Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries.

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Challenges for estimating human norovirus infectivity by viability RT-qPCR as compared to replication in human intestinal enteroids.

Viability RT-qPCR, a molecular detection method combining viability marker pre-treatment with RT-qPCR, has been proposed to infer infectivity of viruses which is particularly relevant for non-culturable viruses or sophisticated cell culture systems. Being human noroviruses (HuNoV) most frequently associated with foodborne outbreaks, this study compared different viability techniques and infectivity in human intestinal enteroids (HIE) to ultimately determine whether the molecular approaches could serve as rapid assays to predict HuNoV inactivation in high-risk food. To this end, the performance of three viability RT-qPCR assays with different intercalating markers ((Viability PCR Crosslinker Kit (CL), propidium monoazide (PMAxx™), and platinum chloride (PtCl4)) in estimating survival of HuNoV exposed to thermal and high pressure (HPP) treatments was compared to replication tested in the HIE cell culture model. A nearly full-length genomic molecular assay coupled with PMAxx™ to infer HuNoV thermal inactivation was also assessed. The experimental design included HuNoV genogroup I.3 [P13], GII.4 Sydney [P16], GII.6 [P7], along with Tulane virus (TV) serving as surrogate. Finally, viability RT-qPCR was tested in HPP-treated strawberry puree, selected as a food matrix with high viral contamination risk. PMAxx™ and CL performed evenly, while PtCl4 affected HuNoV infectivity. Taking all experimental data together, viability RT-qPCR was demonstrated to be an improved method over direct RT-qPCR to estimate viral inactivation at extreme thermal (95 °C) and HPP (450 MPa) exposures, but not under milder conditions as amplification signals were detected. Despite its complexity and limitations, the HIE demonstrated a more robust model than viability RT-qPCR to assess HuNoV infectivity.

Use of Human Intestinal Enteroids for Recovery of Infectious Human Norovirus from Berries and Lettuce.

Norovirus (NoV) is the leading cause of viral foodborne gastroenteritis globally. Currently, the gold standard for detecting NoV in clinical, food, and environmental samples is via molecular-based methods, primarily RT-PCR. Nevertheless, there is a great need for confirmatory assays that can determine the infectivity of viral particles recovered from contaminated matrices. The use of the human intestinal enteroids system (HIEs) has allowed for the expansion of norovirus replication, although it still suffers from limitations of strain preferences and the requirement of high titer stocks for infection. In this study, we wanted to explore the feasibility of using the HIEs to support the replication of NoV that had been recovered from representative food matrices that have been associated with foodborne illness. We first confirmed that HIEs can support the replication of several strains of NoV as measured by RT-qPCR. We subsequently chose two of those strains that reproducibly replicated, GII.4 and GII.6, to evaluate in a TCID50 assay and for future experiments. Infectious NoV could be recovered and quantified in the HIEs from lettuce, frozen raspberries, or frozen strawberries seeded with high titers of either of these strains. While many experimental challenges still remain to be overcome, the results of this study represent an important step toward the detection of infectious norovirus from representative produce items.

Application of whole-genome sequencing for norovirus outbreak tracking and surveillance efforts in Orange County, CA.

Noroviruses are the leading cause of acute gastroenteritis and foodborne illness in the United States. Traditional Sanger sequencing of short genomic regions (~300-600 bp) is the primary method for differentiation of this pathogen; however, whole-genome sequencing (WGS) offers a valuable approach to further characterize strains of this virus. The objective of this study was to investigate the ability of WGS compared to Sanger sequencing to differentiate norovirus strains and enhance outbreak investigation and surveillance efforts. WGS results for 41 norovirus-positive stool samples from 15 different outbreaks occurring from 2012 to 2019 in Orange County, CA, were analyzed for this study. All samples were genotyped with both WGS and Sanger sequencing based on the B-C region. WGS generated nearly full-length viral genome sequences (7029-7768 bp) with 4x to 35,378x coverage. Phylogenetic analysis of WGS data enabled differentiation of genotypically similar strains from separate outbreaks. Single nucleotide variation (SNV) analysis on a subset of strains revealed nucleotide variations (15-79 nt) among isolates from multiple outbreaks of GII.4 Sydney_2015[P31] and GII.17[P17]. Overall, the results demonstrated that coupling norovirus genotype identification with WGS enables enhanced genetic differentiation of strains and provides valuable information for outbreak investigation and surveillance efforts.

