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Norovirus infection is the leading cause of foodborne gastroenteritis worldwide, causing more than 200,000 deaths each year. As a result of a lack of reproducible and robust in vitro culture systems and suitable animal models for human norovirus (HuNoV) infection, the pathogenesis of HuNoV is still poorly understood. In recent years, human intestinal enteroids (HIEs) have been successfully constructed and demonstrated to be able to support the replication of HuNoV. The NLRP3 inflammasome plays a key role in host innate immune responses by activating caspase1 to facilitate IL-1β and IL-18 secretion and N-GSDMD-driven apoptosis, while NLRP3 inflammasome overactivation plays an important role in the development of various inflammatory diseases. Here, we found that HuNoV activated enteric stem cell-derived human intestinal enteroids (HIEs) NLRP3 inflammasome, which was confirmed by transfection of Caco2 cells with full-length cDNA clones of HuNoV. Further, we found that HuNoV non-structural protein P22 activated the NLRP3 inflammasome and then matured IL-1β and IL-18 and processed the cleavage of gasdermin-D (GSDMD) to N-GSDMD, leading to pyroptosis. Besides, berberine (BBR) could ameliorate the pyroptosis caused by HuNoV and P22 by inhibiting NLRP3 inflammasome activation. Together, these results reveal new insights into the mechanisms of inflammation and cell death caused by HuNoV and provide potential treatments. [ABSTRACT FROM AUTHOR]

It is known that the transmission of different foodborne viruses can occur either via discharge of contaminated water close to the production environment or via close contact with animal feces. Cranberries are intimately associated with water throughout their production cycle, and blueberries grow close to the ground which could lead to contact with wildlife. The aim of this study was to evaluate the prevalence of human norovirus (HuNoV GI and GII), hepatitis A virus (HAV) and hepatitis E virus (HEV) in two berries produced commercially in Canada. The detection of HuNoV and HAV on RTE cranberries and of HEV on wild blueberries was evaluated using the ISO method 15216-1:2017. Only 3 of 234 cranberry samples tested positive for HuNoV GI (3.6, 7.4, 5.3 genome copies/g, respectively) and all were negative for HuNoV GII and HAV. PMA pre-treatment and sequencing confirmed the absence of potential intact HuNoV GI particles on cranberries. None of the 150 blueberry samples tested positive for HEV. Overall, the prevalence of foodborne viruses in RTE cranberries and wild blueberries harvested in Canada is low, making these products relatively safe for consumers. [ABSTRACT FROM AUTHOR]

Human norovirus (HuNoV) is the common cause of acute gastroenteritis worldwide. Norovirus-like particles (VLPs) have been applied to analyze virus–host cell interactions. This study established a highly quantitative detection system for HuNoV VLP entry into live cells based on the NanoLuc luciferase complementation. First, we generated a HiBiT-tagged VLP and examined VLP-HiBiT entry into Vero cells expressing LgBiT. The entry was saturable and competed by a non-labeled VLP, indicating the VLP-specific entry into the host cells. Upon comparison of the VLP-HiBiT entry among some cell lines, Caco-2 cells were found to be the most susceptible. The VLP-HiBiT entry was unaffected by treatment at an acidic pH value above 3.0 and was significantly reduced by heat treatment at 75°C for more than 2 min. These results reflected those of previous studies, indicating the stability of VLPs and binding to histo-blood group antigens at various pH ranges and temperatures and cellular tropism. • This study established quantitative detection system for the entry of norovirus. • HiBiT-tagged VLP (VLP-HiBiT) entered into LgBiT-expressing live cells. • VLP-HiBiT entry was unaffected by treatment at pH value above 3.0. • VLP-HiBiT entry was significantly reduced by heat treatment at 75°C for 2 min. • Caco-2 cells were the most susceptible among some cell lines examined. [ABSTRACT FROM AUTHOR]

