We thank Benjamin Knowles, Yan Wei Lim, Andreas Haas, and members of the Viral Dark Matter consortium for their help and constructive input on this manuscript. This research is funded by the National Science Foundation (DEB-1046413) and is part of the Dimensions: Shedding Light on Viral Dark Matter project.

<ol>
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The authors have nothing to disclose.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism. We provided additional tools to manage the large data sets resulting from these technologies, allowing for high throughput processing and analysis24. Lastly, through the comparison of an annotated phage capsid protein, phage thioredoxin, two putative metabolic phage genes, and the average experimental response we propose various strategies to interpret both datasets and gene classes, with emphasis on identification of phenotypic trends and identification of outliers.
As mentioned, both approaches quantitatively measure only half of host metabolism. To interpret the relative function of any of the novel proteins under investigation, data from both methods is required to provide evidence of function. While this is not a focus of our current manuscript, data outputs from each phenomic method is put through combinatory analyses that focus on clustering techniques such as random forest and principal component analysis. Furthermore, hypotheses resulting from the combined analysis must be subsequently validated by traditional genetic methodologies.
Finally, the methods presented are heavily influenced by bacterial physiology and therefore follow the same standards. When undertaking either method, considerations need to be made to ensure independent, clonal groups are experimented with; contamination is prevented; a single variable is being tested; and appropriate controls are being ran simultaneously. Failure to account for these points will result in unclear results, similar to any physiological assay.
Multi-phenotype Assay Plates (MAPs)
The development of MAPs provides a high throughput and adaptable assay compared to technologies currently available (Figure 5A and Tables 1,2). The assay uses supplies, equipment, and fundamental techniques available in all microbiology labs. The incorporation of a computational pipeline, PMAnalyzer24, for subsequent data processing and analysis ensures rapid data interpretation. In addition, both experimental and analytical aspects of the approach can be readily adjusted or tuned for customized purposes. For example, if a large proportion of the data fails to pass filtering outlined in section 4, one can manually sift through the growth curves to identify issues. If the problem arises due to stringent filter parameters, adjustments to the script can be made. Alternatively, if problems are associated with the experimental process (i.e., prolonged condensation; improper transferring of bacterial cells, etc.) then additional replicates can be readily repeated.
As described in Cuevas et al.24, the PMAnalyzer is a single bash program written as a wrapper script that executes the parsing and analysis scripts as a cohesive, automated pipeline. All scripts are freely accessible from a Git repository at 25 by taking the median value for each time point across triplicate data, and subsequently parameterizes the logistic curve to obtain the lag time, maximum growth rate, asymptote, and a novel term, Growth Level. The median value was chosen over the mean in our study to reduce the effect of large outliers, however, the script can be readily adapted to calculate the mean of replicate data. Due to reduced variation (SE) seen across replicate data (Figure 2A) we maintained the use of the median in the PMAnalyzer for fitting a logistic curve. Additionally, the cut off for growth in this study (GL &#8805; 0.4) was determined by comparing how data separated across Growth Level and maximum growth rate (Figure 1A,B). Depending on the instruments and model system used this term may vary, requiring redefinition of this cut off value.
A major advantage of our assay is the ability to compare phenotypes using a single parameter characterizing overall microbial growth, which we define as Growth Level (GL). GL is a harmonic mean, and therefore mitigates the effects of large outliers in the data. The use of a harmonic mean with shifted logistic-fitted values to provide a summary of growth was arrived at through trial and error. Other methods attempted to differentiate growth included: time it took to reach specific curve parameters (half &#181;max, &#181;max, and carrying capacity), the coefficient of determination (R2), and combinations of the R2 multiplied by specific curve parameters. Using a harmonic mean with shifted logistic-fit values for the GL provided the greatest range in evaluating growth, thus it became the method of choice. One consideration to note is that dynamic growth curve patterns have the potential of being lost when using a single parameter or a fitted model. For instance, the individual curve parameters of the logistic curve and the GL are incapable of representing biphasic growth. In a single carbon environment, this effect on growth implies mediation of the viral protein on either conversion of the substrate or shift in substrate utilization. Additional effects potentially lost when not considering multiple growth parameters include: prolonged lag time, proposing an increased burden of viral machinery or products; rapidly accelerating exponential phase, suggesting viral proteins coupled to host energy production pathways; or higher levels of biomass formation, implying viral support in host nutrient uptake and anabolism (data not shown). Thus, plotting nascent growth curves (Figure 2A,B) provides information regarding trends over time whereas the GL takes into account the major variables of the logistic model, providing a single quantitative number to represent overall success of a clone.
When considering the different responses contributed by structural and metabolic genes in the MAPs, it is observed that the different substrate classes in question provide the greatest evidence for protein function. For example, metabolic proteins are often associated with acquisition of limiting nutrients, which are unspecific to host central metabolism16,32. Preliminary MAP experiments reveal that clones harboring putative metabolic phage genes have an increased lag phase when grown on central metabolism carbon sources (Figure 2A). Conversely, clones carrying putative structural genes, which require large proportions of host energy and dNTP pools, result in a false positive response on growth for central and amino acid metabolism carbon substrates. This is likely due to the accumulation of insoluble proteins resulting in host filamentation and/or inclusion bodies, as observed via microscopy (Figure 2A and data not shown). While further analysis is required to validate these preliminary results, the MAPs are capable of retrieving phenotypic responses that correlate to hypothesized functions of specific phage gene classes.
