,404 ~1,179 45 capsid protein and the genome organization of the adenovirus would be the best proof of PpV01 Phycodnaviridae? ~415,000 219 80 such a hypothesis. PBCV-1 Phycodnaviridae 330,743 169 39 A class of self-synthesizing DNA CIV Iridoviridae 212,482 147 39 transposons was recently identified in a Adenovirus Adenoviridae ~35,000 25 36 wide range of eukaryotic organisms (from nematodes to vertebrates)54,55. These trans- Prokaryotic host posable elements were named Mavericks54 STIV Not assigned 17,663 31 33 or Polintons55. Interestingly, Mavericks Bam35 Tectiviridae 14,935 25 29 possess several features that are common PrD1 Tectiviridae 14,927 25 12 to tectiviruses and adenoviruses, including genome organization and gene content55,56 PM2 Corticoviridae 10,079 21 32 (FIG. 2). A typical Maverick is 15–20 kb long, CIV, Chilo iridescent virus; PBCV-1, Paramecium bursaria Chlorella virus type 1; STIV, Sulfolobus turreted is flanked with inverted terminal repeats icosahedral virus, T, triangulation. and encodes approximately 10 protein spe- cies55,56. The most conserved set of encoded two putative proviruses, TKV4 and MVV, a highly conserved scaffolding protein proteins includes a protein-primed Polb, have been recently identified in the genomes (D13) that is homologous to the MCPs of an adenoviral cysteine protease, an ATPase of archaeal species from the second major other NClDVs42,43, and is known to play (similar to the packaging ATPases of internal phylum of Archaea, the Euryarchaeota35. an essential part during the formation of membrane-containing viruses) and an genome-containing, spherical immature integrase55,56. It is important to note that the Eukaryotic viruses. Adenoviruses infect virions47,48. Subsequent transition from imma- ATPase is the second protein that is common higher eukaryotes and have linear dsDNA ture to mature brick-shaped virions seems to to all membrane-containing members of the genomes that are replicated by a protein- be associated with the loss of D13 (REF. 49). PRD1–adenovirus lineage57. These ATPases primed mechanism. Various adenoviruses Intriguingly, D13 was shown to trimerize form a distinct clade in the FtsK–HerA have been subjected to intensive EM and and form an external, honeycomb-like lattice superfamily of cellular chromosome-pumping X-ray analyses36,37. unlike other members of that covers the lipid bilayer of the immature ATPases58 that is evolutionary distinct from the PRD1–adenovirus lineage, adenoviruses virion49. Similarly, a three-dimensional the packaging ATPases of tailed bacteri- do not have an internal membrane (dis- density map of the trimeric D13 homologue ophages and herpesviruses58,59. In addition cussed below). In addition, a high-resolu- in orf virus (Poxviridae family) was found tion X-ray structure of the MCP, as well as a to accommodate the electron-density map of cryo-EM-based reconstruction of the entire the PbCV-1 MCP50, indicating that D13 also Glossary box virion, is available for Paramecium bursaria adopts a double β-barrel fold. These observa- Biosphere Chlorella virus type 1 (PbCV-1; a member tions suggest that the immature virions of The global ecological system that integrates all living of the Phycodnaviridae family)38,39 (FIG. 1a). poxviruses resemble the mature icosahedral organisms. 49,50 The Phycodnaviridae family unites geneti- virions of the PRD1–adenovirus lineage . Capsomer cally diverse viruses that infect both marine Similar morphological transitions might The basic structural unit of the capsid of a virus. and freshwater algae40. Some members of have given rise to viruses of the Ascoviridae the Phycodnaviridae can lysogenize their family. Ascoviruses attack lepidopteran larvae Convergent evolution host cells by integrating into the cellular and pupae, and vary in shape from ovoid The process by which organisms that are not monophyletic independently evolve similar traits as a result of adaptation 41 51 chromosome . to bacilliform depending on the species . to ecological niches or similar environments. Comparative genome analyses of Comparative genomic and phylogenetic anal- large eukaryotic dsDNA viruses exposed yses unexpectedly revealed that ascoviruses Divergent evolution a conserved set of genes for four virus are likely to have evolved from an iridovirus The accumulation of differences between groups that can 52,53 lead to the formation of new species, usually as a result of families — Phycodnaviridae, Iridoviridae, ancestor . the adaptation of different groups of the same species to Asfarviridae, Poxviridae — and the recently different environments. isolated Mimivirus, which suggests that Mavericks: the missing link? these groups share a monophyletic origin. As discussed above, members of the Transposon It has been proposed that these group- Adenoviridae are outliers in the PRD1– A genetic element that can move from one locus of a chromosome to another through a recombinase-mediated (BOX 1) ings should be collectively called the adenovirus lineage . on the one reaction. nucleo-cytoplasmic large DNA viruses hand, the adenoviral MCP is much larger (NClDVs)42,43. Phycodnaviruses, asfarviruses, than the MCPs of the other members Triangulation number iridoviruses and the Mimivirus all possess of the PRD1–adenovirus lineage (FIG. 1a) (T ). Used to describe the number of subunits that exist in a capsid: an icosahedral capsid of triangulation number T an internal membrane that is surrounded and adenoviruses lack lipids as structural will possess 60 T subunits. by a large icosahedral capsid39,44,45, whereas components. on the other hand, adenovi- poxviruses are brick shaped46. However, ruses have the same genome organization Virosphere vaccinia virus (Poxviridae family) encodes and mode of replication as tectiviruses. we The total sum of all viruses.

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60 Box 1 | Capsid‑size variation using Structure Prediction Meta Server , the MCP of PbCV-1 was found to be the An interesting characteristic of the trimeric best template for structural modelling of PY double β‑barrel major capsid proteins (MCPs) is proteins38. In a subsequent analysis, the high their ability to serve as building blocks to form stereochemical quality of a three-dimensional shells of highly variable sizes (FIG. 1b). The X‑ray model of a selected PY protein (FIG. 1a) structure of bacteriophage PRD1 virions (~4 Å resolution) provided the first insights into the indicated that the Maverick protein PY has possible mechanisms of such a scalable the potential to adopt the same fold as the assembly12. The linearly extended protein P30 MCP of PbCV-1: the double β-barrel fold was found to connect the icosahedral vertices (M.K. and D.H.b., unpublished observations). and consequently define the size of the virion. So what are these large transposable Cryo‑electron microscopy analysis revealed a DNA elements of eukaryotes? It is possible similar protein in bacteriophage Bam35 2 that Mavericks represent the remnants of (REF. 29). Interestingly, a mutant of African 1 ancient viruses, as proposed by Pritham swine fever virus (Asfarviridae family) that forms and colleagues56. based on the presence tubular, MCP‑coated membrane structures of the putative MCP and the packaging instead of icosahedral particles has been 3 recently characterized72: a mutation in a single ATPase, the progenitor virus is proposed gene that encodes for a structural protein, to be a member of the PRD1–adenovirus pB438L, was responsible for this change. This is lineage. Retention and high conservation reminiscent of the phenotype observed during Mavericks? of these two genes as part of the Maverick the infection with tape‑measure protein genome, as well as the restricted size of P30‑deficient mutants of bacteriophage PRD1 Nature Reviews | Microbiology the genome itself, are intriguing. one (REF. 73). Additional information is needed to determine whether pB438L of ASFV is an equivalent explanation might be that Mavericks are of the tape‑measure protein of PRD1. still capable of forming virions and can The size of the capsid is proportional to the interior volume and consequently to the size of sometimes spread as genuine viruses. An the genome, which must be fitted into the capsid (TABLE 2). Strategies used by members of the alternative scenario is that the possible 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 ancestor virus of Mavericks, in contrast to shown in the figure. The numbers denote the different strategies: alteration of the triangulation (T) the two evolutionary schemes proposed number by viruses of the nucleo‑cytoplasmic large DNA virus (NCLDV) group (1); the use of larger above for NClDVs and adenoviruses building blocks (pseudohexamers) and removal of the internal membrane by adenoviruses (2) and (BOX 1), evolved a third tactic to avoid the conservation of the original capsid size by Mavericks at the expense of transition to a immune system of the evolving eukaryotic predominantly intracellular persistence as integrated transposable elements (3). host (see the figure in BOX 1). Instead of The beauty of the scalable system used by PRD1 is its predicted capacity to change the interior coding for a larger capsid that would accom- volume depending on the length of the genetic material to be packaged by altering the T number modate a larger genome, Mavericks changed (see the figure). The ability to package larger genomes might be crucial for successful infection. their life style to persist inside the host cell Although some scientists favour the hypothesis of reductive evolution to explain the emergence of without destroying it. However, unless this 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 change in life style occurred recently, this were parasitizing the same hosts, the Mimivirus genome evolved through multiple gene and scenario does not explain the conservation of segmental genome‑duplication events75,76. Similar conclusions were drawn from a recent analysis the two putative genes that encode for struc- of the entire NCLDV group77. These findings suggest that NCLDVs evolved from smaller viruses tural virion proteins or explain the apparent rather than from organisms that were even more complex. conservation of genome length, which is The other possible way to increase the inner capsid volume is to alter the MCP so that it can form likely to be constrained by capsid size. 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 Divergent versus convergent evolution same T number (T = 25) as the tectiviruses, whereas the genome size of adenoviruses is considerably The viral lineage hypothesis assumes that larger (TABLE 2). The larger capacity of the capsid is due to two main reorganizations. First, the origin of viruses in each lineage is mono- compared with PRD1, the two β‑barrels in the adenovirus hexon are further apart, which creates a phyletic and that diverse viral families in a trimer with a width of 97 Å compared with 80 Å in the MCP of PRD1 (REF. 11). Second, the internal divergent evolution membrane of adenoviruses has been removed. However, it should be noted that once the larger lineage arose as a result of . capsid is formed, packaging of the original (smaller) genome would be unlikely to lead to the unfortunately, a lack of significant sequence formation of infectious virions, as a lower genome‑packaging density would not be sufficient to similarity between viral genomes of the drive the initial injection of the viral genome into the host cell78. PRD1-like viruses prevents us from verifying such an assumption. Although it is not pos- sible to reject with certainty that convergent to walker A and walker b motifs, ATPases from the host together with loss of the inter- evolution led to the appearance of the double of these internal membrane-containing nal membrane59. Interestingly, the ATPases β-barrel fold, it should be realized that the viruses contain a conserved and essential encoded by Mavericks possess a P9/A32 protein fold is not the only feature shared by P9/A32 motif, which was proposed to be motif. In addition to the four proteins viruses in the lineage. The most conserved specific to membrane-containing viruses57. mentioned above — Polb, cysteine protease, feature of viruses of the PRD1–adenovirus Adenoviruses seem to use the ATP-binding ATPase and integrase — Mavericks encode lineage is the use of double β-barrel MCPs cassette (AbC)-type ATPase for genome four conserved hypothetical proteins, which to build the protein shell: pseudohexameric packaging, which might have been acquired have been named PX, PY, Pw and PZ55. trimers of the MCP are arrayed as triangular

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Virus or element by contrast, the other category, the viral self ATPase PolB MCP Pro genes21, includes genes that encode the key Frog adenovirus* structural components of the virion. Such ATPase PolB Pro MCP Int components eventually determine how the Maverick-2_XT virion is constructed and are specific to a given viral lineage, which indicates that they Pro PolB ATPase INT MCP are inherited vertically. The probability that Maverick-1_CI so many similarities between viruses within PolB ATPase MCP a viral lineage would arise as a result of PRD1* convergent evolution is low.

