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Molecular Virology of Enteric Viruses (with Emphasis on Caliciviruses)

Human caliciviruses are members of the family Caliciviridae and are responsible for a majority of the outbreaks of acute nonbacterial gastroenteritis. In fact, they now are considered a common cause of sporadic cases of diarrhea in the community (Glass et al., 2000b; Koopmans et al., 2002; Lopman et al., 2002; Hutson et al., 2004). These viruses were implicated in as many as 95% of the reported viral gastroenteritis outbreaks examined over a 4.5-year period in the United States (Fankhauser et al., 2002), and similar high incidence rates have been found in other studies (Maguire et al., 1999; Glass et al., 2000b; Koopmans et al., 2000; Lopman et al., 2003). Common features of the Caliciviridae include the presence of a single major structural protein from which the capsid is constructed and 32 cup-shaped depressions on the surface of the virion arranged in an icosahedral symmetry. The name of the family was derived from the Latin word calix, which means cup or goblet, and refers to the surface hollows (Madeley, 1979). Another major feature of Caliciviridae is the absence of a methylated cap at the 5′ end of the viral RNA. Instead, a small protein (VPg) of ∼10 × 10 to 12 × 10 kDa is covalently linked to the viral RNA and is considered essential for the infectivity of the RNA (Black et al., 1978; Ando et al., 2000). Norwalk virus was the first human enteric calicivirus to be discovered, this after a gastrointestinal outbreak affecting both children and adults in an elementary school in Norwalk, Ohio (Kapikian et al., 1972). Over the next several years, other agents of epidemic viral gastroenteritis were described including Hawaii virus, Montgomery County agent, Snow Mountain virus, Southampton virus, Toronto virus, and so forth. An interim scheme to classify these viruses on a morphological and physicochemical basis was proposed by Caul and Appleton (1982). Some of these viruses were antigenically related to Norwalk virus by IEM (immune electron microscopy) and crosschallenge studies, whereas others, like the Sapporo virus (Chiba et al., 1979), were found to be antigenically distinct (Nakata et al., 1996). Human caliciviruses have been classified into two distinct genera namely, Norovirus (previously called “Norwalk-like viruses” (NLVs) or “small round structured viruses”) and Sapovirus (formerly “Sapporo-like viruses,” or SLVs) (Green et al., 2000b; Mayo, 2002). The noroviruses and sapoviruses form distinct phylogenetic clades within Caliciviridae (Berke et al., 1997), and certain features of their viral genome organization distinguish them from

each other and from other genera of the family (Fig. 3.1). These viruses also differ in their epidemiology and host range. For example, noroviruses infect all age groups and are usually responsible for outbreaks of acute gastroenteritis frequently associated with contaminated food or water. Sapoviruses, on the other hand, are associated with sporadic cases of acute gastroenteritis and mainly infect infants and young children (Green et al., 2001), although outbreaks in adults have also been described (Noel et al., 1997).
Enteric caliciviruses of animals, associated with gastroenteritis in pigs, calves, chickens, mink, dogs, and cats, have also been described (Guo et al., 1999b;Liu et al., 1999a;van der Poel et al., 2000;Guo and Saif, 2003). The Caliciviridae has two additional genera, Lagovirus and Vesivirus, each of which includes caliciviruses that infect animals such as the rabbit hemorrhagic disease virus (RHDV) and the vesicular exanthema of swine virus (VESV), respectively. The proposed nomenclature for calicivirus strains is host species from which the virus was isolated/virus genus/virus name/strain  Figure. 3.1 Genome organization of the four different genera of Caliciviridae. The genome of Norovirus and Vesivirus has three open reading frames (ORFs) that encode the nonstructural proteins, the major capsid protein (VP1), and a minor structural basic protein (VP2). The genera Sapovirus and Lagovirus encode the capsid protein contiguous with the large nonstructural polyprotein (ORF1). An additional small overlapping ORF in a +1 frameshift has been described in certain strains of sapoviruses. A subgenomic RNA that covers the entire 3′ end of the genome, from the capsid gene to the 3′ end, has been detected in calicivirusinfected cells.
On examination by negative contrast electron microscopy (EM), caliciviruses show characteristic cup-shaped structures on their surfaces. These cuplike depressions are more prominent in sapoviruses, leading to the characteristic six-pointed "star of David" appearance, especially when viewed along the major two-, three-, and fivefold axes of symmetry. The noroviruses have a polyadenylated, positive-sense, single-stranded RNA genome with three major open reading frames (ORFs) (Jiang et al., 1993b;Lambden et al., 1993). The virions have a buoyant density of 1.33 to 1.41 g/cm 3 in cesium chloride (CsCl 2 ) (Caul and Appleton, 1982;Madore et al., 1986) and usually lack the distinctive calicivirus cuplike morphology when viewed by EM. The sapoviruses have a polyadenylated, positive-sense, single-stranded RNA genome with two (or three) major ORFs (Liu et al., 1995), have a buoyant density of 1.37 to 1.38 g/cm 3 in CsCl (Terashima et al., 1983), and often possess distinctive calicivirus cup-like morphology (Madeley, 1979).
The molecular era of norovirus research started with the successful cloning of the genomes of Norwalk and Southampton viruses obtained from stool samples (Jiang et al., 1990;Lambden et al., 1993). Despite numerous attempts, human calicivirus infections have not been induced in experimental animals nor have these viruses been propagated successfully in cultured cells, thus hampering many aspects of their research (Duizer et al., 2004). Unlike human caliciviruses, some animal caliciviruses, including primate calicivirus (Smith et al., 1983), feline calicivirus (FCV) (Love and Sabine, 1975), and San Miguel sea lion virus (SMSV) (Smith et al., 1973), have been successfully propagated in cell cultures. These viruses have provided a direct approach for the study of virus infections, genome transcription, viral protein translation, and virus replication (Green et al., 2002). In addition, information gained by the study of caliciviruses that grow efficiently in cell cultures, such as FCV and VESV, or that have an animal model and a limited cell culture system (such as RHDV), has been important in the identification of features that are likely to be shared among members of the Caliciviridae (Marin et al., 2000;Morales et al., 2004). of 22 Å. This analysis showed that the particles (38 nm in diameter by this technique) have a distinct architecture and exhibit T = 3 icosahedral symmetry ( Fig. 3.2). The capsid contains 180 copies of the capsid protein assembled into 90 dimers with an arch-like structure. The arches are arranged in such a way that there are large hollows at the icosahedral five-and threefold positions, and these hollows are seen as cuplike structures on the viral surface (Prasad et al., 1994(Prasad et al., , 1996a(Prasad et al., , 1999. To form a T = 3 icosahedral structure, the capsid protein has to adapt to three quasi-equivalent positions, and the subunits located at these positions are conventionally referred to as A, B, and C. The only high-resolution (3.4 Å) structure is that of the Norwalk virus capsid determined by x-ray crystallography (Prasad et al., 1999).
Each subunit or monomeric capsid protein folds into an N-terminal region facing the inside of the capsid, a shell (S) domain that forms the continuous surface of the VLP, and a protruding (P) domain that forms the protrusions (Fig. 3.2). A flexible hinge of eight amino acids links the S and P domains. The P domain is located at the exterior of the capsid and is likely to contain determinants of genotype specificity. The NH 2 -terminal (N) arm, located within the S domain, consists of residues 10 to 49 and faces the interior of the capsid. The part of the S domain that forms a β-barrel consists of amino acids 50 to 225. The entire S domain (amino acids 1 to 225) corresponds to the N-terminal region of the capsid protein that is relatively conserved among noroviruses in sequence comparisons.
Amino acid residues 226 to 530 form the P domain, which corresponds to the C-terminal half of the capsid protein and forms the arch-like structures extending from the shell. The S domain is required for the assembly of the capsid and participates in multiple intermolecular interactions of dimers, trimers, and pentamers, whereas the P domain is mainly involved in dimeric interactions (Prasad et al., 1999). The P domain is considered to have subdomains, P1 formed by amino acids 226 to 278 and 406 to 530, and P2 encompassing amino acids 279 to 405. The P2 subdomain is the most variable region of the capsid protein among noroviruses  and is believed to play an important role in immune recognition and receptor interaction. The isolated P domain forms dimers and binds to histo-blood group antigen receptors, not requiring the formation of VLPs (Tan et al., 2004). In addition, a binding pocket in the P domain is responsible for viral receptor binding, and formation of this pocket involves only intramolecular interactions . Prasad et al. (1999) have demonstrated that, although the S domain has a canonical eight-stranded β-barrel structure, the P2 subdomain in the P domain has a fold similar to that observed in domain 2 of the elongation factor Tu (EF-Tu), a structure never before seen in a viral capsid protein. Moreover, the fold of the P1 subdomain is unlike any other polypeptide observed so far (Bertolotti-Ciarlet et al., 2003).
A comparison of the capsid proteins from various caliciviruses reveals significant variations both in their sequences and in their sizes. In general, the capsid proteins of human caliciviruses are smaller than those of animal caliciviruses (Chen et al., 2004). The Norwalk virus recombinant capsid protein can also self-assemble into smaller VLPs (23 nm) with suspected T = 1 symmetry that is thought to be composed of 60 copies of the capsid protein (White et al., 1997).
It has been postulated that the N-terminal region of the capsid protein or the genomic RNA acts as a switching region that controls variations in the conformation of the coat protein of T = 3 viruses (Rossmann and Johnson, 1989). Norovirus particles are different from other T = 3 viruses because recombinant capsid protein readily forms rVLPs without RNA (Prasad et al., 1999). It has been suggested that the determinants for the T = 3 capsid assembly for Norwalk virus may lie outside of the N-terminus and that the interaction between subunits B and C is not mandatory for the formation of the capsid (Bertolotti- Ciarlet et al., 2002).
Caliciviruses have a linear, single-stranded, positive-sense RNA genome of 7.5-7.7 kb (Green et al., 2001) (see Fig. 3.1). The RNA genome of Norwalk virus, the prototype strain for the genus Norovirus, is 7,654 nucleotides in length and is polyadenylated at the 3′ end (Jiang et al., 1993b). The lack of a cap structure typical of eukaryotic mRNA and the absence of an internal ribosomal entry site suggest that the VPg protein may play a role in the initiation of protein synthesis on calicivirus RNA through unique proteinprotein interactions with the cellular translation machinery (Daughenbaugh et al., 2003). Removal of VPg from calicivirus RNA by proteinase K digestion results in loss of infectivity and dramatically reduces translation of FCV RNA in vitro (Herbert et al., 1997). The genome of calicivirus is organized into two or three major ORFs. The nonstructural proteins encoded in the calicivirus ORF1 were predicted on the basis of their sequence similarities with picornavirus nonstructural proteins (Neill, 1990). Amino acid sequence motifs in common with the poliovirus 2C NTPase, 3C protease, and 3D RNAdependent RNA polymerase (RdRp) were readily identified and provided templates for further characterization of the calicivirus nonstructural proteins. Proteolytic mapping and enzymatic studies of in vitro-translated polyprotein or recombinant protein expression have confirmed the presence of an NTPase (p41), a 3C-like protease (3CL pro ), an RdRp, and the location in the polyprotein of the genome-linked protein VPg (Liu et al., 1996;Dunham et al., 1998;Pfister and Wimmer, 2001). The proposed six nonstructural proteins encoded in the norovirus ORF1 defined so far proceed N to C terminus, p48-NTPase-p22-VPg-3CL pro -RdRp (Ettayebi and Hardy, 2003). It has been recently reported that the 3C-like proteinase (3CL pro ) inhibits host cell translation by cleavage of poly(A)-binding protein (PABP), a key protein involved in the translation of polyadenylated mRNAs (Kuyumcu-Martinez et al., 2004).
In the genera Norovirus and Vesivirus, the capsid structural protein VP1 is encoded in a separate ORF (ORF2), whereas viruses in the genera Sapovirus and Lagovirus encode the capsid protein contiguous with the large nonstructural polyprotein (ORF1) (see Fig. 3.1). Viruses in the latter two genera have two major ORFs (ORF1 and ORF2). In the genus Sapovirus, the ORF1/2 junction consists of a one-or four-nucleotide overlap between the stop codon of ORF1 and the first AUG codon of ORF2. A third ORF has been described in certain strains that overlaps the capsid protein gene in a +1 frameshift, which is not found in the Norovirus genome (Liu et al., 1995;Guo et al., 1999b;Clarke and Lambden, 2000). The presence of a conserved translation initiation motif GCAAUGG at the 5′ end of this overlapping ORF suggests that a biologically active protein may be encoded in this ORF (Schuffenecker et al., 2001). Viruses in the genera Norovirus and Vesivirus have three major ORFs (ORF1, ORF2, and ORF3). In noroviruses, the first and third ORFs are in the same reading frame and Norwalk virus ORF3 encodes a 212-amino-acid minor structural protein of the virion (Glass et al., 2000a). All calicivirus genomes begin with a 5′-end terminal GU. This 5′ end sequence is repeated internally in the genome and most likely corresponds to the beginning of a subgenomic-sized RNA transcript (2.2-2.4 kb) that is coterminal with the 3′ end of the genome and that has been observed in FCV-and RHDV-infected cells as well as packaged into virions (Herbert et al., 1996). A comparison of the 5′-end sequences of representative viruses within each of the four genera and the corresponding repeated internal sequences suggests that this is a characteristic feature of caliciviruses. The synthesis of a subgenomic RNA in calicivirus-infected cells is a major difference between the replication strategy of caliciviruses and picornaviruses, although several of the replicative enzymes share distant homology (Green et al., 2001). It is not clear whether caliciviruses have a picornavirus-like internal ribosomal entry site (IRES) for the initiation of translation but is considered unlikely because translation of the FCV capsid precursor protein begins at the first AUG of the subgenomic RNA (Sosnovtsev and Green, 2003).

