Enhanced detection of pathogenic enteric viruses in coastal marine environment by concentration using methacrylate monolithic chromatographic supports paired with quantitative PCR

AbstractCurrently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10–100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM® C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods

Tomato brown rugose fruit virus in aqueous environments – survival and significance of water-mediated transmission

Tomato brown rugose fruit virus (ToBRFV) has recently emerged as a major disease of tomatoes and peppers. ToBRFV is a seed- and contact-transmitted virus. In Slovenia, ToBRFV RNA was detected in samples of wastewater, river, and water used to irrigate plants. Even though the source of detected RNA could not be clearly established, this raised the question of the significance of the detection of ToBRFV in water samples and experimental studies were performed to address this question. The data presented here confirm that the release of virus particles from the roots of infected plants is a source of infectious ToBRFV particles in water and that the virus can remain infective up to four weeks in water stored at room temperature, while its RNA can be detected for much longer. These data also indicate that irrigation with ToBRFV-contaminated water can lead to plant infection. In addition, it has been shown that ToBRFV circulated in drain water in commercial tomato greenhouses from other European countries and that an outbreak of ToBRFV can be detected by regular monitoring of drain water. A simple method for concentrating ToBRFV from water samples and a comparison of the sensitivity of different methods, including the determination of the highest ToBRFV dilution still capable of infecting test plants, were also investigated. The results of our studies fill the knowledge gaps in the epidemiology and diagnosis of ToBRFV, by studying the role of water-mediated transmission, and provide a reliable risk assessment to identify critical points for monitoring and control

Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay▿

Rotaviruses are one of the major causes of diarrhea in infants and children under 5 years old, especially affecting developing countries. In natural disasters, fecal matter and potable waters can mix, allowing low, yet infective, concentrations of rotavirus to be present in water supplies, constituting a risk for the population. Any of the most commonly detected rotavirus genotypes could originate an outbreak. The development of a fast and sensitive method that could detect the broadest possible range of rotavirus genotypes would help with efficient diagnosis and prevention. We have designed a reverse transcription (RT)-real-time quantitative PCR approach targeted to the rotaviral VP2 gene, based on a multiple-sequence alignment of different human rotaviral strains. To overcome the high nucleotide sequence diversity, multiple forward and reverse primers were used, in addition to a degenerate probe. The performance of the assay was tested on isolates representing the most prevalent human genotypes: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]. The developed method improved classical rotavirus detection by enzyme-linked immunosorbent assay and nested RT-PCR by 5 and at least 1 order of magnitude, respectively. A survey of 159 stool samples indicated that the method can efficiently detect a broad range of rotavirus strains, including different G-P genotype combinations of human, porcine, and bovine origin. No cross-reactivity was observed with other enteric viruses, such as astrovirus, sapovirus, and norovirus

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Copyright © by Biophysical Society. Final full-text version of the paper available at: http://www.biophysj.org/cgi/content/abstract/80/3/1343Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (;26 mN m21) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (;33 mN m21), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101–120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.This work was supported by grants PI-1998–110 and UE-1998–43 from the Basque Government. J.M.M.C., I.E., and I.G.A. were recipients of predoctoral fellowships from the Basque Government.Peer reviewe

Efficient inactivation of MS-2 virus in water by hydrodynamic cavitation

The aim of this study was to accurately quantify the impact of hydrodynamic cavitation on the infectivity of bacteriophage MS2, a norovirus surrogate, and to develop a small scale reactor for testing the effect of hydrodynamic cavitation on human enteric viruses, which cannot be easily prepared in large quantities. For this purpose, 3 mL scale and 1 L scale reactors were constructed and tested. Both devices were efficient in generating hydrodynamic cavitation and in reducing the infectivity of MS2 virus. Furthermore, they reached more than 4 logs reductions of viral infectivity, thus confirming the scalability of hydrodynamic cavitation for this particular application. As for the mechanism of page inactivation, we suspect that cavitation generated OH$^-$ radicals formed an advanced oxidation process, which could have damaged the host\u27s recognition receptors located on the surface of the bacteriophage. Additional damage could arise from the high shear forces inside the cavity. Moreover, the effectiveness of the cavitation was higher for suspensions containing low initial viral titers that are in similar concentration to the ones found in real water samples. According to this, cavitation generators could prove to be a useful tool for treating virus-contaminated wastewaters in the future

Differential interaction of equinatoxin II with model membranes in response to lipid composition

Copyright © by Biophysical Society. Final full-text version of the paper available at: http://www.biophysj.org/cgi/content/abstract/80/3/1343Equinatoxin II is a 179-amino-acid pore-forming protein isolated from the venom of the sea anemone Actinia equina. Large unilamellar vesicles and lipid monolayers of different lipid compositions have been used to study its interaction with membranes. The critical pressure for insertion is the same in monolayers made of phosphatidylcholine or sphingomyelin (;26 mN m21) and explains why the permeabilization of large unilamellar vesicles by equinatoxin II with these lipid compositions is null or moderate. In phosphatidylcholine-sphingomyelin (1:1) monolayers, the critical pressure is higher (;33 mN m21), thus permitting the insertion of equinatoxin II in large unilamellar vesicles, a process that is accompanied by major conformational changes. In the presence of vesicles made of phosphatidylcholine, a fraction of the protein molecules remains associated with the membranes. This interaction is fully reversible, does not involve major conformational changes, and is governed by the high affinity for membrane interfaces of the protein region comprising amino acids 101–120. We conclude that although the presence of sphingomyelin within the membrane creates conditions for irreversible insertion and pore formation, this lipid is not essential for the initial partitioning event, and its role as a specific receptor for the toxin is not so clear-cut.This work was supported by grants PI-1998–110 and UE-1998–43 from the Basque Government. J.M.M.C., I.E., and I.G.A. were recipients of predoctoral fellowships from the Basque Government.Peer reviewe