Caister Academic Press


Enteroviruses: Omics, Molecular Biology, and Control
Edited by: William T. Jackson and Carolyn B. Coyne
A must-read for anyone with an interest in this family of viruses and an important acquisition for all microbiology libraries.
A review of scientific research into Caliciviridae.


from Caliciviruses: Molecular and Cellular Virology by Hansman, GS et al (2010)

Members of the Caliciviridae family (caliciviruses) are positive-sense, single stranded RNA viruses containing four recognized genera: Norovirus, Sapovirus, Lagovirus and Vesivirus. They are ubiquitous in the environment and are a major cause of disease in humans and many animals. Examples include Norwalk virus, a norovirus, thought to be responsible for roughly 90% of epidemic, non-bacterial outbreaks of gastroenteritis in humans around the world. Lack of a suitable cell culture system for human caliciviruses limited studies in previous decades, however the recent application of modern genomic technologies has revolutionized the field, leading to an explosion in calicivirus publications.

Norovirus Epidemiology

Noroviruses are the dominant cause of outbreaks as well as sporadic community cases of viral gastroenteritis in the world. Their very low infectious dose, combined with high levels of shedding and long persistence in the environment make noroviruses extremely infectious. Although generally norovirus related illness is regarded as mild and self-limiting, more severe outcomes are increasingly described among elderly and immuno-compromised patients. The combination of large and difficult to control outbreaks and severe illness in some patients leads to major problems in healthcare settings, such as hospitals and nursing homes. Additionally, some large and diffuse, multi-national and even multi-continent, foodborne-outbreaks have been described for norovirus, affecting up to thousands of people. With structured outbreak surveillance running in a number of regions across the world for the past ten years, it has become clear that the spread of noroviruses is global, although important information from developing countries is missing. At present, norovirus strains belonging to genogroup II genotype 4 (GII.4) are dominant worldwide. In the last ten years, at least three global pandemics involving GII.4 strains of different genetic variants occurred. Although a straightforward culturing method remains lacking for noroviruses, important progress has been made in immunological studies using virus-like particles. Thus it has been shown that the subsequent genetic variants of GII.4 are antigenically distinct, and that the GII.4 noroviruses evolved and continue to do so by a process known as epochal evolution, in which periods of genetic stasis are interrupted by rapid accumulation of mutations and the subsequent emergence of novel genetic variants. In norovirus evolution, this process is directed by population or herd immunity.

Calicivirus Environmental Contamination

The virus family Caliciviridae contains four genera Norovirus, Sapovirus, Lagovirus and Vesivirus. Norovirus and sapovirus cause gastroenteritis in humans, while lagoviruses and vesiviruses mostly infect animals and cause a variety of diseases. Norovirus and sapoviruses can also infect a number of animals including cow and pig, respectively. Noroviruses are the dominant cause of human gastroenteritis around the world, infecting all age groups. Their low infectious dose and stability in the natural environment allows noroviruses to be easily spread. Contamination in food and water destined for human consumption has lead to numerous outbreaks of gastroenteritis. Noroviruses have been detected in shellfish, sandwiches, fruit, ice, drinking water and treated wastewater. Direct transmission from food and water to humans is well documented. Increased monitoring and improvements in detection methods may help to reduce the number of infections but regulations and standards need to be addressed in order to reduce viral contamination in the natural environment.

Genome Organization and Recombination

Recombination was first described in the human caliciviruses in 1997. Since then naturally occurring recombinants have been detected for all four genera of the Caliciviridae and has become an important mechanism in the emergence of new calicivirus variants. Due to similarities in genome organization between the different genera, recombination predomoninantly occurs at the start of the major structural gene which encodes the capsid, VP1. Knowledge of the mechanisms of calicivirus recombination is important as new variants can emerge, with potentially different pathogenesis and virulence.

Proteolytic Cleavage and Viral Proteins

Caliciviruses are icosahedral nonenveloped viruses with a positive-sense single strand RNA genome that does not exceed 8.6 kb. Despite its small size, the virus genome encodes a number of nonstructural proteins that successfully facilitate and regulate mechanisms required for efficient virus amplification. Although caliciviruses show significant genetic diversity, they share a common protein expression strategy. Recent findings have shown that the nonstructural proteins of caliciviruses are produced by autocatalytic cleavage of a polyprotein encoded by ORF1 of the virus genome. A single virus protease structurally similar to a class of viral chymotrypsin-like cysteine proteases mediates these cleavages, and in some caliciviruses, adds to a release of the virus capsid protein. The temporal regulation of viral protein synthesis relies on the specificity of the protease and may be modulated by additional viral and cellular factors. The proteolytic processing results not only in the synthesis of the mature virus proteins, but also their precursors, whose functions have yet to be determined. Almost all calicivirus proteins have been identified as components of the virus replication complexes; however, their roles in replication are not entirely understood and remain an active and crucial target of calicivirus research.

Calicivirus Protein Structures

Sequence analysis and experimentally determined three-dimensional structures of structural and nonstructural proteins from a range of caliciviruses help to provide a molecular framework for understanding many aspects of their replication strategies. Structures of intact virions, virus-like particles and capsid fragments, as well as capsid-receptor complexes help to explain basic mechanisms of capsid assembly and receptor recognition. Structural studies of the recombinant viral proteinase and polymerase in complex with substrates and inhibitors provide a basis for understanding substrate recognition and enzymatic mechanisms, thus setting the stage for the design of new antiviral compounds.

