Chlorine inactivation of human norovirus,murine norovirus and poliovirus in drinking water

Aims: To evaluate the reduction of human norovirus (HuNoV) by chlorine disinfection under typical drinking water treatment conditions. Methods and Results: HuNoV, murine norovirus (MNV) and poliovirus type 1 (PV1) were inoculated into treated water before chlorination, collected from a drinking water treatment plant, and bench‐scale free chlorine disinfection experiments were performed for two initial free chlorine concentrations, 0·1 and 0·5 mg l^?1. Inactivation of MNV reached more than 4 log₁₀ after 120 and 0·5 min contact time to chlorine at the initial free chlorine concentrations of 0·1 and 0·5 mg l^?1, respectively. Conclusions: MNV was inactivated faster than PV1, and there was no significant difference in the viral RNA reduction rate between HuNoV and MNV. The results suggest that appropriate water treatment process with chlorination can manage the risk of HuNoV infection via drinking water supply systems. Significance and Impact of the Study: The data obtained in this study would be useful for assessing or managing the risk of HuNoV infections from drinking water exposure.     

A Broadly Reactive One-Step SYBR Green I Real-Time RT-PCR Assay for Rapid Detection of Murine Norovirus

A one-step SYBR Green I real-time RT-PCR assay was developed for the detection and quantification of a broad range of murine noroviruses (MNVs). The primer design was based on the multiple sequence alignments of 101 sequences of the open reading frame (ORF)1−ORF2 junction of MNV. The broad reactivity and quantitative capacity of the assay were validated using 7 MNV plasmids. The assay was completed within 1 h, and the reliable detection limit was 10 copies of MNV plasmid or 0.063 median tissue culture infective doses per milliliter of RAW264 cell culture-propagated viruses. The diagnostic performance of the assay was evaluated using 158 mouse fecal samples, 91 of which were confirmed to be positive. The melting curve analysis demonstrated the diversity of MNV in the samples. This is the first report of a broadly reactive one-step SYBR Green I real-time RT-PCR assay for detecting of MNVs. The rapid and sensitive performance of this assay makes it a powerful tool for diagnostic applications.     

Development and evaluation of a broadly reactive reverse transcription recombinase polymerase amplification assay for rapid detection of murine norovirus

Background

Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV.

Results

The detection limit of the RT-RPA assay for the detection of MNV was 1?×?10² copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1?×?10² copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA.

Conclusions

A broadly reactive RT-RPA assay was successfully established for MNV detection.

     

Selection,Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.     

Abiotic Stress and Phyllosphere Bacteria Influence the Survival of Human Norovirus and Its Surrogates on Preharvest Leafy Greens

Foodborne outbreaks of human noroviruses (HuNoVs) are frequently associated with leafy greens. Because there is no effective method to eliminate HuNoV from postharvest leafy greens, understanding virus survival under preharvest conditions is crucial. The objective of this study was to evaluate the survival of HuNoV and its surrogate viruses, murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV), on preharvest lettuce and spinach that were subjected to abiotic stress (physical damage, heat, or flood). We also examined the bacteria culturable from the phyllosphere in response to abiotic stress and in relation to viral persistence. Mature plants were subjected to stressors 2 days prior to inoculation of the viruses on leaves. We quantified the viral RNA, determined the infectivity of the surrogates, and performed bacterial counts on postinoculation days (PIDs) 0, 1, 7, and 14. For both plant types, time exerted significant effects on HuNoV, MNV, SaV, and TV RNA titers, with greater effects being seen for the surrogates. Infectious surrogate viruses were undetectable on PID 14. Only physical damage on PID 14 significantly enhanced HuNoV RNA persistence on lettuce, while the three stressors differentially enhanced the persistence of MNV and TV RNA. Bacterial counts were significantly affected by time and plant type but not by the stressors. However, bacterial counts correlated significantly with HuNoV RNA titers on spinach and with the presence of surrogate viruses on both plant types under various conditions. In conclusion, abiotic stressors and phyllosphere bacterial density may differentially influence the survival of HuNoV and its surrogates on lettuce and spinach, emphasizing the need for the use of preventive measures at the preharvest stage.     

