Advanced simulations and screening to repurposing a 3C protease inhibitor against the rupintrivir-resistant human norovirus-induced gastroenteritis.

Human norovirus (HuNoV) causes acute viral gastroenteritis in all age groups, and dehydration and severe diarrhea in the elderly. The World Health Organization reports ∼1.45 million deaths from acute gastroenteritis annually in the world. Rupintrivir, an inhibitory medicine against the human rhinovirus C3 protease, has been reported to inhibit HuNoV 3C protease. However, several HuNoV 3C protease mutations have been revealed to reduce the susceptibility of HuNoV to rupintrivir. The structural details behind rupintrivir-resistance of these single-point mutations (A105V and I109V) are not still clear. Hence, in this study, a combination of computational techniques were used to determine the rupintrivir-resistance mechanism and to propose an inhibitor against wild-type and mutant HuNoV 3C protease through structure-based virtual screening. Dynamic structural results indicated the unstable binding of rupintrivir at the cleft binding site of the wild-type and mutant 3C proteases, leading to its detachment. Our findings presented that the domain II of the HuNoV 3C protease had a critical role in binding of inhibitory molecules. Binding energy computations, steered molecular dynamics and umbrella sampling simulations confirmed that amentoflavone, the novel suggested inhibitor, strongly binds to the cleft site of all protease models and has a good structural stability in the complex system along the molecular dynamic simulations. Our in silico study proposed the selected compound as a potential inhibitor against the HuNoV 3C protease. However, additional experimental and clinical studies are required to corroborate the therapeutic efficacy of the compound.

Norovirus, Hepatitis A and SARS-CoV-2 surveillance within Chilean rural wastewater treatment plants based on different biological treatment typologies.

During the COVID-19 pandemic, wastewater from WWTPs became an interesting source of epidemiological surveillance. However, there is uncertainty about the influence of treatment type on virus removal. The aim of this study was to assess viral surveillance within wastewater treatment plants (WWTPs) based on different biological treatments. Seasonal monitoring (autumn-winter and spring-summer) was conducted in 10 Chilean rural WWTPs, which were based on activated sludge, aerated lagoons, bio-discs, constructed wetlands, vermifilters and mixed systems. Viruses were measured (influent/effluent) by the RT-qPCR technique, using a commercial kit for SARS-CoV-2, NoV GI, NoV GII, and HAV. The detection of SARS-CoV-2 viral variants by genotyping was performed using SARS-CoV-2 Mutation Assays (ThermoFisher Scientific, USA). JC polyomavirus detection (control), as well as a qPCR technique. Results showed that SARS-CoV-2, NoV GI and GII were detected in influents at values between <5 and 462, 0 to 28, and 0 to 75 GC/mL, respectively. HAV was not detected among the studied WWTPs. The monitored WWTPs removed these viruses at percentages between 0 and 100 %. WWTPs based on activated sludge with bio-discs demonstrated to be the most efficient at removing SARS-CoV-2 (up to 98 %) and NoV GI and GII (100 %). Meanwhile, bio-discs technologies were the least efficient for viral removal, due to biofilm detachment, which could also adsorb viral aggregates. A correlation analysis established that solids, pH, and temperature are the most influential parameters in viral removal. Wastewater-based surveillance at WWTP allowed for the detection of Omicron before the Chilean health authorities notified its presence in the population. In addition, surveillance of viruses and other microorganisms could help assess the potential public health risk of wastewater recycling.

Assessing the Removal Efficiency of Murine Norovirus 1, Hepatitis A Virus, and Human Coronavirus 229E on Dish Surfaces Through General Wash Program of Household Dishwasher.

