A rapid and label-free platform for virus capture and identification from clinical samples.

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.

Single Virion Tracking Microscopy for the Study of Virus Entry Processes in Live Cells and Biomimetic Platforms.

The most widely-used assays for studying viral entry, including infectivity, cofloatation, and cell-cell fusion assays, yield functional information but provide low resolution of individual entry steps. Structural characterization provides high-resolution conformational information, but on its own is unable to address the functional significance of these conformations. Single virion tracking microscopy techniques provide more detail on the intermediate entry steps than infection assays and more functional information than structural methods, bridging the gap between these methods. In addition, single virion approaches also provide dynamic information about the kinetics of entry processes. This chapter reviews single virion tracking techniques and describes how they can be applied to study specific virus entry steps. These techniques provide information complementary to traditional ensemble approaches. Single virion techniques may either probe virion behavior in live cells or in biomimetic platforms. Synthesizing information from ensemble, structural, and single virion techniques ultimately yields a more complete understanding of the viral entry process than can be achieved by any single method alone.

Virus purification by CsCl density gradient using general centrifugation.

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Three-dimensional printed magnetophoretic system for the continuous flow separation of avian influenza H5N1 viruses.

As a result of the low concentration of avian influenza viruses in samples for routine screening, the separation and concentration of these viruses are vital for their sensitive detection. We present a novel three-dimensional printed magnetophoretic system for the continuous flow separation of the viruses using aptamer-modified magnetic nanoparticles, a magnetophoretic chip, a magnetic field, and a fluidic controller. The magnetic field was designed based on finite element magnetic simulation and developed using neodymium magnets with a maximum intensity of 0.65 T and a gradient of 32 T/m for dragging the nanoparticle-virus complexes. The magnetophoretic chip was designed by SOLIDWORKS and fabricated by a three-dimensional printer with a magnetophoretic channel for the continuous flow separation of the viruses using phosphate-buffered saline as carrier flow. The fluidic controller was developed using a microcontroller and peristaltic pumps to inject the carrier flow and the viruses. The trajectory of the virus-nanoparticle complexes was simulated using COMSOL for optimization of the carrier flow and the magnetic field, respectively. The results showed that the H5N1 viruses could be captured, separated, and concentrated using the proposed magnetophoretic system with the separation efficiency up to 88% in a continuous flow separation time of 2 min for a sample volume of 200 µL.

Efficient collection of viable virus aerosol through laminar-flow, water-based condensational particle growth.

AIMS: State-of-the-art bioaerosol samplers have poor collection efficiencies for ultrafine virus aerosols. This work evaluated the performance of a novel growth tube collector (GTC), which utilizes laminar-flow water-based condensation to facilitate particle growth, for the collection of airborne MS2 viruses. METHODS AND RESULTS: Fine aerosols (<500 nm) containing MS2 coliphage were generated from a Collison nebulizer, conditioned by a dilution dryer and collected by a GTC and a BioSampler. The GTC effectively condensed water vapour onto the virus particles, creating droplets 2-5 µm in diameter, which facilitated collection. Comparison of particle counts upstream and downstream revealed that the GTC collected >93% of the inlet virus particles, whereas the BioSampler's efficiency was about 10%. Viable counts of the GTC-collected viruses were also one order of magnitude higher than those of the BioSampler (P = 0·003). CONCLUSION: The efficiency of the GTC for the viable collection of MS2 viruses exceeds that of industry standard instrument, the BioSampler, by a factor of 10-100. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that the GTC is an effective collector of viable MS2 aerosols, and concludes the instrument will be an effective tool for studying viable virus aerosols and the inhalation risks posed by airborne viruses.

Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces.