Molecular Diversity of Caliciviruses
Early studies demostrating the great variability of noroviruses soon led to the belief that it was important to distinguish between strains to better understand their epidemiology. Because no antigenic analysis of norovirus strains was available due to the lack of immune reagents, genome characterization by sequence analysis was used to provide an interim system of genotyping . As the genotypes ideally would correlate with serotypes, the sequence of the major structural protein gene was used as the basis for phylogenetic analyses (Ando et al., 2000;Koopmans et al., 2001). To determine which areas of the genome should be analyzed, sequenced regions in the RNA-dependent RNA polymerase, the capsid gene, and part of the 3′ ORF were compiled and analyzed (Green et al., 2000a;Vinje et al., 2000). By a similar approach, Ando et al. (2000) proposed a numerical system for genotypes based on phylogenetic grouping according to genetic relatedness in the major capsid protein.
Noroviruses were classified into two genetic groups, GI and GII, on the basis of sequence homologies across highly conserved regions of the genome, such as the RNA-dependent RNA polymerase and the capsid gene, although subdivision into five genetic groups or genogroups has been proposed recently (Karst et al., 2003) (Table 3.1). Molecular characterization of bovine enteric caliciviruses suggests that they be included into a proposed GIII group, which so far contains only viruses found in cattle (Ando et al., 2000;Oliver et al., 2003;Smiley et al., 2003). The porcine noroviruses cluster within GII (Sugieda and Nakajima, 2002). Phylogenetic analysis places at least two human noroviruses within a proposed genogroup IV: strains Alphatron (GenBank accession no. AF195847) and Ft. Lauderdale (GenBank accession no. AF414426) (Fankhauser et al., 2002).
The recently described murine norovirus has been included into a proposed GV group, the members of which are closer to GII than those of GI by sequence alignment (Karst et al., 2003). In the major capsid protein VP1, human norovirus strains within the same genogroup share at least 60% amino acid sequence identity, whereas most GI strains share less than 50% amino acid identity with GII strains (Green et al., 2001;Koopmans et al., 2003). Within genogroups, noroviruses can be further divided into at least 22 genetic clusters of genotypes based on genetic homology in the complete ORF2 sequence Green et al., 2001;Vinje et al., 2004). Within these broad groupings there are many different genetic variants of noroviruses circulating in the community Green et al., 2000a;Buesa et al., 2002;Lopman et al., 2003Lopman et al., , 2004. Picornavirus serotypes generally have >85% amino acid identity across the VP1 gene, which is in the range of the cutoff for calicivirus genotypes (>80% amino acid identity) (Oberste et al., 1999).
Serologically, the norovirus genotypes have been classified into different antigenic groups on the basis of solid-phase immuneelectron microscopy (SPIEM) studies  or by enzyme immunoassays using typespecific antibodies generated against recombinant capsid proteins (Atmar and Estes, 2001). The molecular diagnosis of noroviruses relies mainly on a relatively small sequence in the RNA polymerase region (Ando et al., 1995;Vinje and Koopmans, 1996;Jiang et al., 1999b) or the 3′ end of ORF1 (Fankhauser et al., 2002). Although robust for generic norovirus detection, RT-PCR (reverse transcription-polymerase chain reaction) assays targeting small regions of the genome that do not code for structural viral proteins will  Green et al. (2001) and Koopmans et al. (2003) neither reflect the epitopes of the viruses important for attachment to the host cell nor correlate with their antigenic properties. Therefore, neither RNA polymerase nor 3′ end of ORF1 is suitable for genotyping, although, in combination with capsid typing, they may become important for identifying norovirus recombinants Vinje et al., 2004). The Sapovirus capsid gene is fused to and is in frame with the polyprotein gene (Lambden et al., 1994;Numata et al., 1997). All Sapovirus strains except a human strain, London/92, and a porcine strain, PEC Cowden, contain an additional ORF predicted in +1 frame, overlapping the N terminus of the capsid gene (Guo et al., 1999b;Jiang et al., 1999a;Clarke and Lambden, 2001). By analogy with noroviruses, the sapoviruses were previously divided into four genotypes, belonging to two genogroups (Liu et al., 1995;Noel et al., 1997;Clarke and Lambden, 2000). More recently, human sapoviruses have been classified into four or five genogroups (Schuffenecker et al., 2001;Farkas et al., 2004) (Table 3.2). In addition, the porcine enteric calicivirus (PEC) strain Cowden has been shown to be related to the Sapovirus genus and to belong to a differentiated cluster (Guo et al., 1999b). Based on capsid sequences, it has recently been proposed to classify the currently known sapoviruses into nine genetic clusters within five genogroups, including one genogroup represented by the PEC strain Cowden (Farkas et al., 2004).
The occurrence of recombination among human caliciviruses in nature was suspected when it was found that the capsid nucleotide sequences of Snow Mountain virus and Melksham virus were almost identical (94%) but their RNA polymerase sequences were significantly different (79%). When Melksham virus was compared with Mexico virus and the Japanese strain Oth-25, no significant difference was found in sequence identities of the capsid and RNA polymerase regions and hence Snow Mountain virus was considered a recombinant virus . Caliciviruses contain subgenomic RNA that covers the entire 3′ end of the genome from the capsid gene to the 3′ end ( Fig. 3.1). It has been hypothesized that the subgenomic RNA could act as an independent unit participating in the recombination events. It remains unknown how subgenomic RNA is involved in virus replication, but it is clear that both genomic and subgenomic RNAs share highly conserved 5′ end sequences and that both RNAs are assembled into virions. It is possible that interaction between genomic and subgenomic RNAs occurs by the same mechanism as that of genomic-genomic interaction, thereby significantly increasing the chance of recombination events (Jiang et al., 1999a). If RNA recombination is a common phenomenon among caliciviruses, a high diversity might be expected in Caliciviridae, which would facilitate the emergence of new variants and make genotyping more difficult. In addition, recombination may permit caliciviruses to escape host immunity, analogous to antigenic shifts in influenza viruses, but by a different molecular mechanism. It has also been reported that accumulation of mutations in the protruding P2 domain of the capsid protein may result in predicted structural changes, including disappearance of helix structure of the protein, and thus a possible emergence of new phenotypes (Nilsson et al., 2003).

Virus Replication
Studies on the replication strategy of human caliciviruses have been hindered by the lack of an efficient cell culture system. Nevertheless, expression of recombinant proteins from cDNA clones has allowed the generation of proteolytic processing maps for the nonstructural proteins of several caliciviruses, like Southampton virus (a norovirus) and RHDV (a lagovirus) (Liu et al., 1996;Wirblich et al., 1996). Analysis of individual recombinant proteins from these noncultivable caliciviruses allowed the identification of NTPase and 3C-like cysteine protease activities for RHDV and noroviruses (Liu et al., 1999b) and a 3D-like RNA-dependent RNA polymerase for RHDV (López-Vazquez et al., 1998). Studies on the replication mechanisms of cultivable caliciviruses, such as feline calicivirus (FCV), have contributed to a better understanding of the basic features of calicivirus replication (Sosnovtsev and Green, 2003). FCV replicates by producing two major types of polyadenylated RNAs: a positive-sense genomic RNA of approximately 7.7 kb and a subgenomic RNA of 2.4 kb (Herbert et al., 1996). The genomic RNA serves as a template for the synthesis of nonstructural protein encoded by ORF1, and the subgenomic RNA serves as a template for the translation of structural proteins (Carter, 1990).