Titanium coating with mussel inspired polymer and bio-orthogonal chemistry enhances antimicrobial activity against Staphylococcus aureus.

Implant-associated infections present severe and difficult-to-treat complications after surgery, related to implant biofilm colonization. Systemic administration of antibiotics cannot reach sufficient concentrations at the infected site and may be toxic. Here we describe how mussel-inspired dendritic material coated on a titanium surface can locally activate a prodrug of daptomycin (pro-dapto) to treat methicillin-resistant Staphylococcus aureus. The mechanism of the prodrug activation is based on bio-orthogonal click chemistry between a tetrazine (Tz) and trans-cyclooctene (TCO). The former is attached to the dendritic polymer, while the later converts daptomycin into a prodrug. Characterization of the material's properties revealed that it is hydrophobic, non-toxic, and stable for a prolonged period of time. We envision that the titanium coated dendritic material will be able to improve the treatment of implant-associated infections by concentrating systemically administered antibiotic prodrugs, thus converting them into active localized medicines.

Genomic Analyses of Human Sapoviruses Detected over a 40-Year Period Reveal Disparate Patterns of Evolution among Genotypes and Genome Regions.

Human sapovirus is a causative agent of acute gastroenteritis in all age groups. The use of full-length viral genomes has proven beneficial to investigate evolutionary dynamics and transmission chains. In this study, we developed a full-length genome sequencing platform for human sapovirus and sequenced the oldest available strains (collected in the 1970s) to analyse diversification of sapoviruses. Sequence analyses from five major genotypes (GI.1, GI.2, GII.1, GII.3, and GIV.1) showed limited intra-genotypic diversification for over 20-40 years. The accumulation of amino acid mutations in VP1 was detected for GI.2 and GIV.1 viruses, while having a similar rate of nucleotide evolution to the other genotypes. Differences in the phylogenetic clustering were detected between RdRp and VP1 sequences of our archival strains as well as other reported putative recombinants. However, the lack of the parental strains and differences in diversification among genomic regions suggest that discrepancies in the phylogenetic clustering of sapoviruses could be explained, not only by recombination, but also by disparate nucleotide substitution patterns between RdRp and VP1 sequences. Together, this study shows that, contrary to noroviruses, sapoviruses present limited diversification by means of intra-genotype variation and recombination.

Evaluation of U.S. Food and Drug Administration Enteric Viruses Microarray for Detection of Hepatitis A Virus and Norovirus in Inoculated Tomatoes, Green Onions, and Celery.

ABSTRACT: Foodborne viral contamination of fresh produce has been associated with numerous outbreaks. Detection of such contaminated foods is important in protecting public health. Here, we demonstrate for the first time the capability of the U.S. Food and Drug Administration Enteric Viruses tiling microarray (FDA-EVIR) to perform rapid molecular identification of hepatitis A virus (HAV) and human norovirus extracted from artificially inoculated fresh produce. Two published viral extraction strategies, total RNA extraction or virus particle isolation, were used to prepare the viral targets. The total RNA extraction method was used on material eluted from tomatoes, using an alkaline Tris-glycine-beef extract (TGBE) buffer. Optimization procedures including DNase treatment and poly(A)-RNA enrichment were adopted to improve microarray sensitivity. For green onions or celery, material was eluted using either glycine buffer or TGBE buffer supplemented with pectinase, respectively, and then virus particles were concentrated by ultracentrifugation. We also assessed the amount of viral RNA extracted from celery using three commercially available kits and how well that RNA performed on FDA-EVIR. Our results confirm that FDA-EVIR can identify common enteric viruses isolated from fresh produce when present as either a single or mixed species of viruses. Using total RNA extraction from tomatoes yielded a limit of detection of 1.0 × 105 genome equivalents (ge) of HAV per array input. The limit of detection for viral RNA obtained using ultracentrifugation was 1.2 × 105 ge of HAV from green onions and 1.0 × 103 ge of norovirus from celery per array input. Extending microarray methods to other food matrices should provide important support to surveillance and outbreak investigations.

