Christine L. Moe

Norwalk virus: How infectious is it?†

Noroviruses are major agents of viral gastroenteritis worldwide. The infectivity of Norwalk virus, the prototype norovirus, has been studied in susceptible human volunteers. A new variant of the hit theory model of microbial infection was developed to estimate the variation in Norwalk virus infectivity, as well as the degree of virus aggregation, consistent with independent (electron microscopic) observations. Explicit modeling of viral aggregation allows us to express virus infectivity per single infectious unit (particle). Comparison of a primary and a secondary inoculum showed that passage through a human host does not change Norwalk virus infectivity. We estimate the average probability of infection for a single Norwalk virus particle to be close to 0.5, exceeding that reported for any other virus studied to date. Infected subjects had a dose‐dependent probability of becoming ill, ranging from 0.1 (at a dose of 103 NV genomes) to 0.7 (at 108 virus genomes). A norovirus dose response model is important for understanding its transmission and essential for development of a quantitative risk model. Norwalk virus is a valuable model system to study virulence because genetic factors are known for both complete and partial protection; the latter can be quantitatively described as heterogeneity in dose response models. J. Med. Virol. 80:1468–1476, 2008.

Human susceptibility and resistance to Norwalk virus infection

Infectious diseases have influenced population genetics and the evolution of the structure of the human genome in part by selecting for host susceptibility alleles that modify pathogenesis. Norovirus infection is associated with ∼90% of epidemic non-bacterial acute gastroenteritis worldwide. Here, we show that resistance to Norwalk virus infection is multifactorial. Using a human challenge model, we showed that 29% of our study population was homozygous recessive for the α(1,2)fucosyltransferase gene (FUT2) in the ABH histo-blood group family and did not express the H type-1 oligosaccharide ligand required for Norwalk virus binding. The FUT2 susceptibility allele was fully penetrant against Norwalk virus infection as none of these individuals developed an infection after challenge, regardless of dose. Of the susceptible population that encoded a functional FUT2 gene, a portion was resistant to infection, suggesting that a memory immune response or some other unidentified factor also affords protection from Norwalk virus infection.

Binding of Norwalk Virus-Like Particles to ABH Histo-Blood Group Antigens Is Blocked by Antisera from Infected Human Volunteers or Experimentally Vaccinated Mice

ABSTRACT Attachment of Norwalk (NV), Snow Mountain (SMV), and Hawaii (HV) virus-like particles (VLPs) to specific ABH histo-blood group antigens was investigated by using human saliva and synthetic biotinylated carbohydrates. The three distinct Norwalk-like viruses (NLVs) have various capacities for binding ABH histo-blood group antigens, suggesting that different mechanisms for NLV attachment likely exist. Importantly, antisera from NV-infected human volunteers, as well as from mice inoculated with packaged Venezuelan equine encephalitis virus replicons expressing NV VLPs, blocked the ability of NV VLPs to bind synthetic H type 1, Leb, and H type 3, suggesting a potential mechanism for antibody-mediated neutralization of NV.

Cellular and humoral immunity following snow mountain virus challenge

ABSTRACT Little is known about the immune response to noroviruses. To elucidate the immunobiology of norovirus infection in humans, 15 volunteers were challenged with Snow Mountain virus (SMV), a genogroup 2 norovirus. We assessed the cellular and humoral immune response and infection by analyzing stool, serum, saliva, and peripheral blood mononuclear cell (PBMC) responses pre- and postchallenge. In contrast to Norwalk virus (NV), SMV infection was not dependent upon blood group secretor status. Nine of 15 volunteers were infected and showed a ≥4-fold increase over the prechallenge anti-SMV serum immunoglobulin G (IgG) titer, mostly subclass IgG1. Although serum IgG elicited by SMV infection was cross-reactive with Hawaii virus (HV), another genogroup 2 norovirus, salivary IgA was less cross-reactive. Neither SMV-elicited serum IgG nor salivary IgA cross-reacted with NV, a genogroup 1 norovirus. Significant increases in serum gamma interferon (IFN-γ) and IL-2, but not IL-6 or IL-10, were noted on day 2 postchallenge. For the majority of volunteers, both infected and uninfected, PBMCs stimulated with norovirus virus-like particles secreted IFN-γ and other Th1 cytokines, suggesting previous norovirus exposure in most volunteers. Like the IgG antibodies, the SMV-activated T cells were cross-reactive with HV but not NV. IFN-γ production was dependent upon CD4+ cells, consistent with a predominant, but not exclusive, Th1 response. To our knowledge, this is the first report characterizing T-cell and cytokine responses following live norovirus challenge.