Virus-Cell Interactions
Human and animal enteric caliciviruses are assumed to replicate in the upper intestinal tract, causing cytolytic infection in the villous enterocytes but not in the crypt enterocytes of the proximal small intestine. Biopsies of the jejunum taken from experimentally infected volunteers who developed gastrointestinal disease after oral administration of Norwalk, Montgomery County, or Hawaii viruses showed histopathologic lesions, consisting of blunting of the villi, crypt cell hyperplasia, infiltration with mononuclear cells, and cytoplasmic vacuolization (Blacklow et al., 1972;Dolin et al., 1975). Experiments with recombinant Norwalk VLPs and human gastrointestinal biopsies showed binding of the rVLPs to epithelial cells of the pyloric region of the stomach and to enterocytes on duodenal villi. Attachment of these rVLPs occurred only to cells as well as to saliva from histo-blood group antigensecreting individuals (Marionneau et al., 2002). It was previously known that RHDV attaches to H type 2 histo-blood group oligosaccharide present on rabbit epithelial cells (Ruvöen-Clouet et al., 2000). Significant attachment and entry of Norwalk rVLPs to differentiated Caco-2 cells has also been demonstrated . Differentiated Caco-2 cells resemble mature enterocytes, express the H antigen, and were derived from a group O individual (Amano and Oshima, 1999).
To date, the Cowden strain of porcine enteric calicivirus (PEC) is the only cultivable enteric calicivirus (Flynn and Saif, 1988). However, in order to replicate, it requires the incorporation of an intestinal content preparation (ICP) from uninfected gnotobiotic pigs as a medium supplement. Different porcine intestinal enzymes such as trypsin, pancreatin, alkaline phosphatase, enterokinase, elastase, protease, and lipase were tested as medium supplements, but none of them alone promoted the growth of PEC in cell cultures (Parwani et al., 1991). It was speculated that some enzymes or factors in porcine ICP could activate the viral receptor, promote signaling of host cells, or may cleave viral capsid for successful uncoating (Guo and Saif, 2003).
Although noroviruses are highly infectious (it has been estimated that less than 10 virions may be enough to infect an adult), studies with volunteers have shown that some subjects remain uninfected despite having been challenged with high infectious doses (Matsui and Greenberg, 2000). These individuals may remain disease-free because of innate resistance or because of preexisting immunity to the virus (Lindesmith et al., 2003). An increased risk of Norwalk virus infection has been associated with blood group O; Norwalk VLPs bind to gastroduodenal cells from individuals who are secretors (Se+) but not to those from nonsecretors (Se−) (Hutson et al., 2002;Marionneau et al., 2002). The gene responsible for the secretor phenotype, FUT2, encodes an α(1,2) fucosyltransferase that produces the carbohydrate H type 1 found on the surface of epithelial cells and in mucosal secretions (Lindesmith et al., 2003). The form of H type 1 secreted depends on additional glycosyltransferases, including the Lewis, A and B enzymes present in epithelial and red blood cells (Marionneau et al., 2001). The recent discovery that noroviruses attach to cells in the gut only if the individuals express specific, genetically determined carbohydrates is a breakthrough in understanding norovirus-host interactions and susceptibility to norovirus disease (Lindesmith et al., 2003;Hutson et al., 2004).
Recent studies also suggest that some animal caliviviruses may cross the species barrier and potentially infect humans. The hypothetical existence of animal reservoirs and the possibility of interspecies transmission have been suggested by phylogenetic links of bovine and porcine viruses within the genera Norovirus and Sapovirus, respectively Dastjerdi et al., 1999;Liu et al., 1999a;van Der Poel et al., 2000). However, information concerning the frequency of interspecies transmission among caliciviruses is limited.
It has recently been demonstrated that gnotobiotic pigs inoculated by the intravenous route with wild-type PEC Cowden developed diarrhea and also presented histological lesions in the duodenum and jejunum similar to those observed in gnotobiotic pigs inoculated orally with wild-type PEC (Guo et al., 2001). Moreover, PEC RNA and low titers of virus antigen were detected in sera from pigs inoculated with wild-type PEC both orally and intravenously (Guo and Saif, 2003). Thus, viremia may occur after natural PEC infection, and acute sera may contain infectious virus. It has been hypothesized that human calicivirus infections might also induce viremia, interacting with erythrocytes in the blood (Guo and Saif, 2003). If this is true, how the enteric caliciviruses reach the bloodstream from the gut and vice versa remains unexplained.

ROTAVIRUSES
Rotaviruses are the leading etiologic agents of viral gastroenteritis in infants and young children worldwide and in the young of a large variety of animal species . Rotavirus infections in humans continue to occur throughout their lives, but the resulting disease is mild and often asymptomatic (Bishop, 1996). In addition to sporadic cases of acute gastroenteritis, outbreaks of rotavirus diarrhea in school-aged children and adults have increasingly been reported (Griffin et al., 2002;Mikami et al., 2004).
Rotaviruses are responsible for an estimated 500,000 deaths each year in developing countries (Parashar et al., 2003). There is an urgent need to develop an effective vaccine and to implement therapeutic strategies to prevent and treat these infections. A better understanding of the molecular mechanisms of rotavirus replication and interaction with the host cell and of antigenic variability of these viruses are fundamental to achieving these goals.
Rotaviruses are classified into at least five groups (A to E), and there are possibly two more groups (F and G) based on the reactivities of the VP6 middle layer protein (Estes, 2001). Group A rotaviruses are most commonly associated with human infections . Within group A, four subgroups (I, II, I + II, and non-I/non-II) are recognized based on the reactivity of VP6 with two monoclonal antibodies. The two outer capsid proteins, VP7 and VP4, form the basis of the current dual classification system of group A rotaviruses into G and P types (Estes, 2001). At least 14 different G-serotypes and 20 P-types have been identified among human and animal rotaviruses, depending on VP7 and VP4, respectively. G serotypes correlate fully with G genotypes as determined by sequence analysis of the VP7 gene. However, to date only 12 of the 20 P genotypes have been correlated with P serotypes (Estes, 2001). Because VP4 and VP7 are coded for by different RNA segments (segment 4 and segments 7-9, respectively), various combinations of G-and P-types can be observed both in humans and in animals. Viruses carrying G1P[8], G2[P4], G3[P8], and G4[P8] represent more than 90% of human rotavirus strains cocirculating in most countries, although other G and P combinations are being isolated in increasing numbers (Cunliffe et al., 1999;Buesa, 2000;Iturriza-Gomara et al., 2000;Adah et al., 2001;Armah et al., 2003;Zhou et al., 2003).
Group B rotaviruses are common animal pathogens infecting pigs, cows, sheep, and rats but have also been found to infect humans (Sanekata et al., 2003). Group C rotaviruses are commonly found in animals including pigs and dogs and can cause outbreaks in humans (Castello et al., 2000).

Structure of the Virion
Rotavirus particles are of icosahedral symmetry, have three concentric layers of protein, and measure ∼1,000 Å in diameter including the spikes (Estes, 2001). The core layer is formed by a protein known as VP2 (which surrounds the entire viral genome) and proteins VP1 and VP3, which are transcription enzymes and are attached as a heterodimeric complex to the inside surface of VP2 at the fivefold symmetry positions (Prasad et al., 1996b) (Fig. 3.3). VP1, the RNA-dependent RNA polymerase, interacts with VP3, guanylytransferase, and methylase (Liu et al., 1992). This innermost layer is composed of 120 copies of VP2, a RNA-binding protein (Labbé et al., 1991). The addition of VP6 to the VP2 layer produces double-layered particles (DLP). VP6 forms 260 trimers interrupted by 132 aqueous channels of three different kinds in relation to the capsid's symmetry. The outer capsid of the triple-layered particles (TLP) is composed of two proteins, VP7 and VP4.
The smooth surface of the virus is made up of 260 trimers of VP7, and 60 spikes emerging from the viral surface consist of dimers of VP4 (Prasad et al., 1988;Yeager et al., 1990). Rotavirus DLP and TLP contain 132 porous channels that allow the exchange of compounds to the inside of the particle. There are 12 type I channels, each located at the icosahedral fivefold vertices of the TLP and DLP. Each of the type I channels is surrounded by five type II channels, and finally 60 type III channels are placed at the hexavalent positions immediately neighboring the icosahedral threefold axis (Prasad et al., 1988;Yeager et al., 1990).
During cell entry, the TLP loses the VP7 and VP4 proteins, and the resulting DLP becomes transcriptionally active inside the cytoplasm (Estes, 2001). VP4 is a nonglycosylated protein of 776 amino acids and has essential functions in the virus cell cycle, including receptor binding, cell penetration, hemagglutinin activity, and permeabilization of cellular membranes (Fiore et al., 1991;Burke and Desselberger, 1996;Gilbert and Greenberg, 1998;Denisova et al., 1999;Zárate et al., 2000b). VP4 is post-transcriptionally cleaved into the larger VP5* and the smaller VP8* subunits, and cleavage of VP4 enhances viral infectivity by several-fold. It has been shown that trypsin cleavage confers icosahedral ordering on the VP4 spikes, which is essential for the virus to enter the cell . Moreover, biochemical studies of recombinant VP4 indicate that proteolysis of monomeric VP4 yields dimeric VP5* (Dormitzer et al., 2001).
When the DLP is located intracellularly, it becomes transcriptionally competent, and new mRNA transcripts are translocated from the particle through type I channels at the fivefold axes (Lawton et al., 1997). Prasad et al. (1988) were the first to propose that these channels in the VP6 layer could be used by mRNA to exit. Cryo-EM studies have confirmed that DLPs maintain their structural integrity during the process of transcription. In a pseudoatomic model of the T = 13 VP6 layer (Mathieu et al., 2001), a β-hairpin motif of VP6 with a highly conserved sequence that protrudes into the mRNA exit channel may play a functional role in the translocation of the nascent transcripts (Lawton et al., 2000). A detailed mutational analysis of the VP6 layer has helped to elucidate the determinants of VP6 required for its assembly on VP2 and how VP6 may affect endogenous transcription (Charpilienne et al., 2002).