The performance of dishwashers in removing live viruses is an important informative value in practical applications. Since foodborne viruses are present in contaminated food surfaces and water environments. Insufficient washing of dishes typically makes a carrier of foodborne viruses. Dishwashers have shown excellent performance in removing bacterial pathogens, but very limited reports related to eliminate foodborne viruses on contaminated dish surfaces. Here, murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and human coronavirus 229E (HCoV-229E) were experimentally inoculated on the dish surfaces (plate, rice bowl, and soup bowl). Plaque assay, 50% tissue culture infectious dose (TCID50), and real-time quantitative polymerase chain reaction (RT-qPCR) were conducted to determine their removal efficiency of them through the general wash program of household dishwashers. Using titration assay, MNV-1 and HAV were reduced by 7.44 and 6.57 log10 PFU/dish, and HCoV-229E was reduced by 6.43 log10 TCID50/dish through the general wash program, achieving a ≥ 99.999% reduction, respectively. Additionally, RT-qPCR results revealed that viral RNA of MNV-1 and HCoV-229E reduced 5.02 and 4.54 log10 genome copies/dish; in contrast, HAV was not detected on any dish surfaces. This study confirmed the performance of household dishwashers in removing pathogenic live viruses through the general wash program. However, residual viral RNA was not sufficiently removed. Further studies are needed to determine whether the viral RNA can be sufficiently removed using combination programs in household dishwashers.

Expanding the Pathogen Panel in Wastewater Epidemiology to Influenza and Norovirus.

Since the start of the 2019 pandemic, wastewater-based epidemiology (WBE) has proven to be a valuable tool for monitoring the prevalence of SARS-CoV-2. With methods and infrastructure being settled, it is time to expand the potential of this tool to a wider range of pathogens. We used over 500 archived RNA extracts from a WBE program for SARS-CoV-2 surveillance to monitor wastewater from 11 treatment plants for the presence of influenza and norovirus twice a week during the winter season of 2021/2022. Extracts were analyzed via digital PCR for influenza A, influenza B, norovirus GI, and norovirus GII. Resulting viral loads were normalized on the basis of NH4-N. Our results show a good applicability of ammonia-normalization to compare different wastewater treatment plants. Extracts originally prepared for SARS-CoV-2 surveillance contained sufficient genomic material to monitor influenza A, norovirus GI, and GII. Viral loads of influenza A and norovirus GII in wastewater correlated with numbers from infected inpatients. Further, SARS-CoV-2 related non-pharmaceutical interventions affected subsequent changes in viral loads of both pathogens. In conclusion, the expansion of existing WBE surveillance programs to include additional pathogens besides SARS-CoV-2 offers a valuable and cost-efficient possibility to gain public health information.

Evaluation of two different concentration methods for surveillance of human viruses in sewage and their effects on SARS-CoV-2 sequencing.

During the current COVID-19 pandemic, wastewater-based epidemiology (WBE) emerged as a reliable strategy both as a surveillance method and a way to provide an overview of the SARS-CoV-2 variants circulating among the population. Our objective was to compare two different concentration methods, a well-established aluminum-based procedure (AP) and the commercially available Maxwell® RSC Enviro Wastewater TNA Kit (TNA) for human enteric virus, viral indicators and SARS-CoV-2 surveillance. Additionally, both concentration methods were analyzed for their impact on viral infectivity, and nucleic acids obtained from each method were also evaluated by massive sequencing for SARS-CoV-2. The percentage of SARS-CoV-2 positive samples using the AP method accounted to 100 %, 83.3 %, and 33.3 % depending on the target region while 100 % positivity for these same three target regions was reported using the TNA procedure. The concentrations of norovirus GI, norovirus GII and HEV using the TNA method were significantly greater than for the AP method while no differences were reported for rotavirus, astrovirus, crAssphage and PMMoV. Furthermore, TNA kit in combination with the Artic v4 primer scheme yields the best SARS-CoV-2 sequencing results. Regarding impact on infectivity, the concentration method used by the TNA kit showed near-complete lysis of viruses. Our results suggest that although the performance of the TNA kit was higher than that of the aluminum procedure, both methods are suitable for the analysis of enveloped and non-enveloped viruses in wastewater by molecular methods.

Cluster of Norovirus Genogroup IX Outbreaks in Long-Term Care Facilities, Utah, USA, 2021.

We report 5 clustered acute gastroenteritis outbreaks in long-term care facilities in Utah, USA, that were linked to healthcare employees working at multiple facilities. Four outbreaks were caused by norovirus genotype GIX. We recommend continued norovirus surveillance and genotyping to determine contributions of this genotype to norovirus outbreaks.

