Biology subjects

Vinje, J.

Publications and source records attributed to Vinje, J..

10 recordsLinked to original sources

bioRxiv2026

Comparison of Human Intestinal Enteroids and Zebrafish Larvae models for replication of Human Norovirus

Human intestinal enteroids (HIE) and zebrafish larvae (ZF) models support the replication of certain strains of human norovirus (HuNoV), the leading cause of viral gastroenteritis worldwide. The replication of 17 HuNoV (9 genotypes) positive stool specimens from patients ranging from 5 months to 83 years old was evaluated in both HIE and ZF models. The yolk of 3-day post-fertilization ZF larvae were microinjected with 3 nL 10% clarified stool suspension. Each day post-infection (dpi), 10 larvae were pooled as one sample, and two specimens were collected daily until 5 dpi. Viral RNA was extracted from harvested larvae and quantified using reverse transcription (RT) droplet digital PCR. For the HIE model, J4FUT2 K1 enteroid monolayers were inoculated with 100-fold dilution of each stool specimen. Viral RNA levels were quantified at 1 and 72 h post-infection using RT real-time PCR. All genogroup (G) I specimens (n=5) replicated in both models showing a 1 to 2.5 log increase in HuNoV RNA. Of the 12 GII specimens tested, 8 replicated in the ZF model and 7 replicated in the HIE model. Specimens that failed to replicate in each model were not the same, although most of them were GII.4 Sydney [P16]. One additional strain, GII.17[P17], replicated only in the ZF model. Among the GII strains, viral RNA increase ranged from 1.6 to 3.3 log in the ZF and 0.4 to 3.68 log in the HIE model. These data highlight variability in HuNoV replication efficiency across models, potentially indicating the influence of model-specific factors on replication. ImportanceHuman intestinal enteroid and ZF models have been independently shown to support replication of different strains of HuNoV, the leading cause of viral gastroenteritis worldwide. However, whether the same HuNoV positive stool specimens replicate similarly in both systems has not been examined. In this study, stool specimens positive for a range of HuNoV genotypes were tested in both models. While specimens from the GI genogroup consistently replicated in both systems, specimens from the GII genogroup showed variable replication, with some replicating in only one model and a couple of specimens not replicating in either model. These findings demonstrate that model specific biological factors influence HuNoV replication and highlight the significance of using complementary model systems to better understand HuNoV pathogenesis.

microbiology↗

bioRxiv2023

Heat Inactivation of Aqueous Viable Norovirus and MS2 Bacteriophage

Human norovirus is a leading cause of acute gastroenteritis often associated with contaminated food or water exposure. Many studies have used morphologically similar viruses, such as MS2 bacteriophage, and molecular detection methods to study the environmental fate and inactivation characteristics, given the historical challenges to culture human norovirus. In this study, we used human intestinal enteroids (HIEs) to analyze the heat inactivation kinetics of viable human norovirus compared to the surrogate MS2 bacteriophage. Norovirus decay rates were 0.22 min-1, 0.68 min-1, and 1.11 min-1 for 50{degrees}C, 60{degrees}C, and 70{degrees}C, respectively, and MS2 bacteriophage decay rates were 0.0065 min-1, 0.045 min-1, and 0.16 min-1 for 50{degrees}C, 60{degrees}C, and 70{degrees}C, respectively. Norovirus and MS2 bacteriophage had significantly different decay rates at all tested temperatures (p = 0.002 - 0.007). No decrease of RNA titers as measured by realtime RT-PCR for both human norovirus and MS2 bacteriophage over time was observed, indicating molecular methods do not accurately depict viable human norovirus after heat inactivation. Overall, our data demonstrate that MS2 bacteriophage is a conservative surrogate to measure heat inactivation and potentially overestimates the infectious risk of norovirus. IMPORTANCENorovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide. Treatments to inactivate norovirus are critical to reducing the risk associated with contaminated food and water. The recent developments to replicate human norovirus in human intestinal enteroids (HIE) enables the evaluation of heat inactivation kinetics of viable norovirus. Historically, cultivable surrogate viruses, such as bacteriophage MS2, have been used to measure the environmental fate of human norovirus. Our findings indicate that compared to human norovirus, MS2 bacteriophage is a conservative surrogate to measure the effect of heat inactivation. Furthermore, this study corroborates that measuring viral RNA titers, as evaluated by PCR methods, does not correlate with persistence of viable norovirus.

microbiology↗

bioRxiv2023

Glycochenodeoxycholic acid and ceramide suppress the antiviral effect of 25-hydroxycholesterol against human norovirus infection in human intestinal enteroids

