Chapter 15: Viruses of Diarrhea

Until the 1970s, proof of viral causation of acute diarrhea was usually based on exclusion of known bacterial or protozoan pathogens and supported by feeding cell-free filtrates of diarrheal stools to volunteers to reproduce the disease. As might be expected, the results of such experiments were variable, and the methods were impractical for routine laboratory diagnosis. One aspect of such infections that proved to be of great help was the frequent association with abundant excretion of virus particles during the acute phase of illness. Virion numbers greater than 10⁸ per gram of diarrheal stool are relatively common, allowing ready visualization with an electron microscope (Figure 15–1). Direct electron microscopy and immunoelectron microscopy were used to detect and identify the presumed causative viruses; the latter method was also be used to detect humoral antibody responses to infection. More recently, polymerase chain reactions (PCRs) and enzyme immunoassays (EIAs) are employed in diagnosis.

Several criteria were used to establish the role of viruses in diarrheal diseases, including detecting viruses in symptomatically ill patients more frequently than in asymptomatic individuals, demonstrating significant antibody response in patients shedding the virus, reproducing the disease by experimental inoculation of nonimmune human or animal hosts, and excluding other known causes of diarrhea such as bacteria, bacterial toxins, and protozoa.

Using the above criteria, four groups of viruses have been clearly established as important causes of gastrointestinal disease: rotaviruses, caliciviruses, astroviruses, and some adenovirus serotypes (“enteric” adenoviruses). Other viruses have also been implicated, but many of the preceding criteria have not been fulfilled; therefore, they are currently regarded as “candidate” causes of gastrointestinal disease.

The currently established viruses are listed in Table 15–1, and all have several features in common, including a tendency toward brief incubation periods; fecal–oral spread by direct or indirect routes; and production of vomiting, which generally precedes or accompanies the diarrhea. The last feature has influenced physicians to use the term acute viral gastroenteritis to describe the syndrome associated with these agents.

The human intestinal rotaviruses were first found in 1973 by electron microscopic examination of duodenal biopsy specimens from infants with diarrhea (Figure 15–2). Since then, they have been found worldwide and are believed to account for 40% to 60% of cases of acute gastroenteritis occurring during the cooler months in infants and in children less than 5 years of age, with most serious disease in 3 to 35 months of age. Worldwide, more than 528 000 deaths in children younger than 5 years of age in 2000 were attributed to rotavirus infections mainly in Sub-Saharan Africa, South Asia, and Southeast Asia, which has dropped to 215 000 in 2013. Four countries, including India, Nigeria, Pakistan, and Democratic Republic of Congo accounted for 49% of rotavirus-related deaths under 5 years of age in 2013, with 22% alone in India. In the United States, more than 400 000 doctor visits, 200 000 emergency room visits, 50 000 to 70 000 hospitalizations, and 20 to 60 deaths were reported before the rotavirus vaccine was introduced in 2006. Now such deaths in the United States are rather infrequent; the annual morbidity rate has significantly dropped. Before the introduction of rotavirus vaccines in 2006, almost all children were infected in the United States before their fifth birthday. The routine use of rotavirus vaccine in infants has significantly reduced rotavirus infection in the United States. These viruses have been detected in intestinal contents and in tissues from the upper gastrointestinal tract.

VIROLOGY

The rotaviruses belong to the family Reoviridae. The genome of rotaviruses is unique in the sense that they have 11 segments of double-stranded RNA. The 11 segments of the genome encode six structural (VP1–VP4 and VP6–VP7) and six nonstructural (NSP1–NSP6) proteins (Figure 15–3A). There are three types of virus particles, including triple layered (previously called double shelled), double layered (previously called single shelled) and single layered (empty capsids, usually lacking genomes) (Figure 15–1A, 15–2). The complete virus particle of rotavirus is a wheel-shaped virus and the name is derived from the Latin rota (“wheel”) because of the outer capsid, which resembles a wheel attached by short spokes to the inner capsid and core (Figures 15–1, 15–2, 15–3A). Eleven segments of double-stranded RNA genome are packaged into an icosahedral capsid making the spherical particles of 65 to 75 nm in diameter in size (smaller forms have also been described) (Figure 15–3B–D). The virus particle has a virion-associated RNA-dependent RNA polymerase, and a double-shelled outer capsid; two segments encode proteins of the outer capsid (VP4 or P and VP7 or G), which are targets for neutralizing antibodies. The major outer capsid proteins are VP4 and VP7. VP4 performs several functions, including viral attachment protein, whereas VP7 is a type-specific antigen and facilitates viral attachment and entry.

