Food, Bioprocessing, and Nutrition Sciences
Non Technical Summary
Human noroviruses are the most important cause of acute gastroenteritis, with about 21 million illnesses occurring every year in the United States. These viruses are most often spread between people, and also via the food supply. Hands and surfaces contaminated with virus are important contributors to disease spread. There are many reasons it is so difficult to control norovirus transmission, i.e., (i) the viruses are released in the millions in vomit and feces, but it takes only a few to cause disease; (ii) they persist in the environment for weeks; and (iii) noroviruses are very resistant to most routinely used sanitizers and disinfectants. Because scientists cannot grow these viruses in the laboratory, it is very difficult to study, detect, or evaluate methods to kill norovirus.The purpose of this project is to develop better detection methods, and to use those to evaluate how well commonly used hand sanitizers and surface disinfectants work against noroviruses. This work will be done in three phases, i.e., (i) use the tools of molecular biology to develop better detection methods; (ii) validate that those methods work; and (iii) apply the methods to determine which sanitizers and disinfectants are best at killing noroviruses. Identifying the most effective products helps the food and healthcare industries to use the best methods to control norovirus in their settings; it also provides consumers with some guidance for protecting themselves and their families. Such work will reduce the numbers of cases of norovirus illness and improve the health of the American public.
Animal Health Component
Research Effort Categories
Goals / Objectives
The purpose of this project is to develop a better means by which to detect and discriminate human norovirus infectivity status, and then apply these methods to evaluate the efficacy of hand sanitizers and surface disinfectants intended for use in food handling environments. Specific objectives are:Objective 1. Develop and optimize novel ligand-bound integrated detection methods as a means by which to discriminate human norovirus infectivity status;Objective 2. Characterize the performance of the novel ligand-bound methods by comparing them to other infectivity determination methods (e.g., RNase-RT-qPCR, integrated antibody and HBGA capture) using one inactivation strategy targeting virus capsid integrity only (heat) and one targeting both capsid and genome integrity (free chlorine); andObjective 3. Use the methods evaluated above, in conjunction with protein analysis and transmission electron microscopy, to evaluate the efficacy and mechanism of action of candidate surface disinfectants and hand sanitizers.
Representative human norovirus strains, two cultivable human norovirus surrogates (murine norovirus and Tulane virus), and a select group of Virus-Like Particles (VLPs) will be used in these studies. Viruses will be detected by RT-qPCR (human strains and surrogates), mammalian cell culture infectivity assay (surrogates), and/or enzyme-linked capture-detection assays (VLPs). One or more forms of each of these methods are documented in the literature. RNA extraction will be done using a modification of guanidinium-silica based methods.In Objective 1, two novel assays to detect noroviruses, will be developed. Both of these will use nucleic acid aptamers previously reported by our group. The first approach will focus on development and optimization of integrated capture-detection methods to evaluate capsid integrity using aptamers and, for comparison purposes, histoblood group antigens (HBGAs) and strain-specific antibodies. The initial ligand binding assays will be done with VLPs and linked to ELISA-type assays for detection. Assay designs will be optimized by investigation of variables associated with reagents and concentrations, blocking and washing conditions, among others. To the extent possible, these assays will be designed to be semi-quantitative, and may be converted to a bead-based capture format so as to be compatible with RT-qPCR detection, making them fully quantitative.The second novel method for infectivity discrimination will target both capsid and genome integrity, and is based on the use of aptamers targeting a different viral protein, VPg. This protein is linked to one end of the viral genome, and is necessary for infection. Briefly, the method will begin with ligand binding (HBGA, antibody, or aptamer) to assure that capsid integrity is intact. This is followed by a combined RNase/proteinase K digestion, which removes incompletely packaged and free viral RNA. The capsid is then lysed to release genomic RNA, and subjected to aptamer-based capture of the VPg, and then amplified by RT-qPCR. Again, substantial optimizations will be necessary. Because this method is directly linked to RT-qPCR, it should be fully quantitative. For both assay types, the goal is to produce methods showing the highest degree of analytical sensitivity with low background, high resolution, and wide detection range.Once the two novel ligand-bound integrated detection methods have been developed, their performance will be validated using cultivable surrogate viruses (Tulane and murine norovirus) in Objective 2. Two treatment types will be evaluated, i.e., heat (which destroys the virus capsid) and chlorine (which destroys the capsid and at high concentrations, impacts the viral genome). Exposure variables (time-temperature or concentration-time, respectively) will be chosen in accordance with the literature. The assays will be done in suspension format and after treatment and neutralization, samples will be evaluated for remaining virus titer by plaque assay; traditional RT-qPCR and RT-qPCR preceded by an RNase treatment; traditional integrated-RT-qPCR methods (using HBGA and/or antibody ligands); and the novel integrated methods (using aptamer ligands) developed in Objective 1. Quantitative or semi-quantitative results will be expressed as log inactivation as a function of temperature and time, or free chlorine concentration and contact time. By establishing correlations between the different methods to discriminate infectious from non-infectious virus, and comparing them directly to infectivity assay, we anticipate being able to identify the most promising infectivity discrimination methods.Of course, the real interest is the use of commercially feasible methods to inactivate human noroviruses. In Objective 3, a variety of disinfectants and sanitizers (having active ingredients of ethanol, quaternary ammonium compounds, hydrogen peroxide, chlorine dioxide, hypochlorous acid, and metal cations, among others) will be evaluated for anti-noroviral efficacy in parallel studies using Tulane virus and human noroviruses/VLPs. Combinations of molecular assays (RT-qPCR); integrated receptor-binding-RT-qPCR assays (both traditional and novel assays described above); protein degradation (SDS-PAGE and Western blot); and structural visualization (transmission electron microscopy) will be used to provide compelling information on degree of destruction of infectious virus particles, and the mechanism of action for each compound tested.Initial screening of product efficacy will be done in suspension as per the ASTM Standard Test Method E 1052-9. These studies, using virus inoculum both with and without addition of 5% soil load, will allow for preliminary evaluation of the impact of contact time and filth on the efficacy of the disinfectant or sanitizer formulation itself, without consideration of application method. This aids in establishing experimental parameters for further studies. Once these parameters have been established, ASTM Standard Test Method E1053-11 will be used to evaluate surface efficacy, or ASTM Standard Test Method E2276-10 to evaluate the anti-noroviral efficacy of hand sanitizers. Studies using human subjects will undergo NCSU Institutional Review Board (IRB) approval prior to initiation.All experiments will be replicated at least in triplicate. Statistical analysis will be performed by one-way ANOVA with multiple comparisons with Tukey's HSD being used for separation of means. A p value of < 0.05 will be considered statistically significant.