Pathogenesis of noroviruses, emerging RNA viruses

Abstract

Human noroviruses in the family Caliciviridae are a major cause of epidemic gastroenteritis. They are responsible for at least 95% of viral outbreaks and over 50% of all outbreaks worldwide. Transmission of these highly infectious plus-stranded RNA viruses occurs primarily through contaminated food or water, but also through person-to-person contact and exposure to fomites. Norovirus infections are typically acute and self-limited. However, disease can be much more severe and prolonged in infants, elderly, and immunocompromised individuals. Norovirus outbreaks frequently occur in semi-closed communities such as nursing homes, military settings, schools, hospitals, cruise ships, and disaster relief situations. Noroviruses are classified as Category B biodefense agents because they are highly contagious, extremely stable in the environment, resistant to common disinfectants, and associated with debilitating illness. The number of reported norovirus outbreaks has risen sharply since 2002 suggesting the emergence of more infectious strains. There has also been increased recognition that noroviruses are important causes of childhood hospitalization. Moreover, noroviruses have recently been associated with multiple clinical outcomes other than gastroenteritis. It is unclear whether these new observations are due to improved norovirus diagnostics or to the emergence of more virulent norovirus strains. Regardless, it is clear that human noroviruses cause considerable morbidity worldwide, have significant economic impact, and are clinically important emerging pathogens. Despite the impact of human norovirus-induced disease and the potential for emergence of highly virulent strains, the pathogenic features of infection are not well understood due to the lack of a cell culture system and previous lack of animal models. This review summarizes the current understanding of norovirus pathogenesis from the histological to the molecular level, including contributions from new model systems.

Keywords: calicivirus; norovirus; pathogenesis.

Figure 1

Norovirus outbreak characteristics. The Centers for Disease Control collected data from 232 norovirus outbreaks between July 1997 and June 2000. The results of this surveillance are summarized here for the source of virus (A) and the type of setting affected by the outbreak (B). Data are reproduced from http://www.cdc.gov/ncidod/dvrd/revb/gastro/norovirus-factsheet.htm.

Figure 2

The intestinal cell tropism of HS66 in gnotobiotic pigs and calves. (A) Paraffin-embedded sections of the duodenum of HS66-infected gnotobiotic pigs prepared 4 dpi were stained with a monoclonal antibody against the GII human norovirus capsid protein (called NS14). Fluorescently conjugated anti-mouse secondary antibody facilitated visualization of capsid through immunofluorescent confocal microscopy (in green). Nuclei were stained with propidium iodide (in red). Data are reproduced from J. Virol. 2006, 80, 10372–10381. (B) Paraffin-embedded sections of the jejunum of HS66-infected gnotobiotic calves prepared 3 dpi were stained with NS14. Viral capsid was visualized through immunohistochemistry. Data are reproduced from J. Virol. 2008, 82, 1777–1786.

Figure 3

The intestinal cell tropism of MNV-1 in wild-type and STAT1^−/− mice. Intestinal sections prepared from MNV-1-infected 129SvEv (A) or STAT1^−/− (B and C) mice were stained with guinea pig polyclonal antibody raised against the MNV-1 Pro:Pol nonstructural protein. Fluorescently conjugated secondary antibody was then used to visualize viral protein through immunofluoresence, in which viral protein is pseudocolored in green and nuclei in blue. Data are reproduced from J. Virol. 2007, 81, 3251–3262.

Figure 4

The HBGA binding interface on norovirus particles is genogroup-specific. Crystal structures have been determined for P dimers of a representative GI norovirus (A) and a representative GII norovirus (B) complexed with HBGA. Surface models are shown here, with P dimers colored gray (one monomer is darker gray than the other) and the three major components of the HBGA binding interface colored green, red, and orange. The HBGA is colored yellow. While the binding sites on both viruses lie within the exposed P2 domain, the exact residues and HBGA binding mode differ. Moreover, note that the HBGA binding interface of Norwalk virus is located within a single monomer while the interface of VA387 spans both monomers. Data are reproduced from PLoS ONE 2009, 4, e5058.

Figure 5

The known carbohydrate binding partners of caliciviruses. At least one member from four of the six calicivirus genera (colored in purple) has been demonstrated to bind a carbohydrate, suggesting that the original calicivirus ancestor was a carbohydrate binder. Similarly, three of the five norovirus genogroups (colored in blue) have been shown to contain members that bind carbohydrates. Two of the three norovirus genogroups containing human members have been shown to specifically bind HBGAs, either A/B/H antigens (A/B) or Lewis antigens (Lewis). Representative virus strains and their known carbohydrate ligands are shown in orange. Data are adapted from PLoS ONE 2009, 4, e5058.

Figure 6

Norovirus genomic organization and protein function. (A) Norovirus genomes are comprised of a single linear piece of positive-sense RNA between 7.4–7.7 kb. They contain three open reading frames, one encoding a polyprotein of 7 nonstructural protein products (colored in green), one encoding the major structural capsid protein VP1 (colored in orange), and one encoding the minor structural protein VP2 (colored in purple). There is a short overlap between ORFs 1 and 2. A subgenomic mRNA that is 3’ co-terminal with full-length genomes is produced during norovirus replication. This mRNA acts as a template for the production of structural proteins; similar to genomic RNA, it is covalently linked to VPg at its 5’ end and polyadenylated at its 3’ end. (B) Known and hypothesized functions of mature norovirus proteins.

Figure 7

Mucosal norovirus immunity wanes over time. Groups of 129SvEv mice (5–14 mice per group) were mock-infected (1°) or inoculated perorally with 10⁴ pfu MNV-1.CW3 (2°). Six weeks or nine months later, all mice were infected with 10⁷ pfu MNV-1.CW3. One day after secondary challenge, animals were perfused, organs were harvested, and viral burden was determined by plaque assay. The data for all mice in each group are averaged. Limits of detection are indicated by dashed lines. Fold-reductions for titers in mice receiving secondary challenge compared to those in mice receiving primary challenge are listed above the black bars.

Figure 8

Epochal evolution of the GII.4 noroviruses. Early GII.4 norovirus strains (Camberwell and Grimsby) persisted for long periods and were followed by shorter periods of stasis before being replaced by a new dominant strain. The evolution of GII.4 viruses appears to have become much more rapid in recent years, with pandemic strains being replaced by a new dominant strain in 1–2 years. FH, Farmington Hills; M, Minerva.