Rotaviruses are classified into seven groups, A to G, based on the internal capsid protein, VP6. Human infections are predominantly caused by group A and less commonly by group B or C. Based on VP4 and VP7 type-specific antigens on the outer capsid, G (VP7 is a glycoprotein) and P (VP4 is protease-sensitive) serotypes have been designated. Five serotypes (G1, G2, G3, G4, and G9), are of major epidemiologic importance because they represent more than 90% of all serotypes detected worldwide. G1 serotype represents more than 75% of the isolates. The outer capsid is proteolytically cleaved in the gastrointestinal tract to generate intermediate infectious subviral particle (ISVP), which activates the virus for infection. Rotaviruses can replicate in the cytoplasm of infected cell cultures in the laboratory and successful propagation of human strains in vitro has been achieved in cell lines.

Rotavirus replication is depicted in Figure 15–4. Rotavirus is transmitted by fecal–oral route, and the virus particle is partially digested in the gastrointestinal tract and activated by protease cleavage resulting in the loss of VP7 and cleavage of VP4 to generate ISVP. The VP4 binds to sialic acid containing glycoproteins on epithelial cells, and the ISVP penetrates the target cells. The generation of ISVP is necessary for rotavirus infection because the double-shelled virus particle, after entering the cells via receptor-mediated endocytosis, is unable to establish infection owing to a dead-end pathway. After entry of the ISVP, the core containing double-stranded RNA genomes and the RNA-dependent RNA polymerase is partially released into the cytoplasm. Rotaviruses use negative-sense RNA strategy for transcription and replication. RNA-dependent RNA polymerase directs the synthesis of early and late mRNAs followed by genome replication by using the negative-strand RNA of the double-stranded RNA genome. Early proteins are produced that are required for virus replication, whereas late proteins are mainly the structural proteins. Rotavirus assembles by associating its core with a nonstructural protein (NS28, a product of NSP4) and by acquiring VP7 and a membrane budding into the endoplasmic reticulum (ER). The virus eventually loses the membrane in the ER and is released upon cell lysis.

Rotaviruses of animal origin are also highly prevalent and produce acute gastrointestinal disease in a variety of species. Very young animals, such as calves, suckling mice, piglets, and foals, are particularly susceptible. The animal rotaviruses can often replicate in cell cultures, and infection across species has been accomplished experimentally; however, there is no evidence that such interspecies spread occurs in nature (eg, animal rotaviruses are not known to affect humans and vice versa).

One unique feature of rotaviruses is the ease with which the 11 RNA segments can undergo reassortment. This has enabled the development of live vaccines that combine genes from readily cultivated animal rotaviruses with human rotavirus genes that encode serotype-specific capsid proteins.

HUMAN ROTAVIRUS INFECTIONS

EPIDEMIOLOGY

Outbreaks of rotavirus infection were common in pre-vaccine era, particularly during the cooler months, among infants and children of less than 5 years of age, but the incidence of clinical illness was highest among 3 to 35 months of age. Older children and adults can also be affected, but attack rates are usually much lower and the disease is milder. Outbreaks among elderly, institutionalized patients have also been recognized.

Although newborn infants can be readily infected with the virus, such infections often result in little or no clinical illness. This finding is illustrated by reported infection rates of 32% to 49% in some neonatal nurseries, but mild illness in only 8% to 28% of the infants. It is unclear whether this transient resistance to disease is a result of host maturation factors or transplacentally conferred immunity. Seroepidemiologic studies have been useful in demonstrating the ubiquity of these viruses and may help to explain the age-specific attack rates. By the age of 5 years, almost all individuals have humoral antibodies, suggesting a high rate of virus infection early in life.

