Pengbo Liu

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.

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.

Hollow-fiber ultrafiltration for simultaneous recovery of viruses, bacteria and parasites from reclaimed water.

Hollow-fiber ultrafiltration (UF) is a technique that has been reported to be effective for recovering a diverse array of microbes from water, and may also be potentially useful for microbial monitoring of effluent from water reclamation facilities. However, few data are available to indicate the potential limitations and efficacy of the UF technique for treated wastewater. In this study, recovery efficiencies were determined for various options available for performing the tangential-flow UF technique, including hollow-fiber ultrafilter (i.e., dialyzer) type, ultrafilter pre-treatment (i.e., blocking), and elution. MS2 and ΦX174 bacteriophages, Clostridium perfringens spores, Escherichia coli, and Cryptosporidium parvum oocysts were seeded into 10-L reclaimed water samples to evaluate UF options. Then a single UF protocol was established and studied using seeded and non-seeded 100-L samples from two water reclamation facilities in Georgia, USA. Baxter Exeltra Plus 210 and Fresenius F200NR dialyzers were found to provide significantly higher microbial recovery than Minntech HPH 1400 hemoconcentrators. The selected final UF method incorporated use of a non-blocked ultrafilter for UF followed by elution using a surfactant-based solution. For 10-L samples, this method achieved recovery efficiencies of greater than 50% recovery of seeded viruses, bacteria, and parasites. There was no significant difference in overall microbial recovery efficiency when the method was applied to 10- and 100-L samples. In addition, detection levels for pathogens in seeded 100-L reclaimed water samples were 1000 PFU HAV, 10,000 GI norovirus particles, <500 Salmonella and <200 Cryptosporidium oocysts. These data demonstrate that UF can be an effective technique for recovering diverse microbes in reclaimed water to monitor and improve effluent water quality in wastewater treatment plants.

Laboratory evidence of norwalk virus contamination on the hands of infected individuals.

ABSTRACT Human norovirus (NoV) outbreak investigations suggest that the hands of infected individuals play an important role in NoV transmission. However, there is no experimental evidence documenting the likelihood and degree of NoV contamination on hands. As part of a clinical trial designed to evaluate the efficacy of high-pressure processing for Norwalk virus (NV) inactivation in oysters, 159 hand rinse samples were collected from 6 infected and 6 uninfected subjects. NV was concentrated from the samples by polyethylene glycol precipitation, followed by RNA extraction using an automated guanidinium isothiocyanate-silica method. NV RNA was detected and quantified using multiple NV-specific reverse transcription-quantitative PCR (RT-qPCR) assays. A total of 25.4% (18/71) of the hand rinse samples collected from 6 infected volunteers were presumptively positive for NV, with an average of 3.86 log10 genomic equivalent copies (GEC) per hand. Dot blot hybridization of PCR products obtained using a different primer set, and DNA sequencing of selected amplicons, provided further confirmation of the presence of NV in the hand rinses. NV contamination was also detected in two hand rinse samples obtained from one uninfected subject. These findings provide definitive evidence of NV contamination on the hands of infected subjects observed under controlled clinical research conditions. Such data support the need for better hand hygiene strategies to prevent NoV transmission.

Norovirus infection in immunocompromised children and children with hospital-acquired acute gastroenteritis

Norovirus (NoV) is a leading cause of acute viral gastroenteritis among children, yet its burden of disease among immunocompromised hosts and its role in hospital acquired infections (HAI) is not well characterized. To determine the prevalence, genotypes, and NoV loads among immunocompromised children and children with HAI, residual stool samples, and clinical data were collected at two major pediatric hospitals in metropolitan Atlanta from 92 children that were immunocompromised and/or had a hospital acquired acute gastroenteritis. NoV was identified in 16.3% (15/92) of all stool specimens; 23.4% (11/47) in immunocompromised only children, and 13.3% (4/30) in children with HAI. All NoV positive cases were genogroup II (GII), and GII.4 was the predominant strain followed by GII.3, GII.12, and GII.13. The average NoV load for immunocompromised patients was 6.3 ± 1.4 log genome equivalent copies (GEC) per gram of stool compared to 5.8 ± 1.1 log GEC among patients with HAI. NoV infections are common among immunocompromised children and children with hospital‐acquired gastroenteritis, underscoring the urgent need for rapid NoV detection system, and highlighting the importance of strict hospital hygiene practices. J. Med. Virol. 86:1203–1209, 2014.

