Jennifer L. Cannon

Surrogates for the Study of Norovirus Stability and Inactivation in the Environment: A Comparison of Murine Norovirus and Feline Calicivirus

Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values < 3 and > 9. FCV was more stable than MNV-1 at 56 degrees C, but both viruses exhibited similar inactivation at 63 and 72 degrees C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4 degrees C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.

Mechanisms of GII.4 norovirus persistence in human populations.

Background Noroviruses are the leading cause of viral acute gastroenteritis in humans, noted for causing epidemic outbreaks in communities, the military, cruise ships, hospitals, and assisted living communities. The evolutionary mechanisms governing the persistence and emergence of new norovirus strains in human populations are unknown. Primarily organized by sequence homology into two major human genogroups defined by multiple genoclusters, the majority of norovirus outbreaks are caused by viruses from the GII.4 genocluster, which was first recognized as the major epidemic strain in the mid-1990s. Previous studies by our laboratory and others indicate that some noroviruses readily infect individuals who carry a gene encoding a functional alpha-1,2-fucosyltransferase (FUT2) and are designated “secretor-positive” to indicate that they express ABH histo-blood group antigens (HBGAs), a highly heterogeneous group of related carbohydrates on mucosal surfaces. Individuals with defects in the FUT2 gene are termed secretor-negative, do not express the appropriate HBGA necessary for docking, and are resistant to Norwalk infection. These data argue that FUT2 and other genes encoding enzymes that regulate processing of the HBGA carbohydrates function as susceptibility alleles. However, secretor-negative individuals can be infected with other norovirus strains, and reinfection with the GII.4 strains is common in human populations. In this article, we analyze molecular mechanisms governing GII.4 epidemiology, susceptibility, and persistence in human populations. Methods and Findings Phylogenetic analyses of the GII.4 capsid sequences suggested an epochal evolution over the last 20 y with periods of stasis followed by rapid evolution of novel epidemic strains. The epidemic strains show a linear relationship in time, whereby serial replacements emerge from the previous cluster. Five major evolutionary clusters were identified, and representative ORF2 capsid genes for each cluster were expressed as virus-like particles (VLPs). Using salivary and carbohydrate-binding assays, we showed that GII.4 VLP-carbohydrate ligand binding patterns have changed over time and include carbohydrates regulated by the human FUT2 and FUT3 pathways, suggesting that strain sensitivity to human susceptibility alleles will vary. Variation in surface-exposed residues and in residues that surround the fucose ligand interaction domain suggests that antigenic drift may promote GII.4 persistence in human populations. Evidence supporting antigenic drift was obtained by measuring the antigenic relatedness of GII.4 VLPs using murine and human sera and demonstrating strain-specific serologic and carbohydrate-binding blockade responses. These data suggest that the GII.4 noroviruses persist by altering their HBGA carbohydrate-binding targets over time, which not only allows for escape from highly penetrant host susceptibility alleles, but simultaneously allows for immune-driven selection in the receptor-binding region to facilitate escape from protective herd immunity. Conclusions Our data suggest that the surface-exposed carbohydrate ligand binding domain in the norovirus capsid is under heavy immune selection and likely evolves by antigenic drift in the face of human herd immunity. Variation in the capsid carbohydrate-binding domain is tolerated because of the large repertoire of similar, yet distinct HBGA carbohydrate receptors available on mucosal surfaces that could interface with the remodeled architecture of the capsid ligand-binding pocket. The continuing evolution of new replacement strains suggests that, as with influenza viruses, vaccines could be targeted that protect against norovirus infections, and that continued epidemiologic surveillance and reformulations of norovirus vaccines will be essential in the control of future outbreaks.

Herd immunity to GII.4 noroviruses is supported by outbreak patient sera.