PolB ATPase MCP Evolutionary origin of the lineage Bam35 A number of hypotheses have been put forward to explain the origin of viruses (see, ATPase MCP STIV* for example, REFS 4,5,21,65). These hypoth- eses converge to one common feature: the Int MCM ATPase MCP viral capsid protein is the only component TKV4 that is unique to the viral world. Extending this idea, Raoult and Forterre66 have even RCR Int ATPase MCP MVV proposed to divide biological entities into ribosome-encoding organisms, which include RCR ATPase MCP eukaryotic, archaeal and bacterial organisms, PM2* and capsid-encoding organisms, which include viruses. Acquisition of the protein RCR ATPase MCP Int Corticoelement shell by a replicon was a cornerstone in the origin of viruses. This event seems to have 5 kb occurred multiple times, giving rise to diverse viral lineages, some of which still exist today. Figure 2 | Genome organization of selected members of the PrD1–adenovirusNature Revie wslineage | Micr obiologfrom ally The origin of capsid proteins is speculative. domains of life. Presented are the genome organizations of members of the PrD1–adenovirus line- 5 age from the Bacterial domain (PrD1, Bam35, PM2 and a corticoelement in the Vibrio splendidus one possibility, as suggested by Koonin et al. , 12B01 genome), the Archaeal domain (Sulfolobus turreted icosahedral virus (STIV), and proviruses is that viral hallmark genes (one of which is TKV4 and MVV) and the Eukarya (Frog adenovirus). The genomic organizations of Maverick-1_CI and the MCP-coding gene) might have descended Maverick-2_XT are also provided, which reside in the genomes of Ciona intestinalis and Xenopus directly from a primordial gene pool, as they tropicalis, respectively79 (see Further information). Genomic sequences were aligned using major are missing from the cellular gene content. capsid protein (MCP)-coding genes. Arrows indicate the direction of transcription. MCP-coding Structural and genomic data allow us genes are coloured blue, ATPase-coding genes are coloured yellow, replicase-coding genes are to extrapolate back in time and suggest an coloured red, recombinase-coding genes are coloured green and adenoviral protease-coding genes evolutionary trail of the double β-barrel pro- are coloured purple). The last five genomes are arbitrarily linearized for a more convenient alignment. teins and consequently viruses that use these Asterisks denote the viruses for which MCP X-ray structures are available. The genomes of Paramecium proteins for their assembly. FIGURE 3 combines 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, information about a number of viral families polymerase B; Pro, protease; rCr, rolling circle replication. that infect hosts from all three cellular domains. It should be noted, however, that the scenario presented is highly hypothetical, and plates to form the icosahedral virus capsid. Structurally similar viruses that can infect is meant only to stimulate further research Importantly, in all these viruses, the axes hosts from different domains of life have dif- and discussions on virus evolution. The of the β-barrels are perpendicular to the ferent gene contents, as interactions with the numbers represent the possible major turning capsid shell. Furthermore, the position of host cell change the gene content of the virus. points in the evolution of the members of genes in the genome is often conserved35. we therefore find it useful to divide viral the PRD1–adenovirus lineage and will be This is most clearly observed for prokaryotic genes into two groups. one category, referred discussed in numerical order. members of the PRD1–adenovirus lineage to as the non-self genes21, includes viral The double β-barrel fold seems to arise (FIG. 2). It should be noted that these viruses genes that are involved in the interaction through gene duplication9,23,31,38. In line with possess genomes of different topologies, with the host and can be exchanged between this proposal was the recent identification of and replication is carried out with the aid of unrelated viruses or even between the virus SH1 and P23-77, two membrane-containing non-homologous proteins, suggesting that and the host chromosome through hori- viruses that infect archaeal and bacterial genome replication and virion assembly zontal gene transfer24. Consequently, their hosts, respectively67,68. The structure of the are not coupled. This was confirmed by the evolutionary histories cannot be followed SH1 and P23-77 virions has been determined finding that four different replication systems with certainty. This category includes genes by cryo-EM and image reconstruction to are used by PM2-like corticoviral elements61. for DNA and RNA metabolism (replication, 9.6 Å and 14 Å resolutions, respectively68,69. Nevertheless, the genes that encode these pro- recombination and transcriptional factors), Analysis of the two virions revealed that hex- teins are often found at equivalent positions host recognition, evasion of the immune agonal capsomers are similarly arranged in a in the genome (FIG. 2). system and release of the virus progeny35,61–64. T = 28 (T represents the triangulation number)

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ab Ascoviridae PolB Ascoviridae Double β-barrel RNA or DNA lineage Asfarviridae primed Asfarviridae Iridoviridae Iridoviridae Poxviridae Poxviridae Phycodnaviridae NCLD Phycodnaviridae NCLD or

STIV Vs Vs Protein primed Mimivirus Mavericks STIV Mimivirus Mavericks TKV4 Adenoviridae Adenoviridae Vertical β-barrel MVV Vertical β-barrel TKV4 superlineage superlineage MVV PRD1 PRD1 Corticoviridae Corticoviridae 4 3 Tectiviridae 3 Tectiviridae 4 P23-77 Bam35 Cellular P23-77 Bam35 2 2 Single β-barrel replication lineage machinery SH1 SH1 1 1

Figure 3 | Possible origin and evolution of viruses that belong to the machinery-based (part b) relationship betweenNa differentture Revie virusws | Micr familiesobiolog thaty vertical β‑barrel superlineage. The schematic represents the hypotheti- are unified into the vertical β-barrel superlineage. The numbers denote the cal evolutionary relationship between double-stranded DNA viruses that key turning points in the evolution of this virus superlineage. The coloured use vertical β-barrel pseudohexamers for icosahedral capsid shell assem- circles represent the different domains of life from which the correspond- bly. It should be noted that the diagram is not a tree that is inferred from ing hosts reside. Bacteria are coloured red, Archaea are coloured green and phylogenetic analysis but rather a summary of the available data on the Eukarya are coloured blue. The organization of the viral genomes (circular depicted viral families. The same diagram is coloured with different shades or linear) is indicated. NCLDV, nucleo-cytoplasmic large DNA virus; PolB, to reflect the major capsid protein-based (part a) and genome replication polymerase B; STIV, Sulfolobus turreted icosahedral virus. icosahedral lattice. The subnanometre reso- error probability, as proposed by Jäälinoja to be carried out through a rolling-circle lution structure of the SH1 virion, together and colleagues69. Furthermore, double mechanism35,70. Notably, none of these with biochemical data, suggested that hex- β-barrel trimers are likely to be more stable (pro)viruses encodes a DNA polymerase agonal capsomers are formed from six indi- than hexamers, which could account for the for independent genome replication, but vidual β-barrels that are oriented normal to absence of decorating or stabilizing proteins. rather they depend on the cellular replica- the capsid surface: the central axis of the bar- The simplest double β-barrel is found in tion machinery. This also seems to be true rels was orientated perpendicular to the cap- corticovirus PM2 and was designated the for SH1, for which no replication protein sid surface and was stabilized by additional minimal double β-barrel31 (FIG. 1a). Structural could be identified71, and P23-77, which decorating proteins69. Importantly, SH1 and comparison of the two PM2 β-barrels with encodes a putative replication initiation P23-77 both encode a putative packaging those of other viruses from the same lineage protein (M. Jalasvuori and J. K. bamford, ATPase with the P9/A32-specific motif that revealed that the two barrels in PM2 are personal communication). by contrast, all is common to all membrane-containing more similar to each other than to any other other members of the PRD1–adenovirus members of the PRD1–adenovirus lineage57 coat protein, which supports the hypothesis lineage, including those that infect bacteria or (M. Jalasvuori and J. K. bamford, personal that the protein arose by gene duplica- eukaryotes, encode Polb (FIG. 3b). It is possible communication). on the basis of these com- tion23,38. Importantly, an interaction with the that the polB gene was acquired only once, mon virion assembly principles, we propose internal membrane in PM2 is achieved by and then diversified into the protein- and that such single β-barrel viruses should be separate viral integral-membrane proteins. RNA or DNA-primed Polb (FIG. 3, branch- grouped together with the double β-barrel All other membrane-containing viruses of ing point 3). Tectiviruses, adenoviruses and viruses into a vertical β-barrel superlineage the lineage possess larger and more elaborate Mavericks encode a protein-primed version (FIG. 3). Such a superlineage would include MCPs that directly interact with the underly- of Polb, whereas all the other members of the all viruses that use similar virion architec- ing viral membrane12,34,38, which suggests that PRD1–adenovirus lineage, the NClDVs and ture regardless of whether the hexagonal they diversified from the minimal double ascoviruses, encode an RNA or DNA-primed capsomer is composed of six monomers or β-barrel fold (FIG. 3, branching point 2). Polb43,52,55. three dimers. PM2, and all other corticoviral elements Diversification of bacterial tectivi- Duplication and subsequent fusion of that can frequently be found integrated into ruses from adenoviruses, Mavericks and the gene that encodes for a vertical β-barrel the genomes of aquatic bacteria, seem to NClDVs was probably associated with the capsid protein probably led to the emergence possess circular genomes that are apparently emergence of the bacteria and Eukarya of viruses with the double β-barrel MCPs replicated through four different mecha- domains (FIG. 3, branching point 4). This (FIG. 3, branching point 1). The use of double nisms61. Circular dsDNA genomes are also scenario is supported by a structure-based β-barrel trimers instead of single β-barrel characteristic of crenarchaeal virus STIV phylogenetic analysis7 of currently available hexamers reduces the number of protein and the euryarchaeal proviruses, TKV4 and double β-barrel MCPs, which revealed that subunits needed to build the hexagonal cap- MVV33,35. Interestingly, the integrated genome MCPs of PbCV-1 and adenovirus cluster somer (from six in SH1 to three in PRD1) of MVV is only slightly larger than that of together31. It is hard to predict the order in and would account for a lower assembly PM2, and replication of both genomes seems which the radiation from the common

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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.

INTRODUCTION

In the triad of infectious disease transmission involving aetiological agents, susceptible hosts and the environment, the role of the environment is the most ambiguous. The environment receives, maintains or protects and transports aetiological agents to susceptible hosts. Viruses may enter the environment in enormous quantities from clinically ill or inapparent carrier hosts; when extant outside the hosts which support their replication, they are the least understood of infectious agents. The greatest prospects for disease control for the future, however, lie in environmental measures to halt or reduce transmission. Conversely, failure to break the chains of transmission will result from failure to protect the environment or to modify it beneficially.

The increase in respiratory disease transmission through population concentrations, in cities in the case of humans and in confinement production units in the case of animals, is the most striking example of disease prevalence. A greater contemporary awareness of the role played by the confined environment in increasing the transmission of some diseases while reducing that of others has prompted more serious study of the environment.

* Department of Microbiology, Immunology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011, United States of America. 734

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.

ADENOVIRIDAE

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.

ARENAVIRIDAE

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.

HERPESVIRIDAE

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 735 simulate the conditions of survival of HSV2 on plastic-coated benches and seats in spa facilities, HSV2 (104-2CCID50/0.5 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 104 PFU/ml 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 736 was assayed for survival on solid fomites kept moist at 25°C. What follows are the maximum times needed to reach 99.99% inactivation: - 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. Mean survival time of ADV in contact with fomites in saliva suspension was 45% 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 55% relative humidity than at 25% 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 737 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, pH 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).

IRIDOVIRIDAE

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).

NORWALK VIRUS

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.

ORTHOMYXOVIRIDAE

Orthomyxoviruses, influenza A and B (103-5 and 1042 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 "clean" 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.

PAPOVAVIRIDAE

Objects used in the treatment of patients with human papillomavirus (HPV) infections were tested for recovery of HPV-DNA (25). HPV-DNA was identified by hybridization on 8 of 16 (50%) surgical gloves, on 23 of 62 (37%) and 1 of 62 (1.6%) 738 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 HPV on fomites remained infectious was not evaluated; however, extreme caution on the part of patients and personnel was suggested.

PARAMYXOVIRIDAE

In studies on paramyxoviridae obtained from infants, a suspension of respiratory syncytial virus (RSV) containing approximately 105-5 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.

PICORNAVIRIDAE

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 106-6 infectious doses/ml was still detectable at titres of > 102 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). 739

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 portion of the viral population was found viable in contaminated milk after pasteurization at 72°C for 15 seconds or after acidification to pH 4.6 (11). FMD 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 autumn 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 pH < 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 standards.

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 4 5 5 5 from 1 x 10 - to 1 x 10 - CCID50/ml. Depending on soil type, moisture content, pH 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 107.5 PFU/cube. 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 106.5 PFU/roach. 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 MF 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 CCID50 per fly. Wash fluids from surfaces of the flies yielded a mean of 124 CCID50 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 740 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 5 6 at total concentrations of 1 x 10 or 1 x 10 CCID50, 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 75% 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 PFU 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 preparation of Thuringer sausage, approximately 85% of the coxsackievirus A9 (104 to 7.5 Xl05 CCTD50) which was added to the sausage was lost during 24 h fermentation at 30°C (30). Additional 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 103 CCID50 of virus/g of sausage still remained of the initial 7.5 x 105 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 cm2. 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. Human 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 pH 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 pH 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. 741

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 pH range (2 to 12) and survived at pH 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 depopulation and disinfection of the premises. Only processes which involved heat treatment 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 unit/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, pH, 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).

3 Surface sea water from the Mediterranean inactivated 10 CCID50/ml 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 105-5 CCID50/g of 6 5 3 5 digestive tract and 10 - CCID50/g of flesh compared to 10 - CCID50/ml of sea water. At seven days, >99.9% 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). 742

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, 98% 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 106-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 105 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.

POXVIRIDAE

In studies involving one- to two-day-old chicks, avipox virus strain used as modified live virus (MLV) vaccine with 106 CCID50/ml of drinking water proved more efficacious than 104 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 6 higher initial concentration of 10 CCID50/ml. 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).

REOVIRIDAE

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 107 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. 743

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.

RETROVIRIDAE

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 104 to 105 syncytial forming units/ml were prepared in isotonic NaCl or NaCl plus 10% plasma. Results indicated that disinfection (103-104 reduction in 2 min) could be achieved using NaDCC 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

Cl2 concentrations of NaDCC and NaOCl solutions of 5,000 ppm of blood to effect complete inactivation within 2 min.

RHABDOVIRIDAE

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.