The Genome
The genome of rotavirus (simian rotavirus strain SA11) consists of 11 segments of double-stranded RNA (dsRNA) with conserved 5′ and 3′ ends, ranging from 667 bp (segment 11) to 3,302 bp (segment 1) in size, totaling 6,120 kDa. Six structural proteins (VP1, VP2, VP3, VP4, VP6, VP7) and six nonstructural proteins (NSP1 to NSP6) are encoded.The coding assignments, functions, and many properties of the proteins encoded by each of the 11 genome segments are now well established (Table 3.3). Protein assignments have been determined by in vitro translation using mRNA or denatured dsRNA and by analyses of reassortant viruses (Estes and Cohen, 1989;Estes, 2001). Cryo-EM analysis of rotavirus has provided first visualization of the structural organization of the viral genome (Prasad et al., 1996b).The dsRNA forms a dodecahedral structure in which the RNA double helices, interacting closely with the VP2 inner layer, are packed around the transcription complexes located at the icosahedral vertices ( Fig. 3.3D). VP2 has RNA binding properties and may be responsible for the icosahedral ordering and for the closely interacting portions of the RNA. A study by Pesavento et al. (2001) demonstrated reversible condensation and expansion of the rotavirus genome within the capsid interior under various chemical conditions. This condensation is concentric with respect to the particle center, and a dark mass of density is consistently seen in the center of each of the particles. At high pH in the presence of ammonium ions, the genome condenses to a radius of 180 Å from the original radius of 220 Å, and when brought back to physiological pH the genome expands to its original radius (Pesavento et al., 2001). This study suggests that VP2, through its RNA-binding properties, plays an important role in maintaining appropriate spacing between the RNA strands in the native expanded state. A plausible and elegant model for the structural organization of the genome that emerges from these studies is that each dsRNA segment is spooled around a transcription enzyme complex at the fivefold axes, inside the innermost capsid layer (Prasad et al., 1996b;Pesavento et al., 2001Pesavento et al., , 2003.

Mechanisms of Evolution and Strain Diversity
Rotaviruses, like many other RNA viruses, display a great degree of diversity. As well as showing different G and P types and a variety of combinations, there is also intratypic variation. Three mechanisms, singly or in combination, are important for the evolution and diversity of rotaviruses, although it is yet unclear what their relative contributions are to the burden of the disease.

Antigenic Drift
The rate of mutation within rotavirus genes is relatively high because RNA replication is error-prone. The mutation rate has been calculated to be <5 × 10 −5 per nucleotide per replication, which implies that on average a rotavirus genome differs from its parental genome by at least one mutation (Blackhall et al., 1996). Point mutations can accumulate and give rise to intratypic variation as identified by the existence of lineages within the VP7 and VP4 genes of particular G and P types. Point mutations can lead to antigenic changes, which may result in the emergence of escape mutants (Palombo et al., 1993;Maunula and von Bonsdorf, 1998;Iturriza-Gómara et al., 2002).

Antigenic Shift
Shuffling of gene segments through reassortment can occur during dual infection of one cell. Reassortment can therefore contribute to the diversity of rotaviruses, and there is increasing evidence that reassortment takes place in vivo (Ramig, 2000). There is evidence that reassortment, through alteration in protein-protein interactions, possibly leads to changes in conformational epitopes and may contribute to the evolution of antigenic types (Chen et al., 1992;Lazdins et al., 1995). Interspecies transmission and subsequent reassortment have enormous potential to increase the diversity of cocirculating rotaviruses. In addition, human rotavirus genes encoding proteins to which the human population is immunologically naïve may allow a rapid spread of the reassortant strain (Iturriza-Gomara et al., 2000).

Gene Rearrangement
The concatemerization or truncation of genome segments and their ORFs has the potential to contribute to the evolution of rotaviruses through the production of new proteins with altered functions (Desselberger, 1996).

Genome Replication
The RNA polymerase activity of DLPs catalyzes synthesis of 11 mRNAs of rotaviruses, which range in size from ∼0.7 to 3.3 kb. With the exception of gene 11, they are monocistronic (Estes, 2001). The nascent transcripts are extruded through channels present at the fivefold axes of the DLPs (Lawton et al., 1997). The 5′-and 3′-untranslated regions (UTRs) are 9 to 49 and 17 to 182 nucleotides in length, respectively. The viral mRNAs serve as templates for the synthesis of minus-strand RNA to form dsRNA molecules (Chen et al., 1994). The synthesis of viral dsRNA and the assembly of cores and DLPs occur in viroplasms in the cytoplasm (Estes, 2001). There are three species of RNA-containing replication intermediates (RIs) in the infected cells: pre-core RIs, which contain the structural proteins VP1 and VP3; core-RIs, with VP1, VP2, and VP3; and double-layered RIs, which contain VP1, VP2, VP3, and VP6. The 11 genomic segments are produced and packaged in equimolar amounts in rotavirus-infected cells, demonstrating that RNA packaging and replication are coordinated processes (Patton and Gallegos, 1990). The absence of naked dsRNA in infected cells suggests that packaging takes place before replication (Patton et al., 2003).
The only primary sequences that are conserved among the rotavirus mRNAs are located within the UTRs. Because all 11 mRNAs are replicated by the same VP1-VP2-VP3 polymerase complex, the viral mRNAs almost certainly must share common cis-acting signals recognized by the polymerase (Patton et al., 2003). The most remarkable feature of the 3′ ends of rotavirus mRNAs and of other members of the Reoviridae is the absence of a poly(A) tail. Instead, all rotavirus genes and mRNAs end with the same short sequence UGACC, which is conserved among all the segments in group A rotaviruses. Site-specific mutagenesis has revealed that it is the 3′-CC of the 3′ consensus sequence that is most critical for minus-strand synthesis (Chen et al., 2001).
In addition, it has been demostrated that the promoter for minus-strand synthesis is formed by base-pairing in cis of complementary sequences proximal to the 5′-and 3′-ends of the viral mRNAs (Chen and Patton, 1998). The 3′-consensus sequence also contains a cis-acting signal that acts as a translation enhancer, whose activity is mediated by NSP3 that specifically recognizes the last four to five nucleotides of the 3′-consensus sequence (Poncet et al., 1994). NSP3 also interacts with the initiation factor eIF4GI and facilitates the circularization of viral mRNAs in polysomes, thus increasing the efficiency of viral gene expression (Piron et al., 1998).
The development of a cell-free system that supports the synthesis of dsRNA from exogenous mRNA represents an important milestone in the study of rotavirus replication, providing a mechanism by which the elements in viral mRNAs that promote minus-strand synthesis can be analyzed. This system is based in virion-derived cores that have been disrupted or "opened" by incubation in hypotonic buffer (Chen et al., 1994). Although several investigators have tried to develop a reverse genetics system for rotaviruses, this goal has not been achieved so far (Patton et al., 2003). Our understanding of even the most basic mechanisms that occur during rotavirus replication would be greatly enhanced by the development of the reverse genetics approach.

Cell Infection
Rotaviruses have a specific tropism in vivo infecting primarily the mature enterocytes of the villi of the small intestine. Recent reports suggest that extraintestinal spread of the virus takes place during infection, indicating a wider tropism than previously considered (Blutt et al., 2003;Mossel and Ramig, 2003). Rotaviruses can bind to a wide variety of cell lines, although only a subset of them (including cells of renal and intestinal origin, and transformed cell lines from breast, stomach, lung, and bone) is efficiently infected (Ciarlet et al., 2002a;López and Arias, 2004). Most studies on rotavirus replication have been carried out using the simian kidney epithelial MA104 cells, which are routinely used to produce progeny virus. However, new investigations into the pathophysiological mechanisms of rotavirus infection are now being performed using in vitro polarized cells such as the human intestinal HT-29 and Caco-2 cells (Servin, 2003).
Rotaviruses enter the cell by a complex multistep process in which different domains of the viral surface interact with different cell surface molecules that act as receptors for the attachment of the viruses (Guerrero et al., 2000;López and Arias, 2004). Some animal rotavirus strains interact with sialic acid (SA) residues to attach to the cell surface, and the infectivity of these strains is diminished by the treatment of cells with neuraminidase (NA). By contrast, many animal strains and some human strains are NA-resistant .The interaction of rotavirus with SA has been shown to depend on the VP4 genotype of the virus and not the species of origin (Ciarlet et al., 2002b). Ganglioside GM3 has been suggested to act as the SA-containing receptor for the porcine rotavirus strain OSU (Rolsma et al., 1998), and ganglioside GM1 (NA-resistant) has been identified as the receptor for the NA-resistant human rotavirus strains KUN and MO (Guo et al., 1999a). The VP8* domain of VP4 is involved in interactions with SA, whereas VP5* is implicated in interactions with integrins. The interaction of rotavirus with integrin α2β1 has been shown to be mediated by the DGE integrin-recognition motif, located at amino acids 308 to 310 of VP4, within VP5* (Zárate et al., 2000a). VP4 also contains the tripeptide IDA at amino acids 538 to 540, which is ligand motif for integrin α4β1 (Coulson et al., 1997). However, the functionality of this site has not been demonstrated. Integrin αvβ3 is also involved in the cell entry of several rotavirus strains at a postattachment step (Graham et al., 2003). Besides, cellsurface heat shock cognate protein hsc70 has also been implicated as a postattachment receptor for both NA-sensitive and NA-resistant rotavirus strains (Guerrero et al., 2002). Studies with polarized epithelial cell lines show that the viral entry of SA-dependent strains is restricted to the apical membrane, whereas SA-independent strains enter either apically or basolaterally .
It has been suggested that lipid rafts might play an important role in the cell entry of rotavirus (Isa et al., 2004), probably serving as platforms to allow an efficient interaction of cell receptors with the viral particle (Manes et al., 2003;López and Arias, 2004). Rotavirus infection in polarized, fully differentiated Caco-2 cells is followed by a defect in brush-border hydrolase expression (Jourdan et al., 1998). Sucrase-isomaltase activity and apical expression are specifically decreased by rotavirus infection without any apparent cell destruction (Jourdan et al., 1998). In addition, viral infection induces an increase in intracellular calcium concentration, damages the microvillar cytoskeleton, and promotes structural and functional injuries at the tight junctions in cell-cell junctional complexes of cultured Caco-2 cells without damaging the integrity of the monolayers (Brunet et al., 2000;Obert et al., 2000).