Mussel-Inspired Multivalent Linear Polyglycerol Coatings Outperform Monovalent Polyethylene Glycol Coatings in Antifouling Surface Properties.

Biofouling constitutes a major challenge in the application of biosensors and biomedical implants, as well as for (food) packaging and marine equipment. In this work, an antifouling surface coating based on the combination of mussel-inspired dendritic polyglycerol (MI-dPG) and an amine-functionalized block copolymer of linear polyglycerol (lPG-b-OA11, OA = oligo-amine) was developed. The coating was compared to a MI-dPG surface which was postfunctionalized with commercially available amine-terminated polyethylene glycol (HO-PEG-NH2) of similar molecular weight. In the current work, these coatings were compared in their chemical stability, protein fouling characteristics, and cell fouling characteristics. The lPG-b-OA11-functionalized coating showed high chemical stability in both phosphate buffered saline (PBS) and sodium dodecyl sulfate (SDS) solutions and reduced the adhesion of fibrinogen from human plasma with 99% and the adhesion of human serum albumin with 96%, in comparison to the bare titanium dioxide substrate. Furthermore, the proliferation of human umbilical vein endothelial cells (HUVECs) was reduced with 85% when the lPG-b-OA11 system was compared to bare titanium dioxide. Additionally, a reduction of 94% was observed when the lPG-b-OA11 system was compared to tissue culture polystyrene.

Inter- and Intra-Host Nucleotide Variations in Hepatitis A Virus in Culture and Clinical Samples Detected by Next-Generation Sequencing.

The accurate virus detection, strain discrimination, and source attribution of contaminated food items remains a persistent challenge because of the high mutation rates anticipated to occur in foodborne RNA viruses, such as hepatitis A virus (HAV). This has led to predictions of the existence of more than one sequence variant between the hosts (inter-host) or within an individual host (intra-host). However, there have been no reports of intra-host variants from an infected single individual, and little is known about the accuracy of the single nucleotide variations (SNVs) calling with various methods. In this study, the presence and identity of viral SNVs, either between HAV clinical specimens or among a series of samples derived from HAV clone1-infected FRhK4 cells, were determined following analyses of nucleotide sequences generated using next-generation sequencing (NGS) and pyrosequencing methods. The results demonstrate the co-existence of inter- and intra-host variants both in the clinical specimens and the cultured samples. The discovery and confirmation of multi-viral RNAs in an infected individual is dependent on the strain discrimination at the SNV level, and critical for successful outbreak traceback and source attribution investigations. The detection of SNVs in a time series of HAV infected FRhK4 cells improved our understanding on the mutation dynamics determined probably by different selective pressures. Additionally, it demonstrated that NGS could potentially provide a valuable investigative approach toward SNV detection and identification for other RNA viruses.

Testing for Human Norovirus and Recovery of Process Control in Outbreak-Associated Produce Items.

The development of rapid and sensitive detection methods for human noroviruses (HuNoV) in produce items is critical, especially with the recent rise in outbreaks associated with this food commodity. In this study, 50-g portions of various produce items linked to a norovirus outbreak (celery, cucumber, lettuce, grapes, and radish) were artificially inoculated with murine norovirus (MNV-1) and concentrated either by ultracentrifugation or polyethylene glycol (PEG) precipitation after elution with an alkaline Tris-glycine-beef extract buffer supplemented with pectinase. As a viral concentration step following virus elution and clarification, ultracentrifugation yielded a faster method (<8 h, including reverse transcription quantitative PCR), with MNV-1 recoveries similar to or better, than those obtained with PEG precipitation. The addition of polyvinylpyrrolidone to the elution buffer, to remove polyphenolic inhibitors, improved MNV-1 recoveries by over two- and fivefold for cucumber and grapes, respectively. However, despite MNV-1 recoveries ranging from 10 to 38% as calculated with 10-fold diluted RNA, contaminating HuNoV was not detected in any of the outbreak-associated samples tested. For store-bought produce samples, the limit of detection for artificially seeded HuNoV GII.4 was determined to be 103 copies per 50 g, with reproducible detection achieved in grapes, radish, and celery. The results support the use of ultracentrifugation as an alternative approach to PEG precipitation to concentrate norovirus from a variety of produce items.