Norovirus infection is the leading cause of foodborne gastroenteritis worldwide, causing more than 200,000 deaths each year. As a result of a lack of reproducible and robust in vitro culture systems and suitable animal models for human norovirus (HuNoV) infection, the pathogenesis of HuNoV is still poorly understood. In recent years, human intestinal enteroids (HIEs) have been successfully constructed and demonstrated to be able to support the replication of HuNoV. The NLRP3 inflammasome plays a key role in host innate immune responses by activating caspase1 to facilitate IL-1β and IL-18 secretion and N-GSDMD-driven apoptosis, while NLRP3 inflammasome overactivation plays an important role in the development of various inflammatory diseases. Here, we found that HuNoV activated enteric stem cell-derived human intestinal enteroids (HIEs) NLRP3 inflammasome, which was confirmed by transfection of Caco2 cells with full-length cDNA clones of HuNoV. Further, we found that HuNoV non-structural protein P22 activated the NLRP3 inflammasome and then matured IL-1β and IL-18 and processed the cleavage of gasdermin-D (GSDMD) to N-GSDMD, leading to pyroptosis. Besides, berberine (BBR) could ameliorate the pyroptosis caused by HuNoV and P22 by inhibiting NLRP3 inflammasome activation. Together, these results reveal new insights into the mechanisms of inflammation and cell death caused by HuNoV and provide potential treatments.

It is known that the transmission of different foodborne viruses can occur either via discharge of contaminated water close to the production environment or via close contact with animal feces. Cranberries are intimately associated with water throughout their production cycle, and blueberries grow close to the ground which could lead to contact with wildlife. The aim of this study was to evaluate the prevalence of human norovirus (HuNoV GI and GII), hepatitis A virus (HAV) and hepatitis E virus (HEV) in two berries produced commercially in Canada. The detection of HuNoV and HAV on RTE cranberries and of HEV on wild blueberries was evaluated using the ISO method 15216-1:2017. Only 3 of 234 cranberry samples tested positive for HuNoV GI (3.6, 7.4, 5.3 genome copies/g, respectively) and all were negative for HuNoV GII and HAV. PMA pre-treatment and sequencing confirmed the absence of potential intact HuNoV GI particles on cranberries. None of the 150 blueberry samples tested positive for HEV. Overall, the prevalence of foodborne viruses in RTE cranberries and wild blueberries harvested in Canada is low, making these products relatively safe for consumers.

ABSTRACT Human norovirus (HuNoV) is the leading cause of epidemic and sporadic acute gastroenteritis worldwide. HuNoV transmission occurs predominantly by direct person-to-person contact, and its health burden is associated with poor hand hygiene and a lack of effective antiseptics and disinfectants. Specific therapies and methods to prevent and control HuNoV spread previously were difficult to evaluate because of the lack of a cell culture system to propagate infectious virus. This barrier has been overcome with the successful cultivation of HuNoV in nontransformed human intestinal enteroids (HIEs). Here, we report using the HIE cultivation system to evaluate the virucidal efficacy of an olanexidine gluconate-based hand rub (OLG-HR) and 70% ethanol (EtOH70%) against HuNoVs. OLG-HR exhibited fast-acting virucidal activity against a spectrum of HuNoVs including GII.4 Sydney[P31], GII.4 Den Haag[P4], GII.4 New Orleans[P4], GII.3[P21], GII.17[P13], and GI.1[P1] strains. Exposure of HuNoV to OLG-HR for 30 to 60 s resulted in complete loss of the ability of virus to bind to the cells and reduced in vitro binding to glycans in porcine gastric mucin. By contrast, the virucidal efficiency of EtOH70% on virus infectivity was strain specific. Dynamic light scattering (DLS) and electron microscopy of virus-like particles (VLPs) show that OLG-HR treatment causes partial disassembly and possibly conformational changes in VP1, interfering with histo-blood group antigen (HBGA) binding and infectivity, whereas EtOH70% treatment causes particle disassembly and clumping of the disassembled products, leading to loss of infectivity while retaining HBGA binding. The highly effective inactivation of HuNoV infectivity by OLG-HR suggests that this compound could reduce HuNoV transmission. IMPORTANCE Human noroviruses (HuNoVs) are highly contagious and cause nonbacterial acute gastroenteritis in all age groups worldwide. Since the introduction of rotavirus vaccines, HuNoVs have become the leading cause of diarrheal illness in children. These viruses are very stable in the environment and resistant to common disinfectants. This study evaluated the virucidal efficacy of a new disinfectant, olanexidine-based hand rub (OLG-HR), against HuNoV strains in an ex vivo human intestinal stem cell-derived enteroid (HIE) cultivation system. Exposure of multiple HuNoV strains to OLG-HR for 30 to 60 s resulted in complete loss of infectivity and binding to HBGAs, possibly due to partial disassembly and conformational changes in the major virus capsid (VP1). By comparison, the virucidal efficiency of EtOH70% was strain specific, leading to loss of infectivity while retaining HBGA binding. These findings show the utility of the ex vivo HIE cultivation system to test the effectiveness of disinfectants and report a highly effective product.

