This work was supported by the Austrian Science Fund (FWF, P20619 and P22988 to B.Z.).

Microwave assisted sample preparation
<ol>
	<li>Before the start of sample preparation one needs to program the automatic microwave tissue processor for electron microscopy (Leica EM AMW; Leica Microsystems, Vienna, Austria) with the following protocols (Table 1) for microwave assisted sample preparation for TEM: 
		<img src="/files/ftp_upload/2950/2950table1.jpg" alt="Table 1" /> 
		Table 1. Protocol for sample preparation using microwave irradiation. The different columns show (from left to right): 
		<ol>
			<li>Vial number (Vial nr.) represents the order in which the vial is loaded into the carousel of the processor.</li>
			<li>Step of the actual process.</li>
			<li>Reagents in the vials.</li>
			<li>Duration of the actual step.</li>
			<li>Maximum temperature which is reached in the vial before the microwave irradiation is turned off.</li>
			<li>Microwave irradiation setting: Continuous= rapid temperature increase, holding the set temperature; Slope=gentle temperature increase, final temperature reached at the end; Pulsed=Rapid temperature increase, power turned off until the temperature dropped 5&deg;C, power resumed to reach temperature.</li>
			<li>Maximum power of the microwave irradiation.</li>
		</ol>
	</li>
	<li>Freshly prepare the solutions for the different steps described in the sample preparation protocol (for Agar 100 epoxy resin see 1.7), fill them into the designated vials according to the programmed protocol (Table 1), load the vials on the carousel, then insert the carousel into the microwave tissue processor, and finally load the first vial into the mono-mode chamber.</li>
	<li>Cut out small sections of leaves (1mm2) from Nicotiana tabacum infected with Tobacco Mosaic Virus (TMV) with a razor blade on a modeling wax plate in a drop of 3% glutaraldehyde (Agar Scientific Ltd., Stansted, England) in 60mM S&oslash;rensen phosphate buffer (pH 7.2) at room temperature.</li>
	<li>Transfer the sections with fine tweezers immediately into the designated baskets with a mesh width of approximately 200&mu;m. Stack the baskets on top of each other and insert them into the mono-mode chamber. Care must be taken that the samples are constantly covered with fixative solution during loading and stacking of the baskets so that they do not dry out.</li>
	<li>Start the previously programmed microwave assisted sample preparation protocol for fixation, dehydration and infiltration. </li>
	<li>While sample preparation is performed automatically by the microwave tissue processor continue with negative staining with the remaining plant material as described in section 3 (negative staining).</li>
	<li>Freshly prepare Agar 100 epoxy resin by mixing the following components as described: fill 24g Agar 100, 16g dodecenyl succinic anhydride, and 10 g methyl nadic anhydride (for all components see Agar Scientific Ltd., Stansted, England) in a plastic cup, heat it to 40&deg;C and mix it well. Add 1.2g of benzyl dimethylamine and mix thoroughly. Fill Agar 100 epoxy resin into the designated polymerization forms just before the sample preparation protocol comes to an end (e.g. during step 22 in table 1). </li>
	<li>After the protocol is finished (after step 22 in table 1) release the stacked baskets containing the infiltrated samples from the mono-mode chamber into the last vial of the carousel. Remove the carousel from the microwave device, unstack the baskets and load them by using fine tweezers into the designated polymerization forms. Care must be taken that the samples are always covered with Agar 100 epoxy resin during unstacking and loading so that they do not dry out.</li>
	<li>Stack the polymerization forms on top of each other. Care must be taken that the samples are always covered with Agar 100 epoxy resin during stacking and loading so that they do not dry out.</li>
	<li>Remove previously used vials from the carousel of the microwave tissue processor, load it with the stacked baskets and insert the carousel into the microwave tissue processor. </li>
	<li>Start the previously programmed polymerization protocol (table 1).</li>
	<li>While polymerization is carried out automatically by the microwave tissue processor, examine negatively stained grids with a transmission electron microscope [e.g. Philips CM10 TEM, FEI (formerly Philips), Eindhoven, The Netherlands] and perform image analysis as described in section 3 and 4 (negative staining and image analysis).</li>
	<li>After the protocol is finished remove the polymerization forms from the mono-mode chamber, unstack the polymerization forms and remove the polymerized blocks containing the samples. They are now ready to be sectioned with a microtome.</li>
