Norovirus

Human norovirus, previously known as Norwalk virus, was first identified in stool specimens collected during an outbreak of gastroenteritis in Norwalk, OH, and was the first viral agent shown to cause gastroenteritis (1). Illness due to this virus was initially described in 1929 as “winter vomiting disease” due to its seasonal predilection and the frequent preponderance of patients with vomiting as a primary symptom (2).

The 1968 outbreak that led to the identification of the virus affected 50% of students at an elementary school in Norwalk and manifested primarily as nausea, vomiting, diarrhea, and low-grade fever (3). Among primary cases, 98% complained of nausea, and 92% vomited, while 58% had abdominal cramps, 52% complained of lethargy, 38% had diarrhea, and 34% had fever. The occurrence of secondary cases in 32% of family contacts allowed the estimation of a 48-h incubation period. The illness lasted ∼24 h, with complete recovery in all cases.

While no pathogen could be identified in the Norwalk outbreak, oral administration of filtrates prepared from rectal swabs from affected individuals to healthy adult male prisoners at the Maryland House of Correction in Jessup, MD, resulted in symptoms in 2 of 3 subjects (4). In the 2 subjects who became ill, the incubation period was 48 h, as was the duration of symptoms. Mild diarrhea, with 4 to 6 loose stools, lasted 24 h, while low-grade fever lasted only 8 to 12 h. Other symptoms were anorexia, moderately severe abdominal cramping, malaise, headache, and nausea (but no vomiting). A complete return to health occurred by 96 h. The filtrate of a stool sample from 1 of the 2 symptomatic experimentally infected volunteers was then administered to a further 9 subjects, 7 of whom became ill. Two of the seven subjects vomited (one subject, who vomited ∼20 times in 24 h, required parenteral fluid replacement) but failed to develop diarrhea, while two had diarrhea without vomiting, and three had both diarrhea and vomiting. Four of the seven subjects had low-grade fever that lasted 8 to 12 h, and all seven subjects complained of malaise and headache. Symptoms resolved after a mean duration of 33 h.

While the index outbreak and the human volunteer studies that followed provided an accurate picture of the manifestations of norovirus infection in previously healthy children and adults at a time when the etiologic agent was unknown, it was an incomplete one. Further observations, discussed later in this review, have provided a more complex and nuanced view of the infection. Human noroviruses are the leading cause of epidemic gastroenteritis in all age groups and have been associated with high-profile outbreaks in hospitals, nursing homes, cruise ships, and the military (5, 6). It is estimated that each year, noroviruses are responsible for 64,000 diarrheal episodes requiring hospitalization, 900,000 clinic visits among children in industrialized nations, and ∼200,000 deaths of children <5 years old in the developing world (7).

The Norwalk virus agent (the original prototype virus is referred to as Norwalk virus in this review) was originally visualized by using immunoelectron microscopy (1), revealing 27-nm virus-like particles (Fig. 1). Efforts to cultivate the pathogen in cell culture and to develop an animal model were unsuccessful (8); therefore, the evolving literature focused on describing the physical characteristics of this small, round-structured virus in clinical specimens and on the serologic response to infection (9, 10). Although virion morphology, as well as protein and nucleic acid composition, was similar to that of members of the Caliciviridae family (10 – 13), clear taxonomic classification was not achieved until the whole genome sequence was obtained and compared to sequences from other caliciviruses (14, 15).

The Caliciviridae family of small, nonenveloped, positive-stranded RNA viruses is now comprised of five genera, including Norovirus, Sapovirus, Lagovirus, Nebovirus, and Vesivirus (16). The Norovirus and Sapovirus genera contain the human enteric viruses of the same names as well as a number of viruses that cause primarily enteric diseases in other animals, such as murine and canine noroviruses.