The human intestinal enteroid (HIE) cell culture system with the support of glycine-conjugated bile acid glycochenodeoxycholic acid (GCDCA) and ceramide (C2) facilitate successful replication of several norovirus strains. Here we investigate how the presence of GCDCA/C2 impacts gene expression of norovirus-infected HIE and the impact of 25 hydroxycholesterol (25-HC), a key regulator of cholesterol homeostasis and bile acid production on norovirus replication. In absence of GCDCA/C2, 0.01 and 0.1 M 25-HC suppressed virus (GII.4 Sydney[P16]) replication by 1.3 log and 1.1 log respectively (p<0.05). In the presence of GCDCA/C2, 5 M 25-HC was required to achieve a 1 log decrease (p<0.05) in viral titers demonstrating that 25-HC restricts norovirus replication in HIE. RNA sequence analysis showed that during human norovirus infection, 25-HC downregulated expression of genes (CYP3A4, APOB, APOA1, and ABCG1) involved in cholesterol metabolism and transport as well as interferon stimulated genes such as ISG15 and IFIT1. GCDCA/C2 counteracts the suppressive effect of 25-HC expression of some genes related to these pathways including APOA4 and CYP27A1 however, other cholesterol genes such as APOA1 were further suppressed in the presence of GCDCA/C2. ImportanceNorovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and currently, there are no effective therapeutic strategies against this highly contagious pathogen. Our study provides insights into the effect of bile during norovirus infection, highlight the role of the cholesterol/oxysterol pathways during human norovirus replication, and demonstrate the potential utility of oxysterols in developing norovirus therapeutics.

genetics↗

bioRxiv2022

Mucosal and systemic neutralizing antibodies to norovirus and rotavirus by oral immunization with recombinant rotavirus in infant mice

Rotaviruses (RVs) preferentially replicate in the small intestine, frequently cause severe diarrheal disease, and following enteric infection generally induce variable levels of protective systemic and mucosal immune responses in humans and other animals. Rhesus rotavirus (RRV) is a simian RV that was previously used as a human RV vaccine and has been extensively studied in mice. Although RRV replicates poorly in the suckling mouse intestine, infection induces a robust and protective antibody response. The recent availability of plasmid-based RV reverse genetics systems has enabled the generation of recombinant RVs expressing foreign proteins. However, recombinant RVs have not yet been experimentally tested as potential vaccine vectors to immunize against other gastrointestinal pathogens in vivo. This is a missed opportunity because several live-attenuated RV vaccines are already widely administered to infants and young children worldwide. To explore the feasibility of using RV as a dual vaccine vector, we rescued a replication-competent recombinant RRV harboring bicistronic gene segment 7 that encodes both the native RV NSP3 protein and a human norovirus (HuNoV) VP1 protein from the predominant genotype GII.4 (rRRV-HuNoV-VP1). The rRRV-HuNoV-VP1 expressed HuNoV VP1 in infected cells in vitro and importantly, elicited both systemic and local antibody responses to HuNoV following oral infection of suckling mice. Serum IgG and fecal IgA from infected suckling mice bound to and neutralized both RV and HuNoV. These findings have encouraging practical implications for the design of RV-based next-generation multivalent enteric vaccines to target HuNoV and other human enteric pathogens while providing immunity to RV. Significance statementMucosal immunity is a key component of protection against many pathogens. Robust and effective mucosal immune responses are generally induced following infection with a replication-competent pathogen at a mucosal surface. Several studies have attempted to develop viral vector-based enteric mucosal vaccines; however, the most advanced of these are still in clinical development. Here, we successfully induced systemic and mucosal antibody responses against both rotavirus and norovirus following inoculation of a recombinant rotavirus expressing the human norovirus major capsid protein. These responses are likely to correlate with protective immunity. Live-attenuated rotavirus vaccines have already proven safe and effective worldwide. These findings confirm the potential utility of using rotaviruses as a dual enteric vaccine platform for other important human enteric pathogens.

microbiology↗

bioRxiv2020

Actin cytoskeletal and focal adhesion organisation in U2OS cells on polymer nanostructures

Cells in their natural environment are embedded in a complex surrounding consisting of biochemical and biomechanical cues directing cell properties and cell behaviour. Nonetheless, in vitro cell studies are typically performed on flat surfaces, with clear differences from the more complex situation cells experience in vivo. To increase the physiological relevance of these studies, a number of advanced cellular substrates for in vitro studies have been applied. One of these approaches include flat surfaces decorated with vertically aligned nanostructures. In this work, we explore how U2OS cells are affected by arrays of polymer nanopillars fabricated on flat glass surfaces. We focus on describing changes to the organisation of the actin cytoskeleton and in the location, number and shape of focal adhesions. From our findings we identify that the cells can be categorised into different regimes based on their spreading and adhesion behaviour on nanopillars. A quantitative analysis suggests that cells seeded on dense nanopillar arrays are suspended on top of the pillars with focal adhesions forming closer to the cell periphery compared to flat surfaces or sparse pillar arrays. This change is analogous to similar responses for cells seeded on soft substrates. Overall, we show that the combination of high throughput nanofabrication, advanced optical microscopy, molecular biology tools to visualise cellular processes and data analysis can be used to investigate how cells interact with nanostructured surfaces and will in the future help to create culture substrates that induce particular cell function.

biophysics↗

bioRxiv2019

Electron beam lithography fabrication of SU-8 polymer structures for cell studies