Virus contamination and infectivity in beach environment: Focus on sand and stranded material.

To assess the exposure of beachgoers to viruses, a study on seawater, sand, and beach-stranded material was carried out, searching for human viruses, fecal indicator organisms, and total fungi. Moreover, for the first time, the genome persistence and infectivity of two model viruses was studied in laboratory-spiked sand and seawater samples during a one-week experiment. Viral genome was detected in 13.6 % of the environmental samples, but it was not infectious (Human Adenovirus - HAdV, and enterovirus). Norovirus and SARS-CoV-2 were not detected. The most contaminated samples were from sand and close to riverine discharges. In lab-scale experiments, the infectivity of HAdV5 decreased by ~1.5-Log10 in a week, the one of Human Coronavirus-229E disappeared in <3 h in sand. The genome of both viruses persisted throughout the experiment. Our results confirm viral contamination of the beach and suggest HAdV as an index pathogen for beach monitoring and quantitative risk assessment.

Changes in norovirus incidence in Victoria, Australia, during the COVID-19 pandemic, 2020-2021.

There were 142 norovirus positive outbreaks in Victoria for the 2020-2021 calendar years; however, almost half of these (48.6%) occurred in Q1 (January-March) of 2021. For the two-year period, 69.0% of all norovirus positive outbreaks were in childcare settings, and the predominant genotype was GII.P16/GII.2 (64.9%) followed by GII.P31/GII.4_2012 (20.9%). Norovirus incidence was particularly low in 2020 (n = 26) and close to average in 2021 (n = 116), but genotype diversity was low in both years. With the thought that 2022 will approach a more normal aspect to socialising and travel, norovirus incidence in 2022 may be predicted to increase above typical levels.

Impact of COVID-19 Nonpharmaceutical Interventions on the Extent of Norovirus Contamination in Oyster Production Areas in Ireland during Winter 2020 to 2021.

ABSTRACT: A significant decrease in norovirus prevalence and concentration was observed in oyster production areas in Ireland during winter 2020 to 2021. Oyster production areas impacted by human wastewater discharges that had been undergoing norovirus surveillance since 2018 were investigated. Samples collected in the winter seasons of 2018 to 2019 and 2019 to 2020, prior to when the COVID-19 pandemic interventions were applied, showed a prevalence of 94.3 and 96.6%, respectively, and geometric mean concentrations of 533 and 323 genome copies per g, respectively. These values decreased significantly during the winter of 2020 to 2021 (prevalence of 63.2% and geometric concentration of below the limit of quantification), coinciding with the control measures to mitigate the transmission of severe acute respiratory syndrome coronavirus 2 of the genus Betacoronavirus. Divergence between norovirus GI and GII prevalence and concentrations was observed over the 3-year monitoring period. Norovirus GII was the dominant genogroup detected in winter 2020 to 2021, with over half of samples positive, although concentrations detected were significantly lower than prepandemic winters, with a geometric mean concentration of below the limit of quantification.

Of masks and methylene blue-The use of methylene blue photochemical treatment to decontaminate surgical masks contaminated with a tenacious small nonenveloped norovirus.

BACKGROUND: In the context of the SARS-CoV-2 pandemic, reuse of personal protective equipment, specifically that of medical face coverings, has been recommended. The reuse of these typically single-use only items necessitates procedures to inactivate contaminating human respiratory and gastrointestinal pathogens. We previously demonstrated decontamination of surgical masks and respirators contaminated with infectious SARS-CoV-2 and various animal coronaviruses via low concentration- and short exposure methylene blue photochemical treatment (10 µM methylene blue, 30 minutes of 12,500-lux red light or 50,000 lux white light exposure). METHODS: Here, we describe the adaptation of this protocol to the decontamination of a more resistant, non-enveloped gastrointestinal virus and demonstrate efficient photodynamic inactivation of murine norovirus, a human norovirus surrogate. RESULTS: Methylene blue photochemical treatment (100 µM methylene blue, 30 minutes of 12,500-lux red light exposure) of murine norovirus-contaminated masks reduced infectious viral titers by over four orders of magnitude on surgical mask surfaces. DISCUSSION AND CONCLUSIONS: Inactivation of a norovirus, the most difficult to inactivate of the respiratory and gastrointestinal human viruses, can predict the inactivation of any less resistant viral mask contaminant. The protocol developed here thus solidifies the position of methylene blue photochemical decontamination as an important tool in the package of practical pandemic preparedness.

