Evaluation of a Surface Sampling Method for Recovery of Human Noroviruses Prior to Detection Using Reverse Transcription Quantitative PCR.

Human noroviruses are the most common cause of acute viral gastroenteritis, and the environmental persistence of these viruses contributes to their transmissibility. Environmental sampling is thus an important tool for investigating norovirus outbreaks and for assessing the effectiveness of cleaning and decontamination regimens. The purpose of this study was to evaluate a sampling material (wipes) for their efficacy at recovering human norovirus from hard surfaces and foods. Dilutions of a human norovirus GII.4 stool specimen derived from an outbreak were applied to hard surfaces (stainless steel and ceramic) and the surfaces of representative foods (green pepper, apple, tomato, and cheese). The viruses were recovered at various times postinoculation using the wipes, followed by RNA extraction and reverse transcription quantitative PCR. Recovery efficiency ranged from 74% to almost 100% for all artificially inoculated hard surfaces and for most fresh produce surfaces. Less efficient recovery was observed for cheese. Viral RNA could be recovered from select surfaces for up to 7 days postinoculation, with a <1 log reduction in genome copy number. In field tests, 24 (11%) of 210 environmental samples collected during winter 2012 from restrooms in North Carolina were presumptively positive for human norovirus, and six of these samples were confirmed as GII.4 by sequencing. These wipes may be a valuable tool for investigations of norovirus outbreaks and studies of norovirus prevalence.

Aerosolization of a Human Norovirus Surrogate, Bacteriophage MS2, during Simulated Vomiting.

Human noroviruses (NoV) are the leading cause of acute gastroenteritis worldwide. Epidemiological studies of outbreaks have suggested that vomiting facilitates transmission of human NoV, but there have been no laboratory-based studies characterizing the degree of NoV release during a vomiting event. The purpose of this work was to demonstrate that virus aerosolization occurs in a simulated vomiting event, and to estimate the amount of virus that is released in those aerosols. A simulated vomiting device was constructed at one-quarter scale of the human body following similitude principles. Simulated vomitus matrices at low (6.24 mPa*s) and high (177.5 mPa*s) viscosities were inoculated with low (108 PFU/mL) and high (1010 PFU/mL) concentrations of bacteriophage MS2 and placed in the artificial "stomach" of the device, which was then subjected to scaled physiologically relevant pressures associated with vomiting. Bio aerosols were captured using an SKC Biosampler. In low viscosity artificial vomitus, there were notable differences between recovered aerosolized MS2 as a function of pressure (i.e., greater aerosolization with increased pressure), although this was not always statistically significant. This relationship disappeared when using high viscosity simulated vomitus. The amount of MS2 aerosolized as a percent of total virus "vomited" ranged from 7.2 x 10-5 to 2.67 x 10-2 (which corresponded to a range of 36 to 13,350 PFU total). To our knowledge, this is the first study to document and measure aerosolization of a NoV surrogate in a similitude-based physical model. This has implications for better understanding the transmission dynamics of human NoV and for risk modeling purposes, both of which can help in designing effective infection control measures.

Persistence of human norovirus RT-qPCR signals in simulated gastric fluid.

Human noroviruses (HuNoV) are a leading cause of foodborne disease and are known to be environmentally persistent. Foods usually become contaminated by contact with fecal material, both on hands and on surfaces. Emerging evidence suggests that HuNoVs are also shed and potentially aerosolized during projectile vomiting, resulting in another source of contamination. The purpose of this study was to compare the persistence of HuNoV in vomitus-like material (simulated gastric fluid, SGF, pH 2.5) to that in a pH neutral buffer (phosphate buffered saline, PBS, pH 7.4) in suspension and on surfaces. Human fecal suspensions containing two HuNoV strains (GI.1 and GII.4) were suspended in SGF and PBS. Suspension and surface samples were held at room temperature, and subsamples were collected from both samples for a period up to 42 days. Subsamples were subjected to RNA isolation, with and without inclusion of an RNase pre-treatment, followed by RT-qPCR amplification. In suspension assays, the genome copy number of HuNoV GII.4 decreased by ≤1.0-1.3 log10 over 42 days, irrespective of suspension buffer. On stainless steel, there was virtually no reduction in HuNoV GII.4 RT-qPCR signal over the 42-days experimental period, regardless of suspension buffer. Overall, the GI.1 RT-qPCR signal dropped more precipitously. In most cases, there were no statistically significant differences (p > 0.05) between persistence in solution or on surfaces when comparing RT-qPCR assays with and without prior RNase treatment. This study suggests that HuNoV suspended in vomitus-like material can persist for long periods, a likely contributor to foodborne transmission.