Quantification of Norwalk virus inocula: Comparison of endpoint titration and real-time reverse transcription-PCR methods

Human noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis. In order to fully characterize features such as persistence and infectious dose, precise quantification of virus concentration is necessary. The purpose of this study was to compare two methods [endpoint titration RT‐PCR and quantitative RT‐PCR (RT‐qPCR)] with respect to quantification of Norwalk virus (NV) in inocula made from purified stock suspensions of human fecal specimens. A full‐length NV RNA transcript was developed to facilitate quantification using RT‐qPCR and provided log linear detection in the range of 49–4.9 × 104 genome equivalent copies (GEC) per reaction. Endpoint titration RT‐PCR was used to estimate PCR detection units, and RT‐qPCR was used to estimate genome copies in two NV inocula (8fIIa and 8fIIb) used in previous human challenge studies. Overall, RT‐qPCR was 1.1–1.6 log10 more sensitive (lower detection limit) than endpoint titration RT‐PCR when the same RNA release method, PCR primers and thermocycle program were used. These findings have important implications for many experimental interpretations, not the least of which is estimating the median infectious dose in human challenge studies. J. Med. Virol. 82:1612–1616, 2010.

Persistence of Norwalk virus, male-specific coliphage, and Escherichia coli on stainless steel coupons and in phosphate-buffered saline.

Human noroviruses (NoVs) are a leading cause of acute gastroenteritis and are frequently transmitted by contaminated food, water, hands, and environmental surfaces. Little is known about their environmental stability and/or which alternative microorganisms can serve as effective surrogates. To examine whether Escherichia coli and male-specific coliphage MS2 can be appropriate surrogates for NoVs, approximately 6.8 log genomic equivalent copies of Norwalk virus (NV), and 6.0 to 6.5 log PFU or CFU of MS2 and E. coli, respectively, were inoculated onto stainless steel coupons and held at 4°C, room temperature (RT), or 37°C over a period of 75 min (E. coli and MS2) to 4 weeks. These three microorganisms were also seeded into phosphate-buffered saline (PBS) and sampled at different time intervals for up to 6 weeks. MS2 and E. coli survived approximately 15 min at 37°C, 45 min at RT, and 60 min at 4°C on the stainless steel surfaces. In contrast, NV RNA titers were reduced by only 2.4 log at 37°C, 1.5 log at RT, and 0.9 log at 4°C after 4 weeks. MS2 and E. coli were able to survive at least 5 weeks in PBS at 4°C and RT, and NV was stable in PBS at 4°C and RT for at least 6 weeks. However, E. coli, MS2, and NV were completely inactivated after 1-, 4-, and 5-week incubations in PBS at 37°C, respectively. These findings indicate that NoVs are highly persistent on environmental surfaces and in PBS solution at different temperatures. While E. coli does not appear to be an appropriate surrogate for NoVs, MS2 could be more relevant for modeling the environmental persistence of NoVs under wet conditions, but not under dry conditions.

Genetic susceptibility to norovirus GII.3 and GII.4 infections in Chinese pediatric diarrheal disease.

