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1
Bartnicki, Eric, Juliana Bragazzi Cunha, Abimbola O. Kolawole, and Christiane E. Wobus. "Recent advances in understanding noroviruses." F1000Research 6 (January 26, 2017): 79. http://dx.doi.org/10.12688/f1000research.10081.1.
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Noroviruses are the leading cause of acute gastroenteritis around the world. An individual living in the United States is estimated to develop norovirus infection five times in his or her lifetime. Despite this, there is currently no antiviral or vaccine to combat the infection, in large part because of the historical lack of cell culture and small animal models. However, the last few years of norovirus research were marked by a number of ground-breaking advances that have overcome technical barriers and uncovered novel aspects of norovirus biology. Foremost among them was the development of two different in vitro culture systems for human noroviruses. Underappreciated was the notion that noroviruses infect cells of the immune system as well as epithelial cells within the gastrointestinal tract and that human norovirus infection of enterocytes requires or is promoted by the presence of bile acids. Furthermore, two proteinaceous receptors are now recognized for murine norovirus, marking the first discovery of a functional receptor for any norovirus. Recent work further points to a role for certain bacteria, including those found in the gut microbiome, as potential modulators of norovirus infection in the host, emphasizing the importance of interactions with organisms from other kingdoms of life for viral pathogenesis. Lastly, we will highlight the adaptation of drop-based microfluidics to norovirus research, as this technology has the potential to reveal novel insights into virus evolution. This review aims to summarize these new findings while also including possible future directions.
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Thorne, Lucy G., and Ian G. Goodfellow. "Norovirus gene expression and replication." Journal of General Virology 95, no. 2 (2014): 278–91. http://dx.doi.org/10.1099/vir.0.059634-0.
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Noroviruses are small, positive-sense RNA viruses within the family Caliciviridae, and are now accepted widely as a major cause of acute gastroenteritis in both developed and developing countries. Despite their impact, our understanding of the life cycle of noroviruses has lagged behind that of other RNA viruses due to the inability to culture human noroviruses (HuNVs). Our knowledge of norovirus biology has improved significantly over the past decade as a result of numerous technological advances. The use of a HuNV replicon, improved biochemical and cell-based assays, combined with the discovery of a murine norovirus capable of replication in cell culture, has improved greatly our understanding of the molecular mechanisms of norovirus genome translation and replication, as well as the interaction with host cell processes. In this review, the current state of knowledge of the intracellular life of noroviruses is discussed with particular emphasis on the mechanisms of viral gene expression and viral genome replication.
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Liu, Guangliang, Shannon M. Kahan, Yali Jia, and Stephanie M. Karst. "Primary High-Dose Murine Norovirus 1 Infection Fails To Protect from Secondary Challenge with Homologous Virus." Journal of Virology 83, no. 13 (2009): 6963–68. http://dx.doi.org/10.1128/jvi.00284-09.
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ABSTRACT Human noroviruses in the Caliciviridae family are the major cause of nonbacterial epidemic gastroenteritis worldwide. Primary human norovirus infection does not elicit lasting protective immunity, a fact that could greatly affect the efficacy of vaccination strategies. Little is known regarding the pathogenesis of human noroviruses or the immune responses that control them because there has previously been no small-animal model or cell culture system of infection. Using the only available small-animal model of norovirus infection, we found that primary high-dose murine norovirus 1 (MNV-1) infection fails to afford protection against a rechallenge with a homologous virus. Thus, MNV-1 represents a valuable model with which to dissect the pathophysiological basis for the lack of lasting protection against human norovirus infection. Interestingly, the magnitude of protection afforded by a primary MNV-1 infection inversely correlates with the inoculum dose. Future studies will elucidate the mechanisms by which noroviruses avoid the induction of protective immunity and the role played by the inoculum dose in this process, ultimately translating this knowledge into successful vaccination approaches.
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Jones, Melissa K., Katrina R. Grau, Veronica Costantini, et al. "Human norovirus culture in B cells." Nature Protocols 10, no. 12 (2015): 1939–47. http://dx.doi.org/10.1038/nprot.2015.121.
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Haga, Kei, Akira Fujimoto, Reiko Takai-Todaka, et al. "Functional receptor molecules CD300lf and CD300ld within the CD300 family enable murine noroviruses to infect cells." Proceedings of the National Academy of Sciences 113, no. 41 (2016): E6248—E6255. http://dx.doi.org/10.1073/pnas.1605575113.
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Norovirus is the leading cause of acute gastroenteritis worldwide. Since the discovery of human norovirus (HuNoV), an efficient and reproducible norovirus replication system has not been established in cultured cells. Although limited amounts of virus particles can be produced when the HuNoV genome is directly transfected into cells, the HuNoV cycle of infection has not been successfully reproduced in any currently available cell-culture system. Those results imply that the identification of a functional cell-surface receptor for norovirus might be the key to establishing a norovirus culture system. Using a genome-wide CRISPR/Cas9 guide RNA library, we identified murine CD300lf and CD300ld as functional receptors for murine norovirus (MNV). The treatment of susceptible cells with polyclonal antibody against CD300lf significantly reduced the production of viral progeny. Additionally, ectopic CD300lf expression in nonsusceptible cell lines derived from other animal species enabled MNV infection and progeny production, suggesting that CD300lf has potential for dictating MNV host tropism. Furthermore, CD300ld, which has an amino acid sequence in the N-terminal region of its extracellular domain that is highly homologous to that of CD300lf, also functions as a receptor for MNV. Our results indicate that direct interaction of MNV with two cell-surface molecules, CD300lf and CD300ld, dictates permissive noroviral infection.
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Bhar, Sutonuka, and Melissa K. Jones. "In Vitro Replication of Human Norovirus." Viruses 11, no. 6 (2019): 547. http://dx.doi.org/10.3390/v11060547.
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Human norovirus (HuNoV) infection is a major cause of gastroenteritis all over the world. Despite this, these non-enveloped RNA viruses are poorly characterized due to the lack of robust and widely available HuNoV culture systems. The two published systems (B cell line and stem cell-derived enteroids) support replication of HuNoVs but the levels of replication are not sufficient for the generation of highly purified virus stocks or the development of culture-based quantification assays. Therefore, improvement of HuNoV in vitro replication is still needed. Murine norovirus and other caliciviruses have provided insights into norovirus replication that paved the way for the development of the current HuNoV culture systems and may also aid in the improvement of these systems. This review will highlight ways in which previous research guided and impacted the development of HuNoV culture systems and discuss ways in which more recent discoveries might be utilized to improve the quality of the HuNoV in vitro replication.
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Vashist, Surender, Luis Urena, Mariam B. Gonzalez-Hernandez, et al. "Molecular Chaperone Hsp90 Is a Therapeutic Target for Noroviruses." Journal of Virology 89, no. 12 (2015): 6352–63. http://dx.doi.org/10.1128/jvi.00315-15.
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ABSTRACTHuman noroviruses (HuNoV) are a significant cause of acute gastroenteritis in the developed world, and yet our understanding of the molecular pathways involved in norovirus replication and pathogenesis has been limited by the inability to efficiently culture these viruses in the laboratory. Using the murine norovirus (MNV) model, we have recently identified a network of host factors that interact with the 5′ and 3′ extremities of the norovirus RNA genome. In addition to a number of well-known cellular RNA binding proteins, the molecular chaperone Hsp90 was identified as a component of the ribonucleoprotein complex. Here, we show that the inhibition of Hsp90 activity negatively impacts norovirus replication in cell culture. Small-molecule-mediated inhibition of Hsp90 activity using 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) revealed that Hsp90 plays a pleiotropic role in the norovirus life cycle but that the stability of the viral capsid protein is integrally linked to Hsp90 activity. Furthermore, we demonstrate that both the MNV-1 and the HuNoV capsid proteins require Hsp90 activity for their stability and that targeting Hsp90in vivocan significantly reduce virus replication. In summary, we demonstrate that targeting cellular proteostasis can inhibit norovirus replication, identifying a potential novel therapeutic target for the treatment of norovirus infections.IMPORTANCEHuNoV are a major cause of acute gastroenteritis around the world. RNA viruses, including noroviruses, rely heavily on host cell proteins and pathways for all aspects of their life cycle. Here, we identify one such protein, the molecular chaperone Hsp90, as an important factor required during the norovirus life cycle. We demonstrate that both murine and human noroviruses require the activity of Hsp90 for the stability of their capsid proteins. Furthermore, we demonstrate that targeting Hsp90 activityin vivousing small molecule inhibitors also reduces infectious virus production. Given the considerable interest in the development of Hsp90 inhibitors for use in cancer therapeutics, we identify here a new target that could be explored for the development of antiviral strategies to control norovirus outbreaks and treat chronic norovirus infection in immunosuppressed patients.
