Chapter 37: Reoviruses, Rotaviruses, and Caliciviruses

Reoviruses are medium-sized viruses with a double-stranded, segmented RNA genome. The family includes human rotaviruses, the most important cause of infantile gastroenteritis around the world (Figure 37-1). Acute gastroenteritis is a very common disease with significant public health impact. In developing countries, it is estimated to cause as many as 1.5 million deaths of preschool children annually, of which rotavirus is responsible for about 600,000 deaths. In the United States, acute gastroenteritis is second only to acute respiratory infections as a cause of disease in families.

Caliciviruses are small viruses with a single-stranded RNA genome. The family contains noroviruses, the major cause of nonbacterial epidemic gastroenteritis worldwide. Astroviruses also cause gastroenteritis.

Important properties of reoviruses are summarized in Table 37-1.

Structure and Composition

The virions measure 60–80 nm in diameter and possess two concentric capsid shells, each of which is icosahedral. ­(Rotaviruses have a triple-layered structure.) There is no envelope. Single-shelled virus particles that lack the outer capsid are 50–60 nm in diameter. The inner core of the ­particles is 33–40 nm in diameter (Figure 37-2). The double-shelled ­particle is the complete infectious form of the virus.

The reovirus genome consists of double-stranded RNA in 10–12 discrete segments with a total genome size of 16–27 kbp, depending on the genus. Whereas rotaviruses contain 11 genome segments, orthoreoviruses and orbiviruses each possess 10 segments and coltiviruses have 12 segments. The individual RNA segments vary in size from 680 (rotavirus) to 3900 bp (orthoreovirus). The virion core contains several enzymes needed for transcription and capping of viral RNAs.

Rotaviruses are stable to heat at 50°C, to a 3.0–9.0 range of pH, and to lipid solvents, such as ether and chloroform, but they are inactivated by 95% ethanol, phenol, and chlorine. Limited treatment with proteolytic enzymes increases infectivity.

Classification

The family Reoviridae is divided into 15 genera. Four of the genera are able to infect humans: Orthoreovirus, Rotavirus, Coltivirus, and Orbivirus. The genera are divided into two subfamilies: Spinareovirinae contains viruses with large spikes at the 12 vertices on the particle (eg, Orthoreovirus), whereas members of the Sedoreovirinae appear more smooth, lacking the large surface projections (eg, Rotavirus).

There are at least seven species or groups of rotaviruses (A–G) plus one recently proposed group (H), of which three species (A, B, C) infect humans. Strains of human and animal origin may fall in the same serotype. Other rotavirus groups and serotypes are found only in animals. Three different serotypes of reovirus are recognized, along with about 100 different orbivirus serotypes and two coltivirus serotypes.

Reovirus Replication

Viral particles attach to specific receptors on the cell surface (Figure 37-3). The cell attachment protein for reoviruses is the viral hemagglutinin (σ1 protein), a minor component of the outer capsid.

After attachment and penetration, uncoating of virus particles occurs in lysosomes in the cell cytoplasm. Only the outer shell of the virus is removed, and a core-associated RNA transcriptase is activated. This transcriptase transcribes mRNA molecules from the minus strand of each genome double-stranded RNA segment contained in the intact core. There are short terminal sequences at both ends of the RNA segments that are conserved among all isolates of a given subgroup. These conserved sequences may be recognition signals for the viral transcriptase. The functional mRNA molecules correspond in size to the genome segments. Most RNA segments encode a single protein, although a few (depending on the virus) encode two proteins. Reovirus cores contain all enzymes necessary for transcribing, capping, and extruding the mRNAs from the core, leaving the double-stranded RNA genome segments inside.

After being extruded from the core, the mRNAs are translated into primary gene products. Some of the full-length transcripts are encapsidated to form immature virus particles. A viral replicase is responsible for synthesizing negative-sense strands to form the double-stranded genome segments. This replication to form progeny double-stranded RNA occurs in partially completed core structures. The mechanisms that ensure assembly of the correct complement of genome segments into a developing viral core are unknown. However, genome reassortment occurs readily in cells coinfected with different viruses of the same subgroup, giving rise to virus particles containing RNA segments from the different parental strains. Viral polypeptides probably self-assemble to form the inner and outer capsid shells.