Application of next generation sequencing toward sensitive detection of enteric viruses isolated from celery samples as an example of produce.

Next generation sequencing (NGS) holds promise as a single application for both detection and sequence identification of foodborne viruses; however, technical challenges remain due to anticipated low quantities of virus in contaminated food. In this study, with a focus on data analysis using several bioinformatics tools, we applied NGS toward amplification-independent detection and identification of norovirus at low copy (<103 copies) or within multiple strains from produce. Celery samples were inoculated with human norovirus (stool suspension) either as a single norovirus strain, a mixture of strains (GII.4 and GII.6), or a mixture of different species (hepatitis A virus and norovirus). Viral RNA isolation and recovery was confirmed by RT-qPCR, and optimized for library generation and sequencing without amplification using the Illumina MiSeq platform. Extracts containing either a single virus or a two-virus mixture were analyzed using two different analytic approaches to achieve virus detection and identification. First an overall assessment of viral genome coverage for samples varying in copy numbers (1.1×103 to 1.7×107) and genomic content (single or multiple strains in various ratios) was completed by reference-guided mapping. Not unexpectedly, this targeted approach to identification was successful in correctly mapping reads, thus identifying each virus contained in the inoculums even at low copy (estimated at 12 copies). For the second (metagenomic) approach, samples were treated as "unknowns" for data analyses using (i) a sequence-based alignment with a local database, (ii) an "in-house" k-mer tool, (iii) a commercially available metagenomics bioinformatic analysis platform cosmosID, and (iv) an open-source program Kraken. Of the four metagenomics tools applied in this study, only the local database alignment and in-house k-mer tool were successful in detecting norovirus (as well as HAV) at low copy (down to <103 copies) and within a mixture of virus strains or species. The results of this investigation provide support for continued investigation into the development and integration of these analytical tools for identification and detection of foodborne viruses.

Complete Genome Sequence of a Human Sapovirus GI.2 Variant, Isolated from a Patient in Ireland in 2016.

We report here the first near-complete genome sequence (7,463 nucleotides) of a human sapovirus GI.2 variant from Dublin, Ireland, detected in an adult with gastroenteritis in 2016.

Complete Genome Sequence of Human Norovirus GII.Pe-GII.4 Sydney from the United States.

We report here the first near-complete genome sequence (7,551 nucleotides) of a human norovirus GII.Pe-GII.4 Sydney variant, detected in a stool sample from an outbreak on a cruise ship in 2013.

Human Gut-On-A-Chip Supports Polarized Infection of Coxsackie B1 Virus In Vitro.

Analysis of enterovirus infection is difficult in animals because they express different virus receptors than humans, and static cell culture systems do not reproduce the physical complexity of the human intestinal epithelium. Here, using coxsackievirus B1 (CVB1) as a prototype enterovirus strain, we demonstrate that human enterovirus infection, replication and infectious virus production can be analyzed in vitro in a human Gut-on-a-Chip microfluidic device that supports culture of highly differentiated human villus intestinal epithelium under conditions of fluid flow and peristalsis-like motions. When CVB1 was introduced into the epithelium-lined intestinal lumen of the device, virions entered the epithelium, replicated inside the cells producing detectable cytopathic effects (CPEs), and both infectious virions and inflammatory cytokines were released in a polarized manner from the cell apex, as they could be detected in the effluent from the epithelial microchannel. When the virus was introduced via a basal route of infection (by inoculating virus into fluid flowing through a parallel lower 'vascular' channel separated from the epithelial channel by a porous membrane), significantly lower viral titers, decreased CPEs, and delayed caspase-3 activation were observed; however, cytokines continued to be secreted apically. The presence of continuous fluid flow through the epithelial lumen also resulted in production of a gradient of CPEs consistent with the flow direction. Thus, the human Gut-on-a-Chip may provide a suitable in vitro model for enteric virus infection and for investigating mechanisms of enterovirus pathogenesis.

Complete Genome Sequence of Human Norovirus Strain GII.P7-GII.6 Detected in a Patient in the United States in 2014.

Homologous recombination is one of the driving forces contributing to the genetic variation of human norovirus, which is an important cause of sporadic and epidemic acute gastroenteritis globally. We report the near-complete genome of the novel recombinant norovirus strain GII.P7-GII.6, detected in an adult with norovirus gastroenteritis in the United States.