In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!

Human noroviruses (HuNoV), a major cause of acute gastroenteritis worldwide, cannot be readily cultured in the lab. Therefore, a feline calicivirus (FCV) is often used as its surrogate to, among other things, test alcohol-based handrubs (ABHR). The more recent laboratory culture of a mouse norovirus (MNV) provides an alternative. While MNV is closer to HuNoV in several respects, to date, no comparative testing of FCV and MNV survival and inactivation on human hands has been performed. This study was designed to address the knowledge gap. The rates of loss in viability during drying on hands were −1.91 and −1.65% per minute for FCV and MNV, respectively. When the contaminated skin was exposed for 20 s to either a commercial ABHR with 62% (v/v) ethanol or to 75% (v/v) ethanol in water, FCV infectivity was reduced by <1 log₁₀ while that of MNV by nearly 2.8 log₁₀. Extending the contact time to 30 s reduced the FCV titer by almost 2 log₁₀ by both test substances and that of MNV by >3.5 log₁₀ by the commercial ABHR while 75% ethanol did not show any noticeable improvement in activity as compared to the 20 s contact. An 80% (v/v) aqueous solution of ethanol gave only a 1.75 log₁₀ reduction in MNV activity after 20 s. The results show significant differences in the ethanol susceptibility of FCV and MNV in contact times relevant to field use of ABHR and also that 62% ethanol was a more effective virucide than either 75% or 80% ethanol. These findings indicate the need for a review of the continuing use of FCV as a surrogate for HuNoV.     

Prevalence of human <Emphasis Type="Italic">Norovirus</Emphasis> by genotype in contaminated groundwater in Korea over the last decade (2007–2016)

This study investigated the occurrence of human Norovirus (HuNoV) by genotype in 1,486 groundwater samples collected from 843 groundwater wells suspected of contamination during 2007–2016, in South Korea. We identified and genotyped 186 HuNoV sequences in 178 HuNoV-positive samples using the RIVM-NoroNet norovirus genotyping tool (NGT) and phylogenetic tree analysis based on RIVM-NoroNet reference sequences. HuNoV GII was more prevalent than GI. The major genotypes detected were HuNoV GII.4 (43.0%), GII.22 (15.6%), GI.5 (10.2%), and GI.1 (8.6%); several genotypes accounted for < 5.0% of all HuNoVs, including GII.17, GI.6, GI.4, GII.6, GI.8, GII.3, GII.13, GI.3, GI.7, GI.2, GI.9, GII.1, GII.8, and GII.10. The prevalence of HuNoVs and number of genotypes detected has drastically decreased over the last decade. HuNoV GII.17, the emerging genotype worldwide including Europe and Asia, appeared in Korean groundwater from 2010, dominated in 2013–2014, and continued to be observed. HuNoV GII.4, the major type occurred last decade from Korean groundwater except 2013–2014, continued to be detected and prevalent similar to HuNoV GII.17 in 2016.     

Development of a concentration method for detection of tobacco mosaic virus in irrigation water

Tobacco mosaic virus (TMV) causes significant yield loss in susceptible crops irrigated with contaminated water. However, detection of TMV in water is difficult owing to extremely low concentrations of the virus. Here, we developed a simple method for the detection and quantification of TMV in irrigation water. TMV was reliably detected at concentrations as low as 10 viral copies/μL with real-time PCR. The sensitivity of detection was further improved using polyethylene glycol 6000 (PEG6000, MW 6000) to concentrate TMV from water samples. Among the 28 samples from Shaanxi Province examined with our method, 17 were tested positive after virus concentration. Infectivity of TMV in the original water sample as well as after concentration was confirmed using PCR. The limiting concentration of TMV in water to re-infect plants was determined as 10² viral copies/mL. The method developed in this study offers a novel approach to detect TMV in irrigation water, and may provide an effective tool to control crop infection.     