Background: Noroviruses (NoVs) are a leading cause of viral diarrhea in young children. Secretor status has been confirmed to be linked with Norwalk virus (NoV GI.1) infection but there is limited information about whether secretor genotypes are associated with pediatric NoV epidemic strains in vivo. Methods: In this study, fecal specimens and serum samples were collected from 124 hospitalized children with acute diarrhea in Xi’an, China. TaqMan real-time reverse transcription polymerase chain reaction was used to detect NoVs in fecal samples, and NoV-positive samples were further verified using conventional reverse transcription polymerase chain reaction and sequenced. DNA was extracted from sera and TaqMan single-nucleotide polymorphism genotyping assay was applied to determine the FUT2 A385T polymorphism. Results: Only NoV GII.3 and GII.4 genotypes were found in NoV-positive samples, and NoVs were detected in 25% (15/60), 40.5% (17/42) and 9.1% (2/22) of children with homozygous secretor genotype (Se385Se385), heterozygous secretor genotype (Se385se385) and homozygous weak secretor genotype (se385se385), respectively. Children with secretor genotypes Se385Se385 and Se385se385 were significantly (P < 0.05) more susceptible to combined NoV GII.3 and GII.4 infections than children with weak secretor genotype se385se385. Conclusions: These findings indicate that secretor positivity is significantly associated with GII.3 and GII.4 infections in Chinese pediatric diarrheal disease and the weak secretor phenotype does not completely protect children from GII.3 and GII.4 infections.

Immunomagnetic separation combined with RT-qPCR for determining the efficacy of disinfectants against human noroviruses.

Little is known about the effectiveness of disinfectants against human noroviruses (NoV) partially because human NoV cannot be routinely cultured in laboratory. The objective of this study was to develop a NoV monoclonal antibody-conjugated immunomagnetic separation (IMS) procedure combined with real-time reverse transcription polymerase chain reaction (RT-qPCR) assays to study the in vitro efficacy of disinfectants against human NoV. Monoclonal antibodies against Norwalk virus (NV, GI.1) and NoV GII.4 were produced using unique NoV capsid proteins, and the antibodies were conjugated to magnetic Dynalbeads. The immunomagnetic beads were used to simultaneously capture intact NoV in samples and effectively remove PCR inhibitors. We examined the efficacy of ethanol, sodium hypochlorite, nine commercially available disinfectants, and one prototype disinfectant using the IMS/RT-qPCR. The sensitivity of this procedure was approximately 100 virus particles for both the NV and GII.4 viruses. The average log reductions in in vitro activities varied between disinfectants. The prototype disinfectant produced an average 3.19-log reduction in NV and a 1.38-log reduction in GII.4. The prototype disinfectant is promising of inactivating NoV. This method can be used to evaluate in vitro activity of disinfectants against human NoV. The IMS/RT-qPCR method is promising as an effective method to remove PCR inhibitors in disinfectants and enable the evaluation of the efficacy of disinfectants.

Urban sanitation coverage and environmental fecal contamination: Links between the household and public environments of Accra, Ghana.

Exposure to fecal contamination in public areas, especially in dense, urban environments, may significantly contribute to enteric infection risk. This study examined associations between sanitation and fecal contamination in public environments in four low-income neighborhoods in Accra, Ghana. Soil (n = 72) and open drain (n = 90) samples were tested for E. coli, adenovirus, and norovirus. Sanitation facilities in surveyed households (n = 793) were categorized by onsite fecal sludge containment (“contained” vs. “uncontained”) using previous Joint Monitoring Program infrastructure guidelines. Most sanitation facilities were shared by multiple households. Associations between spatial clustering of household sanitation coverage and fecal contamination were examined, controlling for neighborhood and population density (measured as enumeration areas in the 2010 census and spatially matched to sample locations). E. coli concentrations in drains within 50m of clusters of contained household sanitation were more than 3 log-units lower than those outside of clusters. Further, although results were not always statistically significant, E. coli concentrations in drains showed consistent trends with household sanitation coverage clusters: concentrations were lower in or near clusters of high coverage of household sanitation facilities—especially contained facilities—and vice versa. Virus detection in drains and E. coli concentrations in soil were not significantly associated with clustering of any type of household sanitation and did not exhibit consistent trends. Population density alone was not significantly associated with any of the fecal contamination outcomes by itself and was a significant, yet inconsistent, effect modifier of the association between sanitation clusters and E. coli concentrations. These findings suggest clustering of contained household sanitation, even when shared, may be associated with lower levels of fecal contamination within drains in the immediate public domain. Further research is needed to better quantify these relationships and examine impacts on health.