Towards next-generation sequencing analytics for foodborne RNA viruses: Examining the effect of RNA input quantity and viral RNA purity.

Detection and identification of viruses in food samples are technically challenging due largely to the low viral copy number in contaminated food items, and the lack of effective culture enrichment methods that are amenable to regulatory applications for many of the common foodborne viruses. Using an Illumina MiSeq platform and two hepatitis A virus (HAV) cell-culture adapted strains as a representative enteric virus species, this study examined the limits of single-stranded RNA (ssRNA) viral detection following next-generation sequencing without pre-amplification of the viral genome. Complete viral genome sequences were obtained from HAV samples of varying purities and with an input as low as 2ng total RNA containing 1.4×10(5) copies of viral RNA. In addition, single nucleotide variations were reproducibly detected over the range of concentrations examined, and their identity confirmed by alternate sequencing technology. In summary, next-generation sequencing technology has the potential for sensitive detection/identification of a viral genome at a low copy number. This study provides a benchmark for metagenomic sequencing application as is required for virus detection in complex food matrices using a culture-independent diagnostic approach.

Optimizing a custom tiling microarray for low input detection and identification of unamplified virus targets.

Viruses are major pathogens causing foodborne illnesses and are often present at low levels in foods, thus requiring sensitive techniques for their detection in contaminated foods. The lack of efficient culture methods for many foodborne viruses and the potential for multi-species viral contamination have driven investigation toward non-amplification based methods for virus detection and identification. A custom DNA microarray (FDA_EVIR) was assessed for its sensitivity in the detection and identification of low-input virus targets, human hepatitis A virus, norovirus, and coxsackievirus, individually and in combination. Modifications to sample processing were made to accommodate low input levels of unamplified virus targets, which included addition of carrier cDNA, RNase treatment, and optimization of DNase I-mediated target fragmentation. Amplification-free detection and identification of foodborne viruses were achieved in the range of 250-500 copies of virus RNA. Alternative data analysis methods were employed to distinguish the genotypes of the viruses particularly at lower levels of target input and the single probe-based analysis approach made it possible to identify a minority species in a multi-virus complex. The oligonucleotide array is shown to be a promising platform to detect foodborne viruses at low levels close to what are anticipated in food or environmental samples.

Review: Approaches to the Viral Extraction, Detection, and Identification of Hepatitis Viruses, HAV and HEV, in Foods.

Although the incidence rate of hepatitis A virus (HAV) infection has been on the decline in developed countries, in part due to immunization availability, high profile outbreaks continue to be reported. Hepatitis E virus has been recognized as an emerging pathogen in industrialized countries. While associated with waterborne illnesses, particularly in undeveloped countries, several animal species have been identified as reservoirs for the virus. Consequently, the potential of zoonotic transmission exists as a function of the consumption of infected animals. In this review we provide a comparative overview of these two virus species with regard to their known virus properties, discuss extraction methodologies, and describe some basic principles and methodology applied toward the isolation of these viruses (as particles or their isolated genomes) from food commodities. We also discuss the challenges that remain as experimental hurdles to extraction of such viruses from food. As HAV has been the most extensively studied with regard to virus detection in foods, it often serves as a model virus for current and future development of sample preparation methodology for foodborne virus detection. Lastly, we discuss the application and role of current and developing technologies in the post-extraction detection and identification of these viruses from foods.

Complete Genome Sequence of Human Norovirus GII.4_2006b, a Variant of Minerva 2006.

In 2006, the National Calicivirus Laboratory at the U.S. Centers for Disease Control and Prevention (CDC) confirmed multistate outbreaks of norovirus infection and identified two new GII.4 norovirus strains (Minerva and Laurens) through partial sequencing of the major capsid (VP1) gene. Here, we report the first complete genome sequence of the GII.4 Minerva isolate.

Enantiopure isoindolinones through Viedma ripening.

Here we demonstrate that deracemization of isoindolinones using Viedma ripening is possible starting from a racemic mixture of conglomerate crystals. Crystals of the enantiopure isoindolinones lose their chiral identity upon dissolution even without the need for a catalyst. This enabled complete deracemization of the reported isoindolinones without a catalyst.