SCRAPIE AGENT

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 105 hamster infectious units thus interred, between 102 and 103 of infectious prions were recovered after three years in the soil. 744

PRIORITY AREAS FOR FURTHER STUDY

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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A REVIEW Enterically infecting viruses: pathogenicity, transmission and significance for food and waterborne infection

M.J. Carter School 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, 1354 7. Waterborne virus infection, 1362 2. Introduction, 1354 7.1 Wastewater treatment and virus survival, 1362 3. Enterically infecting viruses and their targets, 1355 7.2 Potable water treatment and virus survival, 1365 4. The viruses, 1355 8. Food-borne virus transmission, 1366 4.1 Adenoviruses, 1357 8.1 Shellfish, 1367 4.2 Astroviruses, 1358 8.2 Soft fruit and salad vegetables, 1368 4.3 Caliciviruses, 1358 8.3 Other foods, 1369 4.3.1 Noroviruses, 1358 8.4 Food handlers, 1369 4.3.2 Sapoviruses, 1359 9. Person-to-person transmission, 1370 4.4 Hepatitis A and E viruses, 1359 9.1 Person-to-person or food-borne? 1371 4.5 Parvoviruses, 1360 10. Virus diagnosis and detection, 1371 4.6 Picornaviruses, 1360 10.1 Clinical samples, 1371 4.7 Rotavirus, 1360 10.2 Food samples, 1372 5. Pathogenicity of food-borne viruses, 1360 11. Alternative indicators of virus contamination, 1373 5.1 Gastroenteritis, 1360 12. Conclusions, 1373 5.2 A novel virus toxin, 1361 13. Acknowledgements, 1374 5.3 Hepatitis, 1361 14. References, 1374 6. Virus stability, 1362

and considers their behavior in these human-assisted 1. SUMMARY processes. Enterically infecting viruses are ubiquitous agents, mostly inducing silent infections. Several are however associated 2. INTRODUCTION with significant diseases in man from diarrhoea and vomiting to hepatitis and meningitis. These viruses are drawn from a The term virus stems from the Latin virus meaning poison, variety of virus families and have different structures and and in some ways virus contamination of food resembles genetic material, yet all are suited to this means of toxic contamination more than contamination with other transmission: Normally they are shed in high numbers micro-organisms. Viruses are not free living; they are (assisting environmental transit) and exhibit great particle dormant between hosts and have an absolute requirement stability (permitting survival both outside the body and on for living cells in which to replicate. Human viruses require passage through the stomach). Human activities particularly human cells in which to replicate, these are not present in associated with food and water processing and distribution our food and thus such viruses cannot increase in number have the capacity to influence the epidemiology of these during storage. The amount of any contaminating viruses viruses. This review provides a description of viruses should actually decline during storage, and this can be spreading by these means, their significance as pathogens assisted by treatment with chemicals, heat or irradiation. Human viruses do not cause food spoilage and contamin- Correspondence to: Michael J. Carter, School of Biomedical and Molecular Science, ation may provide no visible clues to its presence. These University of Surrey, Guildford GU2 7XH, UK (e-mail: [email protected]). features mean that measures to control bacteria will not

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necessarily control viruses and could actually preserve them. introductions and it is likely therefore that food-borne Food-borne viruses are infectious at very low doses and transmission is underestimated. could be introduced at any point in the food chain. Many are difficult (or currently impossible) to culture and detection is 3. ENTERICALLY INFECTING VIRUSES no simple task. Outbreaks and sporadic occurrences of food- AND THEIR TARGETS borne virus infection continue throughout the world. There are simply too many to mention and there is no complete There are two types of enterically infecting virus – the first data set. It seems likely that the documented outbreaks are are capable of spreading elsewhere in the body. Infection by limited only by our ability to document them. these viruses is often subclinical but they may induce signs The cost of these events is likely to be phenomenal to the and symptoms of disease in nonintestinal tissues. These community; a single food-borne outbreak of hepatitis A viruses include enteroviruses (e.g. polio or Coxsackie, which virus (HAV) exposed up to 5000 persons in Colorado. In may spread to the meninges, central nervous system; this case the costs for medical treatment of those infected skeletal/heart muscle or pancreas) and hepatitis viruses A amounted to approx. $50 000 whilst the cost of tracing and and E spreading to the liver. The second type of virus are controlling this single outbreak cost over half a million US$ true gut inhabitants. These replicate in the enteric tract, (Dalton et al. 1996). The burden of infectious intestinal specific symptoms when they occur, are those of a gastro- disease (IID) in its broader sense is likewise huge, in the UK intestinal infection; usually diarrhoea and vomiting but the the cost per case of norovirus (NoV)-induced gastroenteritis extent of each component is variable. involving a GP visit is estimated at £176 and two persons in every 1000 will make such a visit each year (FSA 2000). The 4. THE VIRUSES interested reader is referred to several recent reviews (Lees 2000; Seymour and Appleton 2001; Sair et al. 2002; Table 1 lists the main viruses associated with enteric Koopmans and Duizer 2004). infection and summarizes their key properties. The most As enteric viruses cannot replicate outside their hosts, all important are illustrated in Fig. 1. Enteric viruses are drawn such virus transmission is in effect person-to-person. from a variety of virus families, they range approximately Environmental transit time between hosts may be brief 10-fold in diameter and 20-fold in terms of genome size and or prolonged. Long-distance travel may take place, e.g. complexity. The major enterically transmitted and thus through water systems or even the air. Long-distance potentially food/waterborne agents comprise (alphabetic- travel is accompanied by exposure to the environment and ally) the human adenoviruses (AdV), astroviruses (HAstV), dilution; thus viruses having prolonged environmental caliciviruses, hepatitis E virus (HEV), parvoviruses, picor- transit times must be very stable to survive and are naviruses [including enteroviruses, kobuviruses and hepati- (usually) shed in very large numbers. Enteric viruses meet tis A (HAV)], and the rotaviruses (RV). Most enteric both requirements; they are acid stable and replicate to viruses are childhood infections; spreading largely person- prodigious titres in the gut before being shed in concen- to-person and assisted by the lower hygiene levels in this trated doses directly into the sewage system. All potentially group. Food-borne transmission may be negligible (AdV) or food-borne viruses can also be transmitted directly from insignificant (RV) in the developed world. However in the person to person via faecal contamination of the environ- undeveloped world spread of these agents by these routes is ment and viewed in this way food is simply another kind poorly characterized. Childhood infection leaves residual of fomite in environmental transmission, it occupies a immunity that may prevent (or mollify) infection over the special niche simply because of its privileged position in rest of an individual’s lifetime. Although this is not terms of its introduction to the body and the potential it universally true, in general viruses causing childhood illness may offer for widespread distribution through trade and are not significant pathogens in healthy adults previously commerce. exposed as children. The IID survey in England (FSA The relative importance of food-borne vs more direct 2000) estimated the incidence of GP consultations for person-to-person transmission is unclear; enteric infections intestinal disease by patient age and causative organism. are ubiquitous, single occurrences are far too numerous to Data from this survey have been reanalysed (Fig. 2) to show mention and statistics usually record only outbreaks (when the proportion of consultations made for each virus in the several people are infected in one location or through one age groups <5 and >5 years. As expected GP consultations common vehicle). However any one outbreak may involve induced by these viruses were biased towards children different types of spread; these viruses have a high <5 years; GP visits by older persons comprised only a small secondary attack rate and person-to-person transmission proportion of the total consultations. There were two will probably follow even if the virus was actually introduced exceptions to this observation; some 25–50% of GP to that setting by food. This can potentially mask food-borne consultations for NoV infection were made by older

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x 1356 ª ..CARTER M.J. 05TeSceyfrApidMicrobiology, Applied for Society The 2005

Table 1 Properties of the major enterically infecting viruses

Name Virus family (genus) Food-borne Size (genome) Features Associated illness

Polio, Coxsackie, Picornaviridae (enterovirus) Yes, mainly water, 28 nm (ssRNA) Little surface detail Mainly asymptomatic, can induce echo, enterovirus present in shellfish Many cultivable in Vero or muscle pains (Bornholm disease), Hela cells cardiomyopathy, meningitis, CNS motor paralysis Aichivirus Picornaviridae (kobuvirus) Yes, shellfish 28 nm (ssRNA) Knob-like projections Gastroenteritis Cultivable Vero cells Hepatitis A virus Picornaviridae (hepatovirus) Yes 28 nm (ss RNA) Little surface detail Hepatitis, mild in the young ora fApidMicrobiology Applied of Journal Fhrk-1 cells Hepatitis E virus Unclassified Mainly water 34 nm (ssRNA) Calicivirus-like structure Hepatitis, severe in pregnancy Unique genetic organization Not cultivable Rotavirus Reoviridae Rare often water 70 nm (dsRNA) Multilayered Diarrhoea – common in the young, Segmented genome incidence decreasing with age, but (11 pieces) increases in the elderly Cultivable Ma104 cells Adenovirus group F, Adenoviridae Not reported 100 nm (dsDNA) Distinctive icosahedral Mild diarrhoea, shedding may be types 40 and 41 Cultivable Graham 293 cells prolonged, mainly affects children ,

98, Saporovirus Caliciviridae (saporovirus) Yes (rare), mainly 34 nm (ssRNA) Cup-like depressions on surface; Gastroenteritis – common in children shellfish none are cultivable believed to be milder in effect 3418,doi:10.1111/j.1365-2672.2005.02635.x 1354–1380, Norovirus Calicivirus (norovirus) Yes 34 nm (ssRNA) Fuzzy surface structure; Explosive projectile vomiting in not cultivable older children/young adults Human astrovirus Astroviridae (mamastrovirus) Occasionally, water 28 nm (ssRNA) Eight serotypes 5 or Mostly infect children, higher and shellfish 6-point star motif serotypes seen in adults. Relatively Appearance variable mild gastroenteritis, but probably Cultivable CaCO2 cells underestimated Wollan, ditchling, Parvoviridae? Yes, shellfish 25 nm (ssDNA) Smooth featureless, poorly Gastroenteritis – widespread Paramatta characterized noncultivable shellfish-associated outbreaks, and cockle agents largely controlled through cooking FOOD-BORNE VIRUS 1357

123

4 5

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

children/adults; the highest of any virus, with astroviruses close behind. 120·0 100·0 80·0 4.1 Adenoviruses 60·0 There are 51 serotypes of AdV; all are large icosahedral 40·0 DNA-containing viruses. About 30% of the serotypes are consultations 20·0 Percentage of total Percentage 0·0 pathogenic in man; most being upper respiratory tract %<4 %>4 pathogens spreading primarily via droplets. However, even Age group the respiratory strains grow well in the gut and are present Fig. 2 Virus identifications in GP consultations for infectious in the faeces. Only types 40 and 41induce gastroenteritis and intestinal disease by age of patient. Viruses identified following GP these are shed in larger numbers. AdV are frequently found consultations have been segregated into the percentage of cases that in faecally polluted waters and have been identified in involving children below 4 years and those involving older children shellfish (Girones et al. 1995; Pina et al. 1998a; Vantarakis and adults. Bias for infection of the young emerges clearly, even in the and Papapetroupoulou 1998; Chapron et al. 2000), but have case of noroviruses. Data are re-analysed from FSA (2000) IID survey not been appreciably associated with food-borne illness, in England. j, NoV; , HastV; , AdV; , RV; (, SaV

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presumably because most adults are immune and children antibody may not prevent clinical disease: in Japan (1995), do not commonly eat shellfish. However outbreaks of other 1500 older children and teachers were affected in a strains associated with conjunctivitis (shipyard eye) and widespread food-borne outbreak of HAstV type 4 (Oishi pharyngitis are commonly associated with exposure to et al. 1994). Finally, astrovirus identification often relies on polluted water, normally through recreational use (Crabtree electron microscopy but virus appearance is not always clear. et al. 1997). Astroviruses may be frequently mistaken for small round Adenoviruses 40 and 41 account for 5–20% of US (parvovirus)-like agents (Willcocks et al. 1991) and even for hospital admissions for diarrhoea, mainly in children below NoVs (Madore et al. 1986). In England the IID survey age 2 years (Uhnoo et al. 1984; Kotloff et al. 1989). conducted between 1992 and 1995 (FSA 2000) found Incubation lasts 3–10 d, and illness (usually a watery astroviruses comprised 12% of all virus identifications with diarrhoea) may last a week. As children age, experience incidences of 125 and 550 per 100 000 person years in the with AdV infection gradually increases the levels of age groups 0–1 and 2–4 years respectively. However, 16% population immunity. Only 20% of children below of GP consultations for astrovirus infection were made by 6 months have antibody to these viruses, but by age 3 for older children and adults (Fig. 2). As this survey identified this has risen to 50%. In the IID survey in England AdV HAstV only by EM it remains possible that astrovirus infections were confined largely to children under 5 years infections in the adult population were underestimated. and accounted for approx. 12% of all viruses identified Culture conditions have been described (Willcocks et al. (FSA 2000). Incidence was determined as 400 and 800 per 1990) and recently an ELISA-based detection kit has been 100 000 person years in the age groups 0–1 and 1–4 years. produced. Infection is not significant in healthy adults although it may increase in significance again in the elderly (Dupuis et al. 4.3 Caliciviruses 1995); fewer than 4% of enteric AdV GP consultations involved persons over 5 years (Fig. 2). AdV are associated Caliciviruses appear under the electron microscope as if with tumours in mice but no such association has ever been covered in cup-like depressions, from which the virus takes made in man. 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 4.2 Astroviruses nomenclature of these viruses has changed several times Astroviruses are usually described as 28 nm rounded particles recently. Formerly they were known by names derived from with a smooth margin. In their centres they may bear a 5 or 6- their morphology (see Fig. 1): the small round structured pointed star motif from which they are named (Astron, a star) viruses, e.g. Norwalk virus appeared fuzzy and indistinct (see Fig. 1). However, the appearance of these agents is whilst the human caliciviruses, e.g. sapporo virus, had a certainly variable, sometimes showing surface projections more obvious calicivirus-like appearance (Caul and Apple- (Appleton and Higgins 1975) and at other times resembling ton 1982). Classification then moved to genomic organiza- caliciviruses (Willcocks et al. 1990). Morphology was subse- tion and the groups were renamed the Norwalk-like viruses quently used to present a classification scheme for many of and the sapporo-like viruses. The nomenclature is now these small viruses associated with enteric infection and hopefully settled with the refinement of these names to the including the caliciviruses (below) (Caul and Appleton 1982). NoV and SaV respectively. Cryo electron microscopy studies have now confirmed the presence of surface projections (Matsui et al. 2001). Human 4.3.1 Noroviruses. Analysis suggests that the NoV are astroviruses comprise eight serotypes (HAst1–8); types 1 and the single most significant cause of IID in the developed 2 are rapidly acquired in childhood; by age 7 years 50% of world. NoVs were first identified following an outbreak of children are seropositive for type 1 and 75% by age 10 years enteric illness amongst children and adults in the town of (Lee and Kurtz 1982; Kurtz and Lee 1984). Exposure to the Norwalk, OH (Alder and Zickl 1969). Although samples higher serotypes (4 and above) may not occur until adulthood. were first collected in 1968, viruses were not clearly Illness is generally mild, lasting 2–3 d after an incubation identified until 1972 when antibody was used to clump the period of similar length. This has led many to dismiss these particles (Kapikian et al. 1972). NoVs are now routinely viruses as causative agents of significant disease in humans. detected by PCR amplification of the RNA-polymerase However astroviruses are the second most commonly gene and by commercial ELISA kits, electron microscopy identified virus in symptomatic children (Herrmann et al. is used as a back up. Sequence analysis of the PCR 1991) and account for 5% of US hospital admissions for products divides the NoV into two genogroups; group 1 diarrhoea – almost entirely of children (Ellis et al. 1984). exemplified by Norwalk virus itself and group 2 by Hawaii Adults may be infected by higher serotypes and childhood virus (Lambden and Clarke 1994; reviewed in Clarke and