ABSTRACT Noroviruses (NoVs) of genogroup II, cluster 4 (GII.4), are the most common cause of outbreaks of acute gastroenteritis worldwide. During the past 13 years, GII.4 NoVs caused four seasons of widespread activity globally, each associated with the emergence of a new strain. In this report, we characterized the most recent epidemic strain, GII.4-2006 Minerva, by comparing virus-like particle (VLP) antigenic relationships and histo-blood group antigen (HBGA) binding profiles with strains isolated earlier. We also investigated the seroprevalence and specificity of GII.4 antibody in the years prior to, during, and following the GII.4 pandemic of 1995 and 1996 using a large collection of acute- and convalescent-phase serum pairs (n = 298) collected from 34 outbreaks. In a surrogate neutralization assay, we measured the blockade of HBGA binding using a panel of GII.4 VLPs representing strains isolated in 1987, 1997, 2002, and 2006 and a GII.3 VLP representing a strain isolated in the mid-1990s. Serum titers required for 50% HBGA blockade were compared between populations. In general, blockade of GII.4 VLP-HBGA binding was greater with convalescent-phase outbreak sera collected near the time of origin of the VLP strain. Heterotypic genotypes did not contribute to herd immunity against GII.4 NoVs based on their inability to block GII.4 VLP binding to HBGA. However, previous exposure to GII.4 NoV followed by infection by GII.3 NoV appeared to evoke an immune response to GII.4 NoV. These results support the hypothesis that herd immunity is a driving force for GII.4 evolution in the U.S. population. The data also suggest that complex patterns of cross-protection may exist across NoV genotypes in humans.

Environmental persistence and transfer of enteric viruses

Non-enveloped enteric viruses, such as Human Norovirus and Hepatitis A Virus, are readily transmitted via the fecal-oral route. Outbreaks are often prolonged due to the ability of these viruses to survive on environmental surfaces, on foods, and in water. Delineation of properties impacting enteric virus transfer and persistence in the environment has been the focus of several recent publications and is the topic of this review. Such information is important for modeling transmission scenarios, identifying risks of food-borne and water-borne virus contamination, and targeting prevention and control efforts for risk mitigation.

Histo-blood group antigen assay for detecting noroviruses in water.

ABSTRACT We evaluated a novel, magnetic-bead-based histo-blood group antigen assay for the recovery of low numbers of norovirus particles. Using this assay, with Norwalk virus seeded in environmental waters as a model, we were able to recover 30 to 300 genomic copies of the virus.

Presence of Antibodies against Genogroup VI Norovirus in Humans

BackgroundNoroviruses are important enteric pathogens in humans and animals. Recently, we reported a novel canine norovirus (CaNoV) in dogs with diarrhea belonging to a new genogroup (GVI). No data are available on exposure of humans to this virus.MethodsSera from 373 small animal veterinarians and 120 age-matched population controls were tested for IgG antibodies to CaNoV by a recombinant virus like particle based enzyme-linked immunosorbent assay.ResultsAntibodies to CaNoV were found in 22.3% of the veterinarians and 5.8% of the control group (p < 0.001). Mean corrected OD450 values for CaNoV antibodies were significantly higher in small animal veterinarians compared to the control group.ConclusionsThese findings suggest that CaNoV may infect humans and small animal veterinarians are at an increased risk for exposure to this virus. Additional studies are needed to assess if this virus is able to cause disease in humans.

Inactivation of salmonella in biofilms and on chicken cages and preharvest poultry by levulinic Acid and sodium dodecyl sulfate.

Surface contamination (skin and feathers) of broilers with Salmonella occurs primarily during growth and transportation. Immediately after transporting chickens, chicken cage doors were sprayed with a foam containing 3% levulinic acid plus 2% sodium dodecyl sulfate (SDS). Samples were collected for Salmonella assay after 45 min. Salmonella on cage doors was reduced from 19% (19 of 100 doors) before treatment to 1% (1 of 100 doors) after treatment, coliform counts were reduced from 6 to 8 to 2 to 4 log CFU/9 cm(2), and aerobic plate counts were reduced from 7 to 9 to 4 to 6 log CFU/9 cm(2). Whole chicken carcasses with feathers were inoculated with 10(8) CFU of Salmonella Enteritidis, soaked for 5 min at 21°C in 72 liters of a treatment or control solution, and assayed for Salmonella. Salmonella counts on chickens treated with water were 6.8 to 8.5 log CFU/9 cm(2), those treated with 50 ppm of calcium hypochlorite were 7.6 to 8.9 log CFU/9 cm(2), and those treated with 3% levulinic acid plus 2% SDS were <1.7 to 2.8 CFU/9 cm(2) (>4-log reduction). Results of biofilm studies on surfaces of various materials revealed that a 3% levulinic acid plus 2% SDS treatment used as either a foam or liquid for 10 min effectively reduced Salmonella populations by 5 and >6 log CFU/cm(2), respectively.

Human norovirus transfer to stainless steel and small fruits during handling.