PATHOGENESIS

Rotaviruses appear to localize primarily in the duodenum and proximal jejunum, causing destruction of villous epithelial cells with blunting (shortening) of villi and variable, usually mild, infiltrates of mononuclear and a few polymorphonuclear inflammatory cells within the villi. The gastric and colonic mucosa is unaffected; however, for unknown reasons, gastric emptying time is markedly delayed. The primary pathophysiologic effects are a decrease in absorptive surface in the small intestine and decreased production of brush border enzymes, such as the disaccharidases. The net result is a transient malabsorptive state, with defective handling of fats and sugars. It may take as long as 3 to 8 weeks to restore the normal histologic and functional integrity of the damaged mucosa. Although the specific gene product associated with virulence is not yet known, some evidence suggests that one nonstructural protein, NSP4, may behave as an enterotoxin in a manner similar to that of the heat-labile enterotoxin (LT) of Escherichia coli and cholera toxin. This may further explain the excess fluid and electrolyte secretion in the acute phase of illness. Viral excretion usually lasts 2 to 12 days but can be greatly prolonged in malnourished or immunodeficient patients with persistent symptoms.

IMMUNITY

Rotavirus infection responds with production of type-specific humoral antibodies that probably does not last for a lifetime after the first infection. Recovery from the first infection provides 38% protection against infection, 77% protection against diarrhea, and 87% protection against severe diarrhea. Subsequent infections provide long-term immunity. In addition, type-specific secretory IgA antibodies are produced in the intestinal tract, and their presence seems to correlate best with immunity to reinfection. Breastfeeding also seems to play a protective role against rotavirus disease in young infants. Secretory IgA antibodies to rotaviruses appear in colostrum and continue to be secreted in breast milk for several months postpartum. Human breast milk mucin glycoproteins have also been shown to bind to rotaviruses, inhibiting their replication in vitro and in vivo.

CLINICAL ASPECTS

MANIFESTATIONS

After an incubation period of 1 to 3 days, there is usually an abrupt onset of vomiting, followed within hours by frequent, copious, watery, brown stools. In severe cases, the stools may become clear; the Japanese refer to the disease as hakuri, the “white stool diarrhea.” Fever, usually low grade, is often present. Vomiting may persist for 1 to 3 days, and diarrhea for 4 to 8 days. The major complications result from severe dehydration, occasionally associated with hypernatremia.

DIAGNOSIS

Diagnosis of acute rotavirus infection is usually by detection of virus particles, antigen or virion RNA in the stools during the acute phase of illness. This can be accomplished by immunologic detection of antigen with EIA methods or virion RNA by RT-PCR. Direct examination of the specimen by electron microscopy can also be done primarily in research setting.

TREATMENT AND PREVENTION

There is no specific treatment for rotavirus infection. Vigorous replacement of fluids and electrolytes is required in severe cases and can be lifesaving. The rotaviruses are highly infectious and can spread quickly in family and institutional settings. Control consists of rigorous hygienic measures, including careful handwashing and adequate disposal of enteric excretions.

Previously developed live attenuated or reassortant rhesus-based rotavirus vaccine was developed and licensed in the United States in 1998, but withdrawn because of some side effects (intussusception). In 2006, a live, oral bovine/human reassortant vaccine that contains five reassortant rotaviruses (RV5) developed from human and bovine strains (RotaTeq developed by Merck) was licensed for routine use in the United States. It is a three-dose series at 2, 4, and 6 months of age. A second live oral vaccine, RV1 (Rotarix) that contains one live, attenuated human strain (developed by GlaxoSmithKline) was licensed in 2008 for a two-dose series, administered at 2 and 4 months. The minimum age for the first dose administration is 6 weeks and maximum age is 14 weeks and 6 days. The minimum interval between doses is 4 weeks and all doses should be completed by 8 months of age. To date, its efficacy after a three-dose series has been excellent, and no safety concerns have arisen. The efficacy of the vaccine in preventing infection is between 85% and 98%. However, rotavirus vaccine should not be given to infants aged 15 months and above due to lack of availability of safety data. While the vaccine is safe, mild problems such as temporary diarrhea or vomiting may occur. In addition, 1 in 20 000 to 1 in 100 000 infants may have intussusception (a bowel blockage) with rotavirus vaccination.