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Lambden 2001). Recently genogroup 2 has been more mechanism underlying this process requires investigation: common in the UK. food-borne transmission, perhaps via international trade Infections occur around the globe and throughout the year should be considered. but may be more common in winter giving rise to its former name winter vomiting disease. Incubation lasts up to 48 h 4.3.2 Sapoviruses. Sapoviruses induce symptomatic and is followed by a self-limiting illness lasting 24–48 h. infections mainly in children. They account for some 3% NoV infection is not regarded as severe in otherwise healthy of hospital admissions for diarrhoea in both the UK and US. adults, but it is debilitating and very unpleasant. In Most children are sero-positive by age 12 and seem to vulnerable groups, the malnourished or elderly it can be become infected between 3 months and 6 years of age. SaV serious and may even precipitate death. It was thought that were found most frequently in children below age 4 in the subclinical and childhood infection was rare but recent England IID with an incidence of 460 per 100 000 person studies have shown this does occur in very young children years in those aged <1 years that fell to 150 in those between (Carter and Cubitt 1995). The IID study in England 2 and 4 years (FSA 2000), <2% of GP consultations for SaV estimated that 1% of children < 1 years would contract infection took place in those >5 years (Fig. 2). Infection is NoV (FSA 2000). particularly common in institutional settings such as schools Norovirus differs from other agents of gastroenteritis in and day care centres. Incubation is between 24–48 h and three ways: first, it causes disease in adults (teenagers and illness is usually mild and short-lived with diarrhoea above), thus NoVs are the most significant diarrhoeal virus predominating. However in those cases when SaV have in terms of working/education days lost. Secondly, it been seen to infect adults then symptoms are very similar to induces a high level of explosive projectile vomiting that those of NoV (Cubitt 1989). may be the first obvious sign of infection. Many cases are identified at work with serious implications if a food handler 4.4 Hepatitis A and E viruses should be infected. Thirdly, although there are probably multiple serotypes of NoV, immunity to all seems to be Epidemic hepatitis has been recognized since ancient times, short-lived. Thus individuals may be protected for only a and its infectious nature was appreciated in the middle ages. few months following an infection before they become However the causative agent was identified only in 1972 infectable once more by the same virus (Parrino et al. 1977). when the newly developed technique of immune electron Some people appear to have an inherent resistance to microscopy permitted the particles to be identified (Kapik- infection; community outbreaks that stemmed from com- ian et al. 1972). Subsequently named HAV to distinguish it munal exposure by swimming pool contamination showed from serum hepatitis (hepatitis B), this agent was found to familial clustering of symptomatic illness, and even in be responsible for the bulk of infectious hepatitis. Symptoms middle age population antibody levels are only 50%. Many were seen mainly in older children and adults but infection of these seronegative individuals remain symptom-free and was common in younger children although in these it tended it is now thought likely that they lack the cell-surface to be symptom free. HAV was found to be a member of the receptor (a carbohydrate antigen) to which the virus must Picornavirus family (see below) and was initially placed in bind to initiate infection (Hutson et al. 2003). Susceptible the genus Enterovirus. However it possessed some unique persons require several bouts of infection by the same virus properties in relation to its genetic structure and replication before antibody levels are boosted sufficiently to afford some procedure and it was subsequently removed to a new genus protection. In the recent IID survey in England NoV (Hepatovirus) of which it is the only member. HAV can be accounted for 30–40% of all viruses identified, they were the cultured in FRhK-4 cells but this is slow and difficult, most commonly identified agent in the community study, especially for primary isolates. and the third most common agent that caused persons to Although responsible for most enterically transmitted seek consultation with their GP (FSA 2000). Several reports hepatitis in the developed world, it was clear that, HAV alone across the world have indicated a rise in NoV detection could never account for all enterically transmitted hepatitis in during 2002–03. These included shipboard outbreaks, the undeveloped world. Thus the concept of enterically multistate occurrences in the US, a sudden rise in outbreaks transmitted non-A, non-B hepatitis grew up. This gap in in Canada and numerous hospital outbreaks throughout the knowledge was filled in 1990 when a new agent, HEV was UK that forced many to close wards or cease new identified by molecular means (Reyes et al. 1990; reviewed admissions. These have been attributed in part to the Bradley 1990, 1992). The genetic organization and particle emergence of a new strain of NoV across the world structure of HEV resembled the Caliciviruses and HEV was characterized by mutations in the polymerase gene (Lopman initially classified in this family. However the detail of the et al. 2003, 2004). This might be more infectious than genomic organization and the enzymic capacities encoded are previous strains and if such an event has occurred then the such that it could not remain in this family. Consequently it

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now forms the only member of a group called the hepatitis Illness develops after an incubation period of 4–7 d and E-like viruses (Green et al. 2000). HEV is rare in the usually presents as diarrhoea and vomiting lasting approx. developed world with cases generally limited to travellers. 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- 4.5 Parvoviruses tion by EM or PAGE (Moosai et al. 1984), antibody-based Parvoviruses are poorly characterized as agents of enteric bead-agglutination or ELISA systems. RV account for some infection. Diagnosis is based solely on electron microscopy. 3Æ5 million cases of diarrhoea p.a. in the US equating to 35% Although associated clearly with gut infection in animals of hospital admissions for diarrhoea (Ho et al. 1988). (e.g. canine parvovirus), the only infectious human parvo- Approximately 120 children die each year in the US from virus characterized to date is a nonenteric agent B19, causing this virus and fatalities in the undeveloped world may a maculopapular rash in children. Parvoviruses have been amount to millions (Parashar et al. 2003). associated with gastroenteritis in primary and secondary Rotaviruses occur in five groups (A–E) but only groups schools in the UK (Wollan and Ditchling agents) and A–C infect humans. Group A is by far the most common Australia (Paramatta agent) (Paver et al. 1973; Christopher with sporadic episodes due to group C, group B is limited et al. 1978; Appleton 1987, 2001). The cockle agent was largely to China. Only group A viruses can be cultured, identified following a large outbreak in England (Appleton these are grown in Ma104 cells. Within each group RV are and Pereira 1977), and was associated with consumption of divided into serotypes based on their surface-exposed contaminated seafood. There is a strong argument that proteins. Within group A there are 14 types of VP7 (termed some/many of these viruses may actually be misidentified G types) and approx. 20 of VP4 (termed P types). This astroviruses, caliciviruses (Willcocks et al. 1991). 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 4.6 Picornaviruses have been infected. Immunity to rotavirus is long-lasting, Picornaviruses infecting the gut were formerly all contained thus sequential exposure leads to accumulated immunity in the Enterovirus genus. Enteroviruses have a rather and frequency of illness decreases with age. Silent secondary featureless appearance under the electron microscope and re-infections can occur (as in parents caring for infants) and include the ECHO Coxsackie and polioviruses. Most grow this provides another means for the virus to spread in the well in laboratory cell cultures such as HeLa or Vero. community. RV were the most commonly identified enteric Formerly much feared, the success of vaccination has pathogen in children <4 years in England and Wales and controlled polio and allowed the WHO to target this virus comprised 25–35% of the virus identification made in this for global elimination early this century. Picornaviruses were study (FSA 2000). Only 11% of GP consultations for thought not to be usually associated with diarrhoeal rotavirus were made by persons >5 years. However this symptoms in humans but this changed in 1993 when raises two points, first not all adult infections are mild and Aichivirus was discovered as the agent responsible for an secondly this small percentage may actually reflect a greater outbreak of shellfish-associated gastroenteritis (Yamashita number of adult GP consultations for rotavirus infection et al. 1993). Aichivirus could be grown in Vero cells and than for NoV (FSA 2000). 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- 5. PATHOGENICITY OF FOOD-BORNE viruses and bears surface projections similar to those of VIRUSES astroviruses. These viruses have now been recognized as a 5.1 Gastroenteritis new genus in the picornavirus family termed the kobu- viruses. Gastroenteritic viruses replicate and destroy the mature enterocyte covering the upper third of the intestinal villi. Undifferentiated, immature cells do not support virus 4.7 Rotavirus replication. Destruction of functional mature cells disrupts Rotaviruses are large RNA-containing viruses belonging to the reabsorption of water from the gut and diarrhoea ensues. the family Reoviridae. Their particles are multilayered and The villi retract in response to damage and decrease the complex, replicative functions may be built into the shell. surface area available for absorption. At the same time the Particles are readily visible and distinctive in the EM where crypt cells undergo rapid division and soon repopulate the outer layer of the capsid can appear like the spokes of a the villi with young, as yet undifferentiated cells. These wheel from which the virus is named (rota, a wheel). immature cells are resistant to virus infection but cannot

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replace the function of those that have been lost; they require (8–10 weeks) and some 15% of HAV cases may follow a time to mature. Thus malabsorption continues until the cells relapsing course over 12 months or more. can develop the necessary ion uptake capabilities. This Hepatitis viruses A and E have been affected by human exuberant cell division in the crypt is central to recovery. activities. In former times, infection occurred early in life, Fortunately in humans viruses do not attack the crypt cells often whilst still protected by maternal antibody. Such themselves. When such infections do occur (e.g. canine endemic infections tended to be mild or subclinical. Both parvovirus) they result in bloody diarrhoea from which viruses are rare where sanitation is improved. This has recovery may not be possible (especially in a young animal). 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 5.2 A novel virus toxin 9%, although seropositivity in the elderly remained high. In Although most viruses do not manufacture toxins some RV contrast, in France where sanitation has been good for many are exceptional and induce the synthesis of a toxin-protein years, 80% of persons over 30 have no antibody to HAV (i.e. termed NSp4 that can induce diarrhoea if administered have never been infected). alone (Ball et al. 1996). NSP4 has no similarity to bacterial This shift in age at infection increases the severity of toxins (Tian et al. 1995). It stimulates transepithelial infection: below 3 years, HAV infection is virtually always chloride secretion via a calcium ion-dependent path; it has subclinical; but symptomatic infections predominate by no effect on cAMP formation and is independent of the 5 years and severity worsens with age (Hadler et al. 1980). cystic fibrosis channel. The mechanism involves stimulation Persons over 50 years of age account for only 12% of the of inositol production (Dong et al. 1997). The protein also cases of HAV but have a case-fatality rate sixfold higher than has direct effects on brush border transport mechanisms average (CDC 1994; Fiore 2004). This delay in infection (Halaihel et al. 2000). It is proposed that at least one form of allows a pool of susceptible individuals to accumulate in the soluble NSp4 is released from infected cells and binds to community and establishing conditions for epidemic spread. neighbouring cells (Zhang et al. 2000). 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 5.3 Hepatitis on average per year between 1987 and 2001, most were mild Hepatitis A and E viruses enter via the gut and may replicate or symptom free but 10–30 000 acute cases were registered there, however both move rapidly to the liver and invade the annually. Mead et al. (1999) estimate that 5% of cases are hepatocytes. Clinical features of both viruses are similar food-borne. The situation in the UK has been summarized although HEV is more severe and may have a fatality rate of (Crowcroft et al. 2001). The existence of susceptible adults 20–40% in late pregnancy. HEV has a longer incubation in some parts of the world is significant in the context of period than HAV (60 d vs 48 d) and a more prolonged food-borne infection since trade that could bring virus- viraemia (Clayson et al. 1995b; Scharschmidt 1995; Reid and contaminated food grown/produced in areas of high ende- Dienstag 1997). The long incubations make identification of micity to areas of low prevalence could pose a threat for the source of infection problematic as contaminated food will adults in those areas (see below). usually have been eaten or disposed of before illness arises. Hepatitis E virus is not significant in the UK or US; most Haemoglobin breakdown by the liver is impaired and a infections are limited to returning travellers. Epidemics of bilirubin (normally shed in the bile and thence in the faeces) HEV are known, often spread by contaminated water; the overflows into the blood. The skin and whites of the eye turn worst cases involved 30 000 people in New Delhi (1955); yellow (jaundice) and faeces become pale. Bilirubin is filtered 100 000 in Xinjiang Uighar, China (1986), and 79 000 in from the blood by the kidneys, and urine becomes dark. Kanpur India (1991) (Grabow et al. 1994; Scharschmidt Virus particles are shed into the bile and thence in the faeces, 1995). More limited shellfish-associated outbreaks occur but in contrast to the cell destruction caused by gastroen- sporadically around the Mediterranean. HEV replicates in teritis viruses there is little virus-induced liver cell damage. pigs (Balayan et al. 1990) and has been found in both wild and HAV interferes only weakly with host cell activities and new domestic cows, goats and pigs (Clayson et al. 1995a). Repli- viruses are released inside membrane-bound packets without cation also occurs in laboratory rats (Maneerat et al. 1996; necessity to lyse the cell. An immune response develops Meng et al. 1996). These findings mean that animals might act 2–3 weeks after infection and leads to immune attack on as reservoirs for infection (Kabrane-Lazizi et al. 1999; Wu infected liver cells. It is this host response rather than the et al. 2000). Seropositivity has been estimated at 2–10% even virus itself that causes the signs of liver damage. Eventually in areas free from human disease; 15% of homeless persons in the immune response eliminates all infected cells (and thus Los Angeles revealed antibody to HEV, possibly through the virus) from the body. Convalescence may be prolonged contact with infected urban rats (Smith et al. 2002). Similarly

