key: cord-021588-ec7udsmw
authors: Craighead, John E.
title: Enteric Viral Disease
date: 2007-05-09
journal: Pathology and Pathogenesis of Human Viral Disease
DOI: 10.1016/b978-012195160-3/50033-9
sha: 
doc_id: 21588
cord_uid: ec7udsmw

nan

Al Kapikian learned immune electron microscopy from the talented British investigator June Alameda, who had perfected the technique into an art form, as much as a skill. In the early 1970s, Kapikian focused on this approach to the search for the elusive viruses believed to be responsible for nonbacterial gastroenteritis. It was not long before he identified the virions of the so-called Norwalk agent in a diarrheal stool from an adult volunteer who had been challenged with a fecal sample from a child ill during an outbreak of diarrhea. This virus and its soon-to-be-discovered close relatives (the so-called Norwalk-like viruses [NLVs]) proved to be important causes of explosive outbreaks of diarrhea in both children and adults.

NLVs were first reported in 1972 (Kapikian et al, 1972; Kapikian, 1994) . Only a year later, Ruth Bishop and her colleagues (1973) found virions of a different size and appearance in cells of the duodenal mucosa of infants with gastroenteritis. These agents proved to be the prototype for a new genus, the rotaviruses (RVs), members of which are infectious for humans and a wide variety of domestic animals. Rotaviruses, classified into Group A, are now recognized to be the major cause of severe diarrheal disease in infants and young children worldwide.

Cholera morbus has plagued and threatened the lives of infants and children since the beginning of recorded history. Morbidity is universal, and the resulting mortality, particularly in developing areas of the world, continues to be tragic. Overall, as many as a third of the deaths in children under the age of 5 in the less-developed countries of the world are attributed to diarrhea. When pathogenic bacteria are excluded, roughly 80% of the episodes of diarrhea (>3 nonsolid stools per day) are either of unknown etiology or are due to viruses.

With the discovery of the enteroviruses (see Chapter 1) and the demonstration of their chronic presence in the stools of many of those who are infected, it was thought that the search for the cause of nonbacterial diarrhea would soon be over. But despite the presence of high concentrations of enteroviruses in the stools of both ill and healthy children, it shortly became apparent that these viruses were not common etiologic agents of enteritis, but merely nonpathogenic "passengers'' in our digestive tracts. Other viruses such as members of many of the common serotypes of the adenoviruses similarly can often be found in the gut, but they too usually fail to cause disease. As an outgrowth of an enormous amount of laboratory work, it was ultimately concluded that the elusive viruses of childhood enteritis are sufficiently fastidious that they cannot be easily grown in tissue culture and in laboratory animals. Thus, in the early 1970s, investigators initiated attempts to identify viral particles in stool extracts using electron microscopy. To accomplish this, the background was stained (so-called negative staining), with the virions in startling contrast, that is, like stars in a dark sky. Should the virions be present in sufficient number, and should their morphology be sufficiently distinctive, infection could be established. By adding specific antibody (or serum from a previously infected patient) to the suspension, the virions would clump with the antibody and an antigenic identification of the virus accomplished. This also proved a means for assaying the relative concentration of antibody in the blood of those who were infected. The technique of negative-staining electron microscopy using clinical enteric specimens is an arduous art form that requires exceptional skill and attention to detail, clearly not a characteristic of many of our species. But, this painstaking approach has now yielded evidence to indicate that viruses of at least six families may contribute to enteric illness in children and in adult citizens whose immunity has waned (Figure 32.1, Table 32 .1). Studies in domestic animals have also shown that diarrheal disease of economic importance is caused by members of these same virus families, possibly by strains of virus that are host-specific. With the development of refined rnolecular and immunological tools, more sensitive and less laborious diagnostic approaches fortunately are now replacing negative-staining electron microscopy of stool specimens. 