In addition to the elucidation of unknown viral proteins, the MAPs are a novel resource to investigate the functional and metabolic diversity of an individual bacterium or a community of bacteria. MAP components are designed for easy alteration to support the growth of a range of bacteria; including marine, auxotrophic, and anaerobic microbes. To facilitate these efforts the defined basal and pre-growth media require additional or adjusted chemical species before a different bacterial genus can be supported in the MAPs. One note in this use of the MAPs is to maintain defined media, prohibiting the use of ingredients such as tryptone, yeast extract and peptone.
Metabolomics
The field of metabolomics is dependent on metabolite databases, which include isolated metabolites identified by mass spectrometry. The core facility chosen here has one of the largest metabolomics databases. Interestingly, more than half of the metabolites resulting from our experimentations were unidentifiable (~65%), while others had never before been recorded in our host, Escherichia coli (examples include: Indole 3 acetic acid33, salicylic acid34, and dihydroabietic acid35). This fact could be attributed to either a strong bias of the database towards plant metabolites, or the specific proteins under investigation. Regardless, the result is a limited number of known metabolites available for data representation and analysis. In the future, multiple metabolomics methods using various databases would allow for greater metabolite coverage.
Presently, both known and unknown metabolites are used when comparing and contrasting our novel viral proteins. Using this approach, we hypothesize that clones harboring functionally similar proteins will share an increased similarity in their complete metabolomic profile. Preliminary metabolomics analysis revealed that while structural and metabolic genes do not clearly separate from one another, those genes exhibiting similar effects on the host when overexpressed do correlate (Figure 6). For example, the annotated Capsid gene clusters closely with the putative metabolic genes highlighted in this study, EDT2440 and EDT2441. Investigations using a publicly available transmembrane topology and signal peptide predictor program showed evidence that both putative metabolic genes harbor a single transmembrane domain. Interestingly 5 out of the 9 clones in the first cluster group (most left portion of the dendrogram) have predicted transmembrane domains using the same topology program. Further investigations are needed, however, it is likely that the metabolites present during the overexpression of these clones are associated with cellular stress response resulting from membrane or structural burdens. This evidence supports that while the metabolomics data possesses an increased amount of noise, the method is capable of highlighting signals that differentiate general effects of genes, both within and across a gene class. To determine whether the method is capable of extracting out specific information of gene function, metabolites were grouped into specific metabolic pathways. The hypothesis being, if a clone affects metabolites specific to a single pathway, then the overexpressed gene is active in that pathway. Prior to the establishment of our metabolomics quality assurance pipeline, preliminary data revealed that over and underrepresented metabolites were typically &#8220;unknown&#8221;, providing little information on the pathways they are associated with (data not shown). Preprocessed metabolomics data, however, reveals that the majority of the metabolite profiles are similar and only a select number of unknown and known metabolite abundances vary across clones, for instance putrescine and uracil (Figure 6). To provide greater resolution of protein function efforts are being made to experimentally compare the novel phage genes against known phage genes, which can be used to fill in the &#8220;holes&#8221; of metabolite based functional characterization. Using this technique, the assigned function of known viral genes provides a reference for the function of the unknown genes. Nonetheless, the limiting factor of metabolomic analysis is the size and relevance of the database. To correct for these limitations, metabolomic databases relatable to this research need to be developed; such as a database of metabolites and their abundances specific to the ASKA collection of E. coli clones in which a single ORF is overexpressed36. Evidence for the need of such databases was provided in 2013 when researchers at the Lawerence Berkeley National Laboratory compiled the first comprehensive database of metabolites specific to entire mutant libraries of model bacteria37. This research provided novel insight into genes required for utilization of specific metabolites, revealing the clear connection between phenotype and genotype.
When considering metabolomics as a tool, it is important to define the processing regime followed at the core facility. An artifact of most experimental procedures is the day-to-day variance associated with the instruments of use. To date all GC-MS analysis implements the use of internal standards that are included in each analytical run; however, addition of project specific internal samples ran each day of experimentation removes additional variance. These considerations must be addressed early to avoid normalization problems and biases. Another solution is to process all samples at a core facility on the same machine and as a single batch, an option available at any core facility.
The various tools both introduced and re-explored in this manuscript provide novel means to screen and characterize functionally unknown phage genes. The simplicity and adaptability of the experimental techniques with the streamline use of computational pipelines assures these methods are applicable to a broad range of research endeavors and fields. Our goal is that the phenomic approaches presented here will aid further investigations of novel phage proteins in addition to systems that are equally functionally undefined.