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HEV has been detected in sewage from areas in which clinical Values for inactivation times/persistence in water vary disease is absent (Pina et al. 1998b). 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 6. VIRUS STABILITY probably measured in weeks (Chung and Sobsey 1993; The stability of an enteric virus is of fundamental signifi- Callahan et al. 1995). Persistence in artificially contaminated cance to its transmission. First, it may limit the period for water has been demonstrated for >1 year (rotavirus and which virus contamination remains a threat in the environ- poliovirus) (Biziagos et al. 1988) and 300 d for AdV 41 ment whether in sea or fresh water or dried onto a surface, (Enriquez et al. 1995). These variations probably reflect and secondly, it governs the efficiency of attempts to differences in the conditions used particularly, type of water, deliberately destroy a virus, e.g. in food or water processing illumination, turbidity and pH, and emphasize the need for (mainly sensitivity to temperature and chlorination). Many standardized studies of all relevant viruses (and common enteric viruses are nonenveloped and contain RNA as their surrogates) under directly comparable conditions. Despite genetic material (exceptions AdV and parvovirus that this variation all these viruses are probably capable of contain DNA). RNA is labile, hydrolysed at both acid and survival for weeks/months at environmental temperatures alkaline pH and destroyed by radiation or enzymic proces- and at low temperatures, sheltered from UV irradiation ses. DNA is more stable but is still sensitive to UV some may persist for years. irradiation. This emphasizes the crucial role of the virus coat proteins that must protect the nucleic acid. The relative 7. WATERBORNE VIRUS INFECTION stabilities of some of the enteric viruses are assembled from a variety of sources and presented in Table 2. There is no All these viruses enter the sewerage system and may survive central data set for all viruses and their frequently used wastewater treatment to contaminate receiving waters. Here surrogates; most studies use only a few viruses under they could pose a threat to recreational users or to restricted conditions and have not considered the effects of consumers of shellfish or other produce eventually destined combined treatments. The environment in which inactiva- for the food chain. In addition, where such waters are later tion proceeds will certainly affect the values determined and abstracted and treated for use as drinking water, contam- this has central significance when considering survival in inating viruses might survive this treatment too. Drinking different foodstuffs (e.g. inside shellfish or in milk) or in the water can also be contaminated after treatment if supplies environment (marine vs fresh water etc.). As each study has are not adequately separated from untreated sources. used slightly different conditions, this table should be regarded as only indicative. 7.1 Wastewater treatment and virus survival Although all these viruses are destroyed by boiling, the thermal stability of some is remarkable. Temperatures above Enteric viruses are usually shed in large numbers, rotavirus, 90C are required to inactivate HAV (Millard et al. 1987) titres can exceed 109 particles per ml and could comprise up and others suggest 100C may be necessary (Croci et al. to 2 mg in every gram of stool. A value between 106–108 per 1999). Inactivation is often biphasic and a residue of ml is common for astrovirus and AdV. Caliciviruses, entero- resistant virus may survive. This probably represents virus and hepatitis viruses are often shed in lower (but still in a protected microenvironment, e.g. aggregated or com- appreciable) numbers; cultivable enteric viruses are ubiquit- bined with materials exerting a protective/stabilizing effect ous in human populations and levels in sewage can exceed (Tierney and Larkin 1978; Larkin and Fassolitis 1979; 104 PFU per litre. Water treatments lacking a tertiary step Bidawid et al. 2000a). (e.g. UV treatment) reduce this load only poorly. Sewage Virus persistence in dried matter depends on the surface sludge production reduces numbers by 95% but many onto which it is dried, the presence of extra (faecal) viruses survive giving levels in receiving waters of up to material and the temperature/humidity of storage. Many 100 PFU per litre if contamination is serious and 1–10 PFU enteric viruses survive for long periods on common surface per 100 l where it is less so (Gerba et al. 1985; Bloch et al. types requiring up to 60 d for a 2 log reduction in titre 1990; Jothikumar et al. 2000; Scipioni et al. 2000; Pina et al. (Abad et al. 1994a). In general reducing the temperature 2001). Most enteric viruses have been detected in waste- and adding faecal contamination promotes virus survival water, treated water and receiving waters over time, usually but viruses may respond differently to relative humidity; by PCR techniques even if viruses are cultivable, e.g. HAV HAV and rotavirus are stabilized at low relative humidity (Dubrou et al. 1991; Goswami et al. 1993; Graff et al. 1993; whilst enteroviruses are stabilized at higher values (Mbithi Tsai et al. 1993; Jaykus et al. 1996; Schwab et al. 1996) and et al. 1991). This probably depends on the type of surface astrovirus (Le Cann et al. 2004). AdV have consistently been onto which the viruses are dried (Abad et al. 1994a). detected in raw sewage and approx. 80% may be enteric

ª 2005 The Society for Applied Microbiology, Journal of Applied Microbiology, 98, 1354–1380, doi:10.1111/j.1365-2672.2005.02635.x ª Table 2 Virus stability values; assembled from references below. No study has included all these viruses under identical conditions and thus data are not always comparable; values 05TeSceyfrApidMicrobiology, Applied for Society The 2005 presented here should be taken as indicative of stability only. Log titre reduction (LTR), measured by infectivity unless otherwise stated. Bacteriophage MS2, a common surrogate for enteric viruses is included for comparison

Persistence/inactivation conditions

pH Thermal Free chlorine UV irradiation Persistence Persistence Persistence Persistence ) Virus stability inactivation (unless stated) (mJ cm 2 ) (dry) food matrices sea water fresh water

Adenovirus Stable Inactivated 2 LTR at 4 LTR at dose 2Æ5 LTR on drying, Not significantly 1 LTR in 40 d 3Æ2 LTR in 60 d ) pH 6–9Æ5* 56 deg, 0Æ5mgml 1 216 (23) further 3 LTR food-borne 15 deg (27) at 20 deg 10 min* (6) for 10 min (20) 10 d (19) 1 LTR in 40 d 3 LTR at at 15 deg (27) ) 1Æ0mgml 1 for 10 min (20) Astrovirus Resists Resists 2 LTR at No data 1 LTR on drying No data Reportedly lower Drinking water: ora fApidMicrobiology Applied of Journal ) pH 3 (4) 50 deg 1 h, 1mgl 1 for then stable for stability than 2 LTR at 4 deg, 60 deg 10 min, 60 d at 4 deg (19) fresh water 3Æ2 LTR at 20 deg 5 min (4) 2Æ0 LTR at both 60 d ) 0Æ5mgl 1 for 20 min (18) Hepatitis A Resists Resists 60 deg Inactivated 4 LTR at dose Survives >1 month Resists 60 deg 2 LTR in Estuarine water: ) virus pH 1Æ0 for for 10 min; by 2–2Æ5mgl 1 16–39 (23); at 25 deg in 42% for 19 min in 28 d at 2 LTR in 28 d 2 h (9) cations stabilize for 15 min (13) 1 LTR at dose humidity (10, 11); oysters (12) 25 deg (11) at 25 deg (11) to 81 deg CT for 99% 36Æ5 (30); 1 LTR 3 LTR in 8 d and 2 LTR at 63 deg PBS: 1 LTR in Inactivated reduction ¼ 8 (17) in 1Æ3 min at then stable to in water or milk 56 d at 25 deg

, )2

98, 98–100 deg (7) 1 LTR at 42 mW cm in 32 d (16) for 60 min (16) ) 0Æ5mgl 1 for sea water (25) 2 LTR in 7 d in 3418,doi:10.1111/j.1365-2672.2005.02635.x 1354–1380, 10 min; 2 LTR creamy wafer ) at 1 mg l 1 cookies 21 deg for 10 min (20); 3 LTR at 4 ppm ClO2 for 10 min (16) Norovirus Resists Resist 60 deg Resists exposure No data – FCV NoV Recoverable Infectivity in 1 LTR in FCV suspension – 1 )1 pH 2Æ7 for 30 min (14) to 6Æ25 mg l ; surrogate from disinfected shellfish not 24 h at 10 deg LTR per day at VIRUS FOOD-BORNE 3 h (14) 30 min (CT 187Æ5) T90 value hospital surfaces; reduced after (illuminated) 37 deg, 4 LTR in inactivated 47Æ85 (30) FCV 2 LTR 1 month 60 d at 4 deg (31) ) at 10 mg l 1 in 15 d at storage at 4 (CT 300) (15); 4 deg; or in deg or over others suggest 30 d at 20 deg, 4 months frozen. CTs 30–60 inactivated in Stable in ice required (see text) 1 d at 37 deg (31) 1 LTR (PCR) ) at 2 mg l 1 chloramine 1363 for 3 h (25) 1364 ª ..CARTER M.J. 05TeSceyfrApidMicrobiology, Applied for Society The 2005 Table 2 (Continued )

Persistence/inactivation conditions

pH Thermal Free chlorine UV irradiation Persistence Persistence Persistence Persistence ) Virus stability inactivation (unless stated) (mJ cm 2 ) (dry) food matrices sea water fresh water

Picornavirus/ Resists Destroyed 2 LTR at free 1 LTR in 1Æ3 min 2 LTR on drying, >3 LTR in water 1 LTR in 96 h at 1 LTR 24–31 h enterovirus pH 3Æ0 (7) 42 deg, chlorine at intensity further 3 LTR in or milk at 12 deg; 1 LTR in in river water ) ) cations 1Æ1–2Æ5mgl 1 42 mWs cm 2 60 d at 4 deg 63 deg, 30 min 22 h at 22 deg (22); in situ 12–20 stabilize to (28) in sea water (25) 1 LTR >670 d at deg (21) 50 deg(8) 1 LTR by 1 LTR at dose 4 deg (26) chloramine, 24Æ1 (30) ) 2mgl 1 for ora fApidMicrobiology Applied of Journal 3 h (25) Picornavirus/ Resists kobuvirus pH 3Æ5 (7) Rotavirus Stable Resists CT values for 4 LTR at 56 (23) 1 LTR on drying; Bovine Rotavirus – Group A virus: group A pH 3–9 (5) 50 deg (3) 2 log 1 further LTR in decay 0Æ5 LTR 2 LTR >64 d inactivation 30 d 4 deg (19) per day natural tapwater or 10 d 0Æ01–0Æ05 (17) sea water (29) river water 20 deg Group B virus: (27); Group B 1Æ8 LTR 10 min virus: 3Æ2 LTR ) 1mgml 1 ; in 60 d 20

, 1Æ5 LTR 10 min deg (1, 2)

98, ) 0Æ5mgl 1 (20)

3418,doi:10.1111/j.1365-2672.2005.02635.x 1354–1380, MS2 Tolerates >1 LTR in 1 LTR by 4 LTR at dose App 3 LTR in low pH 15 s at 72 deg monochloramine 750 253 nm (24) 4 d (16) ) in water 2mgl 1 , 3 h (25) 1 LTR dose or milk (16) >3 LTR by 23Æ04 (30) 10 min in 2 ppm CLO2 (16)