The human norovirus genome is composed of a linear, positive-sense RNA that is ∼7.6 kb in length (14). The genome is covalently linked to the viral protein genome (VPg) at the 5′ end and polyadenylated at the 3′ end (17). There are three open reading frames (ORFs), designated ORF-1, ORF-2, and ORF-3, encoding eight viral proteins (Fig. 2). ORF-2 and ORF-3 encode the structural components of the virion, viral protein 1 (VP1) and VP2, respectively. The mature virion contains 90 VP1 dimers assembled with icosahedral symmetry and arranged in such a fashion as to create hollows or cup-like structures on the virus surface. Hence, calici is derived from the Latin word calyx, or “cup” (16). ORF-1 encodes a polyprotein that is proteolytically processed into the 6 nonstructural proteins, including the norovirus protease and RNA-dependent RNA polymerase (17).

Norovirus has been likened to a “shape-shifter” (18), a mythical creature that can change form or being. This description refers to its diversity, with, as determined by the VP1 amino acid sequence, at least 6 genogroups (genogroup I [GI] to GVI) and >40 genotypes (18, 19), together with its continued evolution, apparently in response to the selective pressure exerted by the human immune system (20). Human norovirus infections are caused, in decreasing order of frequency, by GII (mostly GII.4), GI, and, to a very limited extent, GIV (some genotypes of which also infect pigs) (21, 22). In addition, antibodies to GIII, a bovine norovirus, have been detected in ∼22% of humans (23), and antibodies to GVI (a genogroup that has been identified only in canines) have been detected in 22.6% of small-animal veterinarians and 5.8% of age-matched controls (24).

The genetic diversity of the human noroviruses is apparent from the observation that VP1 amino acid sequences of GII.4 strains differ by 37% to 38% from the prototypic GI.1 Norwalk virus strain and by 5% to 7% within the GII genotype alone, with as much as a 2.8% difference between strains of an individual virus (19, 25). This variability, resulting from both recombination and mutational events, is of a sufficient magnitude that it has led to the suggestion that the use of sequence difference cutoffs is no longer sufficient to classify noroviruses and that the addition of a phylogenetic approach would prove more accurate (26).

The treatment of norovirus gastroenteritis is supportive, involving primarily the reversal of dehydration and electrolyte abnormalities. Antiemetics and antimotility agents may play a role in some patients.

In patients with persisting symptoms, especially neonates, the elderly, and the immunocompromised, the availability of specific therapy would be of value, but no therapy has been clearly demonstrated to be effective. However, a small, blind, placebo-controlled trial of children with viral gastroenteritis found that nitazoxanide administration was associated with a reduced duration of illness, including in a subset of patients with norovirus infection (85). Siddiq and colleagues reported resolution of diarrhea in a norovirus-infected patient with refractory acute myelogenous leukemia and HSCT after administration of nitazoxanide (86).

Enteric administration of human immunoglobulin was associated with resolution of chronic diarrhea in a transplant patient (87). Florescu and colleagues performed a matched case-control study to evaluate the potential benefit of orally administered human immunoglobulin in the treatment of symptomatic norovirus infection in immunocompromised patients. Cases received 25 mg/kg of body weight of human immunoglobulin (Gamunex; Talecris Biotherapeutics, Inc., NC, USA) every 6 h for a total of 8 doses (58). Although the median age of 24 total cases plus controls was 2 years, one-third were adults, and 20 were solid organ transplant recipients. Coinfection with Clostridium difficile or rotavirus was present in 8 patients. Administration of immunoglobulin was associated with a numerical decrease in stool output at 7 days (P = 0.09). However, while this 7-day interval was counted from the time of treatment in cases, it was counted from the time of diagnosis in controls, potentially leading to bias favoring immunoglobulin administration. Despite this potential bias, there was no significant difference in the time to resolution of diarrhea between cases and controls, with the duration being ∼12 days for each group. There was a numerically greater frequency of resolution of diarrhea in cases (P = 0.078), without a statistically or clinically significant difference in the total time to resolution of diarrhea or length of hospital stay. Looking toward a potential future therapy, Chen and colleagues developed a set of norovirus-specific monoclonal antibodies (88).

