scholarly journals Development of a Fluorescent In Situ Method for Visualization of Enteric Viruses

2009 ◽  
Vol 75 (24) ◽  
pp. 7822-7827 ◽  
Author(s):  
Helen Rawsthorne ◽  
Trevor G. Phister ◽  
Lee-Ann Jaykus
Keyword(s):  
Mammalian Cells ◽  
Hepatitis A Virus ◽  
Fluorescent Microscopy ◽  
Enteric Viruses ◽  
Fluorescent Labeling ◽  
Host Cells ◽  
Virus Infectivity ◽  
Murine Norovirus ◽  
Sandwich Type

ABSTRACT Studying the interactions between enteric pathogens and their environment is important to improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate, and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV); the process was confirmed by using a sandwich-type approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots with an emission range of 635 to 675 nm (Q-Dots 655). The assay demonstrated a relative fluorescence of 528 ± 48.1 and 112 ± 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity, nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surfaces of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ.

scholarly journals Mechanisms of Antiviral Action of Plant Antimicrobials against Murine Norovirus

2014 ◽  
Vol 80 (16) ◽  
pp. 4898-4910 ◽  
Author(s):  
Damian H. Gilling ◽  
Masaaki Kitajima ◽  
Jason R. Torrey ◽  
Kelly R. Bright
Keyword(s):  
Antiviral Effect ◽  
Host Cells ◽  
Natural Food ◽  
Virus Infectivity ◽  
Murine Norovirus ◽  
Cell Binding ◽  
Transmission Electron ◽  
Successful Infection ◽  
Lemongrass Oil ◽  
Plant Compounds

ABSTRACTNumerous plant compounds have antibacterial or antiviral properties; however, limited research has been conducted with nonenveloped viruses. The efficacies of allspice oil, lemongrass oil, and citral were evaluated against the nonenveloped murine norovirus (MNV), a human norovirus surrogate. The antiviral mechanisms of action were also examined using an RNase I protection assay, a host cell binding assay, and transmission electron microscopy. All three antimicrobials produced significant reductions (P≤ 0.05) in viral infectivity within 6 h of exposure (0.90 log10to 1.88 log10). After 24 h, the reductions were 2.74, 3.00, and 3.41 log10for lemongrass oil, citral, and allspice oil, respectively. The antiviral effect of allspice oil was both time and concentration dependent; the effects of lemongrass oil and citral were time dependent. Based on the RNase I assay, allspice oil appeared to act directly upon the viral capsid and RNA. The capsids enlarged from ≤35 nm to up to 75 nm following treatment. MNV adsorption to host cells was not significantly affected. Alternatively, the capsid remained intact following exposure to lemongrass oil and citral, which appeared to coat the capsid, causing nonspecific and nonproductive binding to host cells that did not lead to successful infection. Such contrasting effects between allspice oil and both lemongrass oil and citral suggest that though different plant compounds may yield similar reductions in virus infectivity, the mechanisms of inactivation may be highly varied and specific to the antimicrobial. This study demonstrates the antiviral properties of allspice oil, lemongrass oil, and citral against MNV and thus indicates their potential as natural food and surface sanitizers to control noroviruses.

scholarly journals Bacteria facilitate viral co-infection of mammalian cells and promote genetic recombination

2017 ◽  
Author(s):  
A.K. Erickson ◽  
P.R. Jesudhasan ◽  
M.J. Mayer ◽  
A. Narbad ◽  
S.E. Winter ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Recombination ◽  
Enteric Viruses ◽  
Intestinal Bacteria ◽  
Enteric Virus ◽  
Viral Fitness ◽  
Host Cells ◽  
Bacterial Strains ◽  
Viral Recombination ◽  
Viral Genomes

SUMMARYIntestinal bacteria promote infection of several mammalian enteric viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact enteric viruses. We found that most bacterial strains bound poliovirus, a model enteric virus. Given that each bacterium bound multiple virions, we hypothesized that bacteria may deliver multiple viral genomes to a mammalian cell even when very few virions are present, such as during the first replication cycle after inter-host transmission. We found that exposure to certain bacterial strains increased viral co-infection even when the ratio of virus to host cells was low. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated viral fitness restoration through genetic recombination. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections.

