Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.
23 Related JoVE Articles!
Community-based Adapted Tango Dancing for Individuals with Parkinson's Disease and Older Adults
Institutions: Emory University School of Medicine, Brigham and Woman‘s Hospital and Massachusetts General Hospital.
Adapted tango dancing improves mobility and balance in older adults and additional populations with balance impairments. It is composed of very simple step elements. Adapted tango involves movement initiation and cessation, multi-directional perturbations, varied speeds and rhythms. Focus on foot placement, whole body coordination, and attention to partner, path of movement, and aesthetics likely underlie adapted tango’s demonstrated efficacy for improving mobility and balance. In this paper, we describe the methodology to disseminate the adapted tango teaching methods to dance instructor trainees and to implement the adapted tango by the trainees in the community for older adults and individuals with Parkinson’s Disease (PD). Efficacy in improving mobility (measured with the Timed Up and Go, Tandem stance, Berg Balance Scale, Gait Speed and 30 sec chair stand), safety and fidelity of the program is maximized through targeted instructor and volunteer training and a structured detailed syllabus outlining class practices and progression.
Behavior, Issue 94, Dance, tango, balance, pedagogy, dissemination, exercise, older adults, Parkinson's Disease, mobility impairments, falls
Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy
Institutions: University of Maine.
Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.
Basic Protocol, Issue 82, Microscopy, Super-resolution imaging, Multicolor, single molecule, FPALM, Localization microscopy, fluorescent proteins
Evaluating Plasmonic Transport in Current-carrying Silver Nanowires
Institutions: Université de Bourgogne, University of Science and Technology of China, CEMES, CNRS-UPR 8011.
Plasmonics is an emerging technology capable of simultaneously transporting a plasmonic signal and an electronic signal on the same information support1,2,3
. In this context, metal nanowires are especially desirable for realizing dense routing networks4
. A prerequisite to operate such shared nanowire-based platform relies on our ability to electrically contact individual metal nanowires and efficiently excite surface plasmon polaritons5
in this information support. In this article, we describe a protocol to bring electrical terminals to chemically-synthesized silver nanowires6
randomly distributed on a glass substrate7
. The positions of the nanowire ends with respect to predefined landmarks are precisely located using standard optical transmission microscopy before encapsulation in an electron-sensitive resist. Trenches representing the electrode layout are subsequently designed by electron-beam lithography. Metal electrodes are then fabricated by thermally evaporating a Cr/Au layer followed by a chemical lift-off. The contacted silver nanowires are finally transferred to a leakage radiation microscope for surface plasmon excitation and characterization8,9
. Surface plasmons are launched in the nanowires by focusing a near infrared laser beam on a diffraction-limited spot overlapping one nanowire extremity5,9
. For sufficiently large nanowires, the surface plasmon mode leaks into the glass substrate9,10
. This leakage radiation is readily detected, imaged, and analyzed in the different conjugate planes in leakage radiation microscopy9,11
. The electrical terminals do not affect the plasmon propagation. However, a current-induced morphological deterioration of the nanowire drastically degrades the flow of surface plasmons. The combination of surface plasmon leakage radiation microscopy with a simultaneous analysis of the nanowire electrical transport characteristics reveals the intrinsic limitations of such plasmonic circuitry.
Physics, Issue 82, light transmission, optical waveguides, photonics, plasma oscillations, plasma waves, electron motion in conductors, nanofabrication, Information Transport, plasmonics, Silver Nanowires, Leakage radiation microscopy, Electromigration
Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System
Institutions: Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai, Icahn School of Medicine at Mount Sinai.
The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.
Infection, Issue 81, Influenza A virus, Orthomyxoviridae Infections, Influenza, Human, Influenza in Birds, Influenza Vaccines, hemagglutinin, neuraminidase, H7N9, baculovirus, insect cells, recombinant protein expression
Isolation and Quantification of Botulinum Neurotoxin From Complex Matrices Using the BoTest Matrix Assays
Institutions: BioSentinel Inc., Madison, WI.