Viruses that infect Bacteria (a.k.a. bacteriophage or phage) are estimated to exist at more than 1031 virus like particles (VLPs) globally and outnumber all other organisms in an environment1,2. The first metagenomic study investigating the viral communities associated with marine environments focused on quantifying the diversity seen within the viral fraction3. Additionally, Breitbart and colleagues found that over 65% of the viral community sequences shared no homology to any sequences available in public databases. Subsequent metagenomic studies found similar evidence: metagenomes from marine sediments in San Diego, California contain 75% unknown viral sequences4; metagenomes from hypersaline lakes of the Salton Sea contain 98% unknown viral sequences5; and coral&#8211;associated metagenomes contain 95 - 98% unknown viral sequences6. This accumulation of unannotated information has resulted in phage genetic material being &#8220;the dark matter of the biological universe&#8221;7.
Genomic characterization of phage relies on identifying sequence similarity through comparison against existing nucleic acid and protein databases. Because phage-encoded genetic information is predominantly unknown, homology-based methods are ineffective. Within their genome, phages typically encode three major gene types: transcription and replication genes, metabolic genes, and structural genes. The transcription and replication genes (class I/II genes8) include polymerases, primases, endo/exo-nucleases, and kinases. These genes are highly conserved due to their importance in phage infection, transcribing and replicating phage genetic material. Phage polymerases are readily identified using traditional sequence homology methods due to their global conservation9 and have been shown to serve as effective phylogenetic markers10. In contrast, phage metabolic and structural genes (class II/III genes8) are increasingly divergent and often annotated as hypothetical genes.
Phage metabolic genes affect the metabolic capacity of the host and are not necessarily required for viral replication. These genes, often referred to as auxiliary metabolic genes11 (AMGs), appear to modulate host metabolism and allow optimal progression of infection and success of virion maturation. AMGs have been associated with the utilization and uptake of limiting nutrients or in energy production pathways. Some examples include photosystem genes found in the genomes of various cyanophage12-16, genes connected to and regulated by phosphate metabolism17,18, and utilization of the pentose phosphate pathway for phage dNTP biosynthesis18,19. In comparison, structural genes are among the mid to late genes produced during infection and vary across different phage-host systems. The production of structural proteins are dependent on the availability of viral dNTP, and energy pools for their transcription, translation, and assembly8. The capsid and tail fiber structural proteins are deemed as the most divergent of all viral protein-encoding genes and are required for successful virion production. Their divergence is typically attributed to the active role they play in shaping virus-host coevolution20. Divergent proteins, regardless of the gene class, are readily overlooked when using traditional homology and sequence alignment techniques. An effort to correct for the limitations seen with strict sequence comparisons has resulted in bioinformatics tools capable of using sequence characteristics to determine association, such as artificial neural nets21. Artificial neural nets (ANNs) allow for the prediction of structural and metabolic genes, however, require downstream experimental validation to directly characterize gene function.
The objective of this manuscript is to provide phenomic protocols capable of monitoring both catabolic and anabolic metabolism of a host bacterium during the expression of a novel phage gene, functionally predicted through ANNs. The field of phenomics, the biology associated with cellular phenotypes, is well established in systems biology to aid in the investigation of proteins with unknown or pleiotropic function. Phenomic tools are used to link phenotypic information to genotypic information. We hypothesize for putative phage genes that their function(s) can be determined through observing host physiological effects during phage gene expression. To investigate this hypothesis, two quantitative methods were chosen. Multi-phenotype Assay Plates (MAPs) were used to monitor host substrate utilization and the subsequent biomass formation while metabolomics measured host metabolite diversity and relative abundance during growth in specific environmental conditions. Putative structural and metabolic proteins were overexpressed in Escherichia coli and representative results from both experiments are compared. Numerous visual techniques and high throughput processing pipelines are presented to facilitate experimental replication. Lastly, the reproducibility and accuracy of the presented methods are discussed in the context of expected physiological effects for an annotated capsid protein and phage metabolic protein, thioredoxin, plus two putative AMGs.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>0.22&#181;m Sterivex Filter</td>
