8.5.9 Virus infections causing diarrhoea and vomit

8.5.9 Virus infections causing diarrhoea and vomiting 797

8.5.9  Virus infections causing diarrhoea and vomiting 797 FURTHER READING Cello J, Paul AV, Wimmer E (2002). Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural tem- plate. Science, 297, 1016–​18. Centers for Disease Control and Prevention (CDC) (2011). Progress toward poliomyelitis eradication—​Nigeria, January 2010–​June 2011. MMWR Morb Mortal Wkly Rep, 60, 1053–​7. Centers for Disease Control and Prevention (CDC) (2011). Update on vaccine-​derived polioviruses—​worldwide, July 2009–​March 2011. MMWR Morb Mortal Wkly Rep, 60, 846–​50. Dunn G, et al. (2015). Twenty-​eight years of poliovirus replication in an immunodeficient individual: impact on the global polio eradica- tion initiative. PLoS Pathog, 11, e1005114. Hovi T, et  al. (2011). Role of environmental poliovirus surveil- lance in global polio eradication and beyond. Epidemiol Infect, 140, 1–​13. Joint Working Group of the Royal Colleges of Physicians, Psychiatrists, and General Practitioners (1997). Chronic fatigue syndrome, pp. 58. Royal College of Physicians Publication Unit, London. Kew OM, et al. (2002). Outbreak of poliomyelitis in Hispaniola asso- ciated with circulating type 1 vaccine-​derived poliovirus. Science, 296, 356–​9. Knowles NJ, et  al. (2011). Picornaviridae. In:  King AMQ, et  al.
(eds) Virus taxonomy:  classification and nomenclature of viruses,
pp. 855–​80. Ninth report of the International Committee on Taxonomy of Viruses. Elsevier, San Diego, CA. Larson HJ, Ghinai I (2011). Lessons from polio eradication. Nature, 473, 446–​7. Martin J, et al. (2000). Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-​day period of virus excretion. J Virol, 74, 3001–​10. Melnick JL (1996). Enteroviruses:  polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In:  Fields BN, et  al. (eds) Fields virology, 3rd edition, pp. 655–​712. Lippincott-​Raven, Philadelphia, PA. Mendelsohn C, Wimmer R, Racaniello VR (1989). Cellular receptor for poliovirus: molecular cloning, nucleotide sequence and expres- sion of a new member of the immunoglobulin superfamily. Cell, 56, 855–​65. Minor PD (1990). Antigenic structure of picornaviruses. Curr Top Microbiol Immunol, 161, 122–​54. Minor PD (1996). Poliovirus. In: Nathanson N, et al. (eds) Viral patho- genesis, pp. 555–​74. Lippincott-​Raven, Philadelphia. Minor P (2005). Picornaviruses. In: Mahy BW, ter Meulen V (eds) Topley and Wilson’s microbiology and microbial infections. Virology, 10th edition, pp. 857–​87. Hodder Arnold, London. Nathanson N (2008). The pathogenesis of poliomyelitis: what we don't know. Adv Virus Res, 71, 1–​50. Offit PA (2005). The cutter incident, pp. 256. Yale University Press, New Haven. Pallnsch M, Oberste MS, Whitton LS (2013). Enteroviruses:  polio- viruses, coxsackieviruses, echoviruses, and newer enteroviruses. In:  Knipe DM, et  al. (eds) Fields virology, 6th edition, pp. 490–​ 530. Wolters Kluwer Health/​Lippincott Williams & Wilkins, Philadelphia, PA. Racaniello VR (2013). Picornaviridae:  the viruses and their repli- cation. In: Knipe DM, et al. (eds) Fields virology, 6th edition, pp. 453–​89. Wolters Kluwer Health/​Lippincott Williams & Wilkins, Philadelphia, PA. Racaniello VR, Baltimore D (1981). Cloned poliovirus complemen- tary DNA is infectious in mammalian cells. Science, 214, 916–​19. Skern T (2010). 100 years poliovirus: from discovery to eradication: a meeting report. Arch Virol, 155, 1371–​81. Wimmer E (2006). The test-​tube synthesis of a chemical called polio- virus: the simple synthesis of a virus has far-​reaching societal impli- cations. EMBO Rep, 7 Spec No, S3–​S9. Wimmer E, et  al. (2009). Synthetic viruses:  a new opportunity to understand and prevent viral disease. Nat Biotechnol, 27, 1163–​72. Wimmer E, Paul AV (2011). Synthetic viruses:  a new opportunity to understand and prevent viral disease. Annu Rev Microbiol, 65, 583–​609. World Health Organization (2008). Global polio eradication initia- tive. Annual report 2007. Impact of the intensified eradication effort. WHO, Geneva. http://​polioeradication.org/​wp-​content/​uploads/​ 2016/​07/​AnnualReport2007_​English.pdf Yamashita T, et al. (2000). Application of a reverse transcription-​PCR for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans. J Clin Microbiol, 38, 2955–​61. Websites ICTV. Taxonomy. http://​ictvonline.org/​virusTaxonomy.asp World Health Organization. Polio eradication initiative. http://​www. emro.who.int/​entity/​polio/​ and http://​www.polioeradication.org/​ Dataandmonitoring/​Poliothisweek.aspx 8.5.9  Virus infections causing diarrhoea and vomiting Philip R. Dormitzer and Ulrich Desselberger ESSENTIALS Acute gastroenteritis is frequently caused by rotaviruses, human caliciviruses (noroviruses, sapoviruses), astroviruses and enteric adenoviruses (group F): these cause much disease worldwide and considerable mortality, mainly in developing countries. Other vir- uses found in the human gastrointestinal tract are not regularly associated with diarrhoeal disease, except in patients who are im- munosuppressed and in whom herpes simplex virus, cytomegalo- virus, and picobirnaviruses can cause diarrhoea, as can HIV itself. Epidemiology—​(1) Rotaviruses—​a major cause of endemic infantile gastroenteritis worldwide; transmission is by the faecal-​oral route; there is a strict winter peak of infections in temperate climates, but these occur year-​round in tropical and subtropical regions; many animals and birds harbour a large diversity of rotaviruses and may act as a reservoir for human infections. (2) Human caliciviruses—​ the most important cause of non​bacterial gastroenteritis outbreaks worldwide; transmission is by the faecal-​oral and emesis-​oral routes; contaminated food (oysters, green salads, fresh fruit, cold foods, and sandwiches) and water are frequently implicated in outbreaks.