Accurate detection and quantification of botulinum neurotoxin (BoNT) in complex matrices is required for pharmaceutical, environmental, and food sample testing. Rapid BoNT testing of foodstuffs is needed during outbreak forensics, patient diagnosis, and food safety testing while accurate potency testing is required for BoNT-based drug product manufacturing and patient safety. The widely used mouse bioassay for BoNT testing is highly sensitive but lacks the precision and throughput needed for rapid and routine BoNT testing. Furthermore, the bioassay's use of animals has resulted in calls by drug product regulatory authorities and animal-rights proponents in the US and abroad to replace the mouse bioassay for BoNT testing. Several in vitro
replacement assays have been developed that work well with purified BoNT in simple buffers, but most have not been shown to be applicable to testing in highly complex matrices. Here, a protocol for the detection of BoNT in complex matrices using the BoTest Matrix assays is presented. The assay consists of three parts: The first part involves preparation of the samples for testing, the second part is an immunoprecipitation step using anti-BoNT antibody-coated paramagnetic beads to purify BoNT from the matrix, and the third part quantifies the isolated BoNT's proteolytic activity using a fluorogenic reporter. The protocol is written for high throughput testing in 96-well plates using both liquid and solid matrices and requires about 2 hr of manual preparation with total assay times of 4-26 hr depending on the sample type, toxin load, and desired sensitivity. Data are presented for BoNT/A testing with phosphate-buffered saline, a drug product, culture supernatant, 2% milk, and fresh tomatoes and includes discussion of critical parameters for assay success.
Neuroscience, Issue 85, Botulinum, food testing, detection, quantification, complex matrices, BoTest Matrix, Clostridium, potency testing
Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana
Institutions: Fraunhofer USA Center for Molecular Biotechnology.
-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana
plants with Agrobacteria
carrying launch vectors. Optimization of Agrobacterium
cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana
, N. excelsiana
× N. excelsior
) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium
harboring pBID4-GFP (Tobacco mosaic virus
-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium
laboratory strain GV3101 showed the highest protein production compared to Agrobacteria
laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria
strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana
resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).
Plant Biology, Issue 86, Agroinfiltration, Nicotiana benthamiana, transient protein production, plant-based expression, viral vector, Agrobacteria
Characterization of Complex Systems Using the Design of Experiments Approach: Transient Protein Expression in Tobacco as a Case Study
Institutions: RWTH Aachen University, Fraunhofer Gesellschaft.
Plants provide multiple benefits for the production of biopharmaceuticals including low costs, scalability, and safety. Transient expression offers the additional advantage of short development and production times, but expression levels can vary significantly between batches thus giving rise to regulatory concerns in the context of good manufacturing practice. We used a design of experiments (DoE) approach to determine the impact of major factors such as regulatory elements in the expression construct, plant growth and development parameters, and the incubation conditions during expression, on the variability of expression between batches. We tested plants expressing a model anti-HIV monoclonal antibody (2G12) and a fluorescent marker protein (DsRed). We discuss the rationale for selecting certain properties of the model and identify its potential limitations. The general approach can easily be transferred to other problems because the principles of the model are broadly applicable: knowledge-based parameter selection, complexity reduction by splitting the initial problem into smaller modules, software-guided setup of optimal experiment combinations and step-wise design augmentation. Therefore, the methodology is not only useful for characterizing protein expression in plants but also for the investigation of other complex systems lacking a mechanistic description. The predictive equations describing the interconnectivity between parameters can be used to establish mechanistic models for other complex systems.
Bioengineering, Issue 83, design of experiments (DoE), transient protein expression, plant-derived biopharmaceuticals, promoter, 5'UTR, fluorescent reporter protein, model building, incubation conditions, monoclonal antibody
An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings
Institutions: University of KwaZulu-Natal, Durban, South Africa, Jembi Health Systems, University of Amsterdam, Stanford Medical School.
HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure. An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads <1,000 HIV-1 RNA copies/ml. The method described here was validated against a method of HIV-1 drug resistance testing approved by the United States Food and Drug Administration (FDA), the Viroseq genotyping method. Limitations of the method described here include the fact that it is not automated and that it also failed to amplify the circulating recombinant form CRF02_AG from a validation panel of samples, although it amplified subtypes A and B from the same panel.
Medicine, Issue 85, Biomedical Technology, HIV-1, HIV Infections, Viremia, Nucleic Acids, genetics, antiretroviral therapy, drug resistance, genotyping, affordable
A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses
Institutions: Emory University, Emory University.