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Chaudhry, Yasmin, Michael A. Skinner, and Ian G. Goodfellow. "Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase." Journal of General Virology 88, no. 8 (2007): 2091–100. http://dx.doi.org/10.1099/vir.0.82940-0.
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Despite the significant disease burden caused by human norovirus infection, an efficient tissue-culture system for these viruses remains elusive. Murine norovirus (MNV) is an ideal surrogate for the study of norovirus biology, as the virus replicates efficiently in tissue culture and a low-cost animal model is readily available. In this report, a reverse-genetics system for MNV is described, using a fowlpox virus (FWPV) recombinant expressing T7 RNA polymerase to recover genetically defined MNV in tissue culture for the first time. These studies demonstrated that approaches that have proved successful for other members of the family Caliciviridae failed to lead to recovery of MNV. This was due to our observation that vaccinia virus infection had a negative effect on MNV replication. In contrast, FWPV infection had no deleterious effect and allowed the recovery of infectious MNV from cells previously transfected with MNV cDNA constructs. These studies also indicated that the nature of the 3′-terminal nucleotide is critical for efficient virus recovery and that inclusion of a hepatitis delta virus ribozyme at the 3′ end can increase the efficiency with which virus is recovered. This system now allows the recovery of genetically defined noroviruses and will facilitate the analysis of the effects of genetic variation on norovirus pathogenesis.
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Straub, Timothy M., Rachel A. Bartholomew, Catherine O. Valdez, et al. "Human norovirus infection of Caco-2 cells grown as a three-dimensional tissue structure." Journal of Water and Health 9, no. 2 (2010): 225–40. http://dx.doi.org/10.2166/wh.2010.106.
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Human norovirus (hNoV) infectivity was studied using a three-dimensional model of large intestinal epithelium. Large intestine Caco-2 cells were grown in rotating wall vessel bioreactors for 18–21 days at 37°C and then transferred to 24-well tissue culture plates where they were infected with GI.1 and GII.4 human noroviruses collected from human challenge trials and various outbreak settings, respectively. Compared with uninfected cells, transmission micrographs of norovirus-infected cells displayed evidence of shortening or total loss of apical microvilli, and vacuolization. Quantitative reverse transcription real-time PCR (qRT-PCR) indicated an approximate 2–3 log10 increase in viral RNA copies for the infected cells. A passage experiment examined both the ability for continued viral RNA and viral antigen detection. In the passaged samples 1.01 × 106 copies ml−1 were detected by qRT-PCR. Immune electron microscopy using primary antibody to hNoV GI.1 capsids in conjunction with 6 nm gold-labelled secondary antibodies was performed on crude cellular lysates. Localization of antibody was observed in infected but not for uninfected cells. Our present findings, coupled with earlier work with the three-dimensional small intestinal INT407 model, demonstrate the utility of 3-D cell culture methods to develop infectivity assays for enteric viruses that do not readily infect mammalian cell cultures.
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Estes, Mary K., Khalil Ettayebi, Victoria R. Tenge, et al. "Human Norovirus Cultivation in Nontransformed Stem Cell-Derived Human Intestinal Enteroid Cultures: Success and Challenges." Viruses 11, no. 7 (2019): 638. http://dx.doi.org/10.3390/v11070638.
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Noroviruses, in the genus Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.
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Todd, Kyle V., and Ralph A. Tripp. "Vero Cells as a Mammalian Cell Substrate for Human Norovirus." Viruses 12, no. 4 (2020): 439. http://dx.doi.org/10.3390/v12040439.
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Human norovirus (HuNoV) is a principal cause of acute gastroenteritis worldwide, particularly in developing countries. Its global prevalence is underscored by more serious morbidity and some mortality in the young (<5 years) and the elderly. To date, there are no licensed vaccines or approved therapeutics for HuNoV, mostly because there are limited cell culture systems and small animal models available. Recently described cell culture systems are not ideal substrates for HuNoV vaccine development because they are not clonal or only support a single strain. In this study, we show Vero cell-based replication of two pandemic GII.4 HuNoV strains and one GII.3 strain and confirm exosome-mediated HuNoV infection in Vero cells. Lastly, we show that trypsin addition to virus cultures or disruption of Vero cell host genes can modestly increase HuNoV replication. These data provide support for Vero cells as a cell culture model for HuNoV.
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Rutjes, Saskia A., Harold H. J. L. van den Berg, Willemijn J. Lodder, and Ana Maria de Roda Husman. "Real-Time Detection of Noroviruses in Surface Water by Use of a Broadly Reactive Nucleic Acid Sequence-Based Amplification Assay." Applied and Environmental Microbiology 72, no. 8 (2006): 5349–58. http://dx.doi.org/10.1128/aem.00751-06.
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ABSTRACT Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection assays for viruses in water are often repressed by inhibitory factors present in the environment, like humic acids and heavy metals. To study the effect of environmental inhibitors on the performance of nucleic acid sequence-based amplification (NASBA), we developed a real-time norovirus NASBA targeting part of the RNA-dependent RNA polymerase (RdRp) gene. Specificity of the assay was studied with 33 divergent clones that contained part of the targeted RdRp gene of noroviruses from 15 different genogroups. Viral RNA originated from commercial oysters, surface waters, and sewage treatment plants in The Netherlands. Ninety-seven percent of the clones derived from human noroviruses were detected by real-time NASBA. Two clones containing animal noroviruses were not detected by NASBA. We compared the norovirus detection by real-time NASBA with that by conventional reverse transcriptase PCR (RT-PCR) with large-volume river water samples and found that inhibitory factors of RT-PCR had little or no effect on the performance of the norovirus NASBA. This consequently resulted in a higher sensitivity of the NASBA assay than of the RT-PCR. We show that by combining an efficient RNA extraction method with real-time NASBA the sensitivity of norovirus detection in water samples increased at least 100 times, which consequently has implications for the outcome of the infectious risk assessment.
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Lochridge, Vance P., Kathryn L. Jutila, Joel W. Graff, and Michele E. Hardy. "Epitopes in the P2 domain of norovirus VP1 recognized by monoclonal antibodies that block cell interactions." Journal of General Virology 86, no. 10 (2005): 2799–806. http://dx.doi.org/10.1099/vir.0.81134-0.
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Noroviruses cause the majority of epidemic outbreaks of acute viral gastroenteritis worldwide. Human norovirus strains do not grow in cell culture, but recent carbohydrate binding, sequence and structural analyses have begun to define functional domains in the norovirus capsid that may be conserved among multiple antigenic types. The purpose of this study was to localize domains and define sequences in the major capsid protein VP1 that are important for cell interactions. Monoclonal antibodies to genogroups GI.1 and GII.2 reference strains Norwalk virus and Snow Mountain virus, respectively, were generated that blocked binding of recombinant virus-like particles to Caco-2 intestinal cells and inhibited haemagglutination. Peptides that mimicked the mAb binding epitopes were selected from a phage-displayed random nonapeptide library. Anti-recombinant Norwalk virus mAb 54.6 and anti-recombinant Snow Mountain virus mAb 61.21 recognized epitopes located in the protruding P2 domain of VP1. The epitope recognized by mAb 61.21 contained amino acids that are completely conserved among norovirus strains across genogroups, including strains isolated from swine, bovine and murine species. This study identifies the first epitope involved in inhibition of norovirus–cell interactions and supports increasing evidence that interactions between noroviruses and host cells rely on structures in the P2 domain of VP1.