</ol>
2. Trimming and sectioning 
<ol>
	<li>Insert one or more blocks into separate sample holders for ultrathin sectioning with the sample on top sticking about 1cm out of the holder.</li>
	<li>Trim the block with a specimen trimmer for TEM (e.g. Leica Reichert Ultratrim; Leica Microsystems) so that a block face of max. 1mm in length and 200&mu;m in width which contains as much leaf material as possible is achieved (block face size might need to be adjusted to the size of the diamond knife).</li>
	<li>Section the block with an ultramicrotome (e.g. Reichert Leica Ultracut S; Leica Microsystems) by using a diamond knife at a knife angle of 45&deg; (e.g. Diatome Ultra 45, Gr&ouml;pl, Tulln, Austria). Section thickness should be adjusted to about 70 to 90nm and the cutting speed should be around 1mm/s. Pick up multiple sections with a formvar (Agar Scientific Ltd.) coated copper or nickel 200 square mesh grid.</li>
	<li>Post-stain the sections on the grid with lead citrate (Agar Scientific Ltd.; 1.1g lead citrate dissolved in 42ml double distilled water and 8ml 1N NaOH) for 5 minutes in a petri dish partly filled with NaOH to create a CO2-free environment and for 15 minutes with 1% uranyl acetate (Agar Scientific Ltd.) dissolved in distilled water at room temperature. Wash the grids with distilled water for 1 minute in between each post-staining step. Air dry the grids in a grid box.</li>
	<li>Examine sections with a transmission electron microscope (e.g. Philips CM10 TEM, FEI, Eindhoven, The Netherlands). </li>
</ol>
3. Negative staining
<ol>
	<li>Harvest about 100mg of TMV-infected leaf material and prepare crude sap by homogenizing the material for 2 minutes with a razor blade on a microscope slide in 100&mu;l of 60mM S&oslash;frensen phosphate buffer (pH 7.2).</li>
	<li>Transfer 20&mu;l of the resulting homogenate onto the first well of a teflon coated microscope slide with 4 or more wells (alternatively it is also possible to transfer the homogenate on a piece of parafilm).</li>
	<li>Place a formvar coated grid on top of the homogenate with the formvar (Agar Scientific Ltd.) coated side facing towards the drop and incubate it for 5 minutes.</li>
	<li>Wash the grid 2 times for 2 minutes each by placing the grid on top of two drops of 200&mu;l 60mM S&oslash;rensen phosphate buffer (pH 7.2).</li>
	<li>Incubate the grid for 1 minute with a freshly prepared 2% phosphotungstic acid (Agar Scientific Ltd.) solution in 60mM S&oslash;rensen phosphate buffer (pH 6.5).</li>
	<li>Remove the grid and allow it to air dry in a grid box.</li>
	<li>Examine the grid with a transmission electron microscope (e.g. Philips CM10 TEM; FEI, Eindhoven, The Netherlands). Take at least 10 images of randomly chosen negatively stained virions (at least 10 or more virions should be visible on each image) with the transmission electron microscope at a primary magnification of 21000X or higher. Care must be taken that all images have the same magnification.</li>
</ol>
4. Image analysis
<ol>
	<li>Measure the length and width of at least 100 randomly chosen single virus particles on the micrographs taken from the negatively stained samples by using any image analysis computer software [e.g. Cell D (Olympus, Life and Material Science Europe GmbH, Hamburg, Germany) with the particle analysis tool or Optimas 6.5.1- (Media Cybernetics Inc., Bethesda, Maryland, USA)].</li>
	<li>Calculate means and standard deviations in order to achieve an average length and width of the virus particles visualized by negative staining methods and TEM. </li>
	<li>Compare length and ultrastructural features (obtained in 2.5) with sizes of viruses and ultrastructural alterations induced by virus diseases known in the literature in order to clearly identify the virus diseases.</li>
</ol>
5. Representative results: 
After microwave assisted sample preparation typical TMV-induced ultrastructural alterations such as large areas containing virions aligned in parallel form could be observed with the transmission electron microscope in the cytosol of infected Nicotiana tabacum cells (Fig 1A). Additionally, in the crude sap of TMV-infected leaves TMV particles could be observed as flexuous, rod-shaped structures after negative staining (Fig 1B). Image analysis of 100 virus particles revealed an average size for TMV of 280nm in length and 17nm in width (Fig 2). The ultrastructure in TMV-infected cells and the size of virions observed in this study were found to be in accordance with TMV-induced ultrastructural properties in tobacco and the size range of TMV particles previously reported in the literature9-15.