The NSP4 Enterotoxin
The rotavirus nonstructural glycoprotein NSP4 was shown to function as an intracellular receptor that mediates the acquisition of a transient membrane envelope as subviral particles bud into the endoplasmic reticulum (ER). It has been demonstrated that NSP4 binds intracellularly newly made DLPs, interacting with VP6. This receptor role of NSP4 is confirmed by the observation that DLPs bind to ER membranes containing only NSP4 (Taylor et al., 1993;Estes, 2001). Many structural motifs or protein regions have been implicated in the NSP4 biological function. Amino acids 17 to 20 from the C-terminus extreme are necessary and sufficient for inner capsid particle binding (O'Brien et al., 2000) and the region involved in the retention of the NSP4 protein into the endoplasmic reticulum has been mapped between the amino acids 85 and 123 in the cytoplasmatic region of the protein (Mirazimi et al., 2003). Residues at positions 48 to 91, a region that includes a potential cationic amphipatic helix, have been shown to be involved in membrane destabilization Browne et al., 2000). Purified NSP4 or a peptide corresponding to NSP4 residues 114-135 induce diarrhea in young mice after an increase in intracellular calcium levels, suggesting a role for NSP4 in rotavirus pathogenesis (Tian et al., 1994;Ball et al., 1996;Horie et al., 1999). In vitro studies have shown that after rotavirus replication in cells, a functional 7-kDa peptide of NSP4 (amino acids 112-175) is released into the medium from virus-infected cells by a nonclassic, Golgi-independent cellular secretory pathway (Zhang et al., 2000). This endogenously produced peptide binds to an as yet unidentified apical membrane receptor to mobilize intracellular calcium through phospholipase C signaling.
NSP4, or its active NSP4 114-135 peptide, induces age-dependent diarrhea and age-dependent chloride permeability changes in mice lacking the cystic fibrosis transductance regulator (CFTR) that exhibit no functional cAMPdependent secretory pathway (Morris et al., 1999). These observations indicate that NSP4 is a novel secretory agonist, because the classical secretagogues carbachol and the diterpene forskolin that induce chloride changes by activating cyclic adenosine monophosphate, instead of by mobilizing [Ca 2+ ] i as a secondary mediator, fail to cause disease in CFTR knockout mice (Morris et al., 1999). NSP4 or its active peptide may induce diarrhea in neonatal mice through the activation of an age-and Ca 2+ -dependent plasma membrane anion permeability distinct from CFTR. The molecular identity of the responsible channel remains to be determined (Morris and Estes, 2001).
It has been suggested that NSP4 may directly inhibit the functioning of the cellular Na + -dependent glucose transporter SGLT-1 (Halaihel et al., 2000). In addition, extracellular and/or intracellular NSP4 may contribute to diarrheal pathogenesis by altering the dynamics of intracellular actin distribution and intercellular contacts . NSP4 can affect the cytoskeleton in polarized epithelial cells, but how these pleiotropic properties of NSP4 influence the function of NSP4 in morphogenesis or pathogenesis remains unclear (Tafazoli et al., 2001). The significance of immune response to NSP4 in protection against rotavirus infection in humans is still unknown, although it has been shown that NSP4 elicits both humoral and cell-mediated immune responses (Johansen et al., 1999;Ray et al., 2003).

ASTROVIRUSES
Human astroviruses are nonenveloped viruses with a positive-sense, singlestranded, polyadenylated RNA genome about 6,800 nucleotides in length (Matsui, 1997). Astroviruses are members of the Astroviridae family and were originally described in 1975 in association with outbreaks of gastroenteritis in newborns (Appleton and Higgins, 1975). Astroviruses produce infections mainly in young children, although illness rates increase again in the elderly (Lewis et al., 1989). However, they also cause disease in adults and immunocompromised patients (Cubitt et al., 1999;Coppo et al., 2000;Liste et al., 2000). Persistent gastroenteritis in children with no background disease has also been reported, mainly in association with serotype 3 strains (Caballero et al., 2003). Astrovirus infections occur worldwide, and their incidence ranges from 2% to 9% in both developing and developed countries (Gaggero et al., 1998;Bon et al., 1999;Mustafa et al., 2000;Guix et al., 2002). Astroviruses are transmitted by the fecal-oral route; outbreaks have been associated with consumption of sewage-polluted shellfish and ingestion of water from contaminated sources (Pintó et al., 1996;Pintó et al., 2001). The virus is frequently shed in stools in significant numbers at the onset of the illness, which is in contrast with low numbers of calicivirus produced in the stools of infected patients. Unlike caliviruses, astroviruses replicate in vitro in cell lines, and hence detailed studies on their replication are available (Matsui, 1997;Willcocks et al., 1999;Matsui and Greenberg, 2001). Human astroviruses were originally isolated in HEK cells and were subsequently adapted to grow in LLCMK2 cells in trypsin-containing media, although without demonstrable cytopathic effect (CPE). The colonic carcinoma cell line Caco-2, in contrast with LLCMK2 cells, is directly susceptible to fecally derived astrovirus and displays CPE as early as 2 days postinfection (Willcocks et al., 1990;Pinto et al., 1994).
The average diameter of astrovirus particles is approximately 28 nm (Madeley, 1979), although it may vary depending on the source of the virus and the method of preparation for EM (Woode et al., 1984). More detailed ultrastructural analysis of human astrovirus serotype 2 grown in LLCMK2 cells in the presence of trypsin revealed particles with icosahedral symmetry and an array of spikes emanating from the surface (Risco et al., 1995). These particles had an external diameter of 41 nm (including spikes). They did not display the star-like surface characteristically found on fecally shed virus. However, the star-like morphology was inducible after alkaline treatment (pH 10). Intact virions generally band at densities of 1.35 to 1.37 g/ml in CsCl gradients (Caul and Appleton, 1982), although banding at densities of 1.39-1.40 has also been reported (Konno et al., 1982;Midthun et al., 1993). The astroviruses band at a density of 1.32 g/ml in potassium tartrate-glycerol gradient, which preserves better than CsCl the structural integrity of the virus (Ashley and Caul, 1982).
The RNA genomes of several cell culture-adapted human astrovirus strains have been cloned and sequenced (Jiang et al., 1993a;Lewis et al., 1994;Willcocks et al., 1994;Geigenmuller et al., 1997), providing new perspectives for studying the molecular biology of astroviruses. The organization of the genome includes three long open reading frames (ORFs), designated ORF1a, ORF1b, and ORF2. ORF1a (∼2700 nt) is located at the 5′ end of the genome and contains transmembrane helices, a 3C-like serine protease motif, a putative protease-dependent cleavage site, and a nuclear localization signal (Jiang et al., 1993a;Willcocks et al., 1994). ORF1b (∼1550 nt) contains a RNA-dependent RNA polymerase motif, whereas ORF2 encodes the structural proteins (Matsui and Greenberg, 2001). The nonstructural proteins of the virus are translated from the genomic viral RNA as two polyproteins (Jiang et al., 1993a). One of them contains only ORF1a and the other includes ORF1a/1b and is translated via −1 ribosomal frameshifting event between ORF1a and ORF1b (Jiang et al., 1993a). Both proteins are proteolitically processed, generating a variety of polypeptides, although it is unclear whether the viral protease is responsible for all the cleavages (Geigenmuller et al., 2002a(Geigenmuller et al., , 2002bKiang and Matsui, 2002).
A subgenomic RNA (approximately 2,400 nucleotides) that contains ORF2 is found in abundance in the cytoplasm of astrovirus-infected cultured cells (Monroe et al., 1991). The subgenomic RNA is translated as a 87-kDa capsid precursor protein that is believed to give rise to three to five smaller mature capsid proteins in a process that involves trypsin and a putative cellular protease (Monroe et al., 1991;Bass and Qiu, 2000;Mendez et al., 2002). The 87-kDa capsid protein is rapidly converted intracellularly to a 79-kDa form, which is found in smaller amounts in the cell supernatants. Bass and Qiu (2000) identified a trypsin cleavage site in a highly conserved region of the ORF2 product. Trypsin-free particles were minimally infectious in cultured Caco-2 cells but became highly infectious after trypsin treatment but not chymotrypsin treatment. This trypsin-enhanced infectivity correlated with conversion of the 79-kDa capsid protein to three smaller peptides of approximately 26, 29, and 34 kDa. However, the apparent molecular weight reported for the smaller mature proteins has depended on the astrovirus serotype and whether the virus studied was derived from infected cultured cells or from stools (Matsui and Greenberg, 2001). Recently, astrovirus virus-like particles (VLPs) have been generated by cloning the cDNA corresponding to ORF2 from a human astrovirus serotype 2 into vaccinia virus (Dalton et al., 2003). Protein composition of these purified VLPs revealed no substantial difference from that of authentic astrovirus virions when analyzed by Western blotting. Trypsin cleavage seems to be necessary to process the capsid polyprotein into mature structural proteins.

Polioviruses
Enteroviruses replicate in the gastrointestinal tract, but the resulting infection is frequently asymptomatic. Symptoms, when they occur, range from paralysis to fever. Enteroviruses, named after the site of replication, rarely cause gastroenteritis. In addition, in many cases the enterovirus isolated might merely have been a passenger virus unrelated to the disease (Melnick, 1996).
The capsids of enteroviruses (family Picornaviridae) are composed of four structural proteins (VP1-VP4) arranged in 60 repeating protomeric units with icosahedral symmetry. Among the family members, the capsid proteins are arranged similarly, but the surface architecture varies. These differences account for not only the different serotypes but also the different modes of interaction with cell receptors. The basic building block of the picornavirus capsid is the protomer, which contains one copy of each structural protein. The capsid is formed by VP1 to VP3, and VP4 lies on its inner surface. VP1, VP2, and VP3 have no sequence homology, yet all three proteins have the same topology: they form an eight-stranded antiparallel "βbarrel" core structure (Racaniello, 2001). The external loops that connect the beta strands are responsible for differences in the antigenic diversity of enteroviruses. Neutralization sites are more densely clustered on VP1. The structures of many human picornaviruses have been resolved, indicating that they share a number of conserved structural motifs. For example, the capsids of polioviruses, rhinoviruses, and coxsackieviruses have a groove, or canyon, surrounding each fivefold axis of symmetry. In contrast, cardioviruses and aphthoviruses do not have canyons (Racaniello, 2001). Immediately beneath the canyon floor of each protomer is a hydrophobic pocket occupied by a lipid moiety. These molecules, termed pocket factors, have been shown to stabilize the capsid, and their removal from the pocket is a necessary prerequisite to uncoating. On the basis of the electron densities and uncoating studies, the pocket factors are thought to be short-chain fatty acids (Smyth et al., 2003).
The enterovirus positive-sense RNA genome is approximately 7.4 kb long and serves as a template for both viral protein translation and virus replication (Racaniello, 2001). The 5′ end is covalently linked to a VPg protein (for genome-linked virus protein). The genome is organized into a long (∼740 nucleotides) nontranslated region (5′NTR), which contains the internal ribosome entry site (IRES) and precedes a single ORF. The ORF is subdivided into three regions, P1 to P3. The P1 region codes for structural proteins, whereas P2 and P3 regions encode for nonstructural proteins essential for virus replication (2A-C, 3A-D). Translation of the ORF gives rise to a single large polyprotein that is post-translationally modified by virus encoded proteases. Immediately downstream of the protein-coding region is the 3′ nontranslated region (3′-NTR), which plays a role in viral RNA replication, followed by a terminal poly(A) tail (Racaniello, 2001).
The three serotypes of human poliovirus are considered a species within the genus Enterovirus. A redefinition of the criteria for species demarcation within the genus Enterovirus has recently been issued by the International Committee on Taxonomy of Viruses (ICTV). Based on these molecular and biochemical characteristics, Enteroviruses are now classified as human enteroviruses A through D, bovine enterovirus, and porcine enterovirus A and B (King et al., 2000).
Poliovirus infections, once responsible for high morbility and mortality throughout the world, are now under control, and their eradication is a priority for the World Health Organization (WHO). In the past 15 years, since the Global Polio Eradication Initiative was launched by WHO, the number of cases has fallen from an estimated 350,000 cases in 1988 to 1,919 in 2002. In the same time period, the number of polio-infected countries has been reduced from 125 to 7. There is a historic opportunity to forever stop the transmission of poliovirus. If the world seizes this opportunity and acts immediately, no child will ever again know the effects of this devastating disease (Dowdle et al., 2001).