Noroviruses and Sapoviruses, classified in the Caliciviridae family, are small positive-stranded RNA viruses, considered nowadays the leading cause of acute gastroenteritis globally in both children and adults. Although most noroviruses have been associated with gastrointestinal disease in humans, almost 50 years after its discovery, there is still a lack of comprehensive evidence regarding its biology and pathogenesis mainly because they can be neither conveniently grown in cultured cells nor propagated in animal models. However, other members of this family such as Feline calicivirus (FCV), Murine norovirus (MNV), Rabbit hemorrhagic disease virus (RHDV), and Porcine sapovirus (PS), from which there are accessible propagation systems, have been useful to study the calicivirus replication strategies. Using cell cultures and animal models, many of the functions of the viral proteins in the viral replication cycles have been well-characterized. Moreover, evidence of the role of viral proteins from different members of the family in the establishment of infection has been generated and the mechanism of their immunopathogenesis begins to be understood. In this review, we discuss different aspects of how caliciviruses are implicated in membrane rearrangements, apoptosis, and evasion of the immune responses, highlighting some of the pathogenic mechanisms triggered by different members of the Caliciviridae family.

Human noroviruses (HuNoVs) are the major cause of non-bacterial acute gastroenteritis worldwide. RT-qPCR is a widely used method to detect HuNoVs. However, the method is unable to extract a virus from environmental samples and to discriminate between infectious and non-infectious viruses. In this study, we explored a new in situ capture RT-qPCR (ISC-RT-qPCR) methodology to estimate the infectivity of HuNoV in environmental and food samples. This assay was based on capturing encapsidated HuNoV by viral receptors, followed by in situ amplification of the captured viral genomes by RT-qPCR. We demonstrated that ISC-RT-qPCR did not capture and enable signal amplification of the heat-denatured Tulane virus (TV) and HuNoVs. Therefore, ISC-RT-qPCR provides better estimates for infectivity of HuNoV than RT-qPCT. We then utilized the ISC-RT-qPCR to detect HuNoV in environmental water samples and food samples, as compared to a conventional RT-qPCR procedure. The presence of HuNoV was examined in 36 oyster samples from retail markets using by both assays for detection. The detection rates of HuNoV in gill, digestive glands, and other tissues were 33.3%, 25%, and 19.4%, respectively, by ISC-RT-qPCR; and were 5.6%, 11.1%, and 11.1%, respectively, by RT-qPCR. ISC-RTqPCR is more sensitive than RT-qPCR for the detection of HuNoV in oysters. By contrast, the HuNoV detection rate by ISC-RTqPCR is lower for environmental samples. Of the 72 water samples that tested positive for HuNoV by RT-qPCR, only 20 (27.8%) of these tested positive by ISC-RT-qPCR, suggesting that 72.2% of RT-qPCR-positive samples were unlikely to be infectious. A better detection rate by ISC-RT-qPCR in oyster samples indicates the likelihood of infectious HuNoV that accumulated in oysters, and a lower detection rate of HuNoV in environmental water by ISC-RT-qPCR, indicating that the majority of RT-qPCR-positive samples were from non-infectious viral RNA.