798 section 8  Infectious diseases Clinical features and management—​following an incubation period of 1–​2 days, there is sudden onset of watery diarrhoea lasting between 4 and 7 days, vomiting, and varying degrees of dehydration. Other features include abdominal cramps, headache, myalgia, and fever. Treatment is supportive, mainly with oral rehydration solutions or—​in more severe cases—​intravenous rehydration. Continued feeding is recommended, with zinc supplementation in areas where micronu- trient deficiency may be present. Diagnosis—​viral infection can be demonstrated by virus-​specific enzyme-​linked immunosorbent as- says and by viral genome detection using the polymerase chain re- action (for adenoviruses) or reverse transcription-​polymerase chain reaction (RT-​PCR) (for rotaviruses, caliciviruses, and astroviruses). Prevention and control—​two live attenuated oral rotavirus vaccines have been licensed in numerous countries since 2006, one vac- cine licensed in India in 2014 has achieved WHO prequalification; and three more have national licenses in India, China, or Vietnam. In countries where universal mass vaccination of children as part of childhood vaccination schemes has been established, a significant reduction of rotavirus-​associated acute gastroenteritis has been re- corded. In Mexico, introduction of rotavirus vaccines was associated with a substantial drop in overall mortality due to diarrheal disease of any cause in children 23 months of age and younger. Vaccines against human norovirus disease are under development. Outbreak control measures focus on the interruption of person-​to-​person transmis- sion, the removal of common sources of infection (food, water, and so on), and improvement of general environmental hygiene. Introduction Acute gastroenteritis and vomiting in humans is a well-​characterized clinical entity caused by various microbial agents (viruses, bacteria, parasites, and so on). Viral gastroenteritis is a global problem, par- ticularly in infants and young children. Many viruses are found in the human gut, but not all of them produce acute gastroenteritis, and even pathogenic agents of gastroenteritis may also be shed asymptomatically (Table 8.5.9.1). Viral infections normally associated with gastroenteritis are caused by rotaviruses, human caliciviruses (noroviruses, sapoviruses), astroviruses and enteric adenoviruses (group F). Other viruses found in the human gastrointestinal tract (enteroviruses, reo- viruses, non​group F adenoviruses, toroviruses, coronaviruses, parvoviruses) are not regularly associated with diarrhoeal disease. Finally, there are viruses causing diarrhoea in immunosuppressed patients (most commonly those infected with HIV), including herpes simplex virus, cytomegalovirus, and picobirnaviruses. HIV itself can also infect the gut directly. Recently, the study of inter- actions of different microbes infecting the gut has shown that bac- terial flora can modulate the replication of enteric viruses. Such studies of the gut microbiome are attracting increased attention with the aim of better understanding these mechanisms in health and disease. Only the major virus groups regularly causing gastroenteritis in humans are described here in separate sections. Clinical symptoms, diagnosis, treatment, epidemiology, and vaccine development are reviewed under common headings. Rotaviruses Structure Rotaviruses are the major cause of infantile gastroenteritis world- wide and also of acute diarrhoea in the young of many mammalian species. They constitute the genus Rotavirus in the Reoviridae family, with a genome of 11 segments of double-​stranded RNA encoding six structural viral proteins (VP1–​VP4, VP6, VP7) and six non-​ structural proteins (NSP1–​NSP6). All genes are monocistronic, ex- cept for RNA segment 11, which encodes two proteins (NSP5 and NSP6). The icosahedral virion has three concentric protein layers and no lipid envelope (Fig. 8.5.9.1a). In electron micrographs of negatively stained specimens, virions have a characteristic appear- ance as 75-​nm wheel-​like particles (Fig. 8.5.9.2), the name of the virus being derived from Latin rota  =  wheel. The inner layer (consisting of VP2) encloses the genome seg- ments, the viral RNA-​dependent RNA polymerase (RdRp) VP1, and the capping enzyme VP3. The addition of a middle layer consisting of VP6 leads to the formation of transcriptionally ac- tive subviral particles, referred to as the double-​layered par- ticles (DLPs). VP6 is the most immunogenic rotavirus protein. Infectious virions (triple-​layered particles, TLPs) have an add- itional layer, which mediates the translocation of the DLP into the cytoplasm during cell entry. This outermost