Reoviruses produce inclusion bodies in the cytoplasm in which virus particles are found. These viral factories are closely associated with tubular structures (microtubules and intermediate filaments). Rotavirus morphogenesis involves budding of single-shelled particles into the rough endoplasmic reticulum. The “pseudoenvelopes” so acquired are then removed and the outer capsids are added (Figure 37-3). This unusual pathway is used because the major outer capsid protein of rotaviruses is glycosylated.

Cell lysis results in release of progeny virions.

Rotaviruses are a major cause of diarrheal illness in human infants and young animals, including calves and piglets. Infections in adult humans and animals are also common. Among the rotaviruses are the agents of human infantile diarrhea, Nebraska calf diarrhea, epizootic diarrhea of infant mice, and SA11 virus of monkeys.

Rotaviruses resemble reoviruses in terms of morphology and strategy of replication.

Classification and Antigenic Properties

Rotaviruses have been classified into seven species (A–G) plus one tentative species (H) based on antigenic epitopes on the internal structural protein VP6. These can be detected by immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and immune electron microscopy (IEM). Group A rotaviruses are the most frequent human pathogens. Outer capsid proteins VP4 and VP7 carry epitopes important in neutralizing activity, with VP7 glycoprotein being the predominant antigen. These type-specific antigens differentiate among rotaviruses and are demonstrable by neutralization tests. Five predominant serotype strains of rotavirus species A (G1-G4, G9) are responsible for the majority of human disease. Serotype distributions differ geographically. Multiple serotypes have been identified among human and animal rotaviruses. Some animal and human rotaviruses share serotype specificity. For example, monkey virus SA11 is ­antigenically very similar to human serotype 3. The gene-coding assignments responsible for the structural and antigenic ­specificities of rotavirus proteins are shown in Figure 37-4.

Molecular epidemiologic studies have analyzed isolates based on differences in the migration of the 11 genome segments after electrophoresis of the RNA in polyacrylamide gels (Figure 37-5). These differences in electropherotypes can be used to differentiate species A viruses from other groups, but they cannot be used to determine serotypes.

Animal Susceptibility

Rotaviruses have a wide host range. Most isolates have been recovered from newborn animals with diarrhea. Cross-­species infections can occur in experimental inoculations, but it is not clear if they occur in nature. Swine rotavirus infects both newborn and weanling piglets. Newborns often exhibit subclinical infection, perhaps reflecting the ­presence of maternal antibody; overt disease is more common in weanling animals.

Propagation in Cell Culture

Rotaviruses are fastidious agents to culture. Most group A human rotaviruses can be cultivated if pretreated with the proteolytic enzyme trypsin and if low levels of trypsin are included in the tissue culture medium. This cleaves an outer capsid protein and facilitates uncoating. Very few non–group A rotavirus strains have been cultivated.

Pathogenesis

Rotaviruses infect cells in the villi of the small intestine ­(gastric and colonic mucosa are spared). They multiply in the cytoplasm of enterocytes and damage their transport mechanisms. One of the rotavirus-encoded proteins, NSP4, is a viral enterotoxin and induces secretion by triggering a calcium-dependent signal transduction pathway. Damaged cells may slough into the lumen of the intestine and release large quantities of virus, which appear in the stool (up to 10¹² particles per gram of feces). Viral excretion usually lasts from 2 to 12 days in otherwise healthy patients but may be prolonged in those with poor nutrition and immunocompromised patients. Diarrhea caused by rotaviruses may also be due to impaired sodium and glucose absorption as damaged cells on villi are replaced by nonabsorbing immature crypt cells. It may take from 3 to 8 weeks for normal function to be restored.

Clinical Findings and Laboratory Diagnosis

Rotaviruses cause the major portion of diarrheal illness in infants and children worldwide but not in adults (Table 37-2). There is an incubation period of 1–3 days. Typical symptoms include watery diarrhea, fever, abdominal pain, and vomiting, leading to dehydration.

In infants and children, severe loss of electrolytes and fluids may be fatal unless treated. Patients with milder cases have symptoms for 3–8 days and then recover completely. However, viral excretion in the stool may persist up to 50 days after onset of diarrhea. Asymptomatic infections with seroconversion occur. In children with immunodeficiencies, rotavirus can cause severe and prolonged disease.