Although the caliciviruses were the first to be clearly associated with outbreaks of gastroenteritis, considerably less is known about their biology than about that of the rotaviruses. Caliciviruses belong to Caliciviridae family. Two genera, Norovirus and Sapovirus, infect humans. Caliciviruses were first associated with an outbreak in Norwalk, Ohio, in 1968, and their role was confirmed by production of disease in volunteers fed fecal filtrates. The original virus was thus called the Norwalk agent, and similar viruses have been given names such as Hawaii agent, Montgomery County agent, Ditchling agent, and so on.

VIROLOGY

Caliciviruses are small, naked capsid, icosahedral symmetry, positive-sense RNA-containing particles 27 to 38 nm in diameter; their appearance is similar to that of parvoviruses and hepatitis A virus (Figure 15–1B). The viral capsid is made up of two proteins, VP1 and VP2. The nonstructural proteins include viral protease and viral RNA-dependent RNA polymerase. The virus replicates in the cytoplasm like other positive-sense RNA viruses by using its viral RNA polymerase for transcription and replication, and virus assembly in the cytoplasm and release upon cell lysis. At present, two genera of caliciviruses that cause diarrhea are noroviruses (the family prototype) and sapoviruses. Norovirus particles are round, whereas other calicivirus particles are star shaped. The viruses appear to be extremely hardy; their infectivity persists after exposure to acid, ether, and heat (60°C for 30 minutes). After 48 years of the identification of norovirus as a causative agent of diarrhea, it can now be grown in intestinal epithelial cells in the laboratory.

Five different Norovirus serotypes or genotypes (GI-GV) have been identified, with three genotypes (GI, GII, and GIV) infecting humans, as demonstrated by immunoelectron microscopy with convalescent sera from affected patients and genetic analysis. Knowledge of the antigenic characteristics and biology of these viruses was hampered by the inability to grow them in the laboratory. However, development of the new tissue culture system to grown norovirus in the laboratory may enhance the knowledge about the genetics and pathogenesis of noroviruses.

CALICIVIRUS INFECTIONS

EPIDEMIOLOGY

Calicivirus (norovirus) infection occurs worldwide with an estimated 685 million cases, including 200 million in children below age 5 years and 50 000 child deaths every year mainly in developing countries. In the United States, between 19 to 21 million cases of noroviruses are reported annually, including 1.7 to 1.9 million outpatient visits, 400 000 emergency room visits, 56 000 to 71 000 hospitalizations, and 570 to 800 deaths mainly in young children and the elderly. Generally, they are the most common cause of nonbacterial gastroenteritis in adults. However, with the implementation of the rotavirus vaccine in the United States, norovirus has become the leading cause of viral gastroenteritis in children below 5 years of age. They can infect any time of the year but are most common during winter months. Sharp family and community outbreaks are common and can occur in any season. The noroviruses have been particularly a major issue in closed settings, such as cruise ships, hospitals, nursing homes, and schools. Moreover, norovirus is responsible for causing more than 90% of the diarrhea outbreaks on the cruise ships. The major sources of transmission include contaminated food, person to person, water, and unknown source. Caliciviruses are much more common causes of gastrointestinal illness in older children and adults. This difference in age-specific predilection is perhaps reflected in serosurveys, which have shown that the prevalence of antibodies rises slowly, reaching approximately 50% by the fifth decade of life, a striking contrast to the frequent acquisition of antibodies to rotaviruses early in life. Because rotavirus infection has significantly reduced due to vaccination, norovirus has now become a leading cause of viral diarrhea in infants and children in the United States. Transmission is primarily by fecal–oral route; outbreaks have also been associated with consumption of contaminated water, uncooked shellfish, and other foods. Sharp outbreaks include older children and adults.

PATHOGENESIS

Both the pathogenesis and the pathology are similar to those described for rotaviruses, except that no enterotoxic features have yet been described for caliciviruses. Biopsy of the intestinal tissue shows that the intestinal mucosa is intact but there are histological changes such as broadening and blunting of the villi, shortening of the microvilli, enlarged and pale mitochondria, increased cytoplasmic vacuolization, and intercellular edema. These mucosal changes usually revert to normal within 2 weeks of onset of illness. Virus shedding in the feces generally lasts no more than 3 to 4 days.