A number of antigenically distinct, nonenveloped, round, 27-to 32-nm RNA viruses classified into a newly proposed genus of the calcivirus family have been found to cause sporadic outbreaks of transient severe enteritis in both children and adults. The etiological role of these viruses as a cause of intestinal disease was established by demonstrating a temporal association of naturally occurring infections (as demonstrated by stool examination using electron microscopy) with illness and by experimental induction of disease in both human volunteers and experimental animals (Hall et al, 1984) . The NLVs are provisionally divided for classification purposes into two distinct groups based on genetic analysis (Table  32 .2). The relative clinical importance of the viruses of the various groups listed in Table 32. 2 has yet to be established, but they are believed to be responsible for a substantial proportion of the nonbacterial outbreaks of acute vomiting and diarrhea that occur in families and institutions. Although survey information is incomplete, infections with NLV are thought to occur worldwide. In developing countries, the majority of children are infected during the first 10 years of life ( Figure 32 .2), whereas in North America infection is relatively uncommon in children, and by the fifth decade of life only 50% of the population possess serological evidence of past infection. It is not clear what proportion of these infections are accompanied by clinical illness, and we possess only limited information on the persistence of serum antibodies during convalescence from infection. Thus, NLV-related disease may occur more commonly than serological surveys of the population now suggest.

Outbreaks of illness develop as a result of a contaminated common source, such as a water supply or food, or as a consequence of person-to-person transmission (Fankhauser et ah, 1998) . The mean incubation period is approximately 40 hours, and symptoms persist for 12 

( to 24 hours (Table 32 .3). The clinical course observed in two volunteers is shown in Figure 32 .3) (Dolin ei al., 1971) . Volunteer studies have yielded important histological and ultrastructural documentation of the profound but relatively transient changes that occur in the mucosa of the small intestine during the course of infections with NLVs (Agus ei al, 1973; Schreiber ei al, 1973 Schreiber ei al, , 1974 Dolin ei al, 1975) . Unfortunately, biopsy material from these experimental subjects is, by necessity, limited; thus, the extent and distribution of lesions in the digestive tract is unknown. Of obvious significance is the reduction in the height of the intestinal villi, accompanied by blunting of the villi and evidence of crypt hypertrophy. The individual lining cells of the small intestine exhibit vacuolization of the cytoplasm with loss of polarity. These findings indicate a marked increase in enterocyte replacement as documented by mitotic counts in the epithelium of the crypts. Although cell infiltrates are found in the lamina propria and the core of the villi, inflammation is customarily not a prominent feature of the lesion. Ultrastructurally, the mucosal cells remain intact, but intercellular edema Reprinted with permission from Kapikian (1994) .

is prominent and the microvilli of the brush border are significantly shortened as shown by electron microscopy. Dilatation of both the endoplasmic reticulum and mitochondria of the mucosal cells is seen, and endocytotic vesicles are evident. Ultrastructural studies thus far have not defined the cell site of replication of the virus, most probably because the virions are relatively small, that is, in a size range that would not readily allow one to distinguish them from ribosomes. Immunohistochemistry documents a substantial reduction in disaccharides and alkaline phosphatase in the epithelium of the gut. Intestinal biopsies obtained from volunteers 5 to 6 days after the ingestion of NLVs (i.e., 2-4 days after clinical recovery) continue to show shortening of villi and crypt hypertrophy, but the inflammatory response in the lamina propria and submucosa appear to be reduced. At this time, there remains a significant reduction in the surface area of the gut, and a striking increase in mitotic activity of the epithelium is seen.

RVs are the most important cause of severe and often life-threatening diarrheal disease in infants and young children worldwide (Moulton ei al, 1998) . Although adults are infected, their symptoms are customarily mild, with asymptomatic infections being common. However, severe infections tend to occur in older persons (Hrdy, 1987; Wenman ei al, 1979; Abbas and Denton, 1987; Lewis ei al, 1989 ) and in persons with attenuated immunity (Kaljot ei al, 1989; Dryden and Shanson, 1988; Eiden ei al, 1985; Wood ei al, 1988) , but RVs do not appear to be a major contributing factor to the diarrheal disease that so commonly afflicts those with AIDS.