layer consists of two proteins, VP4 and VP7. VP7 forms a shell, which is shed in the low calcium environment of the cytoplasm. VP4 forms spikes, which are important for attachment and membrane penetration. Crystal structures, electron cryomicroscopy image reconstruc- tions, and functional studies have provided evidence that a fold-​ back rearrangement of the VP4 spike, which is mounted on the VP6 layer and secured by the surrounding VP7 shell, is required for membrane penetration during entry (Fig. 8.5.9.1b). To achieve Table 8.5.9.1  Virus infections of the human gut Viruses found as Genus (family) Regular cause of diarrhoea and vomiting Rotaviruses (Reoviridae)a Human caliciviruses (Caliciviridae) a Group F adenoviruses (Adenoviridae) Astroviruses (Astroviridae) Occasional cause of diarrhoea and vomiting Enteroviruses (Picornaviridae)b Reoviruses (Reoviridae) Adenoviruses other than Group F (Adenoviridae) Toroviruses (Coronaviridae) Coronaviruses (Coronaviridae) Parvoviruses (Parvoviridae) Cause of diarrhoea in immunodeficient patients Human immunodeficiency virus (Retroviridae) Herpes simplex virus (Herpesviridae) Cytomegalovirus (Herpesviridae) Picobirnaviruses (Birnaviridae) a Not all infections cause disease (see text). b Outbreaks of diarrhoea caused by echovirus type 11 infections have been reported (see Chapter 8.5.8), and Aichi virus is an endemic cause of diarrhoea in Asia and of traveller’s diarrhoea.

8.5.9  Virus infections causing diarrhoea and vomiting 799 maximal infectivity of the virion, the VP4 spike must be cleaved by intestinal trypsin or cellular proteases. Classification Most commonly, rotaviruses are classified according to the im- munological reactivity and genomic sequences of three of their structural components (VP6, VP7, and VP4), although a more recent and comprehensive classification differentiates all 11 gene segments of rotaviruses into genotypes. Specific sequences and epitopes on the middle-​shell protein VP6 allow at least 7–​8 groups (A–​G/​H, possibly I) to be distinguished. Group A rotaviruses cause the vast majority of human infections and acute gastroenteritis and have been divided into subgroups on the basis of additional de- terminants on VP6. Group B rotaviruses have caused epidemics of diarrhoea affecting adults and children, mainly in China and India. Group C rotaviruses generally cause milder diarrhoeal disease. The remaining groups are only known to infect non​human hosts. Both surface proteins, VP4 and VP7, elicit and are the targets of neutralizing antibodies. A dual-​type classification system has been devised for group A rotaviruses, which differentiates glycoprotein (G) types (VP7-​specific) and protease-​sensitive protein (P) types (VP4-​specific). For G types, serotype and genotype are equivalent; for P types serotype and genotype may be different, and genotypes are more commonly used. For example, G1P[8]‌ is G serotype and genotype 1, P genotype 8. At least 12 G types and 15 P types have been found in humans. However, at present, rotaviruses carrying a relatively restricted number of G types (G1–​G4, G9, and G12) and P types (P[4], P[6] and P[8]) cause most human disease. Rotavirus genomes undergo point mutations continuously (due to the high inherent error rate of the viral polymerase), with one of the conse- quences being antigenic drift. In addition, rotaviruses can exchange (reassort) genome segments during mixed infections, providing a further mechanism to introduce genetic diversity (antigenic shift). Zoonotic rotavirus infections of humans are well documented, and previously rare serotypic variants have become established among strains pathogenic to humans (mainly in tropical and subtropical regions). Hence, rotavirus epidemiology continues to evolve by various mechanisms, and eradication of rotaviruses is not feasible. Although the mechanisms of rotavirus genetic diversity resemble those of influenza viruses, rotaviruses do not undergo the frequent waves of global strain replacement and occasional pandemic shifts observed with influenza viruses. Fig. 8.5.9.2  Rotavirus particles in the faeces of a child admitted to hospital with acute gastroenteritis. Negative staining with aqueous 2% potassium phosphotungstate, pH 7.0. Scale bar represents 100 nm. Four different morphologies of particles are shown: (a) triple-​layered particle containing RNA; (b) triple-​layered particle without RNA (empty, core penetrated with stain); (c) double-​layered particle containing RNA; and (d) double-​layered empty particle. Courtesy of M. Jenkins, Regional Virus Laboratory, East Birmingham Hospital. From Desselberger U (1992). Reoviruses. In: Greenwood D, Slack R, Peutherer J (eds) Medical microbiology, 14th edition, p. 620. Churchill Livingstone, Edinburgh, with permission of the publisher. VP4 VP7 VP6 VP2 RNA (a) (b) VP8* VP7 VP6 VP2 VP5* (c-c) Fig. 8.5.9.1  (a) The icosahedral rotavirus virion has three layers: (1) an inner VP2 layer that contains the genome, polymerase, and capping enzyme; (2) a middle VP6 layer; and (3) an outer VP7 layer. The VP4 spike is anchored in the VP6 layer and protrudes through the VP7 layer (b). For efficient infection, VP4 must be cleaved by proteases into the VP8* fragment, which includes the receptor binding ‘head’ of the spike, and the VP5* fragment, which combines with the N-​terminal region of VP8* to form the remainder of the spike and includes hydrophobic loops involved in the disruption of a host cell membrane during entry. Panel (a): based on Dormitzer P, et al. (2004). Structural rearrangements in the membrane penetration protein of a non-​enveloped virus. Nature, 430, 1053–​58;