Norovirus Intestinal Infection and Lewy Body Disease in an Older Patient with Acute Cognitive Impairment.

We present a case report on an older woman with unspecific symptoms and predominant long-term gastrointestinal disturbances, acute overall health deterioration with loss of autonomy for daily activities, and cognitive impairment. Autopsy revealed the presence of alpha-synuclein deposits spread into intestinal mucosa lesions, enteric plexuses, pelvic and retroperitoneal nerves and ganglia, and other organs as well as Lewy pathology in the central nervous system (CNS). Moreover, we isolated norovirus from the patient, indicating active infection in the colon and detected colocalization of norovirus and alpha-synuclein in different regions of the patient's brain. In view of this, we report a concomitant norovirus infection with synthesis of alpha-synuclein in the gastrointestinal mucosa and Lewy pathology in the CNS, which might support Braak's hypothesis about the pathogenic mechanisms underlying synucleinopathies.

Rotavirus and Norovirus Infections in Children Under 5 Years Old with Acute Gastroenteritis in Southwestern China, 2018-2020.

OBJECTIVE: Rotaviruses and noroviruses are important causes of acute gastroenteritis in children. While previous studies in China have mainly focused on rotavirus, we investigated the incidence of norovirus in addition to rotavirus in Southwestern China. METHODS: From January 2018 to December 2020, cases of rotavirus or norovirus infections among children under five ages with acute gastroenteritis were evaluated retrospectively. RESULTS: The detection rate of rotavirus was 24.5% (27,237/111,070) and norovirus was 26.1% (4649/17,797). Among 17,113 cases submitted for dual testing of both rotavirus and norovirus, mixed rotavirus/norovirus infections were detected in 5.0% (859/17,113) of cases. While there was no difference in norovirus incidence in outpatient compared to hospitalized cases, rotavirus was detected two times more in outpatients compared to hospitalized cases (26.6% vs.13.6%; P < 0.001). Both rotavirus and norovirus infections peaked in children aged 12-18 months seeking medical care with acute gastroenteritis (35.6% rotavirus cases; 8439/23,728 and 32.5% norovirus cases; 1660/5107). Rotavirus infections were frequent between December and March of each year while norovirus was detected earlier from October to December. Our results showed significant correlation between virus detection and environmental factors such as average monthly temperature but not relative humidity. In addition, we observed a reduction in the detection rates of rotavirus and norovirus at the beginning of the SARS-CoV-2 pandemic in 2020. CONCLUSION: Our results indicate that rotavirus and norovirus are still important viral agents in pediatric acute gastroenteritis in Southwestern China.

Norovirus-VLPs expressing pre-erythrocytic malaria antigens induce functional immunity against sporozoite infection.

Despite the development of prophylactic anti-malarial drugs and practices to prevent infection, malaria remains a health concern. Preclinical testing of novel malaria vaccine strategies achieved through rational antigen selection and novel particle-based delivery platforms is yielding encouraging results. One such platform, self-assembling virus-like particles (VLP) is safer than attenuated live viruses, and has been approved as a vaccination tool by the FDA. We explore the use of Norovirus sub-viral particles lacking the natural shell (S) domain forming the interior shell but that retain the protruding (P) structures of the native virus as a vaccine vector. Epitope selection and their surface display has the potential to focus antigen specific immune responses to crucial epitopes. Recombinant P-particles displaying epitopes from two malaria antigens, Plasmodium falciparum (Pf) CelTOS and Plasmodium falciparum (Pf) CSP, were evaluated for immunogenicity and their ability to confer protection in a murine challenge model. Immune responses induced in mice resulted either in sterile protection (displaying PfCelTOS epitopes) or in antibodies with functional activity against sporozoites (displaying PfCSP epitopes) in an in vitro liver-stage development assay (ILSDA). These results are encouraging and support further evaluation of this platform as a vaccine delivery system.