Human norovirus (HuNoV) is the leading cause of epidemic and sporadic acute gastroenteritis worldwide. HuNoV transmission occurs predominantly by direct person-to-person contact, and its health burden is associated with poor hand hygiene and a lack of effective antiseptics and disinfectants. Specific therapies and methods to prevent and control HuNoV spread previously were difficult to evaluate because of the lack of a cell culture system to propagate infectious virus. This barrier has been overcome with the successful cultivation of HuNoV in nontransformed human intestinal enteroids (HIEs). Here, we report using the HIE cultivation system to evaluate the virucidal efficacy of an olanexidine gluconate-based hand rub (OLG-HR) and 70% ethanol (EtOH 70% ) against HuNoVs. OLG-HR exhibited fast-acting virucidal activity against a spectrum of HuNoVs including GII.4 Sydney[P31], GII.4 Den Haag[P4], GII.4 New Orleans[P4], GII.3[P21], GII.17[P13], and GI.1[P1] strains. Exposure of HuNoV to OLG-HR for 30 to 60 s resulted in complete loss of the ability of virus to bind to the cells and reduced in vitro binding to glycans in porcine gastric mucin. By contrast, the virucidal efficiency of EtOH 70% on virus infectivity was strain specific. Dynamic light scattering (DLS) and electron microscopy of virus-like particles (VLPs) show that OLG-HR treatment causes partial disassembly and possibly conformational changes in VP1, interfering with histo-blood group antigen (HBGA) binding and infectivity, whereas EtOH 70% treatment causes particle disassembly and clumping of the disassembled products, leading to loss of infectivity while retaining HBGA binding. The highly effective inactivation of HuNoV infectivity by OLG-HR suggests that this compound could reduce HuNoV transmission. IMPORTANCE Human noroviruses (HuNoVs) are highly contagious and cause nonbacterial acute gastroenteritis in all age groups worldwide. Since the introduction of rotavirus vaccines, HuNoVs have become the leading cause of diarrheal illness in children. These viruses are very stable in the environment and resistant to common disinfectants. This study evaluated the virucidal efficacy of a new disinfectant, olanexidine-based hand rub (OLG-HR), against HuNoV strains in an ex vivo human intestinal stem cell-derived enteroid (HIE) cultivation system. Exposure of multiple HuNoV strains to OLG-HR for 30 to 60 s resulted in complete loss of infectivity and binding to HBGAs, possibly due to partial disassembly and conformational changes in the major virus capsid (VP1). By comparison, the virucidal efficiency of EtOH 70% was strain specific, leading to loss of infectivity while retaining HBGA binding. These findings show the utility of the ex vivo HIE cultivation system to test the effectiveness of disinfectants and report a highly effective product.

Noroviruses and Sapoviruses, classified in the Caliciviridae family, are small positive-stranded RNA viruses, considered nowadays the leading cause of acute gastroenteritis globally in both children and adults. Although most noroviruses have been associated with gastrointestinal disease in humans, almost 50 years after its discovery, there is still a lack of comprehensive evidence regarding its biology and pathogenesis mainly because they can be neither conveniently grown in cultured cells nor propagated in animal models. However, other members of this family such as Feline calicivirus (FCV), Murine norovirus (MNV), Rabbit hemorrhagic disease virus (RHDV), and Porcine sapovirus (PS), from which there are accessible propagation systems, have been useful to study the calicivirus replication strategies. Using cell cultures and animal models, many of the functions of the viral proteins in the viral replication cycles have been well-characterized. Moreover, evidence of the role of viral proteins from different members of the family in the establishment of infection has been generated and the mechanism of their immunopathogenesis begins to be understood. In this review, we discuss different aspects of how caliciviruses are implicated in membrane rearrangements, apoptosis, and evasion of the immune responses, highlighting some of the pathogenic mechanisms triggered by different members of the Caliciviridae family., (Copyright © 2019 Peñaflor-Téllez, Trujillo-Uscanga, Escobar-Almazán and Gutiérrez-Escolano.)