Yeager M, et al. (1990). Three-​dimensional structure of rhesus rotavirus by cryoelectron microscopy and image reconstruction. J Cell Biol, 110, 2133–​44.
Panel (b): from Settembre EC, et al. (2011). Atomic model of an infectious rotavirus particle. EMBO J, 30, 408–​16. With permission of the authors and the publisher.

800 section 8  Infectious diseases Replication Studies of rotavirus replication have been enabled by adaptation of many rotavirus strains to growth in mammalian cell culture and the ability to assign gene functions through a classic genetic approach in which rotavirus strains exchange genome segments through reassortment during mixed infection of single cells. Recently, ­reverse genetics systems for rotaviruses based only on plasmids have been developed. The primary targets of rotavirus infection are the mature epi- thelial cells at the tips of the villi of the small intestine. The VP8* head of the VP4 spike mediates attachment to cells. Some strains bind cell surface sialic acids; most strains that infect humans bind histo-​blood group antigens. Human polymorphism in histo-​blood group antigens, therefore, influences susceptibility to some rotavirus strains. After attachment, productively entering rotavirus particles are enveloped in vesicles derived from closely fitting membrane in- vaginations that form around the bound particles. Formation of the membrane invaginations appears to be driven by interactions be- tween rotavirus surface proteins and cell membrane glycolipids. As the VP7 shell is released from the TLP, a jack-​knifing rearrangement of the VP5* fragment of the VP4 spike, analogous to the rearrange- ments of enveloped virus fusion proteins, appears to drive disrup- tion of the vesicle so that the DLP is delivered into the cytoplasm. In the cytoplasm of an infected cell, the DLP extrudes 11 dif- ferent newly synthesized mRNAs without releasing the genome segments. One viral non​structural protein, NSP3, binds to the non​polyadenylated 3′ ends of viral mRNAs, substituting for the host poly(A) binding protein in circularizing mRNA by binding the translation initiation factor eIF4G, which is bound to the 5´ end of the RNA. NSP3 also shuts off host translation by depleting eIF4G pools. New DLPs assemble in cytoplasmic inclusion bodies, termed viroplasms. Because each infectious unit corresponds to a small number of virus particles, it is likely that one of each of the 11 genome segments is packaged into each new DLP. The mechanism of this specific and highly selective packaging is likely to involve complex RNA-RNA and RNA-protein interactions involving the rotavirus non-structural protein NSP2. Viroplasms form complexes with the cellular organelle lipid droplets. DLPs released from viroplasms bind to the virally encoded glyco- protein NSP4, which is integrated into the endoplasmic reticulum membrane. When budding into the endoplasmic reticulum lumen, DLPs acquire a transient envelope, which is lost as the outermost protein layer is added to complete the formation of virions. Virions are released from infected enterocytes after transport to the cell surface by a vesicular transport pathway that bypasses the Golgi apparatus. Rotavirus replication in the gut results in very high concentra- tions of viral particles (up to 1011/​ml) in faeces at the peak of acute diarrhoea. The physical hardiness of the shed particles ensures their efficient transmission to new hosts. Pathogenesis The pathogenesis of rotavirus diarrhoea is complex. Viral infec- tion causes direct damage to the enteric epithelium, resulting in the blunting and denudation of villi. The villous damage is repaired by cells emerging and differentiating from the crypts of the gut epithelium, which shows a reactive hyperplasia. Loss of functioning absorptive cells leads to a degree of malabsorption and osmotic fluid loss. However, there also appears to be a secretory component to rotavirus diarrhoea. By raising intracellular calcium concentra- tions in infected cells, NSP4 activates a plasma membrane anion channel causing fluid secretion. There is evidence that a fragment of NSP4 is released from infected cells, acting as a viral enterotoxin to induce a secretory state of uninfected cells. The enteric nervous system also plays a role in pathogenesis. Enteric nervous system inhibitors diminish fluid secretion in the gut of rotavirus-​infected animals. Rotaviruses infect and stimulate enterochromaffin cells to release serotonin (5-​HT) which activates brain structures involved in nausea and vomiting. Immune response A primarily serotype-​specific humoral immune response is elicited after neonatal or primary rotavirus infection. However, during the first 2 years of life children are repeatedly infected with rotaviruses, leading to multiple serotype-​specific, and also partially heterotypic, protection. The presence of rotavirus-​specific secretory IgA copro-​ antibodies seems to correlate best with protection against disease, although