RVs are members of a genus classified in the reovirus family. The Latin roia refers to the spoke-wheel appearance of these nonenveloped 70-nm RNA virions in negatively stained electron micrographs. Based on antigenic analysis of the virion coat proteins, five groups (A-E) have been established. The majority of human infections are due to viruses of group A in regions of the world where intensive studies have thus far been conducted. Overall, the prevalence of group B and C infections is low, and they appear only sporadically in developed countries. Widespread epidemics of group B viruses have been reported from the People's Republic of China; however, overall, infection by group B and C viruses in China as measured by presence of antibodies in the blood serum of members of the general population remains low, that is, -10% (Qui et al, 1986) . Because these viruses are highly mutable, stool specimens from individuals may yield a diversity of variants that differ one from another on the basis of minor changes in the amino-acid makeup of the virion coat protein. The relative pathogenicity of these genetically diverse agents is unknown. Although RVs from domestic animals have been shown experimentally to infect children, there is little evidence to indicate that animals are a significant reservoir for human infections.

In developed countries, RV infections typically occur between the ages of 6 and 24 months. In one study conducted in North America, 40% of children were infected with at least one strain of RV during the first year of life (Ward and Bernstein, 1994) (Figure 32.4) . However, fewer than a third of these infections were symptomatic (Wenman et al, 1979) . In developing countries and in persons residing under poor socioeconomic conditions, as many as 30% of children are infected by 6 months of age (Moulton et al, 1998) , and many infants experience their first encounter with RVs in the newborn nursery during the first week of life (Bishop, 1994) . This high prevalence of infection during early life doubtlessly represents both the common occurrence of RV infections in persons residing in these environments (thus facilitating person-to-person spread) and the lack of acquired secretory IgA to the virus in the gut.

The clinical features of RV infections -fever, vomiting and diarrhea -are known to all mothers. Yet, because symptoms usually are m^ercifuUy short-lived (24 to 48 hr), the maintenance of fluid and electrolyte balance is rarely a problem in otherwise healthy infants (Table 32 .4). In developing countries, however, the threat to life is more imposing, particularly since infections so frequently occur among the very young. Illness in adults is less severe (Table 32 .5), if it occurs at all. Indeed, 60 to 80% of infections in adults are asymptomatic. This may reflect age-related resistance (as documented in animals) (Ciarlet et al, 1998) or the acquisition of a spectrum of serotype-specific IgA antibodies in the gut resulting from previous natural infection. As the years pass, immunity may wane among the elderly, in part due to less frequent encounters with the virus in their home setting. Older folks more often develop severe diarrheal illnesses when infected with RVs, and deaths are reported (Hrdy 1987) .

RVs appear to replicate exclusively in the mucosal lining cells of the villi of the small intestine and not elsewhere in the gut (Figures 32.5 and 32.6 ). Up to lO^^ virions can be found in a gram of feces during the acute illness! This extraordinarily high concentration of virus in diarrheal stools no doubt accounts in part for the ease of transmissibility of these viruses among close contacts. Since the virions are also relatively resistant to environmental stresses, they can persist in water supplies and food. Infection occurs exclusively by the oral route.

As might be expected, relatively few morphologic studies have been carried out on enteric biopsies from infants and children with documented RV infections. Davidson and Barnes (1979) examined duodenal biopsies obtained 24 to 120 hours after the onset of symptoms from children 2 to 33 months of age. Abnormalities were found in 40% of the biopsies. There was blunting of villi, along with an increase in crypt depth and flattening of the epithelial cells lining the villi. Inflammatory cells were present in the lamina propria in 2 of the 17 infants studied. The pathological changes were said to resemble those seen in celiac disease with loss of villi, prominent crypt hypertrophy, and an infiltrate of inflammatory cells. RV particles were found in the enterocytes of these patients by electron microscopy. Histochemical evaluation of the gut mucosa Reprinted with permission from Uhnoo et al. (1986) . "Statistically significant (p > 0.05) difference between hospitalized and nonhospitalized patients. Reprinted with permission from Wenman et al. (1979) . Figure 32 .6. Reprinted with permission from Saif et al. (1978) .

