Caliciviruses Associated with Gastroenteritis Properties of Caliciviridae

The family Calicwiridae derives its name from the 32 cup-shaped {catix, cup) surface depressions that give the virion its unique appearance (Fig. 24-1A).

Fig. 24-1 Negatively stained electron micrographs of virions of Cahrwiridae (A) and Astroviridae (B) Bars, 100 nm (A, Courtesy Drs M Szymanski and P J- Middleton; B, courtesy Dr D Snodgrass.)

The virion is 30-38 nm in diameter, slightly larger than the picornaviruses. The icosahedral capsid is constructed from 90 capsomers, each being a dimer of a single species of polypeptide. Although relatively resistant to inactivation by heat (60°C), stomach acid (pH 3), or minimal levels of chlorination of drinking water, the virions of several caliciviruses, including the hepatitis E virus, are particularly vulnerable to proteolysis and tend to lose their capsid definition on purification or storage

The plus sense linear ssRIMA genome of 7.5-7.7 kb resembles that of the picornaviruses in that a VPg protein is covalently attached to its 5' terminus (except in the case of hepatitis E virus which is capped instead), the 3' terminus is polyadenylated, and the nonstructural proteins include an RNA-dependent RNA polymerase, an ATPase/helicase, and a protease. However, the calicivirus genome has not one but three open reading frames, the long 5' ORF encoding the nonstructural proteins, the middle ORF the capsid protein (sometimes involving a frameshift), and the 3' ORF a protein of uncertain function (Table 24-1).

Classification

Because the human caliciviruses (and most of the other viruses causing gastroenteritis in humans) were discovered by electron microscopy (EM) and defied cultivation, the CauJ and Appleton system for classifying various small, round putative gastroenteritis viruses was based on EM morphology. T he system drew a distinction between (I) "typical" caliciviruses, which were 30-35 nm icosahedral virions with cup-shaped indentations, frequently presenting as a six-pointed Star of David with a dark, slain-filled central hollow (Fig. 24-1 A) and (2) the "amorphous small round-structured viruses," of which the proiotype was the Norwalk agent, which tended to have a wider size range (27-40 nm) and an indistinct ragged or "feathery" outline more difficult to define. In retrospect, it appears that both groups belong to the genus Cnlicmirus and that the differences observed in size and outline might reflect (a) bound coproantibody and/or (b) susceptibility of enteric caliciviruses to proteolytic degradation. Nevertheless, there may be important epidemiologic differences between the "typical" enteric caliciviruses, which tend to be associated with gastroenteritis in infants, and the Norwalk group, occurring mainly in adults and other children. The Norwalk group includes several viruses, falling into at least three "types" or "serogroups" typified by Norwalk, Snow Mountain, and Otofuke/Hawaii agents, but the antigenic relationships among them, or between them and the several strains of "typical" enteric caliciviruses, are not yet clear.

Viral Replication

Calicivirus replication is confined to the cytoplasm. Genomic RNA serves as the mRNA for the polyprotein representing the nonstructural proteins, which are derived from the precursor by proteolytic cleavage. For most caliciviruses a separate subgenomic mRNA encodes the capsid protein. Because subgenomic minus sense RNA has also been reported it is possible that the subgenomic mRNA (as well as genomic RNA) may be capable of replication via a complementary minus strand. It now appears that there may be a 3' nested set of overlapping subgenomic mRNAs, reminiscent of coronavirus strategy. Virions assemble in the cytoplasm, sometimes forming para-crystalline arrays associated with the cyfoskeleton.

Clinical Features

The disease caused by the Norwalk agent has been the most comprehensively studied, in volunteers as well as in natural outbreaks. Following a very short incubation period (generally 24-48 hours), the illness runs its course in 12-60 hours. Nausea, vomiting, diarrhea, and abdominal cramps are prominent features, with vomiting being most common in children and diarrhea in adults; headache, myalgia, and low fever are variable features.

Pathogenesis and Immunity

In Norwalk viral gastroenteritis the tips of the villi in the jejunum slough off but rapidly regenerate, there is infiltration of mononuclear cells and polymorphs into the mucosa, transient malabsorption, and delayed gastric empty-