A reduction of immunosuppressive therapy is indicated whenever feasible. In one case report, a reduction in immunosuppressive therapy together with a change from a calcineurin inhibitor (tacrolimus) to an inhibitor of mTOR (sirolimus) was associated with resolution of chronic diarrhea in a double-transplant recipient (HSCT and lung) (89). Similarly, Engelen and colleagues altered the immunosuppressive therapy of a heart transplant recipient with chronic norovirus-associated diarrhea by substituting everolimus for tacrolimus, with subsequent prompt resolution of diarrhea (90).

Attempts at prophylaxis have been unsuccessful to date. Prophylactic administration of bovine lactoferrin failed to prevent diarrhea, including that due to norovirus, in children (91). In this randomized, placebo-controlled trial in Peru, 544 previously weaned children 12 to 18 months of age were visited daily for 6 months, and stool samples were collected monthly and during episodes of diarrhea. Norovirus nucleic acid was detected in 34.2% of stool samples from lactoferrin recipients and in 35.5% of stool samples from placebo recipients. The prophylactic administration of “probiotic fermented” milk containing a strain of Lactobacillus casei failed to prevent norovirus infection in elderly residents of a health service facility in Japan (92).

Norovirus is a well-described cause of epidemic gastroenteritis in both adult and pediatric populations across a wide range of geographic regions (93 – 97). The U.S. Centers for Disease Control and Prevention (CDC) estimates that norovirus is responsible for 60% of acute gastroenteritis cases (with a known cause), or 21 million cases, in the United States each year. With the addition of molecular methods, norovirus has also been increasingly implicated in sporadic disease (98). A systematic review of all reports of norovirus detected by reverse transcriptase PCR (RT-PCR) attributed 5 to 31% of cases of gastroenteritis in hospitalized patients and an additional 5 to 36% of cases in all patients seeking outpatient evaluation to norovirus (7). The CDC estimates that norovirus is responsible for 400,000 emergency department visits and 71,000 hospitalizations annually, although these numbers may be underestimates due to the limited number of patients who seek medical attention for viral gastroenteritis symptoms (99).

The general population is broadly vulnerable to disease across all age groups, but the majority of morbidity and mortality occurs at the extremes of age. The fecal-oral route is the main mode of transmission, although several other modalities have been described. These modalities include transmission via aerosolized viral particles in vomitus (100, 101) and through food, water, and environmental contamination (102). Some studies have associated certain norovirus genotypes with particular modes of transmission. For example, Vega and colleagues, reviewing norovirus trends in the United States from 2009 to 2013, demonstrated that GII.4 was more likely to be associated with person-to-person transmission, especially in long-term-care facilities (LTCFs) and hospital settings, whereas GI.7 and GII.12 were more frequently associated with foodborne disease (103). The environmental durability of norovirus leads to persistence of the pathogen in clinical settings and other closed-space environments, thus complicating complete disinfection and allowing for recurrent outbreaks (104, 105).

Norovirus outbreaks have been reported in a variety of settings and are uniquely suited to areas of close living quarters, shared dining facilities, and difficult environmental maintenance.

Rapid identification of an outbreak is the key to effective infection control. While molecular methods offer a definitive way to establish etiology, these diagnostic tests may not be available in some clinical settings due to time delays or resource limitations. Kaplan's clinical and epidemiologic criteria (Table 5), developed prior to the advent of molecular methods, can be used to rapidly identify norovirus outbreaks (168). The utility of Kaplan's criteria was reevaluated in 2006, and these criteria continued to prove useful in identifying norovirus outbreaks where molecular diagnostics were not easily accessible (169).