scholarly journals Grape Seed Extract for Control of Human Enteric Viruses

2011 ◽  
Vol 77 (12) ◽  
pp. 3982-3987 ◽  
Author(s):  
Xiaowei Su ◽  
Doris H. D'Souza
Keyword(s):  
Room Temperature ◽  
Hepatitis A Virus ◽  
Antimicrobial Properties ◽  
Enteric Viruses ◽  
Grape Seed Extract ◽  
Grape Seed ◽  
Seed Extract ◽  
Dependent Manner ◽  
Murine Norovirus ◽  
Human Enteric Viruses

ABSTRACTGrape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ∼7 log10PFU/ml or ∼5 log10PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (∼7 log10PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log10PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log10PFU/ml; MS2 by 1.13, 1.43, and 1.60 log10PFU/ml; and HAV by 1.81, 2.66, and 3.20 log10PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (P< 0.05) in a dose-dependent manner. GSE treatment of low titers (∼5 log10PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.

scholarly journals Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices

2021 ◽  
Author(s):  
Sophie Butot ◽  
Luca Galbusera ◽  
Thierry Putallaz ◽  
Sophie Zuber
Keyword(s):  
Electron Beam ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Enteric Virus ◽  
High Energy ◽  
Energy Electron ◽  
Murine Norovirus ◽  
Sesame Seeds ◽  
Freeze Dried ◽  
Dose Dependent

AbstractThe objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qβ, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.

scholarly journals Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

2016 ◽  
Vol 82 (7) ◽  
pp. 2086-2099 ◽  
Author(s):  
Elbashir Araud ◽  
Erin DiCaprio ◽  
Yuanmei Ma ◽  
Fangfei Lou ◽  
Yu Gao ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Receptor Binding ◽  
Thermal Inactivation ◽  
Hepatitis A Virus ◽  
Enteric Viruses ◽  
Heat Stability ◽  
Magnetic Bead ◽  
Murine Norovirus ◽  
Foodborne Viruses ◽  
Porcine Gastric Mucin

ABSTRACTHuman enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.

Inactivation of Norovirus by Lemongrass Essential Oil Using a Norovirus Surrogate System

2017 ◽  
Vol 80 (8) ◽  
pp. 1293-1302 ◽  
Author(s):  
Ye Won Kim ◽  
Hyun Ju You ◽  
Soyoung Lee ◽  
Bomi Kim ◽  
Do Kyung Kim ◽  
...  
Keyword(s):  
Essential Oil ◽  
Viral Replication ◽  
Host Cells ◽  
Virus Infectivity ◽  
Coat Proteins ◽  
Murine Norovirus ◽  
Human Norovirus ◽  
Plaque Reduction Assay ◽  
Reduction Assay ◽  
Lemongrass Essential Oil

ABSTRACT This study investigated the effect of lemongrass essential oil (LGEO) on the infectivity and viral replication of norovirus. Murine norovirus 1 (MNV-1), a surrogate of human norovirus, was preincubated with LGEO and then used to infect RAW 264.7 cells in a plaque reduction assay. LGEO exhibited a significant reduction in MNV-1 plaque formation in both time- and dose-dependent manners. The quantification of viral genome by quantitative real-time PCR showed similar results in line with those of the plaque reduction assay. It was revealed that citral, a single compound in LGEO, showed dramatic reduction in MNV-1 infectivity (−73.09% when using a treatment of 0.02%, v/v). The inhibitory activity of LGEO on viral replication was further investigated in HG23 cells that harbored a human norovirus replicon. LGEO treatment significantly reduced viral replication in HG23 cells, which suggests that LGEO may have dual inhibitory activities that inactivate viral coat proteins required for viral infection and suppress norovirus genome replication in host cells. In animal experiments, oral administration of murine norovirus preincubated with LGEO significantly suppressed virus infectivity in vivo. Collectively, these results suggest that LGEO, in particular the LGEO component citral, inactivates the norovirus and its subsequent replication in host cells. Thus, LGEO shows promise as a method of inhibiting norovirus within the food industry.

scholarly journals Graphene-enabled, spatially controlled electroporation of adherent cells for live-cell super-resolution microscopy

2019 ◽  
Author(s):  
Seonah Moon ◽  
Wan Li ◽  
Ke Xu
Keyword(s):  
Mammalian Cells ◽  
Single Layer ◽  
Super Resolution ◽  
Fluorescent Labeling ◽  
Live Cells ◽  
Biological Research ◽  
Super Resolution Microscopy ◽  
Affinity Probes ◽  
Intracellular Targets