Adult contacts may be infected, as evidenced by seroconversion, but they rarely exhibit symptoms, and virus is infrequently detected in their stools. A common source of infection is contact with pediatric cases. However, epidemics of severe disease have occurred in adults, especially in closed populations, as in a geriatric ward. Group B rotaviruses have been implicated in large outbreaks of severe gastroenteritis in adults in China and southeastern Asia (Table 37-2).

Laboratory diagnosis rests on demonstration of virus in stool collected early in the illness and on a rise in antibody titer. Virus in stool is demonstrated by enzyme immunoassays (EIAs) or IEM. The EIA test is more sensitive than the IEM. Genotyping of rotavirus nucleic acid from stool specimens by the polymerase chain reaction (PCR) is the most sensitive detection method. Serologic tests can be used to detect an antibody titer rise, particularly ELISA.

Epidemiology and Immunity

Rotaviruses are the single most important worldwide cause of gastroenteritis in young children. Estimates range from 3 to 5 billion for annual diarrheal episodes in children younger than 5 years of age in Africa, Asia, and Latin America, resulting in as many as 1 million deaths. Developed countries have a high morbidity rate but a low mortality rate. Typically, up to 50% of cases of acute gastroenteritis of hospitalized children throughout the world are caused by rotaviruses.

Rotavirus infections usually predominate during the winter season. Symptomatic infections are most common in children between ages 6 months and 2 years, and transmission appears to be by the fecal–oral route. Nosocomial infections are frequent.

Rotaviruses are ubiquitous. By age 3 years, 90% of ­children have serum antibodies to one or more types. This high prevalence of rotavirus antibodies is maintained in adults, suggesting subclinical reinfections by the virus. Rotavirus reinfections are common; it has been shown that young children can suffer up to five reinfections by 2 years of age. Asymptomatic infections are more common with successive reinfections. Local immune factors, such as secretory immunoglobulin A (IgA) or interferon, may be important in ­protection against rotavirus infection. Asymptomatic infections are common in infants before age 6 months, the time during which protective maternal antibody acquired ­passively by newborns should be present. Such neonatal infection does not prevent reinfection, but it does protect against the development of severe disease during reinfection.

Treatment and Control

Treatment of gastroenteritis is supportive to correct the loss of water and electrolytes that may lead to dehydration, acidosis, shock, and death. Management consists of replacement of fluids and restoration of electrolyte balance either intravenously or orally as feasible. The infrequent mortality from infantile diarrhea in developed countries is the result of the routine use of effective replacement therapy.

In view of the fecal–oral route of transmission, wastewater treatment and sanitation are significant control measures.

An oral live attenuated rotavirus vaccine was licensed in the United States in 1998 for vaccination of infants. It was withdrawn a year later because of reports of intussusception (bowel blockages) as an uncommon but serious side effect associated with the vaccine. In 2006, an oral live attenuated pentavalent human–bovine reassortant rotavirus vaccine was licensed in the United States, followed by licensing of an oral live attenuated monovalent human rotavirus vaccine in 2008. Both vaccines are safe and effective, and neither is associated with intussusception. Similar to other live attenuated vaccines, immunization of immunocompromised individuals or their family members should be avoided as vaccine strains can cause disease in these patients. A safe and effective vaccine remains the best hope for reducing the worldwide burden of rotavirus disease.

The viruses of this genus, which have been studied most thoroughly by molecular biologists, are not known to cause human disease.

Classification and Antigenic Properties

Reoviruses are ubiquitous, with a very wide range of mammalian, avian, and reptilian hosts. Three distinct but related types of reovirus have been recovered from many species and are demonstrable by neutralization and hemagglutination-inhibition tests. Reoviruses contain a hemagglutinin for human O or bovine erythrocytes.

Epidemiology

Reoviruses cause many inapparent infections because most people have serum antibodies by early adulthood. Antibodies are also present in other species. All three types have been recovered from healthy children, from young children during outbreaks of minor febrile illness, from children with enteritis or mild respiratory disease, and from chimpanzees with epidemic rhinitis.

Human volunteer studies have failed to demonstrate a clear cause-and-effect relationship of reoviruses to human illness. In inoculated volunteers, reovirus is recovered far more readily from feces than from the nose or throat.