<img src="/files/ftp_upload/2950/2950fig1.jpg" alt="Figure 1" /> 
Figure 1. Transmission electron micrographs of TMV-infected leaf cells and virions. A) Image shows the ultrastructure of TMV-infected mesophyll leaf cells of Nicotiana tabacum after microwave assisted plant sample preparation. Note the large area of parallel aligned virions which accumulated in the cytosol. C=chloroplast with starch (St), M=mitochondrion, N=nucleus, V=vacuole, Bar=2&mu;m. B) Image shows virions which were detected by negative staining in the sap of infected leaves.
<img src="/files/ftp_upload/2950/2950fig2.jpg" alt="Figure 2" /> 
Figure 2. Relative size of virions. Relative distribution of length and width of TMV-particles after negative staining the sap of infected leaves as they appeared in the electron microscope. Mean values (mean length/width &plusmn; standard deviation) were calculated from 100 virions.

<ol>
	<li>Bozzola, J. J., Russell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Microscopy. , (1999).</li><li>Kuo, J., Kuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+plant+tissues+for+ultrastructural+study.">Processing plant tissues for ultrastructural study.</a> Methods in Molecular Biology, Electron Microscopy, Methods and Protocols. 369, 47-65 (2007).</li><li>Schroeder, J. A., Gelderblom, H. R., Hauroeder, B., Schmetz, C., Milios, J., Hofstaedter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-assisted+tissue+processing+for+sameday+EM-diagnosis+of+potential+bioterrorism+and+clinical+samples.">Microwave-assisted tissue processing for sameday EM-diagnosis of potential bioterrorism and clinical samples.</a> Micron. 37, 577-590 (2006).</li><li>Webster, P., Kuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-assisted+processing+and+embedding+for+transmission+electron+microscopy.">Microwave-assisted processing and embedding for transmission electron microscopy.</a> Methods in Molecular Biology, Electron Microscopy, Methods and Protocols. 369, 47-65 (2007).</li><li>Zechmann, B., Zellnig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+assisted+rapid+plant+sample+preparation+for+transmission+electron+microscopy.">Microwave assisted rapid plant sample preparation for transmission electron microscopy.</a> J. Microsc. 233, 258-268 (2009).</li><li>Ler&#237;a, F., Marco, R., Medina, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+antigenic+preservation+of+plant+samples+by+microwave-enhanced+fixation,+using+dedicated+hardware,+minimizing+heat-related+effects.">Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects.</a> Micr. Res. Techniq. 65, 86-100 (2004).</li><li>Giberson, R. T., Austin, R. L., Charlesworth, J., Adamson, G., Herrera, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+and+digital+imaging+technology+reduce+turnaround+times+for+diagnostic+electron+microscopy.">Microwave and digital imaging technology reduce turnaround times for diagnostic electron microscopy.</a> Ultrastruct. Pathol. 27, 187-196 (2003).</li><li>Cavusoglu, I., Minbay, F. Z., Temel, S. G., Noyan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+polymerisation+with+microwave+irradiation+for+transmission+electron+microscopy.">Rapid polymerisation with microwave irradiation for transmission electron microscopy.</a> Eur. J. Morph. 39, 313-317 (2001).</li><li>Ermolina, I., Morgana, H., Greena, N. G., Milnerb, J. J., Feldmanc, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectric+spectroscopy+of+tobacco+mosaic+virus.">Dielectric spectroscopy of tobacco mosaic virus.</a> Biochim. Biophys. Acta. 1622, 57-63 (2003).</li><li>Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+atomic+force+microscopy+study+for+the+assembly+structures+of+tobacco+mosaic+virus+and+their+size+evaluation.">An atomic force microscopy study for the assembly structures of tobacco mosaic virus and their size evaluation.</a> Langmuir. 13, 4150-4161 (1997).</li><li>Milne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplication+of+tobacco+mosaic+virus+in+tobacco+leaf+palisade+cells.">Multiplication of tobacco mosaic virus in tobacco leaf palisade cells.</a> Virology. 28, 527-532 (1966).</li><li>Reunov, A. V., Gnutova, I. V., Lapshina, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+tobacco+mosaic+virus+strains+on+the+ultrastructure+of+tobacco+leaf+parenchymal+cells.">Effect of tobacco mosaic virus strains on the ultrastructure of tobacco leaf parenchymal cells.</a> Biol. Bull. 33, 409-415 (2006).</li><li>Sachse, C., Chen, J. Z., Coureux, P. D., Stroupe, M. E., F&#228;ndrich, M., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+electron+microscopy+of+helical+specimens:+a+fresh+look+at+tobacco+mosaic+virus.">High-resolution electron microscopy of helical specimens: a fresh look at tobacco mosaic virus.