the exact correlates of protection remain to be determined. Rotavirus-​specific cytotoxic T-​cell responses are capable of clearing infections, but appear to be less important than humoral immune responses in protecting against repeated infections. The abun- dant antibody that is produced against VP6 during infection does not neutralize extracellular virus. However, anti-​VP6 IgA, which is transported across enterocytes for secretion into the gut lumen, can inhibit viral replication heterotypically (within group A) by binding DLPs in the cytoplasm (‘intracellular neutralization’). The role of anti-​VP6 IgA in protecting against infection in humans is not yet known, but it has become apparent that heterotypic protec- tion mechanisms (after both natural infection and vaccination) play a considerable role. Small llama-​derived VP6-​specific antibody fragments inhibit rotavirus replication in vitro, presumably by a similar mechanism to that of anti-​VP6 IgA. In a double-​blind, placebo-​controlled trial in Bangladesh in male infants with severe rotavirus-​associated diarrhoea and no other pathogens detected, the addition of one of these VP6-​specific antibody fragments to standard oral rehy- dration solution treatment reduced stool output by a statistically significant 22.5%. Human caliciviruses Structure and classification These viruses were first recognized as the cause of gastroenteritis during outbreaks in Norwalk, Ohio, in the late 1960s. Norwalk virus particles are spherical and measure 27–​35 nm in diameter. Norwalk virus and Norwalk-​like viruses are all members of the Caliciviridae family. Their 7.7-​kb genome consists of single-​stranded RNA of positive polarity. Cup-​shaped depressions on the surface of virions have given the name to this viral family (Latin calix  =  goblet, cup) (Fig. 8.5.9.3c, d). The T = 3 icosahedral viral capsid is formed from a single protein, which has a shell (S) domain that makes the icosa- hedral contacts and a protruding domain (P) that makes twofold

8.5.9  Virus infections causing diarrhoea and vomiting 801 contacts. The P2 subdomain is the furthest protruding and most variable part of the virus, and it contains receptor binding sites. Phylogenetic trees of full-​length sequences of caliciviral cDNAs have led to their classification into five genera: viruses of the genera Norovirus and Sapovirus infect humans, whereas viruses of the genera Vesivirus, Lagovirus, and Nebovirus only infect animals. Until recently viruses of the Norovirus genus were often termed ‘small round structured viruses’ and those of the Sapovirus genus ‘classical caliciviruses’. Noroviruses and sapoviruses are genetically very di- verse (consisting of at least five genogroups/​clades each and 3–​>20 genetic clusters/​genotypes within them) and constantly evolve. Replication Details of the replication of human caliciviruses have been deduced from those of animal caliciviruses, because, historically, there has been no reproducible in vitro cell culture system for the human caliciviruses. Recently, replication of human noroviruses has been demonstrated in stem cell-derived human intestinal enteroid mono- layer cultures, in the presence of bile. The viruses seem to interact with species-​specific receptors, and a single protein precursor is cotranslationally and posttranslationally cleaved in a way similar to that observed for the polyprotein of picornaviruses. A reverse genetics system for murine norovirus has been developed. Murine norovirus also replicates in cell culture and in mice, thus providing a useful animal model for norovirus infection. Immune response Although calicivirus infections elicit humoral and cell-​mediated immune responses in humans (with an antibody response mainly directed to the P2 portion of the capsid antigen), they do not seem to give full protection against subsequent infection. Like human rotaviruses, noroviruses use certain histo-​blood group antigens act as receptors. Humans who are ‘secretors’ of such antigens are more susceptible to infection with some norovirus strains than ‘non​ secretors’, depending, in part, on strain-​specific receptor usage. Due to the genetic variability in human susceptibility to some norovirus strains, pre-​existing antibody may not necessarily correlate with protection from reinfection. The level of carbohydrate receptor-​ blocking antibodies in sera from previously exposed human ‘se- cretors’ does correlate with protection from severe disease upon reinfection, although correlates of protection following immuniza- tion and natural infection may differ. Astroviruses Structure and classification Astroviruses are members of the family Astroviridae. They possess a 6.8-​kb genome of single-​stranded RNA of positive polarity. So far, eight serotypes have been distinguished that correlate well with major differences in genome sequences (i.e. genotypes). Astrovirus particles have a characteristic appearance by electron microscopy (Fig. 8.5.9.3e), which has been refined by crystallographic charac- terization of the capsid spike. Replication Human astroviruses grow well in particular cell cultures. After viral absorption to unidentified cellular receptors, receptor-​mediated endocytosis, and uncoating in the cytoplasm, full-​length and subgenomic RNAs are synthesized. These direct the