IMMUNITY

Patients and experimentally infected volunteers respond to infection with the production of humoral antibodies, which persist for long time; their role in protection from reinfection, however, appears minimal. Reinfection and illness with the same serotype occur, and the role of local (mucosal) antibody (IgA) has not been well defined. It is possible that nonimmune or genetic factors are essential for protection.

CLINICAL ASPECTS

The incubation period is 12 to 48 hours (0.5-2 days), followed by abrupt onset of vomiting and diarrhea, a syndrome clinically indistinguishable from that caused by rotaviruses. Patients infected with noroviruses experience more vomiting than sapoviruses. The most common complication is dehydration. Respiratory symptoms rarely coexist, and the duration of illness is relatively brief (usually 1-2 days). These viruses can be detected by electron microscopy or immunoelectron microscopy in stools during the acute phase of illness. In addition, EIA and PCR methods have been developed. As with rotavirus infection, there is no specific treatment other than fluid and electrolyte replacement. Prevention requires good hygienic measures. Currently, there is no vaccine available.

Astroviruses belong to the family Astroviridae. Astroviruses have a shape that resembles a five- or six-pointed star (Figure 15–1C). These have been known since 1975. In recent years, astroviruses have been acknowledged as causes of often-mild gastroenteritis outbreaks, primarily among toddlers, school children, and elderly nursing home residents. Eight human serotypes, 1 to 8, of astroviruses have been identified.

Astroviruses are star-shaped, 28 to 38 nm, naked capsid, icosahedral, positive-sense RNA viruses. The virions are spherical, and the shape and genome resembles that of some calicivirus members. The genome of 6.8 to 7.9 nucleotides encodes a full length and a subgenomic RNA. Subgenomic RNA encodes structural proteins, whereas full-length RNA encodes RNA-dependent RNA polymerase. Astroviruses are acid stable, heat resistant for a short period of time, and resistant to a range of detergents and lipid solvents. The replication cycle of the astroviruses is not fully characterized because of lack of a reliable cell culture system. However, astroviruses have been propagated in primary human embryonic kidney cells with fecal extracts containing astroviruses. The virus most likely replicates similar to other positive-sense RNA viruses in the cytoplasm by first translating the RNA genome into a polyprotein followed by proteolytic cleavage into individual proteins, including RNA-dependent RNA polymerase, which then transcribes mRNA and genomic RNA. Virus assembly takes place in the cytoplasm and release upon cell lysis.

Similar to other viruses of diarrhea, astroviruses are also transmitted via fecal–oral route through contaminated food, water, or fomites and are spread worldwide. The incubation period is 1 to 2 days and the virus is shed in feces. Symptoms include copious, watery diarrhea, nausea, vomiting, fever, malaise, anorexia, and abdominal pain for up to 2 to 3 days, especially in toddlers, children, and elderly. Adults generally do not get sick until they are infected with a very high dose of the virus. The virus was identified in intestinal epithelial cells, suggesting that the virus probably replicates in these cells. Viral pathogenesis data from humans are limited. The virus is shed for a long time in immunocompromised individuals. Viral RNA can be detected by RT-PCR and antibodies by EIA. There is no specific treatment or vaccine. Similar measures such as those taken for other diarrheal viruses are required.

Some adenoviruses serotypes (double-stranded DNA, naked capsid virus), most of which are exceedingly difficult to cultivate in vitro (in contrast to those associated with respiratory diseases and discussed in Chapter 9), are now recognized as significant intestinal pathogens. These adenovirus serotypes may account for an estimated 5% to 15% of all viral gastroenteritis in young children. These include serotypes 40, 41, and perhaps 38. These adenoviruses mainly infect infants aged around less than 2 years. They are transmitted by fecal–oral route and the incubation period is 8 to 10 days and the symptoms of gastroenteritis last for 5 to 12 days. The diagnosis can be done by antigen detection, PCR, virus isolation, and serology. Treatment and prevention strategies are similar to those of other diarrheal viruses.

Other agents that have been associated with gastrointestinal diseases include coronavirus- like agents, toroviruses (coronavirus), and some group A coxsackieviruses (the latter primarily cause gastrointestinal symptoms in severely immunocompromised patients). This list may grow in the future; however, until more is learned about their biology, epidemiologic behavior, and impact on human health, they remain “candidate” viruses for now.