Data sources: 1. Vondefecht et al. (1986); 2. Terrett et al. (1987); 3. Estes (1991); 4. Monroe et al. (2000); 5. Mertens et al. (2000); 6. Russel et al. (1967); 7. King et al. (2000); 8. Dorval et al. (1989); 9. Scholz et al. (1989); 10. McCaustland et al. (1982); 11. Sobsey et al. (1988); 12. Peterson et al. (1978); 13. Coulepsis (1987); 14. Dolin et al. (1972); 15. Keswick et al. (1985); 16. Miriam and Cliver (2000); 17. Hoff (1986); 18. Abad et al. (1997); 19. Abad et al. (1994a); 20. Abad et al. (1994b); 21. O’Brien and Newman (1977); 22. Bitton (1978); 23. Cotton et al. (2001); 24. Someer et al. (2003); 25. Shin and Sobsey (1998); 26. Gantzer et al. (1998); 27. Enriquez et al. (1995); 28. Clark et al. (1993); 29. Loisy et al. (2004); 30. Nuanualsuwan et al. (2002); 31. Doultree et al. (1999). *Nonenteric virus strains. FOOD-BORNE VIRUS 1365

forms. These appear to survive sewage treatment well and 1988; Payment et al. 1997). The assurance of drinking water are frequently detected even in waters that lack enterovirus quality begins with the source water, which should be contamination. This has led many to suggest that AdV may obtained from sources as far removed from potential be appropriate sentinels for the indication of faecal pollution contamination as possible. In the US subsequent treatment in water (Enriquez et al. 1995; Pina et al. 1998a; Wyn-Jones aims to reduce levels of contaminating virus by 99Æ99%. and Sellwood 2001). Filtration can achieve an initial 10-fold reduction with a Noncultivable viruses have also been detected. NoV have further 1000-fold achieved by active disinfection (e.g. been found in sewage influent and effluent waters using PCR chlorine, chlorine dioxide, ozone or UV irradiation). Of techniques (Lodder et al. 1999), serial dilution of influent these, chlorination is the most widespread; peak levels are waters suggested that levels may exceed 107 detectable virus usually around 1 mg l)1 for 60–240 min (Grabow 1990; units although this cannot be readily correlated with particle Thurston-Enriquez et al. 2003) and water entering the US numbers or infectivity (Wyn-Jones et al. 2000). Estimates distribution system should have a residual level of using quantitative PCR obtained similar values (Laverick 0Æ2mgl)1 to comply with requirements to control coliform et al. 2004). A study of treated water suggested that NoV bacteria. Exposure to disinfectant is expressed by the contact levels were virtually undiminished in primary treatment and time (CT) parameter, concentration of agent (mg l)1) were reduced by only a little over 1 log in the final multiplied by time (min). CT values for a 4 log inactivation (secondary) effluent (Cross 2004). Similarly astroviruses of enteric viruses fall in the range of 4–400 mgÆmin l)1 at a ) have been detected in sewage treatment plant inlet and contact concentration of 0Æ4mgl 1 at 5C. However the effluent waters, indicating a reduction of approx. 2 log during rates of virus destruction and thus CT values required are processing; effluent waters still contained 105 detectable temperature dependent, doubling for every 10C rise and astrovirus genomes per litre (Le Cann et al. 2004). HEV was potentially defective around 0C. Increasing the pH from 6 found in sewage by molecular means (Pina et al. 1998b), and to 9 reduces the efficiency of free chlorine threefold, ozone survives at least some wastewater treatments although its or chlorine dioxide are unaffected. Turbidity has the removal has not been quantitated (Jothikumar et al. 1993). greatest effect, shielding viruses from UV radiation, pro- Viruses surviving water treatment and entering receiving moting aggregation and also mopping up virucide. Increas- waters could persist and pose a risk for recreational users of ing the turbidity from 1 to 10 Nephelometric Turbidity this water and studies have inferred potential exposure to Units decreases free chlorine effectiveness eight times both NoV (Gray et al. 1997) and HAstV (Myint et al. 1994) (LeChavallier et al. 1981; Hoff 1986; HECS 2003). Thur- from this source. Studies have indicated that the risks of ston-Enriquez et al. (2003) re-evaluated the efficiency of disease resulting from exposure to recreational water may be chlorine and found viruses to be 30 times more resistant as high as 1/1000 for AdV (Crabtree et al. 1997) and when aggregated. If this is so then procedures commonly in perhaps higher for rotavirus (Gerba et al. 1996). use in the US should destroy most but possibly not all The mechanisms of virus inactivation/removal during aggregated viruses (Thurston-Enriquez et al. 2003). wastewater treatment are not clearly understood and the Norovirus was found relatively insensitive to chlorine; in environment itself probably alters the effectiveness of each volunteer studies the virus survived exposure to 6Æ25 mg l)1 process. Physical removal seems to be significant and much for 30 min (CT 187Æ5) and required 10 mg l)1 for destruc- virus (perhaps 95%) appears to be removed via activated tion (CT 300 mgÆmin l)1) (Keswick et al. 1985). These sludge or complexing with mineral particles. Active entero- values imply that NoV could survive some water chlorin- virus has been recovered from sludge (Albert and Schwa- ation procedures. However, these experiments are difficult rzbrod 1991) and HAV was detected by both antigen capture to do and rely on clinical samples as NoV cannot be and PCR suggesting that particles were intact (Graff et al. cultivated. Keswick et al. (1985) used a crude sample of very 1993). EU requirements for heat-treatment before sludge is high titre that may well have protected the virus. A spread onto land should control virus contamination from protective effect due to such factors was later noted this source if followed [EU directive on sewage sludge in (Meschke and Sobsey 2002). When purified, NoV has the agriculture (86/278/EEC) implemented under the sludge same sensitivity to chlorine as poliovirus (Shin and Sobsey (use in agriculture) regulations 1989] but there may be fewer 1998; Meschke and Sobsey 2002). A PCR-based study found controls in the less developed world and these might a CT value of 30–60 to be sufficient to inactivate the virus contaminate crops destined for export. (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 7.2 Potable water treatment and virus survival form in all locations; exogenous material and aggregates are Tap water may account for some 14–40% of gastrointestinal always likely to be present. Rotavirus is inactivated illness and thus efforts to ensure its safety are vital (Payment efficiently by chlorine (CT 112Æ5at3Æ75 mg l)1) and for

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this virus waterborne infections in the developed world are 8. FOOD-BORNE VIRUS TRANSMISSION usually linked to contamination of clean water post- treatment (e.g. Hopkins et al. 1984). Food-borne transmission may be divided into two areas: Enteric viruses have been found in drinking water in primary contamination food materials are already (1–20 PFU per 1000 l) (Payment 1988; Gerba and Rose contaminated before they are harvested, e.g. shellfish 1990; Payment et al. 1997). Studies in France showed that grown in contaminated waters, or soft fruits irrigated/ the presence of astrovirus RNA in tap water was correlated sprayed with contaminated water. Secondary contamin- with an increased risk of intestinal disease (Gofti-Laroche ation occurs at harvest or during processing and empha- et al. 2003). However it is not at all clear what an sizes the role of the food handler preparing foods for acceptable level of virus contamination might be; a risk of others with whom he/she does not come into direct ) infection of 1 · 10 4 per person per year has been accepted contact. Food handlers in this context include field in both the US and the Netherlands (USA EPA 1991; harvesters, production plant workers right through to Staatscourant 2001). Using a model based on rotavirus professional chefs and caterers. Contamination from these dose–response data, Regli et al. (1991) calculated that persons involves not only transfer of virus from infected ) ) 2Æ22 · 10 7 viruses l 1 would be consistent with this risk persons but also their use of polluted water or materials in factor. A 4 log reduction achieved from even highly processing. It is worth noting that transfer of virus from rotavirus-contaminated source water (Raphael et al. 1985) an infected individual to food would be counted as a food- would meet this level (Gerba et al. 1996). Estimates based borne infection if it takes place in the commercial/ on enterovirus levels suggest that higher reductions might industrial setting, but as person-to-person infection if it be required (Regli et al. 1991). Such assessments are occurs in the home. variable, poor culture efficiency would underestimate the It is difficult to assess the extent of food-borne transmis- levels present and increase risk to the consumer; uneven sion in the community, first because person-to-person and distribution of contamination would cause an uneven risk food-borne routes may overlap (above), but secondly across the country and differences in levels of consumption because it is technically difficult to detect viruses in foods, (and the proportion that is boiled before drinking) also apart from shellfish most are not routinely tested. This has affect risk. Finally models based on infection may be less led to two assumptions, first most food-borne outbreaks in relevant than models based on symptomatic illness and which a vehicle is not demonstrated are likely to have arisen could thus exaggerate the risk (Gerba et al. 1985; Gale by contamination from food-handlers – at or close to the 1996; Payment et al. 1997; Haas et al. 1999; Hurst et al. point of serve – and the second is that most (if not all) cases 2001). occurring in the community (i.e. outside outbreak situations) Most documented instances of drinking water contamin- probably arise from person-to-person transmission. These ation by viruses have been attributed to contamination of assumptions should be reconsidered because both disguise previously clean water. This can result simply from or negate potential instances of primary or distant secondary contamination of drinking water supplies, e.g. through contamination. heavy rainfall or flood overwhelming treatment works and Estimates of the incidence of food-borne disease have permitting untreated or partially treated sewage to contam- relied on statistics obtained from outbreaks. In recent years, inate wells (Cannon et al. 1991; Kukkula et al. 1997, 1999). reports of viral gastroenteritis, particularly NoV outbreaks Failures in treatment processes themselves can also allow have shown a dramatic increase in England and Wales; 418 contamination, e.g. pressure failure (Kaplan et al. 1982), cases were identified in 1992 and 2387 in 1996. In 1994 UK insufficient disinfection, or exceptionally high levels of virus reports of NoV outbreaks outnumbered those of Salmonella overwhelming a correctly applied procedure (Payment 1988; for the first time. This increase may be partially explained Gerba and Rose 1990; Payment et al. 1997; Bitton 1999; by increased awareness and improvements in virus identi- Gofti-Laroche et al. 2003). Virus leakage from sewers or fication techniques, thus more recently attempts have been septic tanks can contaminate clean water (Hedberg and made to estimate the extent of community illness. Mead Osterholm 1993). Waterborne outbreaks of enteric virus et al. (1999) estimated that 67% of food-borne illness in the infection are common; between 1980 and 1994 the US US was caused by viruses and that 40% of NoV infections; recognized 28 outbreaks and 11 195 cases of waterborne 1% astrovirus, 1% rotavirus and 5% of HAV infections, illness, of these 9000 cases were NoV and nearly 400 HAV respectively, were food-borne. NoVs were responsible for (HECS 2003). None of these viruses are appreciably affected 23 million cases per year with over 9 million food-borne by freezing (which may actually preserve infectivity for long events. NoV then emerged as the most significant periods) and thus infection is also spread when ice is food-borne infection in the US. Annually NoV was manufactured and distributed using contaminated water estimated to be responsible for 20 000 hospitalizations (Cannon et al. 1991). and 120 deaths.

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world to Hong Kong (ProMED-mail 2002a; 20020331.3850), 8.1 Shellfish and in 2004 Chinese frozen oysters were implicated in a Enterically transmitted viruses are shed into sewage and (presumed NoV) outbreak in Singapore (ProMED-mail become rapidly diluted as they travel through the water 2004; 20040107.0075). In 1993 a multistate outbreak of system. Filter-feeding molluscan shellfish (particularly NoV illness occurred in the US affecting up to 186 000 cockles, mussels and oysters) partially reverse this dilution people (Berg et al. 2000). This was traced to contaminated because they are filter-feeders. They are harvested from oysters harvested in the Gulf of Mexico. Oysters in Europe close-to-shore locations where water will contain virus accumulate a mixture of all viruses present in their environ- inputs from sewage effluents; viruses become concentrated ment, but in these Gulf oysters only one strain was present. within the shellfish and may be retained for some time. This indicated that input virus must have originated from a Levels of virus in shellfish may be 100–1000-fold higher restricted number of infected persons and was possibly than concentrations of viruses in their surrounding water. attributable to incorrect disposal of faeces or vomitus at sea. European regulation divides shellfish harvesting waters This remarkable occurrence indicates firstly just how into different categories depending on the levels of faecal efficiently the shellfish can accumulate virus from their pollution (Table 3). Very badly contaminated waters may not environment and secondly just how significant commercial be used to harvest shellfish for human consumption at all, trade and distribution can be in the dissemination of whilst intermediate levels may be used provided that the infection. It further demonstrates the power of molecular shellfish are relayed into cleaner waters, heat-treated or epidemiology in linking occurrences that would otherwise depurated before harvest. Depuration is a process in which not necessarily have been connected. Sporadic outbreaks the animals are kept in a recirculation tank; water is passed occur throughout the developed world via consumption of over the shellfish, disinfected by UV treatment and recycled. virus-contaminated shellfish (reviewed by Lees 2000). The shellfish gradually purge their bodies of faecal indicator Many shellfish are subject to minimal cooking (if any), bacteria and may be sold when these reach a target level. It is linking this to their known ability to concentrate environ- now clear that bacteria are not an adequate indicator of the mental viruses the fact that they are the most commonly presence of enteric viruses, and numerous virus infections identified source of food-borne virus infection should not be have been documented from shellfish compliant with these surprising. The most significant viruses in this context are regulations (Chung et al. 1996; Griffin et al. 1999; reviewed NoV first reported as an Australia-wide outbreak (Murphy by Lees 2000). Bacteria are purged more rapidly than viruses. et al. 1979), and hepatitis viruses. Recently Aichivirus has Schwab et al. (1998) found that a 95% reduction in been associated with infection from this source in Japan but Escherichia coli was achieved by 48 h depuration but NoV reported cases are few. However the viruses most commonly was reduced by only 7% in the same period. The efficiency of detected in shellfish are enteroviruses (reviewed by Gerba and virus removal is influenced by a number of factors chiefly Goyal 1978). These seldom give rise to symptomatic disease temperature (Dore et al. 2000). Most gastroenteritis viruses although some cases have been reported (Cliver 1997). are more common in winter (even if not shellfish transmitted) Virus concentration by shellfish is nonspecific and thus and increased community illness coupled with less efficient their consumption can expose the consumer to a cocktail of purging by the shellfish could increase the risks associated viruses that might have different effects when present with shellfish consumption at these times. Viruses retain together. Limitations on current technology mean that infectivity well in shellfish and no loss of infectivity was during investigations is it usual to seek only a few defined observed over 1 month in refrigerated storage (Tierney et al. virus types (those that succeed in inducing clinical disease). 1982) or 4 months when frozen (Di Girolamo et al. 1970). However the possibility of multiple simultaneous infection Trade in shellfish permits long-distance transmission: in brought about by the consumption of shellfish should not be 2002 oysters from Cork Bay spread NoV infection around the overlooked, viruses might act synergistically to increase the