A Field Study of the Microbiological Quality of Fresh Produce

The Centers for Disease Control and Prevention has reported that foodborne disease outbreaks associated with fruits and vegetables increased during the past decade. This study was conducted to characterize the routes of microbial contamination in produce and to identify areas of potential contamination from production through postharvest handling. We report here the levels of bacterial indicator organisms and the prevalence of selected pathogens in produce samples collected from the southern United States. A total of 398 produce samples (leafy greens, herbs, and cantaloupe) were collected through production and the packing shed and assayed by enumerative tests for total aerobic bacteria, total coliforms, total Enterococcus, and Escherichia coli. These samples also were analyzed for Salmonella, Listeria monocytogenes, and E. coli O157:H7. Microbiological methods were based on methods recommended by the U.S. Food and Drug Administration. For all leafy greens and herbs, geometric mean indicator levels ranged from 4.5 to 6.2 log CFU/g (aerobic plate count); less than 1 to 4.3 log CFU/g (coliforms and Enterococcus); and less than 1 to 1.5 log CFU/g (E. coli). In many cases, indicator levels remained relatively constant throughout the packing shed, particularly for mustard greens. However, for cilantro and parsley, total coliform levels increased during the packing process. For cantaloupe, microbial levels significantly increased from field through packing, with ranges of 6.4 to 7.0 log CFU/g (aerobic plate count); 2.1 to 4.3 log CFU/g (coliforms); 3.5 to 5.2 log CFU/g (Enterococcus); and less than 1 to 2.5 log CFU/g (E. coli). The prevalence of pathogens for all samples was 0, 0, and 0.7% (3 of 398) for L. monocytogenes, E. coli O157:H7, and Salmonella, respectively. This study demonstrates that each step from production to consumption may affect the microbial load of produce and reinforces government recommendations for ensuring a high-quality product.

Norovirus Infectivity in Humans and Persistence in Water

ABSTRACT To examine the long-term infectivity of human norovirus in water, 13 study subjects were challenged at different time points with groundwater spiked with the prototype human norovirus, Norwalk virus. Norwalk virus spiked in groundwater remained infectious after storage at room temperature in the dark for 61 days (the last time point tested). The Norwalk virus-seeded groundwater was stored for 1,266 days and analyzed, after RNase treatment, by reverse transcription-quantitative PCR (RT-qPCR) to detect Norwalk virus RNA contained within intact capsids. Norwalk virus RNA within intact capsids was detected in groundwater for 1,266 days, with no significant log10 reduction throughout 427 days and a significant 1.10-log10 reduction by day 1266. Purified Norwalk virus RNA (extracted from Norwalk virus virions) persisted for 14 days in groundwater, tap water, and reagent-grade water. This study demonstrates that Norwalk virus in groundwater can remain detectable for over 3 years and can remain infectious for at least 61 days. (ClinicalTrials.gov identifier NCT00313404.)

Improved detection of human enteric viruses in foods by RT-PCR.

Human enteric viruses (including hepatitis A virus (HAV) and Norwalk-like viruses (NLVs)) are now recognized as common causes of foodborne disease. While methods to detect these agents in clinical specimens have improved significantly over the last 10 years, applications to food samples have progressed more slowly. In an effort to improve the sensitivity and speed of virus detection from non-shellfish food commodities by reverse transcription-polymerase chain reaction (RT-PCR), we (i) evaluated multiple RNA extraction methods; (ii) compared alternative NLV primer sets; and (iii) developed a one-step RT-PCR method. Hamburger and lettuce samples, processed for virus concentration using a previously reported filtration-extraction-precipitation procedure, were inoculated with HAV or NV. Several RNA extraction methods (guanidinium isothiocyanate, microspin column, QIAshredder Homogenizer, and TRIzol) and primer pairs were compared for overall RNA yield (microg/ml), purity (A(260)/A(280)), and RT-PCR limits of detection. The use of TRIzol with the QIAshredder Homogenizer (TRIzol/Shred) yielded the best RT-PCR detection limits (<1 RT-PCR amplifiable units/reaction for NV), and the NVp110/NVp36 primer set was the most efficient for detecting NV from seeded food samples. A one-step RT-PCR protocol using the TRIzol/Shred extraction method and the NVp110/NVp36 or HAV3/HAV5 primer sets demonstrated improved sensitivity (>10-fold) over the routinely used two-step method. HAV RNA was detected by RT-PCR at initial inoculum levels corresponding to <10 and <100 PFU per 300 microl sample concentrate (corresponding to 6 g food sample) for hamburger and lettuce, respectively. NV RNA was detected by RT-PCR at initial inoculum levels <5 and <50 RT-PCR amplifiable units per 300 microl concentrate (corresponding to 6 g food sample) for hamburger and lettuce, respectively. Residual RT-PCR inhibitors were effectively removed as evidenced by the ability to detect viral RNA in food concentrates without prior dilution. The methods reported here show promise for rapid, sensitive detection of human enteric viruses in foods.