The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro
. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro
replication of HIV-1 as influenced by the gag
gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag
gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro
replication of chronically derived gag-pro
sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.
Infectious Diseases, Issue 90, HIV-1, Gag, viral replication, replication capacity, viral fitness, MJ4, CEM, GXR25
Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity
Institutions: Case Western Reserve University.
Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.
Environmental Sciences, Issue 85, Coexistence, community assembly, environmental drivers, plant-soil feedback, soil heterogeneity, soil microbial communities, soil patch
Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules
Institutions: Princeton University.
The aim of de novo
protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo
protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (http://www.proteinwisdom.org), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.
Genetics, Issue 77, Molecular Biology, Bioengineering, Biochemistry, Biomedical Engineering, Chemical Engineering, Computational Biology, Genomics, Proteomics, Protein, Protein Binding, Computational Biology, Drug Design, optimization (mathematics), Amino Acids, Peptides, and Proteins, De novo protein and peptide design, Drug design, In silico sequence selection, Optimization, Fold specificity, Binding affinity, sequencing
Microfluidic Chip Fabrication and Method to Detect Influenza
Institutions: Boston University , Boston University .
Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.
Bioengineering, Issue 73, Biomedical Engineering, Infection, Infectious Diseases, Virology, Microbiology, Genetics, Molecular Biology, Biochemistry, Mechanical Engineering, Microfluidics, Virus, Diseases, Respiratory Tract Diseases, Diagnosis, Microfluidic chip, influenza virus, flu, solid phase extraction (SPE), reverse transcriptase polymerase chain reaction, RT-PCR, PCR, DNA, RNA, on chip, assay, clinical, diagnostics
Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
Institutions: Emerald Bio, Emerald Bio, Emerald Bio, Emerald Bio, Emerald Bio, Emerald Bio, Emerald Bio, Emerald Bio, Emerald Bio.
Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1
. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2
. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.
Infection, Issue 76, Structural Biology, Virology, Genetics, Medicine, Biomedical Engineering, Molecular Biology, Infectious Diseases, Microbiology, Genomics, high throughput, multi-targeting, structural genomics, protein crystallization, purification, protein production, X-ray crystallography, Gene Composer, Protein Maker, expression, E. coli, fermentation, influenza, virus, vector, plasmid, cell, cell culture, PCR, sequencing
Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens
Institutions: University of California, San Francisco, University of California, San Francisco.
The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1
. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5
. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7
, a novel rhinovirus clade5
, XMRV (a retrovirus linked to prostate cancer)8
, avian bornavirus (the cause of a wasting disease in parrots)9
, and a novel cardiovirus in children with respiratory and diarrheal illness10
. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.
Immunology, Issue 50, virus, microarray, Virochip, viral detection, genomics, clinical diagnostics, viral discovery, metagenomics, novel pathogen discovery
ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza
DNA microarrays have emerged as a powerful tool for pathogen detection.1-5
For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11
The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1
replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11
, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is ˜1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.
Immunology, Issue 52, microarrays, colorimetric detection, ampliPHOX, diagnostic, low-density, pathogen detection, influenza
Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays
Institutions: Health canada, The State Food and Drug Administration, Beijing, University of Ottawa, King Abdulaziz University, Public Health Agency of Canada.
Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2
. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3
. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4
, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5
. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7
. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6
, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7
. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8
. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used.
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.
Immunology, Issue 50, Virology, influenza, hemagglutinin, neuraminidase, quantification, universal antibody
Avian Influenza Surveillance with FTA Cards: Field Methods, Biosafety, and Transportation Issues Solved
Institutions: Wageningen University, Linnaeus University, Simon Fraser University .
Avian Influenza Viruses (AIVs) infect many mammals, including humans1
. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2
. Human pandemics of flu originally stem from AIVs3
. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4
. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5
, dispersal can also partly be attributed to wild birds6, 7
. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12
. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14
and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17
. Recently, Kraus et al
introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19
preserve RNA on a dry storage basis20
and render pathogens inactive upon contact21
. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al
a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.