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Lochridge, Vance P., and Michele E. Hardy. "A Single-Amino-Acid Substitution in the P2 Domain of VP1 of Murine Norovirus Is Sufficient for Escape from Antibody Neutralization." Journal of Virology 81, no. 22 (2007): 12316–22. http://dx.doi.org/10.1128/jvi.01254-07.
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ABSTRACT Noroviruses cause epidemic outbreaks of acute viral gastroenteritis worldwide, and the number of reported outbreaks is increasing. Human norovirus strains do not grow in cell culture. However, murine norovirus (MNV) replicates in the RAW 264.7 macrophage cell line and thus provides a tractable model to investigate norovirus interactions with host cells. Epitopes recognized by monoclonal antibodies (MAbs) against the human norovirus strains Norwalk virus and Snow Mountain virus (SMV) identified regions in the P domain of major capsid protein VP1 important for interactions with putative cellular receptors. To determine if there was a relationship between domains of MNV VP1 and VP1 of human norovirus strains involved in cell binding, epitope mapping by phage display was performed with an MNV-1-neutralizing MAb, A6.2.1. A consensus peptide, GWWEDHGQL, was derived from 20 third-round phage clones. A synthetic peptide containing this sequence and constrained through a disulfide linkage reacted strongly with the A6.2.1 MAb, whereas the linear sequence did not. Four residues in the A6.2.1-selected peptide, G327, G333, Q334, and L335, aligned with amino acid residues in the P2 domain of MNV-1 VP1. This sequence is immediately adjacent to the epitope recognized by anti-SMV MAb 61.21. Neutralization escape mutants selected with MAb A6.2.1 contained a leucine-to-phenylalanine substitution at position 386 in the P2 domain. The predicted location of these residues on VP1 suggests that the phage peptide and the mutation in the neutralization-resistant viruses may be in close proximity to each other and to residues reported to be important for carbohydrate binding to VP1 of human norovirus strains.
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Cromeans, Theresa, Geun Woo Park, Veronica Costantini, et al. "Comprehensive Comparison of Cultivable Norovirus Surrogates in Response to Different Inactivation and Disinfection Treatments." Applied and Environmental Microbiology 80, no. 18 (2014): 5743–51. http://dx.doi.org/10.1128/aem.01532-14.
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ABSTRACTHuman norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions. At pH 2, FCV was inactivated by 6 log10units, whereas MNV, TuV, and AiV were resistant. All CSV were completely inactivated at 56°C within 20 min. MNV was inactivated 5 log10units by alcohols, in contrast to 2 and 3 log10units for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log10units by 1,000 ppm chlorine, in contrast to 1 log10unit for the other CSV. All CSV except FCV, when dried on stainless steel surfaces, were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at ≥300 MPa, and TuV at 600 MPa, while AiV was completely resistant to HHP up to 800 MPa. By reverse transcription-quantitative PCR (RT-qPCR), genogroup I (GI) noroviruses were more sensitive than GII noroviruses to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, TuV and MNV were the most resistant CSV overall and therefore are the best candidates for studying the public health outcomes of norovirus infections.
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Koromyslova, Anna D., and Grant S. Hansman. "Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly." Journal of Virology 89, no. 5 (2014): 2718–30. http://dx.doi.org/10.1128/jvi.03176-14.
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ABSTRACTHuman noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ∼10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemblein vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability.IMPORTANCEWe characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral.
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Van Dycke, Jana, Jasper Rymenants, Johan Neyts, and Joana Rocha-Pereira. "Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Identification of active compounds." Antiviral Chemistry and Chemotherapy 29 (January 2021): 204020662110268. http://dx.doi.org/10.1177/20402066211026852.
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Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. As potent and safe anti-norovirus small molecules are urgently needed, we here describe the detailed protocol for a set of assays that will allow the identification of novel norovirus inhibitors.
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Changotra, Harish, Yali Jia, Tara N. Moore, et al. "Type I and Type II Interferons Inhibit the Translation of Murine Norovirus Proteins." Journal of Virology 83, no. 11 (2009): 5683–92. http://dx.doi.org/10.1128/jvi.00231-09.
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ABSTRACT Human noroviruses are responsible for more than 95% of nonbacterial epidemic gastroenteritis worldwide. Both onset and resolution of disease symptoms are rapid, suggesting that components of the innate immune response are critical in norovirus control. While the study of the human noroviruses has been hampered by the lack of small animal and tissue culture systems, our recent discovery of a murine norovirus (MNV) and its in vitro propagation have allowed us to begin addressing norovirus replication strategies and immune responses to norovirus infection. We have previously demonstrated that interferon responses are critical to control MNV-1 infection in vivo and to directly inhibit viral replication in vitro. We now extend these studies to define the molecular basis for interferon-mediated inhibition. Viral replication intermediates were not detected in permissive cells pretreated with type I interferon after either infection or transfection of virion-associated RNA, demonstrating a very early block to virion production that is after virus entry and uncoating. A similar absence of viral replication intermediates was observed in infected primary macrophages and dendritic cells pretreated with type I IFN. This was not due to degradation of incoming genomes in interferon-pretreated cells since similar levels of genomes were present in untreated and pretreated cells through 6 h of infection, and these genomes retained their integrity. Surprisingly, this block to the translation of viral proteins was not dependent on the well-characterized interferon-induced antiviral molecule PKR. Similar results were observed in cells pretreated with type II interferon, except that the inhibition of viral translation was dependent on PKR. Thus, both type I and type II interferon signaling inhibit norovirus translation in permissive myeloid cells, but they display distinct dependence on PKR for this inhibition.
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Thackray, Larissa B., Christiane E. Wobus, Karen A. Chachu, et al. "Murine Noroviruses Comprising a Single Genogroup Exhibit Biological Diversity despite Limited Sequence Divergence." Journal of Virology 81, no. 19 (2007): 10460–73. http://dx.doi.org/10.1128/jvi.00783-07.
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ABSTRACT Viruses within the genus Norovirus of the family Caliciviridae are the major cause of acute, nonbacterial gastroenteritis worldwide. Human noroviruses are genetically diverse, with up to 57% divergence in capsid protein sequences, and comprise three genogroups. The significance of such genetic diversity is not yet understood. The discovery of murine norovirus (MNV) and its ability to productively infect cultured murine macrophages and dendritic cells has provided an opportunity to determine the functional consequences of norovirus diversity in vitro and in vivo. Therefore, we compared the full-length genomes of 21 new MNV isolates with five previously sequenced MNV genomes and demonstrated a conserved genomic organization consisting of four open reading frames (ORFs) and a previously unknown region of nucleotide conservation in ORF2. A phylogenetic analysis of all 26 MNV genomes revealed 15 distinct MNV strains, with up to 13% divergence at the nucleotide level, that comprise a single genotype and genogroup. Evidence for recombination within ORF2 in several MNV genomes was detected by multiple methods. Serological analyses comparing neutralizing antibody responses between highly divergent strains suggested that the MNV genogroup comprises a single serotype. Within this single genogroup, MNV strains exhibited considerable biological diversity in their ability to grow in culture and to infect and/or persist in wild-type mice. The isolation and characterization of multiple MNV strains illustrate how genetic analysis may underestimate the biological diversity of noroviruses and provide a molecular map for future studies of MNV biology.
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Chan, Jenny C. M., Kirran N. Mohammad, Lin-Yao Zhang, Sunny H. Wong, and Martin Chi-Wai Chan. "Targeted Profiling of Immunological Genes during Norovirus Replication in Human Intestinal Enteroids." Viruses 13, no. 2 (2021): 155. http://dx.doi.org/10.3390/v13020155.