Table 24-1

Properties of Caliciviruses

Single genus, Cnl:avmis, includes hepatitis F and human caliciviruses causing gastroenteritis Nonerivelopod spherical virion, 30-38 nm diameter, vulnerable to proteolysis Icosahedral capsid with 32 cup-shaped depressions, 90 capsomers, one capsid protein Linear plus sense ssRNA genome, 7.5 to 7 7 kb, polyadenylafed ,it 3' terminus, prolein at 5'

terminus (except hepatitis L, tapped), infectious Three open reading frames: 5', nonstructural proleins, middle, capsid piolein, 3', unknown Cytoplasmic replication, genomic RNA ORF1 translated into polyprotein, cleaved into nonstruc-lural proteins, subgenomic mRNA encodes capsid protein ing. Acquired immunity is poor, lasting for a year or so at best. Indeed, when volunteers were challenged with Norwalk virus twice, at intervals of 2-3 years, those who had contracted gastroenteritis on the first occasion and had developed antibody were the very ones who became sick the second time. This was interpreted to mean (1) that prior infection confers no significant long-term immunity and (2) that unknown genetic or physiologic factors may predispose certain individuals but not others to gastroenteritis following exposure to virus. Paradoxically, it appears that antibody to the"typical" enteric caliciviruses is acquired early in life and protects adults against reinfection.

Laboratory Diagnosis

Unfortunately, human caliciviruses are extremely fastidious and can still not be grown reproducibly in cultured cells. Nevertheless, reliance on the cumbersome and insensitive diagnostic modality of IEM is now unnecessary following the recent molecular cloning of the genomes of a number of human enteric caliciviruses. For example, using baculovirus as an expression vector for the Norwalk genome, insect cells produce empty Norwalk virions which serve as antigen for immunoassays to probe for antibody, or as immunogen to make monoclonal antibodies (MAbs) to probe for antigen. EIAs based on such cloned reagents are being developed. Probes and primers have been produced for use in PCR, but special precautions must be taken first to inactivate RNases with the virion-disrupting agent guanidine isothiocyanate, and then to adsorb the viral RNA to glass beads or powder to enable soluble inhibitors of reverse transcriptase to be washed away. It must also be remembered that, since asymptomatic enteric viral infections are common and mixed infections not rare, and PCR assays for different agents may differ in sensitivity, PCR results must be interpreted with caution. The first-line diagnostic assay should be EIA for antigen deiection. For epidemiologic investigation of an outbreak, EIA or RIA can be used to demonstrate a rising titer of antibody, with the specificity or cross-reactivity of the assay being determined by the choice of antigen employed; IgM capture assays circumvent the need to recall the patient for a second bleed.

Epidemiology

The Norwalk group of agents are uncommon in infants in developed countries but are seen in older children and adults, about half of all adults revealing serological evidence of past infection. Common-source outbreaks frequently occur via fecal contamination of water or food, such as salads, in restaurants, schools, camps, cruise ships, geriatric nursing homes, etc. Several major outbreaks have been traced to consumption of uncooked or partially cooked oysters, cockles, clams, or other shellfish that have been harvested from sewage-polluted estuaries. Others have been attributed to discharge of sewage into drinking water supplies, or into pools and lakes in which people swim. Secondary spread occurs from person to person within households, with transmission for up to 2 days after the resolution of symptoms. Some 40% of outbreaks of gastroenteritis in adults in the United States have been attributed to Norwalk virus. Unlike bacterial gastroenteritis or food poisoning, viral gastroenteritis occurs year-round. In developing countries these viruses are encountered much earlier in life, with the pattern of endemic exposure resembling that for the typical caliciviruses (see below).

The typical caliciviruses are endemic worldwide, infecting infants from 3 months of age until kindergarten age; most children have been infected with at least one calicivirus before leaving primary school. Outbreaks also occur in day-care centers, orphanages, maternity hospitals, schools, nursing homes, etc., with high attack rates.

Control

Control of person-to-person spread of endemic gastroenteritis requires a long-term national commitment and resources to improve the standards of sanitation, water supply, and personal hygiene (see Chapter 15), which is currently out of the reach of many impoverished countries. Common-source calicivirus outbreaks can be reduced by enforcement of legislation relating to the preparation and supply of food for public consumption. Of course viruses, unlike bacteria, cannot multiply in food or water. Careful investigation of Norwalk virus outbreaks in the United States identified an ill food-handler as the probable source in the majority of instances. Because even caliciviruses are inactivated at temperatures well below that of normal cooking, uncooked foods such as salads are the greatest risk. Shellfish concentrate viruses from fecally contaminated water and are a notorious source of caliciviruses and hepatitis viruses if served raw or inadequately cooked, accounting for fully half of the Norwalk outbreaks in the United States investigated by the Centers for Disease Control. Depuration (prolonged flushing with clean water) is only partially effective in reducing the problem. Caliciviruses are also relatively resistant to chlorination of water. The U.S. Environmental Protection Agency guidelines for municipal water systems recommend residual chlorine concentrations of at least 0.2 mg/liter, and in many localities peak levels of 5 mg/liter are used. Although these levels are adequate to destroy most bacteria and viruses, the Norwalk agent can survive at chlorine concentrations below 10 mg/liter.

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