Inanimate surfaces are regarded as key vehicles for the spread of human norovirus during outbreaks. ISO method 15216 involves the use of cotton swabs for environmental sampling from food surfaces and fomites for the detection of norovirus genogroup I (GI) and GII. We evaluated the effects of the virus drying time (1, 8, 24, or 48 h), swab material (cotton, polyester, rayon, macrofoam, or an antistatic wipe), surface (stainless steel or a toilet seat), and area of the swabbed surface (25.8 cm(2) to 645.0 cm(2)) on the recovery of human norovirus. Macrofoam swabs produced the highest rate of recovery of norovirus from surfaces as large as 645 cm(2). The rates of recovery ranged from 2.2 to 36.0% for virus seeded on stainless-steel coupons (645.0 cm(2)) to 1.2 to 33.6% for toilet seat surfaces (700 cm(2)), with detection limits of 3.5 log10 and 4.0 log10 RNA copies. We used macrofoam swabs to collect environmental samples from several case cabins and common areas of a cruise ship where passengers had reported viral gastroenteritis symptoms. Seventeen (18.5%) of 92 samples tested positive for norovirus GII, and 4 samples could be sequenced and had identical GII.1 sequences. The viral loads of the swab samples from the cabins of the sick passengers ranged from 80 to 31,217 RNA copies, compared with 16 to 113 RNA copies for swab samples from public spaces. In conclusion, our swab protocol for norovirus may be a useful tool for outbreak investigations when no clinical samples are available to confirm the etiology.

Measuring the effectiveness of gaseous virus disinfectants.

The efficacy of gaseous disinfection is critical for prevention and treatment of microbial contamination in biotechnological facilities. For an evaluation of gaseous disinfection efficacy, a down-scaled laboratory model was established, using currently available carrier tests and a custom-made dry fog box. A mixture of peroxyacetic acid and hydrogen peroxide (PAA/HP) was investigated as example, at concentrations between 0.4 and 2.9 mL/m(3) for up to 3 h for inactivation of a panel of lipid-enveloped and non-lipid-enveloped viruses. The influenza viruses were most sensitive to PAA/HP treatment and minute virus of mice was most resistant. Bovine viral diarrhea virus and reovirus III showed intermediate stability and similar inactivation kinetics. Use of the dry fog box circumvents dedicating an entire lab for the investigation, which renders the generation of data more cost-effective and allows for production of highly reproducible kinetic data.

Review: progress in the diagnosis of dengue virus infections and importance of point of care test: a review.

It is an urgent need of highly sensitive, specific and economical diagnostic tools for early and fast diagnosis of highly challenging dengue virus infections. Many laboratory methods including virus detection, genome detection, antigen detection and serological detection of such short-lived viremia were explored but promising outcomes for economical immunochromatographic tests have been reported in this review. With the trend of fast, easy operation, rapid diagnostic tests (RDT) based on immunochromatographic assays are of great importance due to point of care test (POCT) in the dengue endemic regions where it is short of laboratory equipments and cold storage conditions. Such kind of point of care diagnosis is more efficient, fast and user friendly. Moreover, the development of highly advance RDT is dependent on the use of anti-dengue monoclonal antibodies highly specific for particular analyte/antigen.

Real-Time Detection of HIV-2 by Reverse Transcription-Loop-Mediated Isothermal Amplification.

Currently, there are no FDA-approved nucleic acid amplification tests (NAATs) for the detection or confirmation of HIV-2 infection. Here, we describe the development of a real-time assay for the detection of HIV-2 DNA and RNA using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and the ESEQuant tube scanner, a portable isothermal amplification/detection device.

Medical devices; immunology and microbiology devices; classification of John Cunningham Virus serological reagents. Final order.

The Food and Drug Administration (FDA) is classifying John Cunningham Virus (JCV) serological reagents into class II (special controls). The Agency is classifying the device into class II (special controls) in order to provide a reasonable assurance of safety and effectiveness of the device.

Analytical and Clinical Performance of the NeuMoDx™ Platform for Cytomegalovirus and Epstein-Barr Virus Viral Load Testing.