Experimentally induced rotavirus infection in gnotobiotic piglets. Electron micrographs of villous epithelium. Note distended cisternae of the rough endoplasmic reticulum and rarefaction of cytoplasm in panel A. The affected cell is situated between two seemingly normal cells. Panel B shows cells with shortened irregular microvilli and a break in the viroplasm-VP border (arrow) (see Figure 32 .5). Reprinted with permission from Saif et al. (1978) . The associated changes in the villus lengthicrypt length ratio is shown for infected (solid black) and control (broken line) animals. Note the protracted recovery time. Reprinted with permission from Crouch and Woode (1978). showed that disaccharidase concentrations were reduced in the epithelium. Repeat biopsies were done on several children during convalescence (3 to 8 weeks), and regeneration of the mucosa was found.

As noted above, a wide variety of wild and domesticated mammals and poultry (Domermuth and Gross, 1985) acquire RV infections naturally. Experimental studies in calves (Reynolds et al, 1985) , lambs (Snodgrass et al, 1977 (Snodgrass et al, , 1979 , and piglets Crouch and Woode, 1978) have provided insight into the location of viral replication in the gut and the associated histologic changes (Figure 32.7) . To a large extent, the changes observed in children (Davidson and Barnes, 1979) were found in these animals. In calves, the lesions tended to be more prominent in the proximal small intestine, whereas in lambs the distal ileum was more severely affected. Table 32 .6 summarizes the results of immunohistochemical studies that document viral replication in the gut of lambs after experimental infection per os. In general, the sites of RV replication correlate with the pathological changes found in the intestinal mucosa (Snodgrass et al, 1977) . As might be expected, the concentrations of virus in the gut of these piglets were highest before lesions in the mucosa appeared (Crouch and Woode, 1978) . 

Reprinted with permission from Snodgrass et al. (1977) . 

Viruses of several additional families have been implicated in human enteritis, but, in general, the disease is relatively mild and not life threatening (Table 32 .1). Information concerned with the pathological effects of these viruses on the gut mucosa is lacking. In many studies, the common occurrence of asymptomatic enteric infections often makes it difficult to establish, on epidemiological grounds, an etiological relationship between the infection and disease; yet, under certain circumstances, these viruses may be pathogenic for humans.

The enteric adenoviruses types 40 and 41 are recognized causes of enteritis, but they account for fewer than 10% of cases (see Chapter 14) (Brandt et al, 1985) . In two recent studies, toroviruses were associated with enteritis manifest in children as both vomiting and diarrhea. Although not severe, the stools commonly were bloody and disease persisted for several days (Koopmans et ah, 1997) . Enteritis due to toroviruses also occurs in cattle and horses (Weiss and Horzinek, 1987; Woode et al, 1982; Jamieson et al, 1998) . Astroviruses and coronaviruses have been implicated as causes of diarrheal disease in humans, but the evidence supporting a cause-and-effect association is weak (Phillips et al, 1982; Lew et al, 1990) . These latter viruses are etiologically responsible for disease in young domestic animals (Mebus et al, 1973; Thake et al, 1973; Gray et al, 1980; Kurtz et al, 1979) (Figures 32.8 and 32 .9). FIGURE 32.9 Degeneration of mucosal lining cell in the midgut of a lamb infected with an astrovirus. Note the changes in the microvilli (arrows) and virions in a lysosome (V). Reprinted with permission from Gray et al. (1980) .