Table 3 Microbiological classification of Class Standard Requirements for marketing for consumption shellfish harvesting waters and requirements for marketing for human consumption (taken Class A <230 E. coli or 300 faecal coliforms May be consumed directly from EC directive 91/492). Virus contamin- Class B 90% compliance <4600 E. coli and Depuration or relaying until ation is not assessed 600 faecal coliforms 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 proportion of such cases is this link actually proved. Soft pictures. fruits and salad vegetables, like shellfish are eaten raw but Shellfish transmission of HAV is well documented and also share other features; first, they all have a high water includes the largest ever outbreak that of Shanghai (1988); content – absorbed from groundwater during growth; 300 000 persons were believed to have been infected secondly, many are eaten without peeling which would through consumption of contaminated clams (Halliday et al. remove external contamination. Consequently, primary 1991). Studies around the world have consistently identified contamination is possible externally by spraying or internally shellfish consumption as a major risk factor for the by uptake of viruses from contaminated irrigation water or contraction of hepatitis. In some studies the risk was fertilizer. Surface splash may be significant for fruits close to equivalent to that of contact with an infected person (Koff the ground (e.g. strawberries) and multi-state outbreaks of et al. 1967; Kiyosawa et al. 1987). HAV is rarely detected in HAV may have arisen in this way (Niu et al. 1992; Hutin food itself, largely because of the long incubation period for et al. 1999). Wastewater may be used for irrigation, this illness which means that no samples of suspect food are especially in dry areas where water is more precious, and likely to be available for testing. Even so shellfish are these communities show elevated incidence of hepatitis virus believed to be the major vehicle for HAV infection and infection although reasons have not been identified (Katze- Salamina and D’Argenio (1998) estimated that up to 70% of nelson et al. 1976). all hepatitis A in Italy was contracted from shellfish. In the Viruses can enter plants through root damage (Katzenel- UK it is recommended that shellfish flesh be raised to 90C son and Mills 1984), and this occurs universally through for 1Æ5 min (Millard et al. 1987) and continuous flow root abrasion with soil particles. Other workers found virus methods now ensure that all shellfish are subjected equally uptake from roots to the leaves (but not the fruits) of tomato to this treatment. Since the implementation of these plants even when virus was introduced below the soil surface recommendations, there have been no reports of outbreaks eliminating the possibilities of aerial contamination (Oron in the UK of either viral gastroenteritis or hepatitis A et al. 1995). Our own data also show that internal contam- associated with shellfish heat-treated in this way (Appleton ination can occur but is probably of low level compared with 2000). Work by Croci et al. (1999) suggested that even these external contamination (M.J. Carter, unpublished observa- standards may not be sufficient to completely eliminate the tions). However, a lower amount of virus inside a crop plant virus and they recommended treatment at 100C for 2 min. could potentially have an effect equivalent to a much higher Hepatitis E is not commonly transmitted by shellfish external dose because it cannot be removed by either peeling (Chan 1995; Stolle and Sperner 1997) but has been or washing and may persist for longer because it is shielded identified in sewage even originating from nonendemic from UV inactivation. Washing is not always efficient as a areas (Pina et al. 1998b). This finding may be related to the means of removing contamination, for instance HAV was seroconversions also identified in such nonendemic regions found to adhere well to produce, especially lettuce (Croci (see above). Thus the possibility of shellfish transmission et al. 2002). It is difficult to detect viruses in plant material must be considered. and in only a few cases has contamination been shown There are few reports of astrovirus transmission via directly, e.g. NoV in raspberries (Gaulin et al. 1999). Most shellfish consumption (Caul 1987) although recently shell- associations are made by epidemiological connection and fish were implicated in a large outbreak involving teachers identification of the infectious agent in clinical samples from and young adults at a school in Japan (Oishi et al. 1994). those affected (reviewed by Seymour and Appleton 2001). This was unusual in that it affected older persons and was Fruit and salad vegetables are traded around the world, attributed to a higher serotype virus. Astroviruses are originating from areas where sunlight permits growth but frequently detected when sought in oysters and the apparent where water quality is not always assured. This trade either lack of resultant illness is presumably attributable to prior as raw materials or as ready-to-eat products can aid immunity remaining from childhood infection. Similar widespread virus dissemination. In 2003 an outbreak of considerations apply to rotavirus contamination. 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 8.2 Soft fruit and salad vegetables Mexico although virus was not identified in the food; After shellfish, food-borne virus illness is most commonly identification was possible only by analysis of the chain linked to salads and soft fruits. These items are almost of importation and transport (ProMED-mail 2003; always subject to handling immediately before serving and 20031128.2946). In 1997 contaminated strawberries were this presents an opportunity for contamination. In virtually used in a school lunch preparation that was distributed to 17 all cases where this type of food is implicated an infected states in the US and may have exposed up to 9000 children food handler is suggested as the origin but in only a small (ProMED-mail 1997; 19970402.0689). A total of 200 cases

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of HAV were reported in Michigan. Similarly, pre-prepared To some extent this may arise from the assumption that salads distributed across the US were responsible for a community illness is entirely or mainly spread by the direct multi-state outbreak of NoV affecting over 300 persons person–person route even though supporting data for this (ProMED-mail 2000; 20000318.0377). Imported raspberries assumption is weak. When investigations have attempted to were responsible for an outbreak in Canada (Gaulin et al. distinguish between person-to-person and food-borne ill- 1999). Viruses are not destroyed by freezing or freeze- nesses in the community the latter can be identified by drying, although there are few studies of the latter process in statistical means and suggest that there are potentially many contaminated food, in the laboratory freeze-drying is an thousands of such instances per year (see below). efficient means of preserving virus infectivity and should be expected to preserve viruses in produce. Both hepatitis A 8.3 Other foods (Reid and Robinson 1987) and NoV (Ponka et al. 1999) have been transmitted by frozen fruit. The growth in the Few other foods are associated with primary contamination worldwide trade in freeze-dried soft fruits (particularly but a recent example is the consumption of raw liver from raspberries and strawberries) offers another potential vehicle wild boar or venison. Both have resulted in the transmission for transmission. of HEV to humans (Matsuda et al. 2003; Tei et al. 2003; There are no data concerning levels or persistence of Tamada et al. 2004). Consumption of raw liver is rare but potential internal virus contamination of fruits and vegeta- virus contamination of utensils used in handling it should be bles, although Sadovski et al. (1978) recovered poliovirus borne in mind. Supermarket pork liver has also tested from cucumbers that had been irrigated with contaminated positive for HEV (Yazaki et al. 2003) – even in countries water 8 d earlier an upper limit to virus persistence was not with no significant HEV infection. This suggests that pork established. In areas of high UV illumination surface liver is not a significant source of infection for humans. contamination may be short-lived in the field, Badawy et al. (1990) studied virus survival on sewage irrigated grass in 8.4 Food handlers southern USA and found 2 log reductions in titre were achieved in just 8–10 h in the summer, slightly longer in the Food handlers may contaminate foods at any point from winter (16–24 h). However viruses can survive well on the harvest to serving. Toilet arrangements (including hand surface of produce once harvested, a number of studies have washing) should be provided for harvesters in the field to indicated survival times of longer than a week and some- prevent contamination of produce as it is picked. Infected times up to 30 d for a range of enteric viruses under typical food handlers may or may not be symptomatic at the time of storage conditions (Konowalchuk and Speirs 1974, 1975a,b; contamination. HAV infections may be mild or asympto- Badawy et al. 1985; Pirtle and Beran 1991; Kurdziel et al. matic but large numbers of particles are nonetheless shed in 2001; Croci et al. 2002). Increasing temperature decreased the faeces. PCR technology has shown that even in virus survival times but conditions usually associated with symptomatic illness, NoV shedding may precede symptoms produce storage were conducive to virus survival. It is not by 10–30 h and may continue (albeit it at a much lower clear what effect microenvironments on a plant surface level) for 1–2 weeks afterwards (Graham et al. 1994; might have, waxy cuticles, hairy projections and curled, Okhuysen et al. 1995; Estes and Leparc-Goffart 1999). crinkled or convoluted leaves can all modify the way that NoV transmission by pre-, post- or asymptomatic food virus contamination might behave, the amount of wetting handlers has been demonstrated (Patterson et al. 1993; will affect the way that a droplet dries whilst convoluted leaf Parashar et al. 1998; Gaulin et al. 1999). Cowden et al. surfaces could shield a virus from UV or increase local (1995) recommend a 48-h period of exclusion following the humidity. These effects are difficult to replicate experi- cessation of symptoms but as virus shedding is likely to mentally. Data from Kurdziel et al. (2001) and Ward and continue for some weeks returnees must follow strict hand Irving (1987) show that there is variability in the times that washing procedures. Inadequate hand washing/personal poliovirus may survive on different artificially contaminated hygiene has been implicated in outbreaks of both NoV and plants stored at 4C. Similarly these potentially sheltered HAV (Bean et al. 1990; Bidawid et al. 2004) and recently its environments suggest that washing processes will likewise effectiveness has been demonstrated. Bidawid et al. (2000b) vary in efficiency even over sections of the same plant. considered the likely levels of hand contamination by HAV, These data suggest that contaminated fruit and vegetables and then estimated the resultant transfer of viruses to foods might account for a large number of food-borne infections. by handling. They concluded that up to 10% (or appprox. A study in the UK indicated that these might present a 1000 particles) could easily be transmitted via the fingers to significant opportunity for virus infections (O’Brien et al. food items (lettuce). If proper washing procedures were 2000). However, the criticism frequently levelled at this followed this transfer fell to 0Æ3–0Æ6%. Other experiments implication is that these infections are simply not observed. have shown that virus transfer rate is high immediately after

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hand contamination but reduces as the virus dries (Larson sandwiches, pies, vegetable salads, gateaux, fish, lobsters and 1985; Ansari et al. 1988; Springthorpe and Sattar 1998). prawns. Consequently there was potential for primary (or at Whilst this might imply that the window for contamination least remote secondary) contamination in 80% of these closes rapidly, it should be borne in mind that virus does cases. survive drying onto the skin and reapplication of moisture (as in handling washed and wet foods) could then lead to 9. PERSON-TO-PERSON TRANSMISSION increased transfers once more. Findings such as these emphasize the importance of proper hygiene in controlling Most enteric viruses are highly contagious, for most the food-borne infection from this source. However, given the infectious dose is believed to lie between 10 and 100 particles likelihood that this will not be achieved in all cases, and for NoV possibly lower. Given rotavirus shedding at a Koopmans and Duizer (2004) attempted to estimate the level of 109 per ml, as little as 0Æ1 ll of stool contamination risks associated with food contaminated in this way. Using could contain an infectious dose. Such tiny quantities are the value of 1000 particles for transfer (above), they reasoned invisible and easily retained on skin. Although hand washing that a 3 log reduction in virus viability would be required to is effective as a disinfectant (particularly alcohol-containing ensure safety after such a contamination event. They then washes) in the case of rotavirus (Estes et al. 1979) infections estimated the effects of various common food-processing are easily transmitted from person to person either directly procedures that might follow handling (e.g. freezing, or via contamination of objects and surfaces. fermentation, acidification, pasteurization, etc.). With the Vomit is also significant; 30 million particles may be shed exception of boiling (and other high temperature treatments) by this route (Reid et al. 1988; Caul 1994) and RV may no procedure achieved a 3 log reduction suggesting that survive for up to 9 d in the air (Sattar et al. 1984). Proximity viable virus would remain in almost all cases to present a to vomit at the time of vomiting may be more significant threat to the consumer. Mariam and Cliver (2000) also than exposure later on; in a hotel NoV outbreak infection showed that HAV would survive common pasteurization was related to distance between the secondary cases and the procedures for milk (30 min at 63C and 15 s at 72C). primary case at the time of the event (Marks et al. 2000) and The sudden onset of projectile vomiting and associated in a hospital setting, persons in the proximity of a patient aerosolization of virus particles is particularly problematic who vomited were at greater risk of acquiring NoV than when this occurs in a food handler. As vomiting may be the those who actually cleaned up the vomit (Chadwick and first overt sign of infection such a situation is virtually McCann 1993). This implies that aerosolization is a potent impossible to prevent. Given the survival characteristics of means of infection of those in the immediate vicinity. these viruses all uncovered food should be destroyed or Airborne transmission is distinct from respiratory transmis- cooked even if at some distance from the event. Decontam- sion as the route of infection remains via the gut rather than ination of catering premises presents similar problems to the respiratory tract. Infection could result from the that of hospitals. A sink into which an infected food handler trapping of aerosolized droplets containing virus in the had vomited was thoroughly cleansed with chlorine bleach. nasal passages and subsequent swallowing of virus. Alter- The following week the same sink was used to prepare salad natively aerosolized virus may travel some distance before and resulted in an outbreak of NoV amongst the consumers settling and contaminating surfaces or uncovered food. (Patterson et al. 1997). Food handlers certainly can con- Spread may be assisted by artificial ventilation (Chadwick taminate foods but this is usually proved in only a minority et al. 1994). of cases. Attempts were made to assign transmission as Person-to-person transmission is clearly favoured by person-to-person or food-borne in an analysis of 2149 closed settings, persons cannot leave cruise-ships, hospitals outbreaks occurring between 1992 and 1995 (ACMSF or care homes and once a virus has been introduced by 1998). About 33% were attributed to NoV, 2% to RV and whatever route efficient spread within that setting is to be 0Æ5% each to astroviruses and small round (parvovirus)-like expected. In 2002 the CDC, Atlanta, recorded 21 outbreaks agents. Although not robust, this analysis suggested that of NoV on 17 ships docking in the US (ProMED-mail many of the NoV outbreaks were food-borne and a food 2002b; 20021212Æ6049); this included one vessel on which vehicle was suggested in 35 cases. Virus was detected in only more than 200 cases were reported. Once a virus is present it two of the suspect foods (both oysters), in the remaining can be very hard to eliminate even by thorough disinfection cases contamination was attributed to a food handler but (Chadwick et al. 2000; Barker et al. 2004). Some ships subsequent investigation confirmed food handler infection in showed serial outbreaks amongst each new set of passengers only 20%. Food vehicles implicated were diverse but coming aboard. This is not surprising as even cleaning in a included a variety of fresh fruits and salads as well as other hospital context may fail – serial astrovirus infections were items that would commonly either contain or be served with experienced in a bone marrow transplant unit despite a salad or fresh fruit garnish. These included items such as vigorous attempts to disinfect the unit between patients