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Norovirus is the leading cause of acute gastroenteritis worldwide. The pathogenesis of norovirus and the induced immune response remain poorly understood due to the lack of a robust virus culture system. The monolayers of two secretor-positive Chinese human intestinal enteroid (HIE) lines were challenged with two norovirus pandemic GII.4 Sydney strains. Norovirus RNA replication in supernatants and cell lysates were quantified by RT-qPCR. RNA expression levels of immune-related genes were profiled using PCR arrays. The secreted protein levels of shortlisted upregulated genes were measured in supernatants using analyte-specific enzyme-linked immunosorbent assay (ELISA). Productive norovirus replications were achieved in three (75%) out of four inoculations. The two most upregulated immune-related genes were CXCL10 (93-folds) and IFI44L (580-folds). Gene expressions of CXCL10 and IFI44L were positively correlated with the level of norovirus RNA replication (CXCL10: Spearman’s r = 0.779, p < 0.05; IFI44L: r = 0.881, p < 0.01). The higher level of secreted CXCL10 and IFI44L proteins confirmed their elevated gene expression. The two genes have been reported to be upregulated in norovirus volunteer challenges and natural human infections by other viruses. Our data suggested that HIE could mimic the innate immune response elicited in natural norovirus infection and, therefore, could serve as an experimental model for future virus-host interaction and antiviral studies.
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Taube, Stefan, John R. Rubin, Umesh Katpally, et al. "High-Resolution X-Ray Structure and Functional Analysis of the Murine Norovirus 1 Capsid Protein Protruding Domain." Journal of Virology 84, no. 11 (2010): 5695–705. http://dx.doi.org/10.1128/jvi.00316-10.
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ABSTRACT Murine noroviruses (MNV) are closely related to the human noroviruses (HuNoV), which cause the majority of nonbacterial gastroenteritis. Unlike HuNoV, MNV grow in culture and in a small-animal model that represents a tractable model to study norovirus biology. To begin a detailed investigation of molecular events that occur during norovirus binding to cells, the crystallographic structure of the murine norovirus 1 (MNV-1) capsid protein protruding (P) domain has been determined. Crystallization of the bacterially expressed protein yielded two different crystal forms (Protein Data Bank identifiers [PDB ID], 3LQ6 and 3LQE). Comparison of the structures indicated a large degree of structural mobility in loops on the surface of the P2 subdomain. Specifically, the A′-B′ and E′-F′ loops were found in open and closed conformations. These regions of high mobility include the known escape mutation site for the neutralizing antibody A6.2 and an attenuation mutation site, which arose after serial passaging in culture and led to a loss in lethality in STAT1−/− mice, respectively. Modeling of a Fab fragment and crystal structures of the P dimer into the cryoelectron microscopy three-dimensional (3D) image reconstruction of the A6.2/MNV-1 complex indicated that the closed conformation is most likely bound to the Fab fragment and that the antibody contact is localized to the A′-B′ and E′-F′ loops. Therefore, we hypothesize that these loop regions and the flexibility of the P domains play important roles during MNV-1 binding to the cell surface.
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Straub, T. M., J. R. Hutchison, R. A. Bartholomew, et al. "Defining cell culture conditions to improve human norovirus infectivity assays." Water Science and Technology 67, no. 4 (2013): 863–68. http://dx.doi.org/10.2166/wst.2012.636.
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Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.
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Holmdahl, Torsten, Mats Walder, Nathalie Uzcátegui, et al. "Hydrogen Peroxide Vapor Decontamination in a Patient Room Using Feline Calicivirus and Murine Norovirus as Surrogate Markers for Human Norovirus." Infection Control & Hospital Epidemiology 37, no. 5 (2016): 561–66. http://dx.doi.org/10.1017/ice.2016.15.
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OBJECTIVETo determine whether hydrogen peroxide vapor (HPV) could be used to decontaminate caliciviruses from surfaces in a patient room.DESIGNFeline calicivirus (FCV) and murine norovirus (MNV) were used as surrogate viability markers to mimic the noncultivable human norovirus. Cell culture supernatants of FCV and MNV were dried in triplicate 35-mm wells of 6-well plastic plates. These plates were placed in various positions in a nonoccupied patient room that was subsequently exposed to HPV. Control plates were positioned in a similar room but were never exposed to HPV.METHODSVirucidal activity was measured in cell culture by reduction in 50% tissue culture infective dose titer for FCV and by both 50% tissue culture infective dose titer and plaque reduction for MNV.RESULTSNeither viable FCV nor viable MNV could be detected in the test room after HPV treatment. At least 3.65 log reduction for FCV and at least 3.67 log reduction for MNV were found by 50% tissue culture infective dose. With plaque assay, measurable reduction for MNV was at least 2.85 log units.CONCLUSIONSThe successful inactivation of both surrogate viruses indicates that HPV could be a useful tool for surface decontamination of a patient room contaminated by norovirus. Hence nosocomial spread to subsequent patients can be avoided.Infect Control Hosp Epidemiol 2016;37:561–566
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Taube, Stefan, Jeffrey W. Perry, Kristen Yetming, et al. "Ganglioside-Linked Terminal Sialic Acid Moieties on Murine Macrophages Function as Attachment Receptors for Murine Noroviruses." Journal of Virology 83, no. 9 (2009): 4092–101. http://dx.doi.org/10.1128/jvi.02245-08.
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ABSTRACT Noroviruses are the major cause of nonbacterial gastroenteritis in humans. However, little is known regarding the norovirus life cycle, including cell binding and entry. In contrast to human noroviruses, the recently discovered murine norovirus 1 (MNV-1) readily infects murine macrophages and dendritic cells in culture. Many viruses, including the related feline calicivirus, use terminal sialic acids (SA) as receptors for infection. Therefore, we tested whether SA moieties play a role during MNV-1 infection of murine macrophages. Competition with SA-binding lectins and neuraminidase treatment led to a reduction in MNV-1 binding and infection in cultured and primary murine macrophages, suggesting a role for SA during the initial steps of the MNV-1 life cycle. Because SA moieties can be attached to glycolipids (i.e., gangliosides), we next determined whether MNV-1 uses gangliosides during infection. The gangliosides GD1a, GM1, and asialo-GM1 (GA1) are natural components of murine macrophages. MNV-1 bound to ganglioside GD1a, which is characterized by an SA on the terminal galactose, but not to GM1 or asialo-GM1 in an enzyme-linked immunosorbent assay. The depletion of gangliosides using an inhibitor of glycosylceramide synthase (d-threo-P4) led to a reduction of MNV-1 binding and infection in cultured and primary murine macrophages. This defect was specifically rescued by the addition of GD1a. A similar phenotype was observed for MNV field strains WU11 (GV/WU11/2005/USA) and S99 (GV/Berlin/2006/DE). In conclusion, our data indicate that MNV can use terminal SA on gangliosides as attachment receptors during binding to murine macrophages.
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Arias, Armando, Dalan Bailey, Yasmin Chaudhry, and Ian Goodfellow. "Development of a reverse-genetics system for murine norovirus 3: long-term persistence occurs in the caecum and colon." Journal of General Virology 93, no. 7 (2012): 1432–41. http://dx.doi.org/10.1099/vir.0.042176-0.
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Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide, yet, due to the inability to propagate HuNoV in cell culture, murine norovirus (MNV) is typically used as a surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus infections in vivo because it establishes persistence in wild-type mice, yet causes symptoms resembling gastroenteritis in immune-compromised STAT1−/− mice. The lack of reverse-genetics approaches to recover genetically defined MNV-3 has limited further studies on the identification of viral sequences that contribute to persistence. Here we report the establishment of a combined DNA-based reverse-genetics and mouse-model system to study persistent MNV-3 infections in wild-type (C57BL/6) mice. Viral RNA and infectious virus were detected in faeces for at least 56 days after inoculation. Strikingly, the highest concentrations of viral RNA during persistence were detected in the caecum and colon, suggesting that viral persistence is maintained in these tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in the minor capsid protein (VP2) and the viral polymerase (NS7), in contrast with adaptive mutations selected during cell-culture passages in RAW264.7 cells that appeared in the major capsid protein (VP1) and non-structural protein NS4. This system provides an attractive model that can be readily used to identify viral sequences that contribute to persistence in an immunocompetent host and to more acute infection in an immunocompromised host, providing new insights into the biology of norovirus infections.