			<td>Fisher Scientific</td>
			<td>SVGP01050</td>
			<td>Millipore</td>
		</tr>
		<tr>
			<td>0.22&#181;m Millex Filters</td>
			<td>Fisher Scientific</td>
			<td>SLGV033RS</td>
			<td>Millipore</td>
		</tr>
		<tr>
			<td>0.22&#181;m SteriCap Filter</td>
			<td>Fisher Scientific</td>
			<td>SCGPS02RE</td>
			<td>Millipore</td>
		</tr>
		<tr>
			<td>0.22 &#181;m Omnipore membrane filters</td>
			<td>Millipore</td>
			<td>JHWP02500</td>
			<td>Millipore</td>
		</tr>
		<tr>
			<td>96 well micro-titer plates&#160;</td>
			<td>VWR</td>
			<td>82050-764</td>
			<td>Standard F-Bottom 96 well Microplates</td>
		</tr>
		<tr>
			<td>2 mL 96 well plate</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Seal Plate Film</td>
			<td>Sigma-Aldrich</td>
			<td>Z369667</td>
			<td></td>
		</tr>
		<tr>
			<td>2 L Nalgene square bottles</td>
			<td>Cole Parmer</td>
			<td>T-06040-70</td>
			<td></td>
		</tr>
		<tr>
			<td>125 mL Nalgene square bottles</td>
			<td>Cole Parmer</td>
			<td>T-06040-50</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4inch Panel Mount Lock Nut, black nylon&#160;</td>
			<td>Cole Parmer</td>
			<td>EW-45509-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Female Luer Thread Style Panel Mount to 200 Series Barb&#160; 1/16inch&#160;</td>
			<td>Cole Parmer</td>
			<td>EW-45500-30</td>
			<td></td>
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			<td>Female Luer Thread Style Panel Mount to 200 Series Barb, 1/8inch</td>
			<td>Cole Parmer</td>
			<td>EW-45500-34</td>
			<td></td>
		</tr>
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			<td>Male Luer Integral Lock Ring to 500 Series Barb, 1/16inch ID tubing</td>
			<td>Cole Parmer</td>
			<td>EW-45505-31</td>
			<td></td>
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			<td>Male Luer with Lock Ring x Female Luer Coupler</td>
			<td>Cole Parmer</td>
			<td>T-45508-80</td>
			<td></td>
		</tr>
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			<td>Barbed Bulkhead Fittings 1/4inch OD&#160;</td>
			<td>Fisher Scientific</td>
			<td>6149-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sanipure Tubing 1/16inch ID x 1/8inch OD&#160;</td>
			<td>SaniPure</td>
			<td>AR400002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sanipure Tubing 1/4inch OD x 1/8inch ID&#160;</td>
			<td>SaniPure</td>
			<td>AR400007</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable Flow Mini Pump (Peristaltic pump)</td>
			<td>Fisher Scientific</td>
			<td>13-876-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Stirrer</td>
			<td>Velp Scientifica</td>
			<td>F203A0160</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>14-512-141</td>
			<td>&#160;Millipore* Filter Forceps&#160;</td>
		</tr>
		<tr>
			<td>Multi-plate spectrophotometer plate reader</td>
			<td>Molecular Devices Analyst GT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filter manifold</td>
			<td>Fisher Scientific</td>
			<td>&#160;XX10 025 02</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Python version 2.7.5</td>
			<td></td>
			<td></td>
			<td><a href="http://www.python.org/">http://www.python.org/</a></td>
		</tr>
		<tr>
			<td>PyLab module</td>
			<td></td>
			<td></td>
			<td><a href="http://wiki.scipy.org/PyLab">http://wiki.scipy.org/PyLab</a></td>
		</tr>
		<tr>
			<td>R version 3.0.1&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.r-project.org/">http://www.r-project.org/</a></td>
		</tr>
		<tr>
			<td>reshape2 library</td>
			<td></td>
			<td></td>
			<td><a href="http://had.co.nz/reshape">http://had.co.nz/reshape</a></td>
		</tr>
		<tr>
			<td>ggplot2 library</td>
			<td></td>
			<td></td>
			<td><a href="http://ggplot2.org/">http://ggplot2.org/</a></td>
		</tr>
		<tr>
			<td>Gene Composer</td>
			<td>PSI Tech Portal</td>
			<td></td>
			<td>http://www.genecomposer.net</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Services:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>West Coast Metabolomics Center</td>
			<td>UC Davis</td>
			<td></td>
			<td><a href="http://metabolomics.ucdavis.edu/">http://metabolomics.ucdavis.edu</a></td>
		</tr>
		<tr>
			<td>DNA 2.0</td>
			<td></td>
			<td></td>
			<td>https://www.dna20.com</td>
		</tr>
	</tbody>
</table>