Virus-Host Interaction and Cellular Receptors of Caliciviruses

Caliciviruses are a diverse virus family with a wide range of host and tissue tropisms. Most calicivirus genera recognize a carbohydrate ligand for attachment, including the A, B, H and Lewis histo-blood group antigens (HBGAs) and heparan sulfate for the human noroviruses, the H type 2 antigen for the rabbit hemorrhagic disease virus (genus Lagovirus), the type B antigen for the Tulane virus (a potential new genus), and sialic acid for feline calicivirus (FCV; genus Vesivirus) and murine norovirus (MNV; genus Norovirus). Following attachment, FCV recognizes also a cell surface protein, the junctional adhesion molecule 1 (JAM-1), as a functional receptor or co-receptor potentially for penetration or entry into host cells. Some human noroviruses interact also with a 105 kDa membrane protein, but its role in viral penetration/entry into host cells remains unknown. The genetic and structural analyses of selected strains of norovirus and FCV have generated new insights into virus-host interactions that chart the course for innovative research in the development of effective strategies to control and prevent calicivirus infection and illness.

Calicivirus Reverse genetics and Replicon Systems

Recently, reverse genetics and replicon systems have been developed and are starting to be used in the elucidation of the calicivirus replication and pathogenicity. Reverse genetics systems are available for feline calicivirus, porcine enteric calicivirus, murine norovirus, rabbit hemorrhagic disease virus and a rhesus monkey calicivirus. For uncultivable caliciviruses, such as human norovirus, cell-based replicon systems have been established. Norovirus replicon systems are used to screen potential antivirals and therapeutic options against norovirus infection. Replicon systems with reporter genes such as those encoding green fluorescent protein or luciferase allows quantitative analysis of cellular and viral factors that promote virus replication. Further studies with reverse genetics and replicon system could yield important information for cell culture adaptation of human noroviruses which is crucial for development of efficient vaccines and antivirals.

Feline Calicivirus

Feline calicivirus (FCV) represents an important pathogen of cats that has been studied extensively on the molecular level. FCV was the first calicivirus for which milestones like a reverse genetics system or the identification of a verified virus receptor were reached. Recently, great efforts were made to investigate unusual mechanisms of translation initiation driven by the RNA bound protein VPg or an RNA structure named TURBS.

Caliciviruses in Swine

Viruses in three of the four established genera of the family Caliciviridae have been detected in pigs (Sapovirus, Norovirus and Vesivirus), making this animal species of particular interest in the study of calicivirus pathogenesis and host range. The Cowden strain of porcine enteric calicivirus (PEC), a sapovirus, was discovered in a diarrheic pig fecal sample in the US in 1980. Since then, sapoviruses have become recognized as a predominant calicivirus detected in pigs. The Cowden PEC strain grows efficiently in a unique cell culture system, and a reverse genetics system has been developed for elucidation of the mechanisms of replication and pathogenesis at the molecular level. Porcine noroviruses share genetic relatedness with those from humans, and recent studies have shown that pigs are susceptible to infection and mild diarrheal disease when experimentally challenged with related human norovirus strains. Research on porcine caliciviruses has yielded new insights into the mechanisms of pathogenesis, replication, and evolution of the family Caliciviridae.

Murine Norovirus Pathogenesis and Immunity

The first murine norovirus, murine norovirus 1 (MNV-1), was discovered in 2003. Since then, numerous murine norovirus strains have been identified and they were assigned a new genogroup in the genus Norovirus. Murine noroviruses share pathogenic properties with human noroviruses. Specifically, they are infectious orally, they spread between mice, and at least one strain, MNV-1, causes mild diarrhea in wild-type hosts. Furthermore, primary MNV-1 infection fails to elicit protection from a secondary challenge with homologous virus in at least some situations, which is similar to the lack of long-term protective immunity elicited by primary human norovirus infection. Investigators have now begun to extend basic knowledge of norovirus infection and immunity using this system. In particular, studies of murine norovirus infection have provided valuable information regarding the critical nature of innate immunity in controlling infection. Mice deficient in components of the interferon signaling pathway are highly susceptible to MNV-1-induced gastroenteritis, systemic infection, and ultimately death. The precise mechanisms by which interferon protects from serious murine norovirus infection are beginning to be elucidated and will provide potential antiviral targets for combating human norovirus infections. In addition, murine norovirus infection of mice provides a useful model with which to define conditions to elicit protective immunity, potentially providing important information for human norovirus vaccine design. For example, repeated exposure to high doses of MNV-1 may provide protection from mucosal re-infection.

Murine Norovirus Translation, Replication and Reverse Genetics

Murine norovirus, currently the only norovirus that replicates efficiently in tissue culture, has offered scientists the first chance to study the entire norovirus life cycle in the laboratory. In addition, the development of reverse genetics for murine norovirus has provided the ideal opportunity for researchers to determine how variation at the genetic level affects pathogenicity in the natural host. Despite differences in the diseases caused by human and murine noroviruses, they possess a significant amount of genetic similarity; hence the general mechanisms of viral genome translation and replication are likely to be highly conserved.

Rabbit Hemorrhagic Disease Virus and Other Lagoviruses

Rabbit hemorrhagic disease virus (RHDV) is a pathogen of rabbits that causes major problems throughout the world where rabbits are reared for food and clothing, make a significant contribution to ecosystem ecology, and where they support valued wildlife as a food source. The high mortality caused by RHDV has driven research in protecting rabbits from infection. However, RHDV is an unusual calicivirus in that it has served also as an important model in the family Caliciviridae by providing a range of beneficial outcomes as diverse as the creation of virus-like particles (VLPs) for vaccine and therapeutics delivery, the elucidation of calicivirus replication and structural features at the molecular level, and the biological control of a vertebrate pest.


Further reading