production of protein precursors, which are posttranslationally cleaved. Some pro- teins are translated by –​1 ribosomal frameshifting. Replication takes place purely in the cytoplasm. Enteric adenoviruses Structure and classification Adenoviruses are non​enveloped icosahedral viruses possessing a genome of linear double-​stranded DNA of approximately 35 kbp in size. Their capsid measures between 70 and 80 nm in diameter and consists of 240 hexons and 12 pentons, which form the base of each projecting fibre at a fivefold vertex of the icosahedral virus particle (Fig. 8.5.9.3b). Human adenoviruses occur in more than 50 distinct serotypes, ordered in six subgroups (A–​F). Adenoviruses of sub- group F, consisting of serotypes 40 and 41, are regularly associated with gastroenteritis. Adenoviruses of different groups (causing re- spiratory tract infections) are also found frequently in the human gut but are not regularly associated with diarrhoea. Replication Adenoviruses attach to susceptible cells via the fibre proteins and enter via receptor-​mediated endocytosis. Phased early and late gene transcription of the viral DNA in the cell nucleus is followed by trans- lation and morphogenesis in the cytoplasm, and numerous particles are released after cell death. The virally encoded early proteins E1A and E1B induce host cells to enter the S phase, prevent apoptosis, and inhibit antiviral responses. Late adenovirus gene expression blocks (a) (c) (d) (e) (g) (b) (f) Fig. 8.5.9.3  Electron micrographs of (a) rotavirus, (b) enteric adenovirus, (c) Norwalk-​like virus, (d) sapovirus, (e) astrovirus, (f) enterovirus, and (g) parvovirus. Negative staining with 3% phosphotungstate, pH 6.3; bar represents 100 nm. Courtesy of Dr J. Kurtz, Oxford Public Health Laboratory (astroviruses) and
Dr J. Gray, Clinical Microbiology and Public Health Laboratory, Cambridge
(all other viruses). Reproduced from Zuckerman A, Banatvala J, Pattison J (eds) (2000). Principles and practice of clinical virology, 4th edition, p. 236. Wiley &
Sons, Chichester, with permission of the publisher.

802 section 8  Infectious diseases RNA transcription from cellular DNA. Some adenoviruses seem to decrease the expression of major histocompatibility complex class 1 antigens on the surface of infected cells, thus reducing susceptibility to adenovirus-​specific cytotoxic T cells. There is a serotype-​specific humoral immune response providing homotypic protection. Viral gastroenteritis Clinical features The onset of acute viral gastroenteritis follows a short incubation period of 1–​2 days. It is sudden, with watery diarrhoea lasting be- tween 4 and 7 days, vomiting, and varying degrees of dehydration. Over one-​third of children with rotavirus infection have a fever of more than 39°C. Fewer children have a high fever after infection with caliciviruses, and the duration of diarrhoea after infection with caliciviruses is, as a rule, shorter (1–​2 days) than after infec- tion with rotaviruses or enteric adenoviruses (4–​7 days). Disease due to calicivirus infection may be accompanied by abdominal cramps, headache, and myalgia. In rotavirus infection all degrees of severity are seen. Inapparent infections are not infrequent, par- ticularly in neonates, in whom the infection is caused by so-​called nursery strains. It is uncertain whether the asymptomatic nature of rotavirus infection in neonates is due to infection with particular strains or depends on maturational factors, such as changes in the expression on enterocytes of potential rotavirus receptors or the presence of maternal antibodies that provide partial protection. Rotavirus infections are frequently accompanied by respiratory symptoms, but there is no strong evidence that rotavirus replicates in the respiratory tract. Viraemia may be common among patients with rotavirus gastroenteritis and associated with more severe disease. In immunodeficient children, rotavirus may replicate at extraintestinal sites, and chronic gut infections with rotaviruses, adenoviruses, noroviruses, and astroviruses have been observed, accompanied by virus shedding over weeks and even months. Diagnosis The diagnosis of rotavirus, astrovirus, and enteric adenovirus infec- tions is relatively easy for a well-​equipped clinical laboratory, as large numbers of particles are shed during the acute phase of the illness. In contrast, human caliciviruses replicate for a shorter period and are shed at lower concentrations. Diagnosis is commonly carried out by virus-​specific enzyme-​linked immunosorbent assays (ELISAs) and more recently by viral genome detection using the polymerase chain reaction (PCR) (for adenoviruses) and reverse transcription-​PCR (RT-​PCR) (for rotaviruses, caliciviruses, and astroviruses). PCRs are extremely sensitive diagnostic tools, allowing both viral detection and typing. Aliquots of PCR amplicons can also be sequenced, and the information used to establish phylogenetic trees. Such trees are becoming increasingly important not only for virus classification but also for epidemiological studies and surveillance (see next). Electron microscopy of negatively stained specimen suspensions is a ‘catch all’ method that can diagnose less common viral enteric pathogens that are not detected by standard assays, such as non-​group A rotaviruses. Besides