Development of a Quantitative Competitive Reverse Transcription Polymerase Chain Reaction (QC-RT–PCR) for Detection and Quantitation of Chikungunya Virus

Chikungunya is one of the most important emerging arboviral infections of public health significance. Due to lack of a licensed vaccine, rapid diagnosis plays an important role in early management of patients. In this study, a QC-RT–PCR assay was developed to quantify Chikungunya virus (CHIKV) RNA by targeting the conserved region of E1 gene. A competitor molecule containing an internal insertion was generated, which provided a stringent control of the quantification process. The introduction of 10-fold serially diluted competitor in each reaction was further used to determine sensitivity. The applicability of this assay for quantification of CHIKV RNA was evaluated with human clinical samples, and the results were compared with real-time quantitative RT–PCR. The sensitivity of this assay was estimated to be 100 RNA copies per reaction with a dynamic detection range of 10² to 10¹⁰ copies. Specificity was confirmed using closely related alpha and flaviviruses. The comparison of QC-RT–PCR result with real-time RT–PCR revealed 100% concordance for the detection of CHIKV in clinical samples. These findings demonstrated that the reported assay is convenient, sensitive and accurate method and has the potential usefulness for clinical diagnosis due to simultaneous detection and quantification of CHIKV in acute-phase serum samples.     

A novel simple one-step single-tube RT-duplex PCR method with an internal control for detection of bovine viral diarrhoea virus in bulk milk, blood, and follicular fluid samples.

A simple one-step single-tube RT-PCR method was developed for detection of bovine viral diarrhea virus (BVDV) in bulk milk, blood and follicular fluid samples. A set of universal primers (UTR DL1/4) was designed for RT-PCR to detect BVDV type I and II viruses simultaneously and was tested for efficacy in comparison to published primers for two type strains, 42 field isolates, and 95 diagnostic samples. The sensitivity (100%) and specificity (96.2%) of the RT-PCR assay, with the universal primers for 95 diagnostic samples, were equal to those of virus isolation. An internal control targeting a bovine actin gene was also included in the same reaction tube to monitor RNA preparation and RT-PCR procedure. A total of 632 specimens (378 bulk milk, 140 blood, and 112 follicular samples) were tested in the year 2000 by the RT-duplex PCR assay in parallel with virus isolation. The one-step single-tube RT-duplex PCR with the universal primers UTR DL1/4 was sensitive, specific, less complicated and cost effective for detection of BVDV in various types of specimens.     

Quantification of enterovirus RNA in sludge samples using single tube real-time RT-PCR

We have developed a quantitative RT-PCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man technology with the ABI Prism 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5' noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 x 10(7)). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.     

Ultrasensitive Norovirus Detection Using DNA Aptasensor Technology

DNA aptamers were developed against murine norovirus (MNV) using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Nine rounds of SELEX led to the discovery of AG3, a promising aptamer with very high affinity for MNV as well as for lab-synthesized capsids of a common human norovirus (HuNoV) outbreak strain (GII.3). Using fluorescence anisotropy, AG3 was found to bind with MNV with affinity in the low picomolar range. The aptamer could cross-react with HuNoV though it was selected against MNV. As compared to a non-specific DNA control sequence, the norovirus-binding affinity of AG3 was about a million-fold higher. In further tests, the aptamer also showed nearly a million-fold higher affinity for the noroviruses than for the feline calicivirus (FCV), a virus similar in size and structure to noroviruses. AG3 was incorporated into a simple electrochemical sensor using a gold nanoparticle-modified screen-printed carbon electrode (GNPs-SPCE). The aptasensor could detect MNV with a limit of detection of approximately 180 virus particles, for possible on-site applications. The lead aptamer candidate and the aptasensor platform show promise for the rapid detection and identification of noroviruses in environmental and clinical samples.