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Dycke, Jana Van, Jasper Rymenants, Johan Neyts, and Joana Rocha-Pereira. "Assessment of the anti-norovirus activity in cell culture using the mouse norovirus: Early mechanistic studies." Antiviral Chemistry and Chemotherapy 29 (January 2021): 204020662110251. http://dx.doi.org/10.1177/20402066211025175.
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Human norovirus is the main cause of viral gastroenteritis, resulting annually in ∼ 700 million infections and 200,000 deaths, of whom most are children <5 years. Mouse norovirus-infected macrophages are the most widely used in vitro system to screen and characterize the antiviral effect of norovirus-targeting small molecules. We have previously established antiviral assays using this system, identified novel inhibitors and performed additional studies in order to have a first insight into their mechanism of action. After the identification of novel small molecules with anti-norovirus activity (part 1 of this protocol), we here describe the logical next step which entails the generation of early information of their mode of action. This information together with a continuous improvement of the potency of compounds will contribute to the optimization of a compound class towards in vivo efficacy and a successful preclinical development.
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Pommepuy, M., F. Dumas, M. P. Caprais, et al. "Sewage impact on shellfish microbial contamination." Water Science and Technology 50, no. 1 (2004): 117–24. http://dx.doi.org/10.2166/wst.2004.0035.
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Coastal areas are frequently contaminated by microorganisms of human origin, due to high population density and low seawater renewal. To evaluate the impact of wastewater input on shellfish quality, a study was conducted in Brittany (France) over a period of 20 months. A hydrodynamic model was used to simulate wastewater impact on microbial water quality. To validate the model, wastewater from the three main sewage treatment plants and shellfish from three sites were sampled monthly. Bacterial indicators (E. coli), F-RNA phages were searched for by culture and noroviruses by RT-PCR and hybridisation. These microorganisms were detected in the three effluents and clams, with no marked seasonal variation. The microbial concentrations in the two oyster beds, distant from the effluent outfall, were low, and only three of the samples were positive for norovirus. For simulation, the winter wastewater inputs of E. coli and phages were calculated and an estimation for norovirus flux was made from the epidemic situation in the population. The microbial behaviour was included in the model by a decay-rate factor. Results from the model calculations were found to be very similar to E. coli and phage concentrations observed in shellfish. For noroviruses, the model indicated that shellfish distant from the wastewater input were under the detection limit of the RT-PCR method. This study demonstrated the use of modelisation to interpret norovirus contamination in various areas.
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Nguyen, Huang Minh Tue, Mi-Kyung Park, Sangdo Ha, In-Soo Choi, Changsun Choi, and Jinjong Myoung. "Cell Culture Models of Human Norovirus: the End of the Beginning?" Microbiology and Biotechnology Letters 45, no. 2 (2017): 93–100. http://dx.doi.org/10.4014/mbl.1706.06001.
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Mumphrey, Shannon M., Harish Changotra, Tara N. Moore, et al. "Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses." Journal of Virology 81, no. 7 (2007): 3251–63. http://dx.doi.org/10.1128/jvi.02096-06.
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ABSTRACT Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease.
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Hyde, Jennifer L., Stanislav V. Sosnovtsev, Kim Y. Green, Christiane Wobus, Herbert W. Virgin, and Jason M. Mackenzie. "Mouse Norovirus Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway." Journal of Virology 83, no. 19 (2009): 9709–19. http://dx.doi.org/10.1128/jvi.00600-09.
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ABSTRACT Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.
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Leuthold, Mila M., Kevin P. Dalton, and Grant S. Hansman. "Structural Analysis of a Rabbit Hemorrhagic Disease Virus Binding to Histo-Blood Group Antigens." Journal of Virology 89, no. 4 (2014): 2378–87. http://dx.doi.org/10.1128/jvi.02832-14.
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ABSTRACTRabbit hemorrhagic disease virus (RHDV) is a member of theCaliciviridaefamily (Lagovirusgenus). RHDV is highly contagious and attaches to epithelial cells in the digestive or respiratory tract, leading to massive lesions with high mortality rates. A new variant of RHDV (termed RHDVb) recently has emerged, and previously vaccinated rabbits appear to have little protection against this new strain. Similar to human norovirus (Caliciviridae,Norovirusgenus), RHDV binds histo-blood group antigens (HBGAs), and this is thought to be important for infection. Here, we report the HBGA binding site on the RHDVb capsid-protruding domain (P domain) using X-ray crystallography. The HBGA binding pocket was located in a negatively charged patch on the side of the P domain and at a dimeric interface. Residues from both monomers contributed to the HBGA binding and involved a network of direct hydrogen bonds and water-mediated interactions. An amino acid sequence alignment of different RHDV strains indicated that the residues directly interacting with the ABH-fucose of the HBGAs (Asp472, Asn474, and Ser479) were highly conserved. This result suggested that different RHDV strains also could bind HBGAs at the equivalent pocket. Moreover, several HBGA binding characteristics between RHDVb and human genogroup II norovirus were similar, which indicated a possible convergent evolution of HBGA binding interactions. Further structural studies with other RHDV strains are needed in order to better understand the HBGA binding mechanisms among the diverse RHDV strains.IMPORTANCEWe identified, for the first time, the HBGA binding site on an RHDVb P domain using X-ray crystallography. Our results showed that RHDVb and human genogroup II noroviruses had similar HBGA binding interactions. Recently, it was discovered that synthetic HBGAs or HBGA-expressing enteric bacteria could enhance human genogroup II norovirus infection in B cells. Considering that RHDVb and genogroup II norovirus similarly interacted with HBGAs, it may be possible that a comparable cell culture system also could work with RHDVb. Taken together, these new findings will extend our understanding of calicivirus HBGA interactions and may help to elucidate the specific roles of HBGAs in calicivirus infections.
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CANNON, JENNIFER L., EFSTATHIA PAPAFRAGKOU, GEUNWOO W. PARK, JASON OSBORNE, LEE-ANN JAYKUS, and JAN VINJÉ. "Surrogates for the Study of Norovirus Stability and Inactivation in the Environment: A Comparison of Murine Norovirus and Feline Calicivirus." Journal of Food Protection 69, no. 11 (2006): 2761–65. http://dx.doi.org/10.4315/0362-028x-69.11.2761.
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Human noroviruses (NoVs) are the leading cause of food- and waterborne outbreaks of acute nonbacterial gastroenteritis worldwide. As a result of the lack of a mammalian cell culture model for these viruses, studies on persistence, inactivation, and transmission have been limited to cultivable viruses, including feline calicivirus (FCV). Recently, reports of the successful cell culture of murine norovirus 1 (MNV-1) have provided investigators with an alternative surrogate for human NoVs. In this study, we compared the inactivation profiles of MNV-1 to FCV in an effort to establish the relevance of MNV-1 as a surrogate virus. Specifically, we evaluated (i) stability upon exposure to pH extremes; (ii) stability upon exposure to organic solvents; (iii) thermal inactivation; and (iv) surface persistence under wet and dry conditions. MNV-1 was stable across the entire pH range tested (pH 2 to 10) with less than 1 log reduction in infectivity at pH 2, whereas FCV was inactivated rapidly at pH values <3 and >9. FCV was more stable than MNV-1 at 56°C, but both viruses exhibited similar inactivation at 63 and 72°C. Long-term persistence of both viruses suspended in a fecal matrix and inoculated onto stainless steel coupons were similar at 4°C, but at room temperature in solution, MNV-1 was more stable than FCV. The genetic relatedness of MNV-1 to human NoVs combined with its ability to survive under gastric pH levels makes this virus a promising and relevant surrogate for studying environmental survival of human NoVs.
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Kim, MinJung, and GwangPyo Ko. "Quantitative characterization of the inhibitory effects of salt, humic acid, and heavy metals on the recovery of waterborne norovirus by electropositive filters." Journal of Water and Health 11, no. 4 (2013): 613–22. http://dx.doi.org/10.2166/wh.2013.187.