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(Cubitt et al. 1999). Conventional cleaning of surfaces can 10. VIRUS DIAGNOSIS AND DETECTION fail to remove detectable NoV virus (Green et al. 1998; 10.1 Clinical samples Barry-Murphy et al. 2000). An assessment of the impact of these viruses in human disease requires the provision of a sensitive and reliable 9.1 Person-to-person or food-borne? detection method for application to clinical samples. Rout- Unsurprisingly, contact with an infected person is a major ine reagents for the detection of rotavirus, HAV and risk factor for contraction of these viruses: secondary attack poliovirus have been available for some time but technology rates of up to 95% have been reported for HAV within the for the other viruses of interest has only recently moved home (Villarejos et al. 1982). HEV appears to infect less well from the specialist laboratory into widespread use. As a in the domestic setting and secondary attack rates are lower result the burden of disease contributed by other gut- (Purcell 1997) even though virus shedding may be more infecting viruses has not been so clearly established. prolonged (Clayson et al. 1995b). Contact with an infected Diagnostic reagents have formerly been hard to provide person was found to constitute the greatest single risk for and in their absence perhaps the simplest method has been infection by NoV in studies in the UK, US and the to examine the faeces using electron microscopy. This is a Netherlands (Mead et al. 1999; FSA 2000; De Wit et al. catch-all method and requires no prior knowledge of the 2003). However this does not mean that all community virus in order to detect it but it is expensive, makes high spread must be via the person-to-person route, because operator demands and its efficiency depends on a virus unlike other viruses (which may be maintained entirely by being readily recognizable; Viruses that are large, of this route), enteric viruses can also spread through food or distinctive appearance, and present in significant numbers water. It is difficult to estimate the contribution of each (>106 particles per ml) are relatively easily observed. route to the total burden of infection in the community. However, small, diffuse or fuzzy viruses can often be Most figures consider only outbreaks, but the IID survey in overlooked. Identification makes high demands on the the UK revealed the true extent of these missed commu- operator and this is even worse when a virus may have nity infections for the first time (FSA 2000). This survey several distinct appearances (see astroviruses). Historically it estimated that for every case of NoV that is laboratory is thought that this method has identified AdV and rotavirus confirmed, over 1500 occur in the community. The report infections quite well but has been less successful in the cases attributes community cases (almost) entirely to person-to- of caliciviruses, astroviruses and parvoviruses. person transmission but presents no data on this point. Most gut-dwelling bacteria are routinely identified by Recently De Wit et al. (2003) have made a thorough attempt culture; however, this route has not been open to the to determine the extent of food-borne transmission in the detection of viruses. All viruses require living cells as hosts community. They found that person-to-person transmission and are totally dependent on the processes that their host was indeed common, much of it occurring in the home and cells are able to provide, e.g. for protein synthesis and in many cases actually involving contamination of food protein processing, but the gut is a very specialized habitat prepared for others. This is not counted as food-borne in virological terms. It contains a multiplicity of different infection (see above). The analysis also showed for the first cell types, each having a different enzyme content and time that person-to-person transmission alone could not surface protein composition; they may even vary in these account for all community illness and the authors concluded properties at different stages of their differentiation. Fur- that 12–16% of infections could be attributed to contam- thermore, they are all bathed in a solution of the various inated food/water entering the household. In the UK there secreted products produced by other specialized cells, are some 9Æ5 million cases of IID annually and has been notably of course proteases and bile salts. These features estimated that some 16% (1Æ52 · 106) are caused by NoV. make the gut a very difficult cellular environment to mimic Assuming that all of these took place in a household of four in culture. A virus may replicate in one type of cell, at one persons and that all persons in each household were stage of its differentiation and may require soluble products infected, this would correspond to a minimum of 380 000 released from quite different cells entirely. It is probably for infected households. Lastly, if 12–16% of these resulted this reason that gut-replicating viruses have been very from incoming contaminated food as above, then contam- difficult to culture in the laboratory. Those that can be inated food could instigate directly some 45–60 000 food- cultivated often require that the culture be supplemented borne cases in the UK per year as a minimum estimate. with proteases (usually trypsin) and in one case (porcine Estimates in the Netherlands using less conservative enteric calicivirus) with duodenal juice, bile salts being the assumptions suggest 80 000 food-borne NoV infections active ingredient (Chang et al. 2004). Specialized cells are could occur annually even in this smaller country (De Wit often needed, e.g. differentiating colonic carcinoma cells for et al. 2003). astroviruses. Viruses that penetrate beyond the gut, invading

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other tissues are often (but by no means always) simpler to either not be cultivated or cultivated only with difficulty, cultivate. The significance of this point cannot be overstated, most detection methods have concentrated on PCR tech- most extracellular parasites will grow reasonably efficiently nology. This has proved extremely successful when applied in a broth which mimics the gut contents, but this is not true to NoV detection in water and shellfish and can even of viruses and there are very good reasons why the provide quantitative information (Laverick et al. 2004; Le characterization and detection of the viruses of gastroenter- Cann et al. 2004). However PCR-based detection has two itis have been problematic. Culture for viruses is not problems, the first is that most of these viruses are RNA generally used even to identify agents present in a faecal viruses, PCR cannot be used unless the RNA has been sample. The chances of using such a procedure for the reverse transcribed (RT) to manufacture a DNA copy and detection of the lower quantities of virus present in food are the efficiency of this RT step is notoriously variable. still more remote. Secondly PCR targets only a short section of the virus In view of the above difficulties in EM detection, reagent genome (usually 200–300 bases) and reveals nothing at all availability in the past and difficulties in culture most about the presence or integrity of the rest of the genome. workers have acknowledged the existence of a diagnostic Strictly then, even quantitative PCR for such a virus detects gap represented by the large proportion of infections for only the levels of cDNA to a specific region of the virus, it which no obvious cause could be found. Even in the IID cannot tell us how the numbers of DNA copies detected survey in England and Wales, when causative agents were relate to the number of RNA copies actually present in each vigorously pursued, target organisms were not identified in case and nor can it tell us whether the target detected was 63% of community samples and 45% of General Practice present as an intact genome inside a viable particle or samples (FSA 2000). Given the difficulties in virus diagno- present as a short section of free RNA released by virus sis, it was always likely that a large part of this diagnostic gap degradation. The first of these difficulties can be tackled by would probably be caused by viruses, but probably by the careful incorporation of internal controls, although these smaller, less well-characterized and less-readily observed may of themselves interfere with target amplification. The viruses. The extent of any such bias in detection has recently second is harder to address. It is often assumed that RNA become clearer with the development of objective and degradation occurs in two phases; virtually none at all whilst sensitive methods for the detection of many viruses: the the particle is intact followed by rapid and total degradation development of recombinant antigens for astroviruses and once the particle is breached. Consequently levels of free especially NoV, and the development of ELISA systems for RNA should be insignificant and most detectable RNA both have at last provided a reproducible supply of antigen would be contained inside an intact particle. However it is from which to prepare and validate diagnostic serological not clear that this assumption is valid; whilst Slomka and reagents as well as to conduct serosurveys of the incidence Appleton (1998) used FCV as a model for NoV and found and age-at-first-infection profiles of these viruses. This type that RNA was indeed rapidly degraded after inactivation of of detection method has been used in the IID survey in the virus in shellfish they also confirmed that some PCR- England and Wales – although not all tests in all cases (FSA positive samples were not cultivable. Similar findings were 2000), and in similar studies in Europe (Koopmans et al. reported for poliovirus and bacteriophage MS2 (Shin and 2000) and the US (Mead et al. 1999). These studies all Sobsey 1998; Sobsey et al. 1999). Recently a study by support the conclusion that virus-associated enteric illness Nuanualsuwan and Cliver (2003) suggests that the situation has been greatly underestimated and currently suggest (in could be yet more complex. These workers inactivated the absence of concrete data) that most, if not this entire calicivirus and picornaviruses (including hepatitis A) by diagnostic void is made up of NoV infections. Similarly data exposure to high temperature (72C), UV radiation and from the FSA survey in England (2000) suggested that for hypochlorite disinfection. They found that infectivity of all every case of NoV actually detected in the diagnostic viruses could be vastly reduced by these means. However, laboratory, on average 1562 have probably occurred in the the RNA inside the particle appeared to survive, the community. 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 10.2 Food samples this failure the RNA remained intact within them and Virus detection in food and water (i.e. preinfection) is even continued to be detected by PCR; furthermore it was still more problematic than detection postinfection, in clinical protected from degradation by hydrolytic enzymes suggest- samples. Levels of virus are orders of magnitude lower and ing that the disjunction between PCR detection and virus the challenge is to detect a potentially infectious dose viability would become worse with time. These findings are (perhaps as low as 10 particles) in a quantity of a size likely of great significance as they suggest that PCR-based to be consumed as a single portion. As these viruses can detection and quantitation would also detect virus particles

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that are inactive and pose no threat to a consumer. This gastroenteritis outbreaks) and are thought to show similar would overestimate the extent of virus contamination. stabilities to the enteric viruses. These data suggest that they Antibody-based detection methods (or PCR detection may be good models for virus contamination in these systems following an antibody-mediated concentration step) should (Dore et al. 1998, 2000). However it should be borne in mind overcome this effect and this has also been observed (Sobsey that indicator organisms such as these can only indicate the et al. 1999). A technical account of the primers and possibility of human virus contamination; they cannot prove conditions required for virus detection is beyond the scope either its presence or its absence. F+-specific bacteriophage of this review but have been summarized elsewhere (Sair can be specifically enumerated using an engineered pilus- et al. 2002). bearing Salmonella typhimurium host that cannot be infected One drawback of PCR-based detection is that the test by non-RNA coliphage (Havelaar and Hogeboom 1983). Use consists of the observation of amplification or failure to of this host yields data relatively free of contaminating amplify. Specificity relies on the choice of primers which somatic coliphage but does not easily separate contamination should amplify only the selected target viruses. This approach of animal origin from that of human. Further work would be is intrinsically unsuitable for the identification of multiple necessary to make such a distinction using oligonucleotide viruses simultaneously. It yields no information on the hybridization (Beekwilder et al. 1995; Hsu et al. 1995). At presence of other virus types not specifically sought by the present there are no data concerning the use of these procedure, or more distantly related to the known viruses organisms as potential indicators of faecal contamination for from which primers were designed. PCR for NoV for instance soft fruits and vegetables. Mariam and Cliver (2000) can identify only 90–95% of EM-positive samples, presum- attempted to use bacteriophage to determine the extent of ably because of inherent sequence variability within the virus virus inactivation likely in response to certain treatments in family. However microarray technology now offers the ability various food processes and Dawson et al. (2005) used phage to use multiple and redundant PCR primers to target and to monitor the survival of viruses on soft fruits. Slomka and amplify a range of viruses present in a sample. Specificity is Appleton (1998) used feline calicivirus in a similar manner. then achieved at the array hybridization stage when hybrid- However these experiments require careful analysis as ization to a microarray plate containing several hundred neither are exactly equivalent to the viruses of interest. probes for individual viruses or groups of viruses detects each Mariam and Cliver (2000) found that both MS2 and UX174 simultaneously. Such detection technologies offer the chance were more labile than HAV in their analysis. However they of defining at last the true extent of virus contamination of nevertheless concluded that these organisms could be useful both food and the environment. 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 11. ALTERNATIVE INDICATORS OF VIRUS of concern to calibrate the response of the indicators in a CONTAMINATION variety of circumstances and such data is urgently required. In view of the difficulties in detecting viruses above, much use has been made of indicator organisms to provide clues to 12. CONCLUSIONS the likelihood of virus contamination (reviewed by Kator and Rhodes 1987). Cultivable enteroviruses such as poliovirus Food-borne viruses are ubiquitous; it is likely that their have been extensively used. This agent has been ubiquitous incidence has been underestimated in the past not only in in sewage-polluted environments because of widespread terms of the number of infections but also in terms of the vaccination in the community and the resultant faecal frequency with which they are food-borne. Sewage treat- shedding from vaccinees. However it is difficult and expen- ment without disinfection should be recognized as inad- sive to routinely extract and assay poliovirus from contam- equate and UV treatments of final effluents should be inated food and water, further the WHO aim to eliminate this encouraged to protect both recreational waters and espe- virus from the world and work with it is already subject to cially shellfish harvesting areas. Intrinsic contamination of control. As a substitute many workers now suggest AdV (see foods other than shellfish needs to be considered and more above). However an alternative approach has been to use work is needed to characterize the uptake, survival and nonhuman viruses such as the bacteriophages (Power and removal properties of viruses in plant tissues. Given the Collins 1989, 1990) and chief amongst these are the male- difficulty of excluding (or even identifying) food handlers specific bacteriophages of the Levivirus family such as MS2. likely to be shedding virus at any one time and the growth These bacteriophages contain an RNA genome of similar size in national/international trade in foods requiring minimal to the picorna-, calici- and astroviruses and have particle sizes cooking we may expect food-borne viruses to increase in in a similar range. Bacteriophage are frequently found in significance in the future. This effect may be of most faecally contaminated materials (and shellfish associated with significance in the case of HAV where the divergence in

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