Kobuviruses
Aichi virus, a cytopathic small round virus, was isolated for the first time in 1989 from fecal samples of patients involved in an oyster-associated gastroenteritis outbreak (Yamashita et al., 1991). Since then, several Aichi virus strains have been isolated in BS-C-1 cells from patients with gastroenteritis (Yamashita et al., 1995). The virus is commonly found in outbreaks of gastroenteritis in Japan and is often associated with the consumption of oysters (Yamashita et al., 1995). Genetic analysis of Aichi virus revealed that it belongs to the Picornaviridae family but that it is different from any other genus such as the Entero-, Rhino-, Cardio-, Aphtho-, Hepato-, Tescho-, or Parechovirus (Yamashita et al., 1998). It has been recently proposed by the ICTV that this virus be assigned to a new genus named Kobuvirus (King et al., 1999). Kobu means "knob" in Japanese; the reference based on the characteristic morphoplogy of the virion.
Aichi virus has been cultivated in BS-C-1 and Vero cells, producing CPE characterized by detachment of cells. It does not replicate in other human cell lines such as HeLa, RD, or HEL cells. By EM, the virion shows a rough surface and measures around 30 nm in diameter. Three capsid proteins of 42, 30, and 22 kDa have been described (Yamashita et al., 1998). Aichi virus possesses a genome of single-stranded RNA of 8,280 nucleotides, excluding a poly(A) tract. It contains a large ORF with 7,299 nucleotides that encodes a polyprotein precursor of 2,432 amino acids, which is preceded by 744 nucleotides and followed by 237 nucleotides and a poly(A). The precise secondary structure of the 5′ nontranslated region (5′-NTR) has not been defined, although an internal ribosomal entry site (IRES) similar to that of other members of the Picornaviridae family has been reported (Sasaki et al., 2001). The complete nucleotide sequence of Aichi virus (GenBank accession no. AB010145) has been determined (Yamashita et al., 1998).
The organization of the deduced amino acid sequence of the polyprotein encoded by the Aichi virus genome is analogous to that of the other picornaviruses. Preceded by a leader (L) protein, there is the P1 region, which corresponds to the structural proteins (VP0, VP3, and VP1, with molecular weights of 42, 30, and 22 kDa, respectively) followed by the P2 and P3 regions, which contain sequences encoding the nonstructural proteins (2A-C, 3A-D). It has been reported that the 2A protein of Aichi virus contains conserved motifs that are characteristic of the H-rev107 family of cellular proteins involved in the control of cellular proliferation (Hughes and Stanway, 2000). Amino acid sequences of the 2C, 3C, and 3D regions are well aligned with the corresponding sequences of other picornaviruses. The 3B protein corresponds to the VPg protein. The 3C protein is the protease and contains conserved motifs characteristic of all picornaviruses . The relationship of kobuviruses to other picornaviruses has been analyzed based on the 3D amino acid sequence (which corresponds to the RNA polymerase) and the polyprotein sequence (Hughes, 2004). Recently, bovine kobuvirus has been isolated and characterized (accession no. AB084788) . The genus Kobuvirus, including bovine kobuvirus and Aichi virus, cluster near the genera Teschovirus, Cardiovirus, Erbovirus, and Aphthovirus (Hughes, 2004).

HEPATITIS A VIRUS
Hepatitis A virus (HAV), the prototype of the genus Hepatovirus in the family Picornaviridae (Minor, 1991), is a hepatotropic virus that represents a significant problem for human health (Hollinger and Emerson, 2001). HAV infection is mainly propagated via the fecal-oral route, and although trans-mission remains primarily from person to person, waterborne and foodborne outbreaks of the disease have been reported (De Serres et al., 1999;Hutin et al., 1999;Fiore, 2004). HAV was originally classified as enterovirus type 72 because its biophysical characteristics are similar to those of enteroviruses. However, later studies demonstrated that HAV nucleotide and amino acid sequences are different from those of other picornaviruses, as are the predicted sizes of several HAV proteins (Cohen et al., 1987b). This virus is difficult to cultivate in cell cultures and usually replicates very slowly without producing CPE. It is resistant to temperatures and drugs that inactivate other picornaviruses and is stable at pH 1. There is only a single serotype of human HAV with one immunodominant neutralization site (Lemon and Binn, 1983). However, a significant degree of nucleic acid variability has been observed among different isolates from different regions of the world (Robertson et al., 1992;Costa-Mattioli et al., 2001). The molecular basis of this genetic variability may be the high error rate of the viral RNAdependent RNA polymerase and the absence of proofreading mechanisms (Sánchez et al., 2003).
The virion is composed of a genome of linear, single-stranded RNA of messenger sense polarity, approximately 7.5 kb in length, and a capsid containing multiple copies of three or four proteins named VP1, VP2, VP3, and a putative VP4, encoded in the P1 region of the genome (Coulepis et al., 1982;Racaniello, 2001). The presence of a fourth protein VP4 has been described repeatedly, but the reported molecular weight (7-14 kDa) does not correspond to that predicted from nucleic acid sequence data (1.5 or 2.3 kDa) (Weitz and Siegl, 1998). The P2 and P3 regions encode for nonstructural proteins associated with replication.
HAV has a buoyant density of 1.32 to 1.34 g/cm 3 in CsCl and sediments with 156S during sucrose gradient centrifugation (Coulepis et al., 1982). Infectious viral particles with higher (1.44 g/cm 3 ) as well as lower (1.27 g/cm 3 ) buoyant densities have also been reported (Lemon et al., 1985). Phylogenetic analysis based on a 168-base segment encompassing the VP1/2A junction region of the HAV genome has established the classification of human and simian isolates into seven different genotypes (I-VII), with genotypes I, II, III, and VII including human isolates (Robertson et al., 1992). About 80% of the human isolates belong to genotype I, which has been subdivided into two subgenotypes, IA and IB (Fujiwara et al., 2003).

The Genome
Detailed analysis of the HAV genome has been accomplished with cloned or RT-PCR-amplified cDNA. Cultured cells can be infected with RNA transcribed from cloned HAV cDNA (Cohen et al., 1987a). HAV contains a genome that differs from that of Caliciviridae in that the genes encoding the nonstructural proteins are located at the 3′ portion of the genome, whereas those encoding the structural proteins are located at the 5′ end (Weitz and Siegl, 1998). The HAV genome is divided into a 5′ nontranslated region of 735 nucleotides, a long open reading frame of 6,681 nucleotides encoding a polyprotein of 2,227 amino acids, and a 3′ nontranslated region 63 nucleotides in length.
The 5′-NTR contains a cis-acting internal ribosome entry site (IRES) that directs initiation of cap-independent translation directed to a particular AUG triplet several hundreds of nucleotides downstream Brown et al., 1994;Ali et al., 2001;Borman et al., 2001;Kang and Funkhouser, 2002). It has been demonstrated that the IRES is located between nucleotides 151 and 734, and that it is able to direct internal initiation of translation in a cap-independent manner . However, a cap-dependent message effectively competes with the IRES of HAV . Translational efficiency of this IRES may be dependent on the availability of intact cellular proteins such as the p220 subunit of the eukaryotic initiation factor eIF-4F and requires association between the cap-binding translation initiation factor (eIF4E) and eIF4G (Ali et al., 2001;Borman et al., 2001). The 5′-NTR is the most highly conserved region in the HAV genome among all strains sequenced to date, with a 95% nucleotide identity. By contrast, the 3′-NTR region shows the highest (up to 20%) degree of variability. The presence of a 23-nucleotide-long cis-acting element has been described that specifically interacts with proteins involved in the establishment and maintenance of the persistent type of infection characteristic for replication of most HAV isolates in cell cultures. Several unidentified cytoplasmic and ribosomal proteins of infected cells bind to the 3′ end of HAV RNA, indicating an intimate and dynamic interaction between host proteins and viral RNA (Kusov et al., 1996). The poly(A) tail is also involved in formation of RNA/protein complexes.
In analogy with other picornaviruses, the coding region can be subdivided into P1, P2, and P3 regions, which specify proteins 1A-D, 2A-C, and 3A-D, respectively. The polyprotein is further processed to the four structural and seven nonstructural proteins by proteinases encoded in and around the 3C region (Probst et al., 1998). Proteins 1A-D correspond to structural proteins VP1-VP4. As in other picornaviruses, the 5′ end of HAV genome is covalently linked to VPg protein, which is specified by 3B, instead of the classical m7G cap structure (Weitz et al., 1986). The 2C gene carries a guanidine resistance marker and is assumed to play a role in viral RNA replication (Cohen et al., 1987b). The region 3D is considered the RNA-dependent RNA polymerase. This region shows the highest degree of homology (29%) with the corresponding sequence in the poliovirus type 1 protein (Cohen et al., 1987b). A region of 18 amino acids considered to be essential for an active polymerase is present in the 3D region. This sequence contains a conserved motif of two asparatate residues flanked by hydrophobic amino acids that might function as a GTP-binding domain (Kamer and Argos, 1984). Replication efficiency seems to be controled by amino acid substitutions in the 2B and 2C regions (Yokosuka, 2000).