</a> J. Mol. Biol. 371, 812-835 (2007).</li><li>Smith, M. L., Lindbo, J. A., Dillard-Telm, S., Brosio, P. M., Lasnik, A. B., McCormick, A. A., Nguyen, L. V., Palmer, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+tobacco+mosaic+virus+particles+as+scaffolds+for+display+of+protein+antigens+for+vaccine+applications.">Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications.</a> Virology. 348, 475-488 (2006).</li><li>Zechmann, B., Zellnig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+TEM+diagnosis+of+plant+virus+diseases.">Rapid TEM diagnosis of plant virus diseases.</a> J. Virol. Meth. 162, 163-169 (2009).</li><li>Giberson, R. T., Demaree, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+processing+techniques+for+electron+microscopy:+a+four-hour+protocol.">Microwave processing techniques for electron microscopy: a four-hour protocol.</a> Meth. Molec. Biol. 117, 145-158 (1999).</li></ol>

No conflicts of interest declared.

Microwave assisted plant sample preparation for TEM has been proven to supply fast and reliable ultrastructural data within a few hours3,4,16. The fine structural preservation of organelles and membranes achieved with the method used in this study was similar to conventional and cryofixed samples5,15 with the advantage of a massive reduction in sample fixation and embedding time from 3 days or longer to about 2 hours. This represents the fastest sample preparation protocol for TEM currently available in the literature. The method described in this study combined microwave assisted plant sample preparation for TEM with negative staining methods which allowed a clear and rapid identification of TMV-induced ultrastructural alterations and the viral agent itself. TMV induced ultrastructural alterations could be investigated after trimming, sectioning and post-staining within about 4 hours after the beginning of fixation in the transmission electron microscope. Using the fully automatically specimen preparation mode freed the researcher to conduct negative staining in the interim in order to determine the size and width of the viral agent. Thus, we can conclude that this method allows a clear and rapid diagnosis of plant virus diseases in about half a day which is of great importance for future use in agriculture and scientific experiments in plant phytopathology. As this method could also be used for the rapid diagnosis of animal and human diseases it has a large potential for future application in medical and veterinary pathology.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent/equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1312</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1022</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agar 100 resin </td>
			<td>Agar Scientific Ltd.</td>
			<td>R1043</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dodecenyl succinic anhydride</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1051</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Methyl nadic anhydride</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1081</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Benzyl dimethylamine</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1060</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1210</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1260A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1213</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formvar</td>
			<td>Agar Scientific Ltd.</td>
			<td>R1202</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td colspan="4">&nbsp;</td>
		</tr>
		<tr>
			<td>Leica EM AMW</td>
			<td>Leica Microsystems</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leica (Reichert) Ultratrim</td>
			<td>Leica Microsystems</td>
			<td>&nbsp;</td>
			<td>Newer model is available</td>
		</tr>
		<tr>
			<td>Leica (Reichert) Ultracut S</td>
			<td>Leica Microsystems</td>
			<td>&nbsp;</td>
			<td>Newer model is available</td>
		</tr>
		<tr>
			<td>Diatome Ultra 45</td>
			<td>Gr&ouml;pl</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Philips CM10 TEM</td>
			<td>FEI (formerly Philips)</td>
			<td>&nbsp;</td>
			<td>Newer model is available</td>
		</tr>
		<tr>
			<td>Cell D</td>
			<td>Olympus Inc.</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Optimas 6.5.1</td>
			<td>Media Cybernetics Inc.</td>
			<td>&nbsp;</td>
			<td>Newer version is available</td>
		</tr>
	</tbody>
</table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K Views.  University of Graz. This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Video: Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...