Several studies, with various degrees of evidence quality, on infection prevention and control practices to interrupt the transmission of norovirus in health care settings have been reported. Many of these results are summarized in the HICPAC (Healthcare Infection Control Practices Advisory Committee) guidelines for the prevention and control of norovirus gastroenteritis outbreaks in health care settings published in 2011 and in a recent review of norovirus infection control measures (233, 234). The three main strategic areas included staff and patient policy development, hand hygiene, and proper environmental disinfection. Despite the breadth of existing literature on norovirus outbreaks and mitigation strategies, a recent survey of infection preventionists showed clear room for improvement in their knowledge of both prevention and control practices (235).

An inability to cultivate norovirus in vitro until very recently and the absence of an animal model that closely resembles human disease, together with viral and human host diversity, have represented major obstacles to our understanding of the pathogenesis of and immune response to norovirus infection. As a consequence, much of our knowledge of the immunology of this highly species-specific virus revolves around studies of MNV and the use of virus-like particles (VLPs) of human norovirus. VLPs are the product of self-assembling viral structures derived from the expression of recombinant VP1. They are structurally and antigenically similar to their corresponding wild virions but lack genomic material.

There are strong public health and economic arguments in support of the potential benefits of the development of an effective norovirus vaccine. Using a simulation model, it was concluded that a vaccine with 50% protective efficacy for 12 months and costing US$50 would save US$1,000 to US$2,000 per case averted in the United States (347). Thus, despite the biological obstacles to vaccine development, which require dealing with the complex matrix of inherited human traits and variability among noroviruses (Table 10), the potential benefits would appear to be well worth the effort. Table 11 summarizes clinical studies on norovirus vaccines.

Most efforts at vaccine development are now focusing on the use of VLPs as the immunogen (348). GII.4 P particles are immunogenic in mice (284, 349), but evidence indicates that GII.4 VLPs are superior to homologous P particles (350). Furthermore, VLP vaccines have a successful history in the prevention of hepatitis B virus and of human papillomavirus infection (351).

Intravenous administration of Norwalk virus to chimpanzees led to the appearance of virus in feces (along with liver and duodenal and jejunal tissues), but all animals remained asymptomatic (25). Animals were resistant to rechallenge with Norwalk virus ≥4 months later, and this appeared to correlate with the antibody response to the virus. Intramuscular inoculation of other chimpanzees with VLPs derived from Norwalk virus protected against intravenous challenge with the same virus 6 and 18 months later, together with the development of blockade antibodies. These data provide evidence of homologous but not heterologous resistance and indicate that norovirus-derived VLP inoculation may produce homologous immunity.

Despite all the potential obstacles to the development of an effective vaccine, the interest is high, and by 2010, Herbst-Kralovetz and colleagues were able to list 12 phase 1 trials that had reported the immunogenicity of plant or insect cell-derived norovirus VLPs after mucosal delivery (352). Both oral and intranasal administrations have been shown to elicit antibody responses (353 – 355). El-Kamary and colleagues reported the results of two of these trials in that same year (355). These investigators administered GI.1-derived VLPs with the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MLA) and mucoadherent chitosan formulated as a dry powder by the intranasal route and found evidence of safety and dose-dependent immunogenicity, both mucosal and systemic (355). The majority of circulating IgA norovirus antigen-specific cells carried markers indicative of homing to mucosal tissues alone or to mucosal and lymphoid tissues. Furthermore, subjects developed significant norovirus IgA and IgG memory B-cell responses (356).

