Diarrhoeal Diseases (Updated February 2009)
Viral gastroenteritis is one of the most common illnesses in humans worldwide and caliciviruses, especially noroviruses (NoV), are one of its major agents. The prototype strain of NoV is the Norwalk virus, which was originally discovered in 1968 in an outbreak of gastroenteritis in an elementary school in Norwalk, Ohio, USA .
The role of human caliciviruses, including noroviruses, as agents of gastroenteric diseases has long been unrecognized and under-appreciated because diagnostic tools were not commonly available as these viruses remain uncultivable by standard cell culture assays. The application of new molecular diagnosis tools such as RT-PCR has shown that they are significant contributors to diarrhoeal disease burden in both children and adults. They appear to be the most common cause of winter gastroenteritis outbreaks in humans, popularly known as "stomach flu" in the UK ("grippe intestinale" in France), and a common cause of sporadic cases and outbreaks of acute gastroenteritis with vomiting, abdominal cramps, diarrhoea, headache and fever which occur in various epidemic settings such as restaurants, schools, day care centers, hospitals, nursing homes and cruise ships       . The implicated vehicles of infection are contaminated water, shellfish, and food contaminated either at its source or by food handlers. Transmission also readily occurs from person-to-person or through contact with contaminated objects. Asymptomatic virus shedding can persist for up to two weeks, and the virus can survive freezing and heating to 60°C, as well as chlorinated waters, permitting its spread in recreational and drinking water as well as in steamed shellfish.
From several studies, it appears that, in industrialized countries, NoV are the second most common agent of non bacterial acute gastroenteritis in children after rotavirus, with an incidence of about 12% in children less than five years of age with severe diarrhoea, and the most common cause of outbreaks of acute viral gastroenteritis in adults, including those that are foodborne      . In the USA alone, NoV may account every year for more than 235 000 clinic visits, 91 000 emergency visits and 23 000 hospitalizations among children less than 5 years of age . The role of NoV in developing countries has been less firmly established. However, in many Asian and African countries, most children appear to acquire serum antibodies to NoV early in life, suggesting that the virus probably plays a pre-eminent role in pediatric diarrhoeal diseases    . In Beijing, China, infants had a seroprevalence rate of 41% at 7 months of age, 65% at 1 year, 85% at 3 years, and 100% at 8-9 years of age. A recent estimate puts at more than 1 million the number of hospitalizations and at at more than 200 000 the number of deaths NoV may cause each year among children less than 5 years of age worldwide .
Human caliciviruses, which form the family Caliciviridae (previously referred to as the Norwalk family of viruses or Small Round Structured Viruses) are 27-35 nm nonenveloped icosahedral viruses whose genome is a single-stranded positive RNA molecule. They include the genera Sapovirus and Norovirus (NoV). The NoV genome contains three open reading frames (ORFs), ORF1, 2, and 3. ORF1 encodes viral nonstructural proteins, including an NTPase, a protease and the viral replicase. ORF2 encodes the 58 kD capsid protein, VP1, and ORF3 a smaller capsid protein, VP2. VP1 is divided into N-terminal region, shell (S) domain, protruding domain (P) and C-terminal region. The P domain is in turn divided into P1-1, P2 and P1-2 domains, with P2 bearing the most important antigenic determinants   . The virus is uncultivable in cell culture but cloning of either VP1 alone or VP1 and VP2 together in a baculovirus expression system leads to the spontaneous formation of virus-like particles (VLPs) that are antigenically similar to intact virions.
Cloning and sequencing of NoV genomes has allowed genetic characterization of the virus and its repartition into genogroups GI to GVII, each in turn subdivided into genotypes and subgenotypes  . Porcine, bovine and murine NoV belong to genogroups II, III and V, respectively. The majority of human NoV outbreaks are caused by viruses belonging to the GII.-3 and GII-4 genotypes (genogroup II genotype 3 and genogroup II genotype 4), whose pandemic spread was recognized in the 1990s  and which appear to continuously undergo antigenic drift and recombination, leading to the emergence of a multiplicity of new virus variants    .
These viruses exhibit a restricted tropism for infection of the gastrointestinal tract of humans. Their receptor has been identified as the ABH histo-blood group antigens (HBGAs) on mucosal surfaces    This explains why individuals who have a defective alpha-1,2 fucosyltransferase (FUT2) enzyme and who, for that reason, are unable to express HBGAs on cell surfaces, are resistant to infection   The surface-exposed, carbohydrate-binding domain of the NoV capsid appears to be under heavy immune selection and to evolve by antigenic drift to escape human immune pressure from herd immunity .
There are no vaccines available against caliciviruses. However, NoV VLPs produced in a baculovirus system are morphologically and antigenically similar to native virions, as judged from electron microscopy and ELISA. They also are stable at low pH, making them attractive as an oral immunogen. NoV VLPs administered by the intranasal or oral routes to mice with or without a mucosal adjuvant (the E coli toxin LT or its detoxified R192G derivative) induced a high serum antibody response as well as faecal IgAs. The safety and immunogenicity of the VLPs was evaluated in a Phase I trial on healthy human volunteers who received two successive 250µg VLP oral doses in bicarbonate buffer. All volunteers showed a >4-fold increase in IgG1 and IgA antibody titers  . The question remains of the extent of protection such a vaccine could provide in the field, in view of the natural diversity and variability of NoVs. A broad multivalent mixture of VLPs would most probably be needed to cover a significant number of virus variants and genotypes  , but the number and serotypes of VLPs to be included is unknown.
The same VLPs were independently used as a test antigen to determine whether immune responses could be generated in volunteers who ingested transgenic potatoes that expressed NoV VLPs. Healthy adult volunteers at the Center for Vaccine Development (CVD), University of Maryland, USA, received 2 or 3 successive 150g doses of transgenic potatoes expressing the 58 kD NoV capsid protein or 3 doses of wild-type potato. Most of the volunteers who ingested the raw transgenic potatoes developed significant increase in the number of specific IgA antibody-secreting cells, 30% developed NoV-specific stool IgAs and 20% specific serum IgGs, but no increase in serum IgG titer was observed after the second dose  . Whether the modest antibody titers obtained would be protective against infection is unknown.