Proteins
The genomes of all picornaviruses encode a single polyprotein, which is coand post-translationally cleaved by virus-encoded proteinase(s). In contrast with well-characterized proteolytic events in the polyprotein of Enterovirus and Rhinovirus, this processing has been difficult to characterize in Hepatovirus. It has been shown that the primary cleavage of HAV polyprotein occurs at the 2A/2B junction, which has been mapped by the N-terminal sequencing of 2B. This primary cleavage of the polyprotein is mediated by 3C pro proteinase, which is the only proteinase known to be encoded by the virus (Martin et al., 1995;Gosert et al., 1996). The P1-2A capsid protein precursor is probably released from the nonstructural protein precursor (P3-2BC) as soon as 3C pro is synthesized, as the full-length polyprotein has not been observed in these studies. A P1-2A precursor produced in a cellfree translation system has been shown to be cleaved in vitro by recombinant 3C pro to generate VP0 (VP4-VP2), VP3, and VP1-2A (Malcolm et al., 1992). This VP1-2A polypeptide is unique to hepatoviruses; it associates with VP0 and VP3 to form pentamers, intermediates in the morphogenesis of HAV particles (Borovec and Andersen, 1993). The mature capsid protein VP1 is subsequently derived from the VP1-2A precursor later in the morphogenesis process. It has been hypothesized that the maturation of VP1 is dependent on 3C pro processing of the VP1-2A precursor (Probst et al., 1998). However, using recombinant vaccinia viruses expressing relevant HAV substrates, it has also been shown that 3C pro is incapable of directing the cleavage of VP1 from the VP1-2A precursor, indicating that maturation of VP1 could not depend on processing by 3C pro proteinase (Martin et al., 1999).

Virus Replication
Hepatitis A virus typically has a protracted and noncytolytic replication cycle in cell cultures and fails to shut down host cell metabolism (Lemon and Robertson, 1993). Even after successful adaptation to grow in cell cultures, replication of HAV is a slow process that terminates in a state of persistent infection (de Chastonay and Siegl, 1987). Cytopathic HAV strains have been recovered only from persistently infected cell cultures. Maximal levels of viral RNA synthesis can be detected at 24 h after infection, and exponential production of progeny virus continues for up to 4 days postinfection. Lysis of infected cells may become apparent within 3 to 9 days postinfection, and yield of progeny virus rarely exceeds 10 7 TCID 50 /ml (Siegl et al., 1984).

HEPATITIS E VIRUS
Hepatitis E virus (HEV), the prototype of the proposed Hepevirus, is a nonenveloped RNA virus, previously classified as a calicivirus but provisionally considered as a separate group of HEV-like viruses (Berke and Matson, 2000). Sequence comparison and phylogenetic analysis suggest that these viruses are more closely related to the Togaviridae (Koonin et al., 1992).
Hepatitis E virus is a major cause of epidemics and sporadic cases of acute hepatitis in Southeast and Central Asia, the Middle East, and many areas of Africa and Mexico . Sporadic cases have also been identified in Europe, Japan, and the United States (Schlauder et al., 1998(Schlauder et al., , 1999Takahashi et al., 2001). Transmission of HEV infection during outbreaks primarily occurs through contaminated water. Provision of clean drinking water is considered the best preventive strategy. The illness is selflimited and no chronic HEV infection has been described, although high mortality rate has been observed in pregnant women, with death rate as high as 25% (Balayan, 1997). HEV has been isolated from humans and swine (Meng et al., 1997(Meng et al., , 1998aHsieh et al., 1999;Garkavenko et al., 2001). Antibodies to the HEV capsid protein have been detected in many animal species, including rats and non-human primates (Kabrane-Lazizi et al., 1999a;Arankalle et al., 2001).
To date, four genotypes have been identified (Tam et al., 1991); a genotype 3 strain of HEV isolated from swine has been passed experimentally to monkeys, and a genotype 3 strain isolated from humans has been passed to swine (Meng et al., 1998b). Intriguingly, attempts to infect swine with other HEV human isolates have failed (Meng et al., 1998a). The question of whether HEV infection is a zoonosis is still being discussed (Meng, 2000). The viral genome is approximately 7.2 kb in length and has three partially overlapping ORFs: ORF1 encodes nonstructural proteins (1,693 amino acids), ORF2 encodes the capsid protein (660 amino acids), and ORF3 encodes a very small protein (123 amino acids) that has been shown to bind in vitro to a number of proteins involved in cellular signal transduction (Korkaya et al., 2001). The virus has not been propagated efficiently in cell cultures. Recently, various regions of the viral genome have been cloned and expressed in vitro. The ORF1 gene encodes the largest protein, which contains motifs characteristic of a methyl-transferase, a papain-like protease, a helicase, and an RNA-dependent RNA polymerase (Koonin et al., 1992). It has been demonstrated that the HEV genome contains a m 7 G cap at its 5′ end (Kabrane-Lazizi et al., 1999b;Zhang et al., 2001). There is still controversy over whether the 5′ cap is critical for HEV replication. Although it has been shown that the cap is required for infectivity of recombinant genomes in vivo, uncapped viral genomes can replicate in transfected HepG2 cells (Panda et al., 2000;Emerson et al., 2001).
The full-length ORF2 gene encodes a 72-kDa capsid protein consisting of 660 amino acids. ORF2 is the major, if not the only protein in the virion, but its real size in infectious virions is not known. When synthesized in insect cells, the initial 72-kDa protein is processed to smaller proteins of approximately 63, 56, and 53 kDa (Robinson et al., 1998). It has been demostrated that antibodies against ORF2 antibodies can neutralize HEV (Mast et al., 1998;Meng et al., 2001). Persons with preexisting anti-ORF2 did not develop hepatitis E after exposure to HEV in an outbreak in Pakistan (Bryan et al., 1994). Similarly, anti-ORF2-positive monkeys did not develop hepatitis after challenge with infectious HEV Arankalle et al., 1999). A number of antigenic determinants have been identified within ORF2 (Khudyakov et al., 1999;Li et al., 2000;Riddell et al., 2000). However, it appears that only antibodies directed against the C-terminus of ORF2 are neutralizing (Mast et al., 1998). The ORF2-encoded 56-kDa protein, truncated at its N-and C-termini, served as a highly reactive antigen in detecting anti-HEV antibodies (Zhang et al., 1997).
The ORF3 protein contains only 123 amino acids, and its biological role has yet to be elucidated. Recombinant ORF3 protein expressed in eukaryotic cells is an immunogenic phosphoprotein that accumulates in the cytoplasm and associates with the cytoskeleton (Zafrullah et al., 1997). This association is dependent on amino acids 1 to 32 of ORF3, which comprise a hydrophobic domain. In addition, ORF3 contains two conserved prolinerich domains. By the yeast two-hybrid assay, it has been demonstrated that the second of these proline-rich domains binds to certain Src homology 3 domains present in a subset of cellular proteins. It has been hypothesized that ORF3 may be a viral regulatory protein involved in cellular signal transduction (Korkaya et al., 2001). Also, recombinant ORF3 protein has been shown to interact in yeast two-hybrid assays and by co-immunoprecipitation assays with the nonglycosylated form of the major capsid protein, ORF2 protein (Tyagi et al., 2002). These observations lead to the hypothesis that ORF3 has a well-regulated role in HEV structural assembly (Emerson and Purcell, 2003). One HEV vaccine candidate, a baculovirus-expressed protein spanning 112-607 amino acids of ORF2, was demonstrated to be safe and immunogenic in volunteers and is currently being evaluated in clinical trials in Nepal (Emerson and Purcell, 2003).

ENTERIC ADENOVIRUSES
Adenoviruses are nonenveloped, icosahedral viruses measuring 70 to 90 nm in diameter. They have a buoyant density in cesium chloride of 1.33 to 1.34 g/cm 3 . The capsid is composed of 252 capsomeres, of which 240 are hexons and 12 are pentons. Inside the capsid is a single molecule of linear, double-stranded DNA (Shenk, 2001). Two genera are recognized within the Adenoviridae family: Mastadenovirus includes viruses that infect mammals, and Aviadenovirus contains viruses that infect birds (Benkö et al., 2000). Adenoviruses are species-specific and generally replicate only in cells derived from their native host. Human adenoviruses are associated with a variety of infectious diseases affecting the respiratory, urinary, and the gastrointestinal tracts and the eyes (Horwitz, 2001). To date, 51 serotypes of human adenoviruses have been recognized, which are classified into six subgroups, A to F, based on immunological properties, oncogenicity in rodents, DNA homologies, and morphological properties (Benkö et al., 2000).
Enteric adenoviruses were originally identified in stool samples of infants with acute gastroenteritis (Flewett et al., 1973) and have been consistently associated with gastroenteritis in children through epidemiological and clinical studies (Uhnoo et al., 1983;Uhnoo et al., 1984). They are responsible for 5% to 20% cases of acute diarrhea in children (Uhnoo et al., 1984;Kotloff et al., 1989;Uhnoo et al., 1990;Bon et al., 1999) and are found in clinical samples throughout the year with little seasonal variation (de Jong et al., 1983).
The enteric serotypes 40 and 41 have been designated as subgroup F adenoviruses. They share the adenovirus group antigen and are distinguished from each other and from other nonenteric serotypes by serology and DNA restriction patterns (Wadell, 1984). These two serotypes are shed in large numbers from the gut of infected patients and were originally described as being noncultivable or "fastidious" adenoviruses, because they could not be cultivated in cell cultures that generally supported the propagation of other adenovirus types. Later, however, it was discovered that enteric adenoviruses could be propagated in Graham 293 cells, a cell line of human embrionic kidney (HEK) cells transformed with adenovirus 5 early (E) region 1 (Graham et al., 1977;Takiff et al., 1981), although at lower levels than other serotypes. This suggests that E1 functions are poorly expressed in cells infected with adenovirus types 40 and 41, and therefore it was postulated that the inability of these serotypes to grow in cell lines normally supportive for other adenovirus types was due to the relative inability of the adenovirus 41 E1A gene to transactivate other adenovirus 41 genes (Takiff et al., 1984;van Loon et al., 1985a). The Graham 293 cell retains the E1A and E1B regions of the adenovirus genome covalently linked to the host DNA. The mechanism of facilitation of the growth of the EAd40 in 293 cells seems to be a function of the E1B-55 kd protein (Mautner et al., 1989). Efficient replication of adenovirus types 40 and 41 has also been achieved in other cell lines, including Hep-2 cells, Chang conjuntiva cells, CaCo-2 cells (Perron-Henry et al., 1988;Pintó et al., 1994), and PLC/PRF/5 cells (a primary liver carcinoma cell line) (Grabow et al., 1992).
The genome of adenovirus 40 has been sequenced (Davidson et al., 1993) (GenBank accession no. L19443) and described in detail (Mautner et al., 1995). The main difference between adenovirus 40 and the other human adenovirus serotypes is the presence of two distinct fiber genes, a single VA gene involved in late translation and a highly divergent E3 region. The growth restriction of adenovirus 41 in cell cultures seems to be less severe than that of serotype 40 because a number of cell lines have been found to support the propagation of serotype 41 but not of serotype 40 (de Jong et al., 1983;Uhnoo et al., 1983;van Loon et al., 1985b). The blockade in adenovirus 41 replication occurs within the early phase of the infectious cycle (Tiemessen et al., 1996). In a study on the ability of adenovirus 40 E1A encoded products (proteins 249R and 221R) for trans-activation (van Loon et al., 1987;Ishino et al., 1988), it was found that the adenovirus 40 E1A promoter does indeed contain transcription factor binding sites sufficient for trans-activation by the adenovirus 5 E1A 289R protein. It is possible that adenovirus 40 has evolved to use components of the RNA processing machinery that are unique to enterocytes (Stevenson and Mautner, 2003). Hence, a better understanding of the replication and pathogenicity of adenovirus 40 will require the development of intestinal cell cultures.