Flat surfaces decorated with micro- and nanostructures are important tools in biomedical research used to control cellular shape, in studies of mechanotransduction, membrane mechanics, cell migration and cellular interactions with nanostructured surfaces. Existing methods to fabricate surface-bound nanostructures are typically limited either by resolution, aspect ratio or throughput. In this work, we explore electron beam lithography based structuring of the epoxy resist SU-8 on glass substrate. We focus on a systematic investigation of the process parameters and determine limits of the fabrication process, both in terms of spatial resolution, structure aspect ration and fabrication throughput. The described approach is capable of producing high-aspect ratio, surface bound nanostructures with height ranging from 100 nm to 4000 nm and with in-plane resolution below 100 nm directly on a transparent substrate. Fabricated nanostructured surfaces can be integrated with common techniques for biomedical research, such as high numerical aperture optical microscopy. Further more, we show how the described approach can be used to make nanostructures with multiple heights on the same surface, something which is not readily achievable using alternative fabrication approaches. Our research paves an alternative way of manufacturing nanostructured surfaces with applications in life science research.

biophysics↗

bioRxiv2019

Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses

Next-generation sequencing is a powerful tool for virological surveillance. While Illumina(R) and Ion Torrent(R) sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. We evaluated the Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 outperformed the older Ion Torrent PGM platform in data quality and cost, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 or Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 or Illumina MiSeq V2) the cost per sample was comparable. These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.

microbiology↗

bioRxiv2019

Preadaptation of pandemic GII.4 noroviruses in hidden virus reservoirs years before emergence

The control of pandemic pathogens depends on early prediction of pandemic variants and, more generally, understanding origins of such variants and factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have suggested that new GII.4 pandemic viruses evolve from previous pandemic variants through substitutions in the antigenic region of the VP1 protein that enable evasion of host population immunity, leading to global spread. In contrast, we show here that the acquisition of new genetic and antigenic characteristics is not the proximal driver of new pandemics. Instead, pandemic GII.4 viruses circulate undetected for years before causing a new pandemic, during which time they diversify and spread over wide geographical areas. Serological data demonstrate that by 2003, some nine years before it emerged as a new pandemic, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically-preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics.\n\nSignificanceNorovirus pandemics and their associated public health and economic costs could be prevented by effective vaccines. However, vaccine development and distribution will require identification of the sources and drivers of new pandemics. We here use phylogenetics and serological experiments to develop and test a new hypothesis of pandemic norovirus emergence. We find that pandemic noroviruses preadapt, diversify and spread worldwide years prior to emergence, strongly indicating that genetic changes are necessary but not sufficient to drive a new pandemic. We instead suggest that changes in population immunity enable pandemic emergence of a pre-adapted low-level variant. These findings indicate that prediction of new pandemics will require surveillance of under-sampled virus reservoirs and that norovirus vaccines will need to elicit broad immunity.

evolutionary biology↗

bioRxiv2019

Influence of nanopillar arrays on fibroblast motility, adhesion and migration mechanisms

Surfaces decorated with high aspect ratio nanostructures are a promising tool to study cellular processes and design novel devices to control cellular behaviour, perform intracellular sensing or deliver effector molecules to cells in culture. However, little is known about the dynamics of cellular phenomenon such as adhesion, spreading and migration on such surfaces. In particular, how these are influenced by the surface properties. In this work, we investigate fibroblast behaviour on regular arrays of 1 micrometer high, polymer nanopillars with varying pillar to pillar distance (array pitch). NIH-3T3 fibroblasts spread on all arrays, and on contact with the substrate engulf nanopillars independently of the array pitch. As the cells start to spread, different behaviour is observed. On dense arrays which have the pitch equal or below 1 micrometer, cells are suspended on top of the nanopillars, making only sporadic contact with the glass support. Cells stay attached to the glass support and fully engulf nanopillars during spreading and migration on the sparse arrays which are characterized by a pitch of 2 micrometers and above. These alternate states have a profound effect on cell migration rates, which are strongly reduced on nanopillar sparse arrays. Dynamic actin puncta colocalize with nanopillars during cell spreading and migration. Strong membrane association with engulfed nanopillars might explain the reduced migration rates on sparse arrays. This work reveals several interesting phenomenon of dynamical cell behaviour on nanopillar arrays, and provides important perspectives on design and applications of high aspect ratio nanostructures.

biophysics↗

bioRxiv2019

Enteropathogen seroepidemiology among children in low-resource settings

Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG response to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) using multiplex bead assays in cohorts from Haiti, Kenya, and Tanzania. By age 2 years, most children had evidence of exposure by IgG response to the pathogens studied. We discovered a shift in IgG distributions for many pathogens as children age, caused by boosting and waning from repeated exposures, which complicates interpretation of seroprevalence among older children. Longitudinal profiles revealed important variation in enteropathogen IgG response above seropositivity cutoffs, underscoring the importance of longitudinal designs to estimate seroincidence rates as a measure of force of infection. In longitudinal cohorts there was a linear relationship between seroprevalence and prospective seroincidence rates, suggesting the two measures provide similar information about variation in pathogen transmission.

epidemiology↗