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The virus adsorption-elution technique (VIRADEL) using electropositively charged filters is used frequently for recovering enteric viruses from water. The filter-absorbed virus is typically eluted, concentrated, and subsequently detected by culture or molecular methods. Human norovirus (HuNoV), one of the most important waterborne pathogens, cannot be cultivated by conventional culture methods and is typically detected using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. However, it is plausible that various inhibitors could be concentrated simultaneously during the VIRADEL process and affect RT-PCR assays. In this study, we evaluated the effect of typical inhibitors, including humic acid, heavy metals, and salt, on the recovery of norovirus by two different electropositive filters: 1MDS and Nanoceram. Known amounts of HuNoV and murine norovirus were inoculated in 1 L of surface water containing various concentrations of humic acid, heavy metals (cadmium and lead), or NaCl. Our results indicate that the presence of heavy metals or salt significantly reduced the recovery of virus from the electropositive filters. Thus, care should be taken when analyzing waterborne norovirus using electropositive filters in environments with high concentrations of heavy metal inhibitors or salts.
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Qu, Lin, Kosuke Murakami, James R. Broughman, et al. "Replication of Human Norovirus RNA in Mammalian Cells Reveals Lack of Interferon Response." Journal of Virology 90, no. 19 (2016): 8906–23. http://dx.doi.org/10.1128/jvi.01425-16.
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ABSTRACTHuman noroviruses (HuNoVs), named after the prototype strain Norwalk virus (NV), are a leading cause of acute gastroenteritis outbreaks worldwide. Studies on the related murine norovirus (MNV) have demonstrated the importance of an interferon (IFN) response in host control of virus replication, but this remains unclear for HuNoVs. Despite the lack of an efficient cell culture infection system, transfection of stool-isolated NV RNA into mammalian cells leads to viral RNA replication and virus production. Using this system, we show here that NV RNA replication is sensitive to type I (α/β) and III (interleukin-29 [IL-29]) IFN treatment. However, in cells capable of a strong IFN response to Sendai virus (SeV) and poly(I·C), NV RNA replicates efficiently and generates double-stranded RNA without inducing a detectable IFN response. Replication of HuNoV genogroup GII.3 strain U201 RNA, generated from a reverse genetics system, also does not induce an IFN response. Consistent with a lack of IFN induction, NV RNA replication is enhanced neither by neutralization of type I/III IFNs through neutralizing antibodies or the soluble IFN decoy receptor B18R nor by short hairpin RNA (shRNA) knockdown of mitochondrial antiviral signaling protein (MAVS) or interferon regulatory factor 3 (IRF3) in the IFN induction pathways. In contrast to other positive-strand RNA viruses that block IFN induction by targeting MAVS for degradation, MAVS is not degraded in NV RNA-replicating cells, and an SeV-induced IFN response is not blocked. Together, these results indicate that HuNoV RNA replication in mammalian cells does not induce an IFN response, suggesting that the epithelial IFN response may play a limited role in host restriction of HuNoV replication.IMPORTANCEHuman noroviruses (HuNoVs) are a leading cause of epidemic gastroenteritis worldwide. Due to lack of an efficient cell culture system and robust small-animal model, little is known about the innate host defense to these viruses. Studies on murine norovirus (MNV) have shown the importance of an interferon (IFN) response in host control of MNV replication, but this remains unclear for HuNoVs. Here, we investigated the IFN response to HuNoV RNA replication in mammalian cells using Norwalk virus stool RNA transfection, a reverse genetics system, IFN neutralization reagents, and shRNA knockdown methods. Our results show that HuNoV RNA replication in mammalian epithelial cells does not induce an IFN response, nor can it be enhanced by blocking the IFN response. These results suggest a limited role of the epithelial IFN response in host control of HuNoV RNA replication, providing important insights into our understanding of the host defense to HuNoVs that differs from that to MNV.
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TIAN, PENG, DAVID YANG, CHRISTINA QUIGLEY, MARISSA CHOU, and XI JIANG. "Inactivation of the Tulane Virus, a Novel Surrogate for the Human Norovirus." Journal of Food Protection 76, no. 4 (2013): 712–18. http://dx.doi.org/10.4315/0362-028x.jfp-12-361.
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Human noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis epidemics. The culturable feline calicivirus and murine norovirus have been used extensively as surrogates to study HuNoV biology, as HuNoV does not grow in vitro. Additional efforts to identify new surrogates are needed, because neither of these common surrogates are truly intestinal pathogens. The newly described Tulane virus (TV) is a typical calicivirus, it is isolated from macaque stools, is cultivable in vitro, and recognizes human histo-blood group antigens. Therefore, TV is a promising surrogate for HuNoVs. In this study, we evaluated the resistance or stability of TV under various physical and environmental conditions by measuring a 50% reduction of tissue culture infective dose (TCID50) by using a TV cell culture system. Due to the nature of this virus, it is hard to produce a high-titer stock through tissue culture. In our study, the maximal reduction in virus titers was 5 D (D = 1 log) in heat-denaturation and EtOH experiments, and 4 D in UV, chlorine, and pH-stability experiments. Therefore in this study, we defined the inactivation of TV as reaching a TCID50/ml of 0 (a 4- to 5-D reduction in TCID50, depending on the detection limit). TV was inactivated after incubation at 63°C for 5 min, incubation at 56°C for 30 min (5 D), exposure to 60 mJ/cm2 of UVC radiation (4 D), or incubation at 300 ppm of free chlorine for 10 min (4 D). TV was shown to be stable from pH 3.0 to 8.0, though an obvious reduction in virus titer was observed at pH 2.5 and 9.0, and was inactivated at pH 10.0 (4 D). TV was resistant to a low concentration of EtOH (40% or lower) but was fully inactivated (5 D) by 50 to 70% EtOH after a short exposure (20 s). In contrast, quantitative real-time PCR was unable to detect, or poorly detected, virus titer reductions between treated and untreated samples described in this study.
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Papafragkou, Efstathia, Joanne Hewitt, Geun Woo Park, Gail Greening, and Jan Vinjé. "Challenges of Culturing Human Norovirus in Three-Dimensional Organoid Intestinal Cell Culture Models." PLoS ONE 8, no. 6 (2013): e63485. http://dx.doi.org/10.1371/journal.pone.0063485.
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Arthur, Sabastine Eugene, and Kristen Elizabeth Gibson. "Environmental persistence of Tulane virus — a surrogate for human norovirus." Canadian Journal of Microbiology 62, no. 5 (2016): 449–54. http://dx.doi.org/10.1139/cjm-2015-0756.
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Human noroviruses (HuNoVs) are the leading cause of acute viral gastroenteritis worldwide. The persistence of HuNoV in the environment contributes significantly to its transmission to humans. Surrogate viruses are used to study HuNoV owing to the lack of a cell culture system for this virus. Here, the persistence of Tulane virus (TV) — a novel HuNoV surrogate — in surface water (SW) and groundwater (GW) as well as on acrylic-based solid (ABS) and stainless steel (SS) surfaces was investigated. After 28 days, TV remained stable in SW (<1 log10 reduction) but was reduced by ≥3.5 to 4 log10 in GW by day 21. TV had a higher rate of reduction on SS compared with ABS, with corresponding D values of 18.5 ± 0.34 and 13.1 ± 0.36 days, respectively. This is the first study to demonstrate the persistence of TV in environmental waters and on fomite surfaces.
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Hyde, Jennifer, and Jason McKenzie. "Pathogenesis and replication of norovirus: following the mouse tail?" Microbiology Australia 33, no. 2 (2012): 74. http://dx.doi.org/10.1071/ma12074.
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The emergence of human noroviruses (NoV) as significant human pathogens over the last decades has highlighted the need to research and understand the replication and pathogenesis of this group of viruses. One of the major hurdles faced by researchers in this field has been the lack of a viable tissue culture system or small animal model with which to study human NoV replication. The discovery of a murine NoV in 2003 and the identification of its tropism for macrophage and dendritic cell lines has provided the opportunity to study aspects of NoV replication and pathogenesis that were previously closed to researchers.