phage phenomics: physiological approaches to characterize novel viral proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Watch this Scientific Journal Video about Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins at JoVE.com

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

phage-phenomics-physiological-approaches-to-characterize-novel-viral

Research

JoVE Journal

Immunology and Infection

12.0K Views.  San Diego State University. Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

Video: Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

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.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

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 ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Forward Genetic Approaches in Chlamydia trachomatis

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its intractability to experimental transformation with recombinant DNA. We developed an approach to perform genetic analysis in C. trachomatis&#160;despite the lack of molecular genetic tools. Our method involves: i.) chemical mutagenesis to rapidly generate comprehensive libraries of genetically-defined mutants with distinct phenotypes; ii.) whole-genome sequencing (WGS) to map the underlying genetic lesions and to find associations between mutated gene(s) and a common phenotype; iii.) generation of recombinant strains through co-infection of mammalian cells with mutant and wild type bacteria. Accordingly, we were able to establish causal relationships between genotypes and phenotypes. The coupling of chemically-induced gene variation and WGS to establish correlative genotype&#8211;phenotype associations should be broadly applicable to the large list of medically and environmentally important microorganisms currently intractable to genetic analysis.

Forward Genetic Approaches in Chlamydia trachomatis

We describe a methodology to perform genetic analysis in Chlamydia based on chemical mutagenesis and whole genome sequencing. In addition, a system for DNA exchange within infected cells is described that can be used for genetic mapping. This method may be broadly applicable to microbial systems lacking transformation systems and molecular genetic tools.

Chlamydia trachomatis, the etiological agent of sexually transmitted diseases and ocular infections, remains poorly characterized due to its ...

Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of discrete plaques (infectious units and cellular dead zones) in cell culture. Here we demonstrate how to perform a basic plaque assay, and how differing overlays and techniques can affect plaque formation and production. Typically solid or semisolid overlay substrates, such as agarose or carboxymethyl cellulose, have been used to restrict viral spread, preventing indiscriminate infection through the liquid growth medium. Immobilized overlays restrict cellular infection to the immediately surrounding monolayer, allowing the formation of discrete countable foci and subsequent plaque formation. To overcome the difficulties inherent in using traditional overlays, a novel liquid overlay utilizing microcrystalline cellulose and carboxymethyl cellulose sodium has been increasingly used as a replacement in the standard plaque assay. Liquid overlay plaque assays can be readily performed in either standard 6 or 12 well plate formats as per traditional techniques and require no special equipment. Due to its liquid state and subsequent ease of application and removal, microculture plate formats may alternatively be utilized as a rapid, accurate and high throughput alternative to larger scale viral titrations. Use of a non heated viscous liquid polymer offers the opportunity to streamline work, conserves reagents, incubator space, and increases operational safety when used in traditional or high containment labs as no reagent heating or glassware are required. Liquid overlays may also prove more sensitive than traditional&#160;overlays for certain heat labile viruses.

Plaque assays are the gold standard for viral quantification, utilizing entrapping overlays on host cellular monolayers to determine viral titers. While various semisolid overlays have traditionally been used, here we demonstrate plaque techniques comparing semisolid overlays to a novel liquid microcrystalline cellulose among several families of viruses.

Plaque assays remain one of the most accurate methods for the direct quantification of infectious virons and antiviral substances through the counting of ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.