Human noroviruses (NoVs) cause an estimated 58% of foodborne illnesses in the United States annually. The majority of these outbreaks are due to contamination by food handlers. The objective of this study was to quantify the transfer rate and degree of contamination that occurs on small fruits (blueberries, grapes, and raspberries) and food contact surfaces (stainless steel) when manipulated with NoV-contaminated hands. Human NoVs (genogroups I and II [GI and GII]) and murine norovirus (MNV-1) were inoculated individually or as a three-virus cocktail onto donor surfaces (gloved fingertips or stainless steel) and either immediately interfaced with one or more recipient surfaces (fruit, gloves, or stainless steel) or allowed to dry before contact. Viruses on recipient surfaces were quantified by real-time quantitative reverse transcriptase PCR. Transfer rates were 58 to 60% for GII NoV from fingertips to stainless steel, blueberries, and grapes and 4% for raspberries under wet conditions. Dry transfer occurred at a much lower rate (<1%) for all recipient surfaces. Transfer rates ranged from 20 to 70% from fingertips to stainless steel or fruits for the GI, GII, and MNV-1 virus cocktail under wet conditions and from 4 to 12% for all viruses under dry transfer conditions. Fomite transfer (from stainless steel to fingertip and then to fruit) was lower for all viruses, ranging from 1 to 50% for wet transfer and 2 to 11% for dry transfer. Viruses transferred at higher rates under wet conditions than under dry conditions. The inoculum matrix affected the rate of virus transfer, but the majority of experiments resulted in no difference in the transfer rates for the three viruses. While transfer rates were often low, the amount of virus transferred to recipient surfaces often exceeded 4- or 5-log genomic copy numbers, indicating a potential food safety hazard. Quantitative data such as these are needed to model scenarios of produce contamination by food handling and devise appropriate interventions to manage risk.

Genetic and Epidemiologic Trends of Norovirus Outbreaks in the United States from 2013 to 2016 Demonstrated Emergence of Novel GII.4 Recombinant Viruses

ABSTRACT Noroviruses are the most frequent cause of epidemic acute gastroenteritis in the United States. Between September 2013 and August 2016, 2,715 genotyped norovirus outbreaks were submitted to CaliciNet. GII.4 Sydney viruses caused 58% of the outbreaks during these years. A GII.4 Sydney virus with a novel GII.P16 polymerase emerged in November 2015, causing 60% of all GII.4 outbreaks in the 2015-2016 season. Several genotypes detected were associated with more than one polymerase type, including GI.3, GII.2, GII.3, GII.4 Sydney, GII.13, and GII.17, four of which harbored GII.P16 polymerases. GII.P16 polymerase sequences associated with GII.2 and GII.4 Sydney viruses were nearly identical, suggesting common ancestry. Other common genotypes, each causing 5 to 17% of outbreaks in a season, included GI.3, GI.5, GII.2, GII.3, GII.6, GII.13, and GII.17 Kawasaki 308. Acquisition of alternative RNA polymerases by recombination is an important mechanism for norovirus evolution and a phenomenon that was shown to occur more frequently than previously recognized in the United States. Continued molecular surveillance of noroviruses, including typing of both polymerase and capsid genes, is important for monitoring emerging strains in our continued efforts to reduce the overall burden of norovirus disease.

The Fate of Murine Norovirus and Hepatitis A Virus During Preparation of Fresh Produce by Cutting and Grating

Human noroviruses and hepatitis A virus (HAV) are commonly associated with outbreaks occurring in restaurant establishments and catered events. Food handlers are major contributing factors to foodborne illnesses initiated in the kitchen setting. In this study, transfer of HAV and murine norovirus (MNV-1), a human norovirus surrogate, between produce (cucumbers, strawberries, tomatoes, cantaloupes, carrots, and honeydew melons) and common kitchen utensils (graters and knives) was investigated. The extent of virus transfer to produce during utensil application, in the presence and the absence of food residue, and the impact of knife surface properties (sharp, dull, serrated) was also investigated. Transfer of MNV-1 and HAV from produce items, initially contaminated with ~5.5 log PFU, to knives and graters during application ranged from 0.9 to 5.1 log PFU. MNV-1 transfer to knives was the greatest for cucumbers, strawberries, and tomatoes, and the least for honeydew melons, while transfer of HAV to knives was greater for tomatoes and honeydew melons than strawberries, cantaloupes, and cucumbers. After preparation of a contaminated produce item, knife cross-contamination easily occurred as viruses were detected on almost all of the seven produce items successively prepared. Produce residues on utensils often resulted in less virus transfer when compared to utensils without residue accumulation. Knife surface properties did not impact virus transfer. The ease of virus transfer between produce and utensils demonstrated by the current study highlights the importance of efforts aimed toward preventing cross-contamination in the kitchen environment.