Immunology, Issue 54, AI, Influenza A Virus, zoonoses, reverse transcription PCR, viral RNA, surveillance, duck trap, RNA preservation and storage, infection, mallard
Aseptic Laboratory Techniques: Plating Methods
Institutions: University of California, Los Angeles .
Microorganisms are present on all inanimate surfaces creating ubiquitous sources of possible contamination in the laboratory. Experimental success relies on the ability of a scientist to sterilize work surfaces and equipment as well as prevent contact of sterile instruments and solutions with non-sterile surfaces. Here we present the steps for several plating methods routinely used in the laboratory to isolate, propagate, or enumerate microorganisms such as bacteria and phage. All five methods incorporate aseptic technique, or procedures that maintain the sterility of experimental materials. Procedures described include (1) streak-plating bacterial cultures to isolate single colonies, (2) pour-plating and (3) spread-plating to enumerate viable bacterial colonies, (4) soft agar overlays to isolate phage and enumerate plaques, and (5) replica-plating to transfer cells from one plate to another in an identical spatial pattern. These procedures can be performed at the laboratory bench, provided they involve non-pathogenic strains of microorganisms (Biosafety Level 1, BSL-1). If working with BSL-2 organisms, then these manipulations must take place in a biosafety cabinet. Consult the most current edition of the Biosafety in Microbiological and Biomedical Laboratories
(BMBL) as well as Material Safety Data Sheets
(MSDS) for Infectious Substances to determine the biohazard classification as well as the safety precautions and containment facilities required for the microorganism in question. Bacterial strains and phage stocks can be obtained from research investigators, companies, and collections maintained by particular organizations such as the American Type Culture Collection
(ATCC). It is recommended that non-pathogenic strains be used when learning the various plating methods. By following the procedures described in this protocol, students should be able to:
● Perform plating procedures without contaminating media.
● Isolate single bacterial colonies by the streak-plating method.
● Use pour-plating and spread-plating methods to determine the concentration of bacteria.
● Perform soft agar overlays when working with phage.
● Transfer bacterial cells from one plate to another using the replica-plating procedure.
● Given an experimental task, select the appropriate plating method.
Basic Protocols, Issue 63, Streak plates, pour plates, soft agar overlays, spread plates, replica plates, bacteria, colonies, phage, plaques, dilutions
Improving IV Insulin Administration in a Community Hospital
Institutions: Wyoming Medical Center.
Diabetes mellitus is a major independent risk factor for increased morbidity and mortality in the hospitalized patient, and elevated blood glucose concentrations, even in non-diabetic patients, predicts poor outcomes.1-4
The 2008 consensus statement by the American Association of Clinical Endocrinologists (AACE) and the American Diabetes Association (ADA) states that "hyperglycemia in hospitalized patients, irrespective of its cause, is unequivocally associated with adverse outcomes."5
It is important to recognize that hyperglycemia occurs in patients with known or undiagnosed diabetes as well as during acute illness in those with previously normal glucose tolerance.
The Normoglycemia in Intensive Care Evaluation-Survival Using Glucose Algorithm Regulation (NICE-SUGAR) study involved over six thousand adult intensive care unit (ICU) patients who were randomized to intensive glucose control or conventional glucose control.6
Surprisingly, this trial found that intensive glucose control increased the risk of mortality by 14% (odds ratio, 1.14; p=0.02). In addition, there was an increased prevalence of severe hypoglycemia in the intensive control group compared with the conventional control group (6.8% vs. 0.5%, respectively; p<0.001). From this pivotal trial and two others,7,8
Wyoming Medical Center (WMC) realized the importance of controlling hyperglycemia in the hospitalized patient while avoiding the negative impact of resultant hypoglycemia.
Despite multiple revisions of an IV insulin paper protocol, analysis of data from usage of the paper protocol at WMC shows that in terms of achieving normoglycemia while minimizing hypoglycemia, results were suboptimal. Therefore, through a systematical implementation plan, monitoring of patient blood glucose levels was switched from using a paper IV insulin protocol to a computerized glucose management system. By comparing blood glucose levels using the paper protocol to that of the computerized system, it was determined, that overall, the computerized glucose management system resulted in more rapid and tighter glucose control than the traditional paper protocol. Specifically, a substantial increase in the time spent within the target blood glucose concentration range, as well as a decrease in the prevalence of severe hypoglycemia (BG < 40 mg/dL), clinical hypoglycemia (BG < 70 mg/dL), and hyperglycemia (BG > 180 mg/dL), was witnessed in the first five months after implementation of the computerized glucose management system. The computerized system achieved target concentrations in greater than 75% of all readings while minimizing the risk of hypoglycemia. The prevalence of hypoglycemia (BG < 70 mg/dL) with the use of the computer glucose management system was well under 1%.