AbstractThe incorporation of exogenous molecules into live cells is essential for both biological research and therapeutic applications. In particular, for the emerging field of super-resolution microscopy of live mammalian cells, reliable fluorescent labeling of intracellular targets remains a challenge. Here, utilizing the unique mechanical, electrical, and optical properties of graphene, a single layer of bonded carbon atoms, we report a facile approach that enables both high-throughput delivery of fluorescent probes into adherent live cells and in situ super-resolution microscopy on the same device. ∼90% delivery efficiencies are achieved for free dyes and dye-tagged affinity probes, short peptides, and whole antibodies, thus enabling high-quality super-resolution microscopy. Moreover, we demonstrate excellent spatiotemporal controls, which, in combination with the ready patternablity of graphene, allow for the spatially selective delivery of two different probes for cells at different locations on the same substrate. We thus open up a new pathway to the microscopic manipulation and visualization of live cells.

Synthesis of sandwich type acyclic tetra-nuclear silver(I)-N-heterocyclic carbene complexes for wound healing applications

2020 ◽  
Vol 75 (9-10) ◽  
pp. 369-376
Author(s):  
Ayesha Riaz ◽  
Muhammad Adnan Iqbal ◽  
Haq Nawaz Bhatti ◽  
Muhammad Shahid
Keyword(s):  
Alkyl Halides ◽  
Wound Contraction ◽  
Spectroscopic Techniques ◽  
Open Chain ◽  
Standard Drug ◽  
Sandwich Type ◽  
Benzimidazolium Salts ◽  
Multistep Reaction

AbstractTwo meta-xylyl linked tetrakis-benzimidazolium salts (L1-L2) as multidentate ligands and two respective silver complexes (C1 and C2) were synthesized. A multistep reaction was done at room temperature, starting with simple benzimidazole and alkyl halides, going through precursors and salt formation by reflux and finally in situ deprotonation of tetrabenzimidazolium salts with Ag2O to yield respective tetra-nuclear Ag(I)-N-heterocyclic Carbene (NHC) complexes. Propyl and butyl groups were bonded at the terminal positions of tetra-azolium open chain salts. Characterization of compounds was done by analytical and spectroscopic techniques. On the basis of spectroscopic data, a chemical structure with open chains having four Ag(I) ions sandwiched between NHC layers was established. Potential of synthesized complexes (C1 & C2) for wound contraction was evaluated and compared with standard wound contraction gel. Percentage wound contraction of both complexes was found very close to that of standard drug used in parallel.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

scholarly journals D936Y and Other Mutations in the Fusion Core of the SARS-CoV-2 Spike Protein Heptad Repeat 1: Frequency, Geographical Distribution, and Structural Effect

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2622
Author(s):  
Romina Oliva ◽  
Abdul Rajjak Shaikh ◽  
Andrea Petta ◽  
Anna Vangone ◽  
Luigi Cavallo
Keyword(s):  
Three Dimensional ◽  
Salt Bridge ◽  
Structural Effect ◽  
Host Cells ◽  
Heptad Repeat ◽  
Genomic Sequences ◽  
Strong Interactions ◽  
Virus Infectivity ◽  
S Protein ◽  
Global Threat

The crown of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is constituted by its spike (S) glycoprotein. S protein mediates the SARS-CoV-2 entry into the host cells. The “fusion core” of the heptad repeat 1 (HR1) on S plays a crucial role in the virus infectivity, as it is part of a key membrane fusion architecture. While SARS-CoV-2 was becoming a global threat, scientists have been accumulating data on the virus at an impressive pace, both in terms of genomic sequences and of three-dimensional structures. On 15 February 2021, from the SARS-CoV-2 genomic sequences in the GISAID resource, we collected 415,673 complete S protein sequences and identified all the mutations occurring in the HR1 fusion core. This is a 21-residue segment, which, in the post-fusion conformation of the protein, gives many strong interactions with the heptad repeat 2, bringing viral and cellular membranes in proximity for fusion. We investigated the frequency and structural effect of novel mutations accumulated over time in such a crucial region for the virus infectivity. Three mutations were quite frequent, occurring in over 0.1% of the total sequences. These were S929T, D936Y, and S949F, all in the N-terminal half of the HR1 fusion core segment and particularly spread in Europe and USA. The most frequent of them, D936Y, was present in 17% of sequences from Finland and 12% of sequences from Sweden. In the post-fusion conformation of the unmutated S protein, D936 is involved in an inter-monomer salt bridge with R1185. We investigated the effect of the D936Y mutation on the pre-fusion and post-fusion state of the protein by using molecular dynamics, showing how it especially affects the latter one.

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