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Chang, Kyeong-Ok, David W. George, John B. Patton, Kim Y. Green, and Stanislav V. Sosnovtsev. "Leader of the Capsid Protein in Feline Calicivirus Promotes Replication of Norwalk Virus in Cell Culture." Journal of Virology 82, no. 19 (2008): 9306–17. http://dx.doi.org/10.1128/jvi.00301-08.
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ABSTRACT The inability to grow human noroviruses in cell culture has greatly impeded the studies of their pathogenesis and immunity. Vesiviruses, in the family Caliciviridae, grow efficiently in cell culture and encode a unique protein in the subgenomic region designated as leader of the capsid protein (LC). We hypothesized that LC might be associated with the efficient replication of vesiviruses in cell culture and promote the replication of human norovirus in cells. To test this hypothesis, a recombinant plasmid was engineered in which the LC region of feline calicivirus (FCV) was placed under the control of the cytomegalovirus promoter (pCI-LC) so that the LC protein could be provided in trans to replicating calicivirus genomes bearing a reporter gene. We constructed pNV-GFP, a recombinant plasmid containing a full-length NV genome with a green fluorescent protein (GFP) in the place of VP1. The transfection of pNV-GFP in MVA-T7-infected cells produced few GFP-positive cells detected by fluorescence microscopy and flow cytometry analysis. When pNV-GFP was cotransfected with pCI-LC in MVA-T7-infected cells, we observed an increase in the number of GFP-positive cells (ca. 3% of the whole-cell population). Using this cotransfection method with mutagenesis study, we identified potential cis-acting elements at the start of subgenomic RNA and the 3′ end of NV genome for the virus replication. We conclude that LC may be a viral factor which promotes the replication of NV in cells, which could provide a clue to growing the fastidious human noroviruses in cell culture.
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Lou, Fangfei, Pengwei Huang, Hudaa Neetoo, et al. "High-Pressure Inactivation of Human Norovirus Virus-Like Particles Provides Evidence that the Capsid of Human Norovirus Is Highly Pressure Resistant." Applied and Environmental Microbiology 78, no. 15 (2012): 5320–27. http://dx.doi.org/10.1128/aem.00532-12.
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ABSTRACTHuman norovirus (NoV) is the leading cause of nonbacterial acute gastroenteritis epidemics worldwide. High-pressure processing (HPP) has been considered a promising nonthermal processing technology to inactivate food- and waterborne viral pathogens. Due to the lack of an effective cell culture method for human NoV, the effectiveness of HPP in inactivating human NoV remains poorly understood. In this study, we evaluated the effectiveness of HPP in disrupting the capsid of human NoV based on the structural and functional integrity of virus-like particles (VLPs) and histo-blood group antigen (HBGA) receptor binding assays. We found that pressurization at 500 to 600 MPa for 2 min, a pressure level that completely inactivates murine norovirus and feline calicivirus, was not sufficient to disrupt the structure and function of human NoV VLPs, even with a holding time of 60 min. Degradation of VLPs increased commensurate with increasing pressure levels more than increasing time. The times required for complete disruption of human NoV VLPs at 700, 800, and 900 MPa were 45, 15, and 2 min, respectively. Human NoV VLPs were more resistant to HPP in their ability to bind type A than type B and O HBGAs. Additionally, the 23-nm VLPs appeared to be much more stable than the 38-nm VLPs. Taken together, our results demonstrated that the human NoV capsid is highly resistant to HPP. While human NoV VLPs may not be fully representative of viable human NoV, destruction of the VLP capsid is highly suggestive of a typical response for viable human NoV.
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Guix, Susana, Miyuki Asanaka, Kazuhiko Katayama, et al. "Norwalk Virus RNA Is Infectious in Mammalian Cells." Journal of Virology 81, no. 22 (2007): 12238–48. http://dx.doi.org/10.1128/jvi.01489-07.
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ABSTRACT Human noroviruses are positive-sense RNA viruses and are the leading cause of epidemic acute viral gastroenteritis in developed countries. The absence of an in vitro cell culture model for human norovirus infection has limited the development of effective antivirals and vaccines. Human histo-blood group antigens have been regarded as receptors for norovirus infection, and expression of the α(1,2) fucosyltransferase gene (FUT2) responsible for the secretor phenotype is required for susceptibility to Norwalk virus (NV) infection. We report for the first time that transfection of NV RNA, isolated from stool samples from human volunteers, into human hepatoma Huh-7 cells leads to viral replication, with expression of viral antigens, RNA replication, and release of viral particles into the medium. Prior treatment of the RNA with proteinase K completely abolishes RNA infectivity, suggesting a key role of an RNA-protein complex. Although overexpression of the human FUT2 gene enhances virus binding to cells, it is not sufficient to allow a complete viral infection, and viral spread from NV-transfected cells to naïve cells does not occur. Finally, no differences in NV RNA replication are observed between Huh-7 and Huh-7.5.1 cells, which contain an inactivating mutation in retinoic acid-inducible gene I (RIG-I), suggesting that the RIG-I pathway does not play a role in limiting NV replication. Our results strongly suggest that the block(s) to NV replication in vitro is at the stage of receptor and/or coreceptor binding and/or uncoating, either because cells lack some specific factor or activation of cellular antiviral responses independent of RIG-I inhibits virus replication.
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Bolton, Stephanie L., Grishma Kotwal, Mark A. Harrison, S. Edward Law, Judy A. Harrison, and Jennifer L. Cannon. "Sanitizer Efficacy against Murine Norovirus, a Surrogate for Human Norovirus, on Stainless Steel Surfaces when Using Three Application Methods." Applied and Environmental Microbiology 79, no. 4 (2012): 1368–77. http://dx.doi.org/10.1128/aem.02843-12.
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ABSTRACTHuman noroviruses are major etiologic agents of epidemic gastroenteritis. Outbreaks are often accompanied by contamination of environmental surfaces, but since these viruses cannot be routinely propagated in laboratory cultures, their response to surface disinfectants is predicted by using surrogates, such as murine norovirus 1 (MNV-1). This study compared the virucidal efficacies of various liquid treatments (three sanitizer liquids, 5% levulinic acid plus 2% SDS [LEV/SDS], 200 ppm chlorine, and an isopropanol-based quaternary ammonium compound [Alpet D2], and two control liquids, sterile tap water and sterile tap water plus 2% SDS) when delivered to MNV-1-inoculated stainless steel surfaces by conventional hydraulic or air-assisted, induction-charged (AAIC) electrostatic spraying or by wiping with impregnated towelettes. For the spray treatments, LEV/SDS proved effective when applied with hydraulic and AAIC electrostatic spraying, providing virus reductions of 2.71 and 1.66 log PFU/ml, respectively. Alpet D2 provided a 2.23-log PFU/ml reduction with hydraulic spraying, outperforming chlorine (1.16-log PFU/ml reduction). Chlorine and LEV/SDS were equally effective as wipes, reducing the viral load by 7.05 log PFU/ml. Controls reduced the viral load by <1 log with spraying applications and by >3 log PFU/ml with wiping. Results indicated that both sanitizer type and application methods should be carefully considered when choosing a surface disinfectant to best prevent and control environmental contamination by noroviruses.
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Perry, Jeffrey W., and Christiane E. Wobus. "Endocytosis of Murine Norovirus 1 into Murine Macrophages Is Dependent on Dynamin II and Cholesterol." Journal of Virology 84, no. 12 (2010): 6163–76. http://dx.doi.org/10.1128/jvi.00331-10.
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ABSTRACT Although noroviruses cause the vast majority of nonbacterial gastroenteritis in humans, little is known about their life cycle, including viral entry. Murine norovirus (MNV) is the only norovirus to date that efficiently infects cells in culture. To elucidate the productive route of infection for MNV-1 into murine macrophages, we used a neutral red (NR) infectious center assay and pharmacological inhibitors in combination with dominant-negative (DN) and small interfering RNA (siRNA) constructs to show that clathrin- and caveolin-mediated endocytosis did not play a role in entry. In addition, we showed that phagocytosis or macropinocytosis, flotillin-1, and GRAF1 are not required for the major route of MNV-1 uptake. However, MNV-1 genome release occurred within 1 h, and endocytosis was significantly inhibited by the cholesterol-sequestering drugs nystatin and methyl-β-cyclodextrin, the dynamin-specific inhibitor dynasore, and the dominant-negative dynamin II mutant K44A. Therefore, we conclude that the productive route of MNV-1 entry into murine macrophages is rapid and requires host cholesterol and dynamin II.