its complexity in a diagnostic environment the main disadvan- tage of electron microscopy is its relatively low sensitivity. The mor- phological appearances of the main viruses pathogenic for humans are shown in Fig. 8.5.9.3. Treatment Treatment is mainly with oral rehydration solutions or, in more se- vere cases, intravenous rehydration. In poorly nourished popula- tions, zinc supplementation, used in addition to oral rehydration, decreases the duration of diarrhoea. The enkephalinase inhibitor racecadotril, used as a supplement to oral rehydration, has been shown to significantly decrease the duration and total fluid loss in rotavirus-​infected children. In severe and prolonged rotavirus infec- tions, particularly in immunocompromised hosts, treatment with oral immunoglobulins can decrease the duration of diarrhoea and virus shedding; however, this is not a routine treatment. Otherwise treatment is symptomatic, but the use of antimotility drugs (codeine phosphate, diphenoxylate, loperamide) in children is not advised. Specific antiviral agents have been tested in animal models of rota- virus infections but have not been developed for human treatment. Epidemiology Rotaviruses Rotavirus infections occur endemically worldwide and in 2013 caused approximately 213 000 infant and childhood deaths annually, mainly in developing countries. Therefore, development of vaccine candidates has been a major goal since the early 1980s (see next). The epidemiology of rotaviruses is complex. Besides chil- dren, elderly patients and patients with immunodeficiencies can be affected. There is a strict winter peak of rotavirus infections in temperate climates, but infections occur year-​round in trop- ical and subtropical regions. Transmission is by the faeco-​oral route. Nosocomial infections on infant hospital wards occur and are difficult to eliminate. Group A rotaviruses of different G and P types are found to cocirculate in various populations within the same geographical location, and the relative incidence of different types changes over time. Various surveys have shown that usually more than 90% of cocirculating strains are types G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8], with the G9 and G12 strains emerging relatively recently. Most mammalian as well as avian spe- cies harbour a large diversity of rotaviruses and may act as a reser- voir for human infections. An animal source is suspected for many of the more unusual human group A rotavirus isolates and pos- sibly for group B rotavirus isolates. The latter caused outbreaks in children and adults in China during the 1980s and have also been isolated from patients with diarrhoea in different regions of India and Bangladesh. Group C rotaviruses are associated with small outbreaks in humans in both developed and developing countries. Human caliciviruses Age-​related seroprevalence studies of human caliciviruses have shown that infection is much more frequent and occurs from younger ages onwards more than previously thought. Approximately 50% of children have been infected by the age of 2 years. In countries where rotavirus vaccination programmes have been established, norovirus infections are now the predominant cause of hospitalization of chil- dren with acute gastroenteritis. The rate of inapparent infection with noroviruses is high, particularly in the young. Human caliciviruses cause outbreaks of acute gastroenteritis, often due to contamination of food or water, and are now recognized as the most important cause of non​bacterial gastroenteritis outbreaks worldwide. Contaminated

8.5.9  Virus infections causing diarrhoea and vomiting 803 oysters, green salads, fresh fruit, cold foods, and sandwiches are often implicated as sources of infection. Outbreaks occur in older children and adults in recreational camps, hospitals, nursing homes, schools, cafeterias, hotels, cruise ships, at banquets, and so on. Human calicivirus outbreaks occur worldwide throughout the year, in con- trast to the regular winter peaks of rotavirus infections in temperate climates. The viruses are highly infectious (i.e. a few virus particles constitute an infectious dose), relatively resistant to inactivation, and spread rapidly. Transmission is by the faecal-​oral route and also by projectile vomiting, which scatters viruses into the environment by aerosol. There is cocirculation of highly divergent genotypes. Astroviruses Endemic infections with astroviruses occur in infants and elderly people, but they can also cause food-​borne outbreaks of diarrhoea. There are at least eight genotypes, correlating well with known sero- types, which cocirculate. Serotype 1 is most frequently found, fol- lowed by serotypes 2 to 4 at intermediate frequencies and serotypes 5–​8 at low frequencies. Seroprevalence studies have indicated that infection by more than one serotype is not unusual. Vaccine development Vaccines, when available, have been confirmed as the best individual and population-​based tools to restrict infection with epidemic vir- uses. Of the gastroenteritis-​inducing viruses, vaccine development has only been intensively directed towards rotaviruses. The first, a live attenuated, quadrivalent rhesus rotavirus (RRV)-​based human reassortant vaccine that contained strains with VP7 from each of human rotavirus