Medicine, Issue 64, Physiology, Computerized glucose management, Endotool, hypoglycemia, hyperglycemia, diabetes, IV insulin, paper protocol, glucose control
Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock
Institutions: Lethbridge Research Centre, Dalian University of Technology, Alberta Agriculture and Rural Development.
Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of animals housed within a confined area enables rapid dissemination of most infectious agents throughout a herd. Rapid containment is key to controlling any infectious disease outbreak, thus depopulation is often undertaken to prevent spread of a pathogen to the larger livestock population. In that circumstance, a large number of livestock carcasses and contaminated manure are generated that require rapid disposal.
Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We designed a bio-contained mortality composting procedure and tested its efficacy for bovine tissue degradation and microbial deactivation. We used materials available on-farm or purchasable from local farm supply stores in order that the system can be implemented at the site of a disease outbreak. In this study, temperatures exceeded 55°C for more than one month and infectious agents implanted in beef cattle carcasses and manure were inactivated within 14 days of composting. After 147 days, carcasses were almost completely degraded. The few long bones remaining were further degraded with an additional composting cycle in open windrows and the final mature compost was suitable for land application.
Duplicate compost structures (final dimensions 25 m x 5 m x 2.4 m; L x W x H) were constructed using barley straw bales and lined with heavy black silage plastic sheeting. Each was loaded with loose straw, carcasses and manure totaling ~95,000 kg. A 40-cm base layer of loose barley straw was placed in each bunker, onto which were placed 16 feedlot cattle mortalities (average weight 343 kg) aligned transversely at a spacing of approximately 0.5 m. For passive aeration, lengths of flexible, perforated plastic drainage tubing (15 cm diameter) were placed between adjacent carcasses, extending vertically along both inside walls, and with the ends passed though the plastic to the exterior. The carcasses were overlaid with moist aerated feedlot manure (~1.6 m deep) to the top of the bunker. Plastic was folded over the top and sealed with tape to establish a containment barrier and eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration. After 147 days, losses of volume and mass of composted materials averaged 39.8% and 23.7%, respectively, in each structure.
JoVE Infectious Diseases, Issue 39, compost, livestock, infectious disease, biocontainment
Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes
Institutions: University of California, Irvine (UCI).
In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.
Cellular Biology, Issue 5, mosquito, malaria, dengue fever, genetics, infectious disease, Translational Research
Predicting the Effectiveness of Population Replacement Strategy Using Mathematical Modeling
Institutions: University of California, Los Angeles.
Charles Taylor and John Marshall explain the utility of mathematical modeling for evaluating the effectiveness of population replacement strategy. Insight is given into how computational models can provide information on the population dynamics of mosquitoes and the spread of transposable elements through A. gambiae subspecies. The ethical considerations of releasing genetically modified mosquitoes into the wild are discussed.
Cellular Biology, Issue 5, mosquito, malaria, popuulation, replacement, modeling, infectious disease
Population Replacement Strategies for Controlling Vector Populations and the Use of Wolbachia pipientis for Genetic Drive
Institutions: Johns Hopkins University.
In this video, Jason Rasgon discusses population replacement strategies to control vector-borne diseases such as malaria and dengue. "Population replacement" is the replacement of wild vector populations (that are competent to transmit pathogens) with those that are not competent to transmit pathogens. There are several theoretical strategies to accomplish this. One is to exploit the maternally-inherited symbiotic bacteria Wolbachia pipientis. Wolbachia is a widespread reproductive parasite that spreads in a selfish manner at the extent of its host's fitness. Jason Rasgon discusses, in detail, the basic biology of this bacterial symbiont and various ways to use it for control of vector-borne diseases.
Cellular Biology, Issue 5, mosquito, malaria, genetics, infectious disease, Wolbachia