OTHER ENTERIC VIRUSES
Although rotaviruses, caliciviruses, astroviruses, and enteric adenoviruses are the most prominent viral pathogens of acute gastroenteritis, other candidates such as coronaviruses, toroviruses, and picobirnaviruses are also considered to have capability to cause diarrhea in humans.

Coronaviruses
Coronavirus is a genus of the Coronaviridae family in the order Nidovirales (Enjuanes et al., 2000). The first reports of coronavirus-like particles in stools of patients with diarrhea were documented by Caul and Clarke (1975), who reported cultivation of these viral particles in human embryo intestinal organ culture and human embryo kidney monolayers (Caul and Clarke, 1975). Since then, the existence of human enteric coronavirus (HEC) has been controversial (Mathan et al., 1975;Caul and Egglestone, 1977). Some investigators have considered these particles as viruses and as potential pathogens in human diarrhea, necrotizing enterocolitis, and malabsorption. Other authors cite their variable size, their presence in stools of normal subjects, and their failure to be cultivated in vitro as evidence that they might represent cell debris or even portions of other microorganisms (Macnaughton and Davies, 1981). However, isolation and propagation of enteric coronaviruses have been reported (Resta et al., 1985;Luby et al., 1999). The enveloped particles are 120-160 nm in diameter and possess a genome of linear, positive-sense, single-stranded RNA of approximately 30 kb in size. The genome is surrounded by the nucleocapsid protein (N) with helical symmetry, which is contained in an envelope, in which the spike (S), envelope (E), hemagglutinin esterase (HE), and membrane (M) proteins are embedded. Proteins are expressed from RNA molecules that are in most cases subgenomic, and all mRNAs carry a leader sequence derived from the 5′ end of the genome (Lai and Holmes, 2001).

Toroviruses
Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-sense RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about the epidemiology and molecular genetics of toroviruses (Koopmans and Horzinek, 1994;Smits et al., 2003).
Toroviruses have not been propagated in cell cultures, with the sole exception so far of the equine Berne virus. Early seroepidemiological surveys based on Berne virus cross-neutralization assays and enzyme-linked immunosorbent assay (ELISA) indicated that toroviruses occur in a wide variety of ungulate hosts (cattle, sheep, goats, and swine) (Koopmans and Horzinek, 1994). Breda virus was detected in the stools of diarrheic calves but could not be isolated in cell cultures (Woode et al., 1982). Thereafter, two antigenically distinct serotypes of Breda virus have been identified, referred to as bovine toroviruses 1 and 2 (BoTV-1 and BoTV-2) (Woode et al., 1985).
Morphologically similar pleomorphic particles characterized by a welldefined fringe around the outer edges have also been found in human stools (Beards et al., 1984;Duckmanton et al., 1997). These viruses have a unique morphology with a helical nucleocapsid in a torus-shaped configuration within an envelope carrying large spikes. In samples from children and adults with diarrhea, torovirus-like particles cross-reactive with Berne virus-specific and Breda virus-specific antisera were detected by immunoelectron microscopy (Beards et al., 1984;Beards et al., 1986;Duckmanton et al., 1997), and torovirus antigens were detected by ELISA (Koopmans et al., 1997). Recently, the nucleotide sequence of the Berne virus genomic RNA has been completed (Smits et al., 2003).
With a genome length of 28 kb, toroviruses are among the largest RNA viruses, rivaled in size only by the coronaviruses. The 5′-most two-thirds of the genome is taken up by two huge overlapping open reading frames, 1a and 1b, encoding polyproteins from which various subunits of the viral replicase/ transcriptase are derived (Snijder et al., 1990a). Downstream of ORF1b, there are four cistrons of 5 kb, 0.7 kb, 1.2 kb, and 0.5 kb (as ordered from 5′ to 3′). These encode the structural proteins, the spike (S), membrane (M), hemagglutinin-esterase (HE), and nucleocapsid (N) proteins, respectively (Snijder and Horzinek, 1993). The structural proteins are translated from a 3′-coterminal nested set of subgenomic mRNAs, produced by discontinuous and nondiscontinuous RNA synthesis (Snijder et al., 1990b;van Vliet et al., 2002). Four genotypes, displaying 30% to 40% sequence divergence, have been recently distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV), equine torovirus Berne, and the putative human torovirus (Smits et al., 2003). It remains unclear, however, whether the toroviruses are host specific and how the torovirus genotypes are geographically distributed.

Picobirnaviruses
Picobirnavirus is the tentative name for a new virus genus in the family Birnaviridae (Leong et al., 2000). Picobirnaviruses were first observed in 1988 in fecal samples of humans and rats (Pereira et al., 1988a(Pereira et al., , 1988b. The viruses have subsequently been found in many mammals and birds (Rosen, 2003) and have been frequently detected in cases of human gastroenteritis associated with human immunodeficiency virus infections (HIV) (Grohmann et al., 1993;Gonzalez et al., 1998;Liste et al., 2000). Association of picobirnavirus with nonbacterial gastroenteritis outbreaks has also been reported recently (Banyai et al., 2003).
Morphological, physicochemical, and genomic characteristics suggest that picobirnaviruses belong to a distinct group of viruses. The small size of the virion and the nature of the viral genome prompted the name picobirnaviruses, referring to pico (small) and birna (two RNA segments). The virion is 25-41 nm in diameter, nonenveloped, and has a buoyant density of 1.38 to 1.42 g/cm 3 (Pereira et al., 1988b;Ludert et al., 1995). However, virions with a diameter of around 35 nm have been found most often (Ludert et al., 1991;Gallimore et al., 1995). The genome consists of two segments of doublestranded RNA with lengths estimated around 2.3-2.6 Kpb and 1.5-1.9 Kpb, respectively (Gallimore et al., 1995;Chandra, 1997). These viruses are noncultivable. They are detected by EM and polyacrylamide gel electrophoresis (PAGE) (Ludert and Liprandi, 1993;Gallimore et al., 1995;Cascio et al., 1996). Picobirnavirus genomic fragments migrate in PAGE within the migration range of the genomic segments of group A rotaviruses (Pereira et al., 1988a;Ludert et al., 1991;Rosen et al., 2000). This fact led to the discovery of these viruses when fecal samples containing both rotaviruses and picobirnaviruses were subjected to PAGE (Pereira et al., 1988a(Pereira et al., , 1988b. Currently, RT-PCR is used to detect human picobirnaviruses in fecal samples (Rosen et al., 2000).
Limited information is available on the genetic sequence of picobirnaviruses. To date, genomic segment 1 of a rabbit and a human picobirnavirus and segment 2 of two human picobirnaviruses have been cloned Rosen et al., 2000). Human picobirnavirus sequences have been determined mostly for the smaller genomic segment that encodes the RNAdependent RNA polymerase (Rosen et al., 2000). The genomic segment 1 has been postulated to encode for the capsid protein Rosen et al., 2000). Based on the sequence data of the RNA polymerase, there are two major genogroups within human picobirnaviruses, for which prototype strains are 1-CHN-97 and 4-GA-91 (Rosen et al., 2000).

SUMMARY
Food-borne and waterborne viruses mainly cause gastroenteritis (caliciviruses, rotaviruses, astroviruses, and enteric adenoviruses), hepatitis (hepatitis A virus, hepatitis E virus), and other diseases (enteroviruses). Other enteric viruses like kobuviruses, coronaviruses, toroviruses, and picornaviruses also cause diarrhea, although the causative role for some of these viruses in humans is still controversial.
Human caliciviruses have been recognized as the leading cause of acute gastroenteritis outbreaks and sporadic cases in children and adults worldwide. Enteric caliciviruses belonging to the Norovirus and Sapovirus genera remain refractory to cell culture propagation. This limitation has hampered our ability to investigate their biology, pathogenesis, and host immunity, although molecular approaches are providing new insights into these areas. The morphology, composition, and structure of several enteric viruses have been elucidated in recent years. Cryo-electron microscopy and x-ray crystallography have been crucial for this purpose. Biochemical and structural studies of virus-like particles produced by recombinant baculoviruses are contributing to better understand the structure-function relationships of the capsid proteins. Viral genome organization is being clarified for all these viruses, as well as their replication and gene expression strategies. Most of the proteins encoded by the viral genomes have been characterized and their functions identified. Sequence analysis of viral genes is currently being applied in molecular epidemiological studies. However, many questions still remain to be answered.The development of efficient reverse genetics systems would be extremely useful in the analysis of the mechanisms of viral replication and of gene expression of the different enteric viruses.
Biochemical characterization of viral interactions with cells and analysis of the functional properties of the viral proteins are providing a better understanding of the pathogenesis of enteric viruses. Rotavirus NSP4 is the first viral enterotoxin to be characterized. Several cell membrane molecules have been identified recently as being receptors for different enteric viruses (i.e., integrins and hsc70 for rotaviruses, ABH histo-blood group carbohydrates for noroviruses). Studies on human susceptibility to norovirus infections have characterized some resistant nonsecretor (Se-) individuals in the population, which is a breakthrough in our knowledge of norovirus-host interactions. Similarly, molecular analyses of orally transmitted viruses causing hepatitis are clarifying the phylogenetic relationships between these viruses and other viral genera, as well as their pathophysiological mechanisms.