serotypes G1 to G4, was licensed in 1998. Although the vaccine offered significant protection from severe, dehydrating disease, it was taken off the market by the manufacturer due to a tem- poral association of immunization with intestinal intussusception. The epidemiological findings have not lead to a satisfactory explan- ation of the association and of possible mechanisms of pathogenesis. Subsequently, additional live attenuated oral rotavirus vaccines have been developed. Rotarix® and Rotateq® have been licensed in many countries worldwide since 2006; Rotavac® was licensed in India in 2014 and received WHO prequalification in 2018. Three additional live attenuated rotavirus vaccines have received national licenses in India, China, or Vietnam. Although these are all oral, live attenuated vaccines, there are some significant differences be- tween them. Rotateq® is a pentavalent vaccine that contains genes encoding the human antigens G1 to G4 and P[8]‌ in monoreassortant viruses on a bovine rotavirus (WC3 strain, G6P7[5]) genetic back- bone. Rotarix® is a monovalent vaccine derived from an attenuated human G1P[8] strain. Rotavac®, developed by an Indian manufac- turer in collaboration with a consortium of international institutions is a monovalent vaccine based on a naturally occurring reassortant strain (116E, G9P[11]). Strain 116E carries a bovine rotavirus VP4 antigen on the background of antigens derived from human rota- viruses. 116E-​like strains were originally isolated from nosocomially infected, asymptomatic neonates in India. The pentavalent Rotateq® was designed to elicit type-​specific anti- bodies against all the rotavirus types that are recognized to cir- culate most frequently in humans. The monomeric Rotarix® and Rotavac® rely on cross-​protection between serotypes. The potential for cross-​protection is supported by two clinical observations: (1) cross-​protection against various rotavirus serotypes is accu- mulated through successive natural infections and; (2) vaccination with one rotavirus type can provide protection, even if subsequent infections are by rotaviruses of a different type. In countries where universal mass vaccination of children as part of childhood vaccination schemes has been accepted, a substantial reduction in rotavirus disease and an unexpected rise of herd im- munity have been recorded. In Mexico, overall childhood mortality from diarrhoea decreased after introduction of the vaccine. Recently, a decrease of the annual infant and childhood mortality worldwide from rotavirus-​associated diarrhoea was recorded. A relatively low risk of intussusception associated with rotavirus vaccination has been detected in some postmarketing studies, which has led the Centers for Disease Control and Prevention to recommend that a history of intussusception in an infant should be a contraindication to rotavirus vaccination. Based on the age-​dependence of the risk of intestinal intussusception, the first dose of the licensed rotavirus vaccines is to be given before 15 weeks of age. Although the efficacy of rotavirus vaccination is lower in im- poverished settings in sub-​Saharan Africa and South East Asia, in 2009 the Strategic Advisory Group of Experts of the World Health Organization (WHO) recommended worldwide use of the vaccine, since ‘vaccine efficacy estimates correlate inversely with disease in- cidence and child mortality strata’. There is ongoing postmarketing surveillance in order to estimate the global impact of the vaccine and also to monitor whether or not novel rotavirus strains may emerge. Extension of rotavirus immunization to the settings of greatest med- ical need is being advanced, in part, by the emergence of rotavirus vaccine manufacturers in developing countries. The factors involved in decreased rotavirus vaccine effectiveness in low-income coun- tries are complex; recognition of these factors will help to gradually improve rotavirus vaccine effectiveness worldwide. Next gener- ation approaches to immunization against rotavirus infection are under investigation, such as the use of virus-​like particles obtained from baculovirus-​recombinant coexpressed rotavirus proteins, en- hancement of rotavirus immunogenicity by microencapsidation, DNA-​based candidate vaccines, and possibly ‘edible vaccines’. A recombinant subunit vaccine, consisting of the VP8* head domain fused to the P2 epitope from tetanus toxin is currently in clinical trials. A bivalent, recombinant virus-​like particle vaccine against norovirus disease is in clinical trials, with a trend towards efficacy seen in a small human challenge study, and a phase 2 immunogen- icity and safety study has been completed. Outbreak control Nosocomial rotavirus outbreaks among paediatric populations (on hospital wards and in day-​care centres) are common. There have been numerous reports of outbreaks of diarrhoea and vomiting occurring in adults and children due to infections with caliciviruses acquired from banquets, travel on cruise ships, cafeterias, schools, hotels, fast-​food restaurants, and so on. Outbreak control measures should focus on the interruption of person-​to-​person transmission, and the removal of common sources of infection (such as food and water) in conjunction with

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