Pathogenesis

Reoviruses have become important model systems for the study of the pathogenesis of viral infection at the molecular level. Defined recombinants from two reoviruses with differing pathogenic phenotypes are used to infect mice. Segregation analysis is then used to associate particular features of pathogenesis with specific viral genes and gene products. The pathogenic properties of reoviruses are primarily determined by the protein species found on the outer capsid of the virion.

Orbiviruses are a genus within the reovirus family. They commonly infect insects, and many are transmitted by insects to vertebrates. About 100 serotypes are known. None of these viruses cause serious clinical disease in humans, but they may cause mild fevers. Serious animal pathogens include bluetongue virus of sheep and African horse sickness virus. Antibodies to orbiviruses are found in many vertebrates, including humans.

The genome consists of 10 segments of double-stranded RNA, with a total genome size of 18 kbp. The replicative cycle is similar to that of reoviruses. Orbiviruses are sensitive to low pH, in contrast with the general stability of other reoviruses.

Coltiviruses form another species within the Reoviridae. The virus particle is 80 nm in diameter with a genome consisting of 12 segments of double-stranded RNA, totaling about 29 kbp. Colorado tick fever virus, transmitted by ticks, is able to infect humans (see Chapter 38). It is found in the southwestern United States and can cause fever, rash, and systemic symptoms in infected patients.

In addition to rotaviruses and noncultivable adenoviruses, members of the family Caliciviridae are important agents of viral gastroenteritis in humans. The most significant members are the noroviruses, the prototype strain being Norwalk virus. Properties of caliciviruses are summarized in Table 37-3.

Classification and Antigenic Properties

Caliciviruses are similar to picornaviruses but are slightly larger (27–40 nm) and contain a single major structural ­protein (Figure 37-6). They exhibit a distinctive morphology in the electron microscope (Figure 37-7). The family ­Caliciviridae is divided into five genera: Norovirus, which includes the Norwalk viruses; Sapovirus, which includes the Sapporo-like viruses; Nebovirus, which includes bovine enteric viruses; Lagovirus, the rabbit hemorrhagic disease virus; and Vesivirus, which includes vesicular exanthem virus of swine, feline calicivirus, and marine viruses found in pinnipeds, whales, and fish. The first two genera contain human viruses that cannot be cultured; the latter two genera contain animal strains that can be grown in vitro. Rabbit hemorrhagic disease virus was introduced in 1995 in Australia as a biologic control agent to reduce that country’s population of wild rabbits.

Human calicivirus serotypes are not defined. Multiple genotypes of noroviruses have been detected. Three genogroups are associated with human gastroenteritis, designated GI, GII, and GIV. Since 2001, genotype GII.4 viruses have caused most viral gastroenteritis outbreaks worldwide. Noroviruses appear to undergo antigenic drift over time, probably in response to population immunity.

Cellular receptors for noroviruses are histo-blood group antigens that are expressed on the mucosal epithelia of the digestive tract. A person’s secretor status is controlled by the fucosyltransferase 2 gene; secretor-negative individuals are resistant to infection with common strains of Norwalk virus.

Clinical Findings and Laboratory Diagnosis

Noroviruses (Norwalk virus) are the most important cause of epidemic viral gastroenteritis in adults (Table 37-2). Epidemic nonbacterial gastroenteritis is characterized by (1) absence of bacterial pathogens, (2) gastroenteritis with rapid onset and recovery and relatively mild systemic signs, and (3) an ­epidemiologic pattern of a highly communicable disease that spreads rapidly with no particular predilection in terms of age or geography. Various descriptive terms have been used in reports of different outbreaks (eg, epidemic viral gastroenteritis, viral diarrhea, winter vomiting disease) depending on the predominant clinical feature.

Norwalk viral gastroenteritis has an incubation period of 24–48 hours. The onset is rapid, and the clinical course is brief, lasting 12–60 hours; symptoms include diarrhea, nausea, vomiting, low-grade fever, abdominal cramps, headache, and malaise. The illness can be incapacitating during the symptomatic phase, but hospitalization is rarely required. Norovirus infections are more likely to induce vomiting than those with Sapporo-like viruses. Dehydration is the most common complication in young and elderly individuals. Viral shedding may persist for as long as 1 month. No sequelae have been reported.

Volunteer experiments have shown that the appearance of Norwalk virus coincides with clinical illness. Antibody develops during the illness and is usually protective on a short-term basis against reinfection with the same agent. Long-term immunity does not correspond well to the presence of serum antibodies. Some volunteers can be reinfected with the same virus after about 2 years.