DNA assays for viral load (VL) monitoring are key tools in the management of immunocompromised patients with cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection. In this study, the analytical and clinical performances of the NeuMoDx™ CMV and EBV Quant Assays were compared with artus CMV and EBV QS-RGQ Kits in a primary hospital testing laboratory. Patient plasma samples previously tested using artus kits were randomly selected for testing by NeuMoDx assays. The NeuMoDx CMV Quant Assay and artus CMV QS-RGQ Kit limits of detection (LoDs) are 20.0 IU/mL and 69.7 IU/mL, respectively; 33/75 (44.0%) samples had CMV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.9503; 20 samples (60.6%) had lower NeuMoDx CMV quantification values versus the artus kit. The LoD of the NeuMoDx EBV Quant Assay and artus EBV QS-RGQ Kit are 200 IU/mL and 22.29 IU/mL, respectively; 16/75 (21.3%) samples had EBV DNA levels above the LoD of both assays. The Pearson correlation coefficient was 0.8990. EBV quantification values with the NeuMoDx assay were higher versus the artus kit in 15 samples (93.8%). In conclusion, NeuMoDx CMV and EBV Quant Assays are sensitive and accurate tools for CMV and EBV DNA VL quantification.

Rapid detection of human immunodeficiency virus types 1 and 2 by use of an improved piezoelectric biosensor.

Disasters can create situations in which blood donations can save lives. However, in emergency situations and when resources are depleted, on-site blood donations require the rapid and accurate detection of blood-borne pathogens, including human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Techniques such as PCR and antibody capture by an enzyme-linked immunosorbent assay (ELISA) for HIV-1 and HIV-2 are precise but time-consuming and require sophisticated equipment that is not compatible with emergency point-of-care requirements. We describe here a prototype biosensor based on piezoelectric materials functionalized with specific antibodies against HIV-1 and HIV-2. We show the rapid and accurate detection of HIV-1 and HIV-2 in both simple and complex solutions, including human serum, and in the presence of a cross-confounding virus. We report detection limits of 12 50% tissue culture infective doses (TCID50s) for HIV-1 and 87 TCID50s for HIV-2. The accuracy, precision of measurements, and operation of the prototype biosensor compared favorably to those for nucleic acid amplification. We conclude that the biosensor has significant promise as a successful point-of-care diagnostic device for use in emergency field applications requiring rapid and reliable testing for blood-borne pathogens.

Evaluation of electropositive filtration for recovering norovirus in water.

The virus adsorption-elution (VIRADEL) technique has been widely used in the recovery of various enteric viruses in water, and an electropositive filter such as 1 MDS has been commonly applied. However, effective methods of monitoring waterborne norovirus (NoV) have not yet been well characterized and optimized. Hence, in this study, the VIRADEL technique was evaluated and optimized for effectively detecting NoV in water by two commonly used electropositive filters (1MDS and NanoCeram filter). Various elution and concentration methods were evaluated by using both murine norovirus (MNV) and human NoV. Among the tested elution buffers, the most effective was 1.5% beef extract plus 0.01% Tween 80 for both 1MDS (67.5%) and NanoCeram (85.7%) microfilters. The recovery rate of GII-4 human NoV was higher by organic flocculation (86.6%) than by polyethylene glycol (PEG) precipitations (11.6~73.6%). When both 1MDS and NanoCeram filters were tested to detect NoV in surface and groundwater, the sensitivity of NoV recovered by these filters appeared to depend on the types and conditions of environmental water. The results of this study will help to set a standard of detection method for NoV in water.

Assembly and characterization of pandemic influenza A H1N1 genome in nasopharyngeal swabs using high-throughput pyrosequencing.

De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.

A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses.

Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity.

Printed monolith adsorption as an alternative to expanded bed adsorption for purifying M13 bacteriophage.

An ordered 3D printed chromatography stationary phase was used to purify M13 bacteriophage (M13) directly from crude cell culture. This new approach, which offers the same advantages as expanded bed adsorption (EBA) with regard to tolerating solids-laden feed streams but without the corresponding issues associated with fluidized bed stability that affect the latter, can be described as "printed monolith adsorption (PMA)". PMA columns (5, 10 and 15 cm length by 1 cm diameter) were made via a wax templating method from cross-linked cellulose hydrogel and functionalized with a quaternary amine ligand. The recovery of M13 was found to be strongly linked to load flow rate, with the highest recovery 89.7% ± 6% for 1.4 × 1011 pfu/mL of resin occurring at 76 cm/h with a 10 cm column length. A recovery of 87.7% ± 5% for 1.49 × 1011 pfu/mL of media was achieved with a 15 cm column length under conditions comparable to a reported EBA process. The PMA process was completed three times faster than EBA because PMA flow rates can readily be adjusted during operation, with high flow rates and low back pressure, which is unique to the ordered monolithic media geometry used. Equilibration, wash, and cleaning steps were carried out at high flow rates (611 cm/h), minimizing process time and were limited only by the volumetric flow rate capacity of the pumps used, rather than column back pressure (<0.1 MPa at 611 cm/hr). Initial capture of M13 appears to occur on the surface of the monolith solid phase (i.e. the mobile phase channel walls) and subsequently, at a slower rate, within the internal pores of the solid phase media. The difference in binding rate between these two sites is likely caused by slow pore diffusion of the large M13 particles into the pores, with similar slow diffusion out of the pores resulting in tailing of the elution peak. The results indicate that PMA is a promising technology for the efficient purification of viruses directly from crude cell culture.