Norovirus Capture with Histo-Blood Group Antigens Reveals Novel Virus-Ligand Interactions

ABSTRACT Noroviruses are genetically diverse, uncultivable, positive-sense RNA viruses and are the most common cause of epidemic acute gastroenteritis in humans in the United States. Recent studies of norovirus attachment in vitro by using recombinant virus-like particles (VLPs) suggest that various norovirus strains exhibit different patterns of attachment to ABH histo-blood group antigens, which are carbohydrate epitopes present in high concentrations on mucosal cell surfaces of the gut. However, attachment of live norovirus strains to histo-blood group antigens has not been investigated to date. Utilizing a newly designed magnetic bead-virus capture method, we characterized histo-blood group antigen attachment properties of various norovirus strains obtained from clinical stool specimens to compare the attachment properties of wild-type virus and VLPs and to further map norovirus attachment. Consistent with previous reports using VLPs, various strains of noroviruses exhibited different patterns of attachment to histo- blood group antigens. Norwalk virus bound specifically to H type 1, H type 3, and Leb. Two genogroup II noroviruses, one representing the Toronto genotype and the other from a novel genotype, bound specifically to Leb. A Desert Shield-like strain did not attach to H types 1, 2, or 3, H type 1 and 3 precursors, Lea, or Leb. Surprisingly, wild-type Snow Mountain virus (SMV) attached specifically to H type 3, which contradicted previous findings with SMV VLPs. On further investigation, we found that stool components promote this attachment, providing the first known observation that one or more components of human feces could promote and enhance norovirus attachment to histo-blood group antigens.

Transmission of Norwalk Virus during a Football Game

BACKGROUND During a college football game in Florida, diarrhea and vomiting developed in many of the members of a North Carolina team. The next day, similar symptoms developed in some of the players on the opposing team. METHODS We interviewed those who ate the five meals served to the North Carolina team before the game and some of the players on the opposing team who became ill. Patients with primary cases were members or staff of the team who had vomiting or diarrhea at least 10 hours after but no more than 50 hours after eating a box lunch served the day before the game. Patients with secondary cases had a later onset of symptoms or had symptoms without having eaten the box lunch. Stool samples were examined by electron microscopy and by a reverse-transcription-polymerase-chain-reaction (RT-PCR) assay. RESULTS The two football teams shared no food or beverages and had no contact off the playing field. Of five meals served to the North Carolina team before the game, only the box lunch was associated with a significant risk of illness (relative risk of illness, 4.1; 95 percent confidence interval, 1.6 to 10.0). The rate of attack among those who ate the box lunch was 62 percent. There were 11 secondary cases among the members and staff of the North Carolina team and 11 such cases among the Florida players. All four stool samples obtained from North Carolina patients were positive for Norwalk-like virus on electron microscopy. All four samples as well as one of two stool samples from players on the Florida team were positive for a Norwalk-like virus of genogroup I on RT-PCR assay; the RT-PCR products had identical sequences. CONCLUSIONS This investigation documents person-to-person transmission of Norwalk virus among players during a football game. Persons with acute gastroenteritis should be excluded from playing contact sports.

Effectiveness of Liquid Soap and Hand Sanitizer against Norwalk Virus on Contaminated Hands

ABSTRACT Disinfection is an essential measure for interrupting human norovirus (HuNoV) transmission, but it is difficult to evaluate the efficacy of disinfectants due to the absence of a practicable cell culture system for these viruses. The purpose of this study was to screen sodium hypochlorite and ethanol for efficacy against Norwalk virus (NV) and expand the studies to evaluate the efficacy of antibacterial liquid soap and alcohol-based hand sanitizer for the inactivation of NV on human finger pads. Samples were tested by real-time reverse transcription-quantitative PCR (RT-qPCR) both with and without a prior RNase treatment. In suspension assay, sodium hypochlorite concentrations of ≥160 ppm effectively eliminated RT-qPCR detection signal, while ethanol, regardless of concentration, was relatively ineffective, giving at most a 0.5 log10 reduction in genomic copies of NV cDNA. Using the American Society for Testing and Materials (ASTM) standard finger pad method and a modification thereof (with rubbing), we observed the greatest reduction in genomic copies of NV cDNA with the antibacterial liquid soap treatment (0.67 to 1.20 log10 reduction) and water rinse only (0.58 to 1.58 log10 reduction). The alcohol-based hand sanitizer was relatively ineffective, reducing the genomic copies of NV cDNA by only 0.14 to 0.34 log10 compared to baseline. Although the concentrations of genomic copies of NV cDNA were consistently lower on finger pad eluates pretreated with RNase compared to those without prior RNase treatment, these differences were not statistically significant. Despite the promise of alcohol-based sanitizers for the control of pathogen transmission, they may be relatively ineffective against the HuNoV, reinforcing the need to develop and evaluate new products against this important group of viruses.