Reverse transcriptase PCR is the most widely used technique for detection of human caliciviruses in clinical specimens (feces, vomitus) and environmental samples (contaminated food, water). Because of the genetic diversity among circulating strains, the choice of PCR primer pairs is very important. There can be up to 100 billion viral genome copies per gram of feces at peak shedding (2–5 days after infection).

Electron microscopy can be used to detect virus particles in stool samples. However, norovirus particles are usually present in low concentration (unless the sample was collected at peak viral shedding) and are difficult to recognize; they can be identified by IEM. ELISA immunoassays based on recombinant virus-like particles can detect antibody responses, with a fourfold or greater rise in IgG antibody titer in acute and convalescent-phase sera indicative of a recent infection. However, the necessary reagents are not widely available, and the antigens are not able to detect responses to all antigenic types of noroviruses.

Epidemiology and Immunity

Human caliciviruses have worldwide distribution. ­Noroviruses are the most common cause of nonbacterial ­gastroenteritis in the United States, causing an estimated 21 million cases annually.

The viruses are most often associated with epidemic outbreaks of waterborne, foodborne, and shellfish-associated gastroenteritis. All age groups can be affected. Outbreaks occur throughout the year, with a seasonal peak during cooler months. Fecal–oral spread is the primary means of transmission. Most outbreaks involve foodborne or person-to-person transmission via fomites or aerosolization of contaminated body fluids (vomitus, fecal material). Outbreaks in closed settings, such as cruise ships and nursing homes, are typical.

Characteristics of norovirus include a low infectious dose (as few as 10 virus particles), relative stability in the environment, and multiple modes of transmission. It ­survives 10 ppm chlorine and heating to 60°C; it can be maintained in steamed oysters.

No in vitro neutralization assay is available to study immunity. Volunteer challenge studies have shown that about 50% of adults are susceptible to illness. Norwalk virus antibody is acquired later in life than rotavirus antibody, which develops early in childhood. In developing countries, most children have developed norovirus antibodies by 4 years of age.

Treatment and Control

Treatment is symptomatic. The low infectious dose permits efficient transmission of the virus. Effective handwashing is probably the most important method to prevent norovirus infection and transmission. Because of the infectious nature of the stools, care should be taken in their disposal. Containment and disinfection of soiled areas and bedding can help decrease viral spread. Careful processing of food and education of food handlers are important because many foodborne outbreaks occur. Purification of drinking water and swimming pool water should decrease norovirus outbreaks. There is no vaccine.

Astroviruses are about 28–30 nm in diameter and exhibit a distinctive starlike morphology in the electron microscope (Figure 37-7F). They contain single-stranded, positive-sense RNA, 6.4–7.4 kb in size. The family Astroviridae contains two genera; all human viruses are classified in the Mamastrovirus genus. At least eight serotypes of human viruses are recognized by IEM and neutralization.

Astroviruses cause diarrheal illness and may be shed in extraordinarily large quantities in feces. The viruses are transmitted by the fecal–oral route through contaminated food or water, person-to-person contact, or contaminated surfaces. They are recognized as pathogens for infants and children, elderly institutionalized patients, and immunocompromised persons (Table 37-2). They may be shed for prolonged periods by immunocompromised hosts.

Animal astroviruses are found in a variety of mammals and birds and have recently been identified in several species of bats. Clinical testing for astroviruses is not commonly performed, but detection can be achieved with electron microscopy, antigen, or RT-PCR methods.

• Reoviruses and rotaviruses are nonenveloped and contain a double-stranded, segmented RNA genome.

• Reoviruses are not known to cause human disease but are important model systems for molecular pathogenesis studies.

• Rotaviruses are the most important cause of diarrheal illness in infants and young children worldwide.

• Genetic reassortment occurs readily with rotaviruses.

• Caliciviruses are small nonenveloped viruses with a ­single-stranded, nonsegmented RNA genome.

• Noroviruses, a genus of caliciviruses, are the major cause of nonbacterial epidemic gastroenteritis in the world.

• Rotaviruses and noroviruses are transmitted primarily by fecal–oral spread; noroviruses are associated with foodborne and waterborne outbreaks.

• Oral live attenuated rotavirus vaccines are available that are safe and effective; there is no norovirus vaccine.