Microfluidics in Single-Cell Virology: Technologies and Applications.

Microfluidics has proven to be a powerful tool for probing biology at the single-cell level. However, it is only in the past 5 years that single-cell microfluidics has been used in the field of virology. An array of strategies based on microwells, microvalves, and droplets is now available for tracking viral infection dynamics, identifying cell subpopulations with particular phenotypes, as well as high-throughput screening. The insights into the virus-host interactions gained at the single-cell level are unprecedented and usually inaccessible by population-based experiments. Therefore, single-cell microfluidics, which opens new avenues for mechanism elucidation and development of antiviral therapeutics, would be a valuable tool for the study of viral pathogenesis.

Mycoplasma detection in a historical arbovirus repository: Commercial kit comparison and implications for improved repository management.

The Centers for Disease Control and Prevention, Arbovirus Reference Collection (ARC) contains viral isolates from both environmental and human sources that are maintained in the laboratory through passage in suckling mouse brain and/or vertebrate and invertebrate cell culture. There has been increased concern regarding the effect of mycoplasma contamination on virus growth and its impact on research and phenotypic analysis. Therefore, quality control testing of virus preparations has become a routine part of the ARC quality assurance program. We compared the performance of three kits - the PCR Mycoplasma Detection Kit (ABM), the VenorGem Mycoplasma Detection Kit (Sigma), and the MycoAlert Mycoplasma Detection Kit (Lonza) - against a reference mycoplasma detection assay from the American Tissue Culture Collection (ATCC) using 744 virus preparations in the ARC, representing 721 unique viruses comprising twelve families and unclassified viruses. We found the ABM kit had the highest sensitivity and specificity, followed by the Sigma kit and Lonza kit, when compared to the ATCC kit. An increase in false positives was observed for the Lonza kit for preparations recently passaged in suckling mouse. Our data supports previously reported observations; that once introduced a specific species of mycoplasma is maintained within a lab.

Human Organs-on-Chips for Virology.

While conventional in vitro culture systems and animal models have been used to study the pathogenesis of viral infections and to facilitate development of vaccines and therapeutics for viral diseases, models that can accurately recapitulate human responses to infection are still lacking. Human organ-on-a-chip (Organ Chip) microfluidic culture devices that recapitulate tissue-tissue interfaces, fluid flows, mechanical cues, and organ-level physiology have been developed to narrow the gap between in vitro experimental models and human pathophysiology. Here, we describe how recent developments in Organ Chips have enabled re-creation of complex pathophysiological features of human viral infections in vitro.

Discovery of Components Acting as the Obstacles in the Detection of Enteric Viruses from Berries.

This study investigated the obstacles in detecting enteric viruses from berry fruits, which are on the one hand often associated with outbreaks of viral enteric disease, and on the other hand recognized as a challenging food matrix for molecular detection of enteric viruses. According to the ISO 15216 protocol, for soft fruit samples, virus extraction is by elution with agitation followed by precipitation with polyethylene glycol/NaCl. As a result, first, the phenolic content in the berry eluate was found to be weakly correlated with the detection of coliphage MS2 spiked in the berry samples. Second and more importantly, it was observed that the gel-like pellets formed after precipitation could entrap considerable portions of viruses from being further purified and recovered for detection, suggesting that the low virus detection sensitivity from berries is largely due to the pectin content with complicated chemical structures in the berry fruits. Future research is needed to solve this problem in a targeted way.