Use of a Hydrogen Peroxide Nebulizer for Viral Disinfection of Emergency Ambulance and Hospital Waiting Room.

Disinfection of hospital facilities and ambulances is an important issue for breaking the chain of transmission of viral pathogens. Hydrogen peroxide has provided promising results in laboratory assays. Here, we evaluate the efficacy of a hydrogen peroxide nebulizer for the inactivation of surrogate MS2 bacteriophage and murine norovirus (MNV) in a patient waiting room and the fully equipped cabin of a medical ambulance. We observed an average 3 log10 titer reduction in both settings, which represents the destruction of over 106 and 109 infectious particles of MNV and MS2 per cm2, respectively. The potential for viral exposure is high for health workers when disinfecting confined and cluttered spaces, so the use of a hydrogen peroxide mist might offer an affordable and efficient solution to minimize the risk of viral contaminations.

rprimer: an R/bioconductor package for design of degenerate oligos for sequence variable viruses.

BACKGROUND: This paper presents a new R/Bioconductor package, rprimer, for design of degenerate oligos and PCR assays for sequence variable viruses. A multiple DNA sequence alignment is used as input data, while the outputs consist of comprehensive tables (data frames) and dashboard-like plots. The workflow can be run directly from the R console or through a graphical user interface (Shiny application). Here, rprimer is demonstrated and evaluated by using it to design two norovirus genogroup I (GI) assays: one RT-qPCR assay for quantitative detection and one RT­PCR assay for Sanger sequencing and polymerase-capsid based genotyping. RESULTS: The assays generated were evaluated using stool samples testing positive for norovirus GI. The RT-qPCR assay accurately amplified and quantified all samples and showed comparable performance to a widely-used standardised assay, while the RT-PCR assay resulted in successful sequencing and genotyping of all samples. Merits and limitations of the package were identified through comparison with three similar freely available software packages. Several features were comparable across the different tools, but important advantages of rprimer were its speed, flexibility in oligo design and capacity for visualisation. CONCLUSIONS: An R/Bioconductor package, rprimer, was developed and shown to be successful in designing primers and probes for quantitative detection and genotyping of a sequence-variable virus. The package provides an efficient, flexible and visual approach to degenerate oligo design, and can therefore assist in virus research and method development.

Efficacy of washing produce in removing human coronavirus OC43 and murine norovirus.

AIMS: Fresh produce is often a vehicle for the transmission of foodborne pathogens such as human norovirus. Thus, it is recommended to wash the surface of produce before consumption, and one of the most common ways to wash produce is by rinsing under running tap water. This study determined the effectiveness of removal of human coronavirus-OC43 (HCoV-OC43), as a surrogate for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and murine norovirus-1 (MNV-1), as a surrogate for human norovirus, from contaminated lettuce, apples and cucumbers. METHODS AND RESULTS: The produce surfaces were artificially inoculated in conjunction with faecal material to represent natural contamination. Rinsing under tap water for 10 s at 40 ml/s removed 1.94 ± 0.44, 1.42 ± 0.00 and 1.42 ± 0.42 log of HCoV-OC43 from apple, cucumber and lettuce respectively. The same washing technique removed 1.77 ± 0.17, 1.42 ± 0.07 and 1.79 ± 0.14 log of MNV-1 from apple, cucumber and lettuce respectively. This washing technique was effective at reducing a significant amount of viral contamination, however, it was not enough to eliminate the entire contamination. There was no significant difference in the reduction of viral load between the two viruses, nor between the three surfaces tested in this study. CONCLUSIONS: Our data suggest that washing under tap water would be an efficient way of reducing the risk of foodborne viral transmission only if the level of contamination is less than 2 log PFU. SIGNIFICANCE AND IMPACT OF STUDY: This study demonstrates that running tap water was effective at reducing the amount of infectious HCoV-OC43 and MNV on produce surfaces, and washing produce continues to be an important task to perform prior to consumption to avoid infection by foodborne viruses, particularly for foods which are eaten raw.

Emerging norovirus GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan, during COVID-19, 2021.