Pathological observations provide insight into the physiological basis for enteritis in those infected with enteropathic viruses. At present, relatively few clinical studies have addressed these issues in detail. Involvement of the upper intestinal tract (duodenum and jejunum) may account for the vomiting that so frequently occurs in NLV infections. Diarrheal disease no doubt is consequent to the profound changes in the intestinal mucosa that occur during the acute illness. Reduction in the surface area of the gut mucosa and functional alterations in the individual enterocytes that line villi reduce absorption of fluids and solids beyond the capacity of the colon to compensate. The loss of disaccharidases in the mucosal cells of the small intestine increases the carbohydrate concentrations of the large bowel content, resulting in the generation of fermentation products.

An outbreak of rotavirus infection in a geriatric hospital

Acute infectious nonbacterial gastroenteritis: Intestinal histopathology. Histologic and enzymatic alterations during illness produced by the Norwalk agent in man

Viral Infections of the Gastrointestinal Tract

Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis

Rotavirus disease, but not infection and development of intestinal histopathological lesions, is age restricted in rabbits

Serial studies of virus multiplication and intestinal damage in gnotobiotic piglets infected with rotavirus

Structural and functional abnormalities of the small intestine in infants and young children with rotavirus enteritis

Transmission of acute infectious nonbacterial gastroenteritis to volunteers by oral administration of stool filtrates

Viral gastroenteritis induced by the Hawaii agent: Jejunal histopathology and serologic response

Hemorrhagic enteritis of turkeys

The microbial causes of diarrhoea in patients infected with the human immunodeficiency virus

Rotavirus RNA variation during chronic infection of immunocompromised children

Molecular epidemiology of "Norwalk-like viruses'' in outbreaks of gastroenteritis in the United States

Ultrastructure of the small intestine in astrovirus-infected lambs

Prevalence of antibody to the Norwalk virus in various countries

Lesions of gnotobiotic calves experimentally infected with a calcivirus-like (Newbury) agent

Epidemiology of rotaviral infection in adults

Human torovirus: A new nosocomial gastrointestinal pathogen

Prevalence of acute enteric viral pathogens in acquired immunodeficiency syndrome patients with diarrhea

Norwalk and Norwalk-like viruses

Visualization by immune electron microscopy of a 27-nm particle associated with acute infectious nonbacterial gastroenteritis

Association of torovirus with acute and persistent diarrhea in children

Astrovirus infection in volunteers

Six-year retrospective surveillance of gastroenteritis viruses identified at ten electron microscopy centers in the United States and Canada. Pediatr

Outbreaks of astrovirus type 1 and rotavirus gastroenteritis in a geriatric inpatient population

Pathology of neonatal calf diarrhea induced by a coronavirus-like agent

Pathophysiology of viral diarrhea

The protective effectiveness of natural rotavirus infection in an American Indian population

Astrovirus within human small intestinal mucosa

Antibody against adult diarrhoea rotavirus among healthy adult population in China

Pathology of natural rotavirus infection in clinically normal calves

Morphogenesis of porcine rotavirus in porcine kidney cell cultures and intestinal epithelial cells

The mucosal lesion of the proximal small intestine in acute infectious nonbacterial gastroenteritis

The small intestinal lesion induced by Hawaii agent acute infectious nonbacterial gastroenteritis

Rotavirus infection in lambs: Pathogenesis and pathology

Small intestinal morphology and epithelial cell kinetics in lamb rotavirus infections

Epithelial cell dynamics in transmissible gastroenteritis of neonatal pigs

Pathogenesis of porcine rotaviral infection in experimentally inoculated gnotobiotic pigs

Clinical features of acute gastroenteritis associated with rotavirus, enteric adenoviruses, and bacteria

Protection against rotavirus disease after natural rotavirus infection

The proposed family, Toroviridae: agents of enteric infections

Rotavirus infection in adults: Results of a prospective family study

Chronic enteric virus infection in two T-cell immunodeficient children

Studies with an unclassified virus isolated from diarrheic calves