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Katpally, Umesh, Christiane E. Wobus, Kelly Dryden, Herbert W. Virgin, and Thomas J. Smith. "Structure of Antibody-Neutralized Murine Norovirus and Unexpected Differences from Viruslike Particles." Journal of Virology 82, no. 5 (2007): 2079–88. http://dx.doi.org/10.1128/jvi.02200-07.
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ABSTRACT Noroviruses (family Caliciviridae) are the major cause of epidemic nonbacterial gastroenteritis in humans, but the mechanism of antibody neutralization is unknown and no structure of an infectious virion has been reported. Murine norovirus (MNV) is the only norovirus that can be grown in tissue culture, studied in an animal model, and reverse engineered via an infectious clone and to which neutralizing antibodies have been isolated. Presented here are the cryoelectron microscopy structures of an MNV virion and the virion in complex with neutralizing Fab fragments. The most striking differences between MNV and previous calicivirus structures are that the protruding domain is lifted off the shell domain by ∼16Å and rotated ∼40° in a clockwise fashion and forms new interactions at the P1 base that create a cagelike structure engulfing the shell domains. Neutralizing Fab fragments cover the outer surface of each copy of the capsid protein P2 domains without causing any apparent conformational changes. These unique features of MNV suggest that at least some caliciviruses undergo a capsid maturation process akin to that observed with other plant and bacterial viruses.
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CANNON, JENNIFER L., ALI AYDIN, AMY N. MANN, STEPHANIE L. BOLTON, TONG ZHAO, and MICHAEL P. DOYLE. "Efficacy of a Levulinic Acid Plus Sodium Dodecyl Sulfate–Based Sanitizer on Inactivation of Human Norovirus Surrogates." Journal of Food Protection 75, no. 8 (2012): 1532–35. http://dx.doi.org/10.4315/0362-028x.11-572.
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Human noroviruses are the most common etiologic agent of foodborne illness in the United States. The inability to culture human noroviruses in the laboratory necessitates the use of surrogate viruses such as murine norovirus (MNV-1) and feline calicivirus (FCV) for inactivation studies. In this study, a novel sanitizer of organic acid (levulinic acid) plus the anionic detergent sodium dodecyl sulfate (SDS) was evaluated. Viruses were treated with levulinic acid (0.5 to 5%), SDS (0.05 to 2%), or combinations of levulinic acid plus SDS (1:10 solution of virus to sanitizer). MNV-1 inoculated onto stainless steel also was treated with a 5% levulinic acid plus 2% SDS liquid or foaming solution. Log reductions of viruses were determined with a plaque assay. Neither levulinic acid nor SDS alone were capable of inactivating MNV-1 or FCV, resulting in a ≤0.51-log reduction of the infectious virus titer. However, the combination of 0.5% levulinic acid plus 0.5% SDS inactivated both surrogates by 3 to 4.21 log PFU/ml after 1 min of exposure. Similarly, MNV-1 inoculated onto stainless steel was reduced by >1.50 log PFU/ml after 1 min and by >3.3 log PFU/ml after 5 min of exposure to a liquid or foaming solution of 5% levulinic acid plus 2% SDS. The presence of organic matter (up to 10%) in the virus inoculum did not significantly affect sanitizer efficacy. The fact that both of the active sanitizer ingredients are generally recognized as safe to use as food additives by the U.S. Food and Drug Administration further extends its potential in mitigating foodborne disease.
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Bae, Jinhee, and Kellogg J. Schwab. "Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater." Applied and Environmental Microbiology 74, no. 2 (2007): 477–84. http://dx.doi.org/10.1128/aem.02095-06.
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ABSTRACT Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log10/day for FCV, 0.13 and 0.10 log10/day for PV, 0.12 and 0.06 log10/day for MS2, and 0.09 and 0.05 log10/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log10/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log10/day) and MNV (0.04 ± 0.03 log10/day) and were significantly different from those of FCV (0.08 ± 0.03 log10/day) and PV (0.09 ± 0.03 log10/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.
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Desselberger, Ulrich. "Caliciviridae Other Than Noroviruses." Viruses 11, no. 3 (2019): 286. http://dx.doi.org/10.3390/v11030286.
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Besides noroviruses, the Caliciviridae family comprises four other accepted genera: Sapovirus, Lagovirus, Vesivirus, and Nebovirus. There are six new genera proposed: Recovirus, Valovirus, Bavovirus, Nacovirus, Minovirus, and Salovirus. All Caliciviridae have closely related genome structures, but are genetically and antigenically highly diverse and infect a wide range of mammalian host species including humans. Recombination in nature is not infrequent for most of the Caliciviridae, contributing to their diversity. Sapovirus infections cause diarrhoea in pigs, humans and other mammalian hosts. Lagovirus infections cause systemic haemorrhagic disease in rabbits and hares, and vesivirus infections lead to lung disease in cats, vesicular disease in swine, and exanthema and diseases of the reproductive system in large sea mammals. Neboviruses are an enteric pathogen of cattle, differing from bovine norovirus. At present, only a few selected caliciviruses can be propagated in cell culture (permanent cell lines or enteroids), and for most of the cultivatable caliciviruses helper virus-free, plasmid only-based reverse genetics systems have been established. The replication cycles of the caliciviruses are similar as far as they have been explored: viruses interact with a multitude of cell surface attachment factors (glycans) and co-receptors (proteins) for adsorption and penetration, use cellular membranes for the formation of replication complexes and have developed mechanisms to circumvent innate immune responses. Vaccines have been developed against lagoviruses and vesiviruses, and are under development against human noroviruses.
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La Rosa, G., S. Fontana, A. Di Grazia, M. Iaconelli, M. Pourshaban, and M. Muscillo. "Molecular Identification and Genetic Analysis of Norovirus Genogroups I and II in Water Environments: Comparative Analysis of Different Reverse Transcription-PCR Assays." Applied and Environmental Microbiology 73, no. 13 (2007): 4152–61. http://dx.doi.org/10.1128/aem.00222-07.
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ABSTRACT Noroviruses have received increased attention in recent years because their role as etiologic agents in acute gastroenteritis outbreaks is now clearly established. Our inability to grow them in cell culture and the lack of an animal model hinder the characterization of these viruses. More recently, molecular approaches have been used to study the genetic relationships that exist among them. In the present study, environmental samples from seawater, estuarine water, and effluents of sewage treatment plants were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for transmission of norovirus genogroups I and II. Novel broad-range reverse transcription-PCR/nested assays targeting the region coding for the RNA-dependent RNA polymerase were developed, amplifying fragments of 516 bp and 687 bp in the nested reactions for genogroups II and I, respectively. The assays were evaluated and compared against widely used published assays. The newly designed assays provide long regions for high-confidence BLAST searches in public databases and therefore are useful diagnostic tools for molecular diagnosis and typing of human noroviruses in clinical and environmental samples, as well as for the study of molecular epidemiology and the evolution of these viruses.
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Haramoto, Eiji, Hiroyuki Katayama, Kumiko Oguma, and Shinichiro Ohgaki. "Application of Cation-Coated Filter Method to Detection of Noroviruses, Enteroviruses, Adenoviruses, and Torque Teno Viruses in the Tamagawa River in Japan." Applied and Environmental Microbiology 71, no. 5 (2005): 2403–11. http://dx.doi.org/10.1128/aem.71.5.2403-2411.2005.
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ABSTRACT The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% ± 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.
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Chan, Martin C. W., Y. P. Wong, and Wai K. Leung. "Cell Culture Assay for Human Noroviruses." Emerging Infectious Diseases 13, no. 7 (2007): 1117–18. http://dx.doi.org/10.3201/eid1307.070131.
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