INTRODUCTION: Norovirus (NoV) is the most common agent causing outbreaks and sporadic cases of acute gastroenteritis among all ages, especially children under 5 years old. During the coronavirus disease 2019 (COVID-19) pandemic, NoV infection has decreased drastically in Japan due to school closures and no outbreak related to NoV infection had been reported. METHOD: In mid-September 2021, NoV outbreak occurred in kindergarten and nursery schools in Maizuru, Kyoto prefecture, Japan. Twenty-six stool samples collected from patients who were diagnosed of NoV gastroenteritis from the outbreak by an immunochromatographic (IC) kit at a pediatric outpatient clinic in Maizuru city during 3 weeks from September 13 to October 8, 2021 were examined for the presence of NoV GII by reverse transcriptase-polymerase chain reaction (RT-PCR), genome sequencing, and phylogenetic analysis. RESULT: All 26 samples were confirmed positive to NoV GII and their genotypes were identified as GII.4 Sydney[P31]. The amino acid substitutions in open reading frame1 (ORF1) and ORF2 genes were found when compared with previously detected sporadic NoV GII.4 Sydney[P31] strains isolated in Japan. The clinical characterization of infected children was described. Most of the children were mild cases and vomiting was the most frequent clinical symptom. CONCLUSION: This study reported a recent emergence of NoV GII.4 Sydney[P31] causing acute gastroenteritis outbreak in children in Japan during the COVID-19 pandemic and suggests a need for further monitoring of NoV GII.4 variants.

Interventions to prevent surface transmission of an infectious virus based on real human touch behavior: a case study of the norovirus.

OBJECTIVES: Infectious viruses (e.g., SARS-CoV-2, norovirus) can transmit through surfaces. Norovirus has infected millions of individuals annually. Interventions on norovirus transmission in high-risk indoor environment are important. METHODS: This study focused on a restaurant in Guangzhou, China. More than 41,000 touches by both diners and staff members were collected using video cameras. A surface transmission model was developed and combined with these real human touch behaviors to analyze the effectiveness of different norovirus prevention strategies. RESULTS: When the virus carrier was a diner, the virus intake fraction of diners in the same table was the highest. Increasing the touch frequency on personal private surfaces would reduce the virus exposure. The virus intake fraction was reduced by 18.4% on average if public surfaces were not touched. Optimization on surface materials could reduce the virus intake fraction by 86.6%. Additionally, disinfecting tablecloths, clothes of diners, and chairs were the three most effective surface disinfection strategies. CONCLUSION: Controlling human touch behavior (e.g., reducing the self-touches on mucous membranes) is more effective than surface disinfection in controlling norovirus transmission, but surface disinfection cannot be ignored because human behavior is difficult to be controlled.

Norovirus 3C-Like protease antagonizes interferon-ß production by cleaving NEMO.

An epidemic owing to Norovirus (NoV) has recently been occurring worldwide. Severe cases of NoV can lead to patient death, resulting in significant public health problems. In the early stages of infection, antagonizing the production of host interferon (IFN) is an important strategy for viruses to establish infection. However, the relationship between NoV and interferon and its mechanism remains unclear. In this study, the 3C-like protease encoded by NoV was found to effectively suppress Sendai virus (SEV)-mediated IFN-ß production by cleaving the NF-κB essential modulator (NEMO). Glutamine 205 is the site of NoV3CLpro-mediated cleavage of NEMO and this cleavage suppresses the ability of NEMO to activate downstream IFN production. These findings demonstrate that NoV3CLpro-induced cleavage limits NEMO to the activation of type I IFN signaling. In summary, our findings indicate that NoV3CLpro is a new interferon antagonist, and enhances our understanding of the escape of innate immunity mediated by NoV3CLpro.

Return of Norovirus and Rotavirus Activity in Winter 2020‒21 in City with Strict COVID-19 Control Strategy, China.

A rapid decrease in viral gastroenteritis during winter 2019-20 and a return of norovirus and rotavirus activity during winter 2020-21 were observed while multiple nonpharmaceutical interventions for coronavirus disease were in effect in Hong Kong. The initial collateral benefit of coronavirus disease countermeasures that reduced the viral gastroenteritis burden is not sustainable.