This GI.1-derived VLP vaccine was subsequently further evaluated. Healthy adults with a functional FUT2 gene, as evidenced by the presence of FUT2 detected by the presence of HBGAs in saliva, were randomized to receive either placebo or the adjuvanted GI.1 VLP vaccine (357). Two doses of each vaccine were administered intranasally 3 weeks apart. Participants were then challenged orally with a dose of Norwalk virus (48 RT-PCR units) that was ∼10-fold higher than the ID₅₀. The vaccine was well tolerated, and 70% of 47 vaccine recipients had a ≥4-fold increase in the serum concentration of Norwalk virus-specific IgA antibody levels, as measured by an enzyme immunoassay. Changes in IgG and IgM antibody levels were limited, as was the level of HBGA blocking antibody. The vaccine was modestly protective against infection after challenge, with 81% of placebo recipients showing evidence of infection compared to 62% (P = 0.05) of those who received the vaccine, and it also reduced virus-specific gastrointestinal symptoms (69% of placebo patients versus 37% of vaccine recipients; P = 0.009). A post hoc analysis found evidence that a serum HBGA blocking antibody titer of ≥1:200 was associated with a >50% relative reduction in the frequency of both viral infection and illness. In addition, antigen-specific memory T cells were generated in all subjects who received the highest dose (100 μg) of vaccine. Thus, this proof-of-concept trial demonstrated the ability of an intranasal vaccine to provide a degree of short-term protection against illness after homotypic challenge.

A systemically administered bivalent vaccine has also been evaluated in humans. Healthy adults were randomized to receive either placebo or a bivalent GI.1/GII.4-derived VLP vaccine with MLA and alum (358). The GII.4 component was a consensus product designed by alignment of the VP1 sequence of 3 GII.4 strains and was shown to be antigenically similar to GII.4 strains that have been circulating for 3 decades (359). Each vaccine was administered in 2 intramuscular doses at an interval of ≥28 days, and subjects were then challenged at least 28 days later with 4,000 RT-PCR genome equivalents of a heterologous GII.4 strain. The primary composite endpoint of reduction in any type of gastroenteritis or norovirus case ascertainment was not achieved. However, fewer of the vaccine recipients (n = 56) than placebo recipients (n = 48) reported vomiting and/or diarrhea of any severity after challenge (20% versus 41.7%; 52% reduction; P = 0.028).

There is interest in the development of norovirus vaccines to be given in combination with other immunogens. A vaccine containing human rotavirus recombinant VP6 as well as VLPs derived from GII.4, GII.12 New Orleans, and GI.3 was immunogenic for all components in BALB/c mice (360, 361). In addition, a bivalent norovirus/hepatitis E virus vaccine has been demonstrated to be immunogenic in mice (362).

Many factors will need to be effectively addressed for the development of a useful norovirus vaccine (318). Given our current understanding of the virus and the host response to it, such a vaccine will almost certainly be multivalent and, as is the case with influenza virus, may require frequent reformulations in response to viral evolution.

Norovirus is an important cause of morbidity due to acute gastroenteritis both within health care institutions and in the broader community. Although mortality is typically limited to the extremes of age, the disease exacts a significant toll on the health care system. Therapeutic management is usually supportive, and advances in molecular diagnostics may lead to the earlier identification of outbreaks and a reduction in person-to-person transmissions, particularly in vulnerable patient populations. Ongoing global reporting initiatives will be enhanced by improved diagnostic methods. Additionally, global control efforts will benefit from the growing knowledge of the clinical implications of various norovirus strains. Several advances into understanding the relationship among the viral strain, the host human blood group antigen type, and disease susceptibility have recently been elucidated, but this work has not yet been extended to clinical practice. The interplay of norovirus and host immunity still poses many unanswered questions. Areas of future research may overcome technical limitations, such as the inability to cultivate norovirus in vitro, and may elucidate a way to directly measure neutralizing antibodies, which could pave the way for vaccine development. The recent demonstration of infectability of B cells by the human norovirus GII.4 Sydney due to the presence of Enterobacter cloacae expressing H antigen may represent an important advance in our ability to understand norovirus replication (394). Several additional factors, such as inherited host variability, noroviral genogroup diversity, and ongoing viral evolution, will continue to complicate the process of vaccine development. Until a broadly effective, sustainable vaccine is developed, outbreak management will depend primarily on infection control efforts.