Institute for Translational Sciences UTMB-NIH grant 1UL1RR029876-01 and Alisha Prather for her assistance with video editing for this manuscript.

1. Animal Preparation
<ol>
 <li>Animal arrival: Upon arrival to the animal biosafety level 2 (ABSL2) facilities, allow animals a minimum of 2 days to acclimate to their new environment. Following this rest period, inspect the animals to evaluate their health and overall physical appearance.
 <ol>
 <li>When utilizing fluorescent reporters it is important to place all animal subjects on an alfalfa free diet to limit the amount of GI autofluorescence and background signal. </li>
 </ol>
 </li>
 <li>Shave animals: To improve the bioluminescent signal detected from the cranial region during imaging; shave the heads of all the animals. Anesthetize the animals with a mixture of oxygen gas and vaporized isoflurane. Once the animals are fully anesthetized, use electric clippers to shave their heads. Once finished with shaving, return the animals to their cages and observe for complete recovery from the effects of the anesthesia. </li>
 <li>Bio Medic Data Systems (BMDS) transponder insertion (to take place following shaving of the mice while the animals are fully anesthetized): For a less invasive means to track temperature implant a preprogrammed BMDS wireless transponder subcutaneously in the dorsal region of each mouse. These transponders allow for wireless identification and temperature recording. Following implantation of the transponder, apply a veterinary-grade tissue adhesive to the wound. </li>
 <li>Baseline collection: Before infection, record a baseline temperature and weight for all animals. Collect blood for plaque reduction neutralization test (PRNT) analysis through retro-orbital (RO) bleed while the mice are under anesthesia. Following blood collection, apply veterinary antibiotic eye ointment to reduce potential secondary bacterial infection. This baseline collection should be completed with consideration to the number of times the animals are placed under anesthesia due to stress on the mice. Future RO collections should be completed after imaging, while the mouse is still under anesthetic. The maximum blood collected from an RO should be around 200 &mu;l. </li>
 <li>Infection: Place mice under anesthesia as described previously. Inoculate through the intranasal route using a dose of 5x106 to 5x107 pfu TC83-Luciferase in a total volume of 40 &mu;l diluted by Phosphate Buffered Saline (PBS) through intranasal infection. Following infection, place the mice within their housing cage and observe recovery. </li>
</ol>
2. Animal Imaging
Disclaimer: Keep mice within their housing unit, the biosafety cabinet (BSC), or the XIC containment box at all times. The approved BSCs for this protocol are a Class II Type B1 or B2. 
<ol>
 <li>Inject all animals with 10 &mu;l per gram of body weight of intraperitoneal (IP) luciferin at a concentration of 15 mg/ml.
 <ol>
 <li>Mice receiving Ampligen are to be injected IP at a dose of 12.5 mg/kg body weight at -4 hr post infection or +48 hr post infection based upon their group. </li>
 </ol>
 </li>
 <li>Prior to injection with luciferin, separate the mice into groups of three to ensure a proper fit within the XIC-3 containment box.
 <ol>
 <li>For all imaging, utilize ocular ointment/lubricant to prevent the drying of the cornea throughout the procedure. </li>
 </ol>
 </li>
 <li>Open the LivingImage software and press Initialize to prepare the imaging box; set auto saving, exposure time to auto (exposure time on auto is a new feature of the LivingImage software 3.2 and newer), view field to C, and filter to open; field of view is based upon the number of mice you are imaging; and filter can be optimized as needed for other imaging projects. </li>
 <li>Confirm that the air charcoal filters have not expired, the oxygen tank is full, and the isoflurane reservoir is filled. Place small strips of electrical tape in the XIC-3 isolation box which assists with reducing animal movement during imaging.</li>
 <li>Inject the mice with luciferin IP as described in section 2.1 and place within the Isoflurane induction chamber (with lid opened) for 3-5 min. </li>
 <li>After 5 min, close the lid of the induction chamber and start the flow of isoflurane. The anesthetic is operated at approximately 3-5% with the oxygen flow rate set at approximately 2.0 L/min. Once fully unconscious wait an additional 30 sec and transfer the mice to the XIC-3 isolation box. Ensure the biosafety cabinet being utilized allows for the safe scavenging of isoflurane as this procedure will involve loss of isoflurane from the induction chamber (the IVIS unit contains its own Passive Fair canisters and the XIC-3 containment box directly connects to the in/out sockets to ensure contained anesthetic flow). 
 <ol>
 <li>Transfer the mice based upon chip/group number into the XIC-3 isolation box with the isoflurane connector attached and open. Position the mice so they are laid out in sternal recumbency with heads gently resting on the tape strips.</li>
 </ol>
 </li>
 <li>Wipe down the XIC containment box with ethanol, spray hands and bottom of the XIC-3 with cavicide, and transfer to the IVIS imaging chamber. Reconnect the anesthesia to the containment box once inside the imaging unit. </li>
 <li>After 10 min following the injection of luciferin, image the mice through the living image software.</li>
 <li>Following initial imaging with an open filter, initiate a DLIT 3-Dimensional imaging using the Sequence analysis and Image Wizard setup for bioluminescent firefly luciferase. This imaging process will take 4-5 min and should be completed at a field of view relevant for the number of mice desired.
 <ol>
 <li>Ex-vivo analysis is possible following necropsy and collection of the brain. Place the brain on a sterile Petri dish, drip 50-100 &mu;l of stock luciferin on top, and place within the imaging box. </li>
 </ol>
 </li>
 <li>Finalize a second open filter image at 15-17 min post infection upon completion of DLIT imaging. Ensure sequence analysis is off and the field of view and filter settings are returned to previous settings. </li>
</ol>
3. Return Mice and Data Analysis
<ol>
 <li>Turn off the isoflurane vaporizer and transfer the XIC containment box back to the biosafety cabinet.</li>
 <li>Place the mice back within their housing unit, close the top, spray the exterior with appropriate disinfectant and place back on the housing rack.
 <ol>
 <li>Ensure mice have fully recovered from anesthesia. </li>
 </ol>
 </li>
 <li>Repeat the above procedure as the experiment requires until imaging has completed. </li>
 <li>Disinfect the BSC, shut down the equipment, and transfer all data to an outside laboratory location for further analysis. </li>
 <li>Analyze the data and generate 3-dimensional images based upon IVIS results utilizing the LivingImage software.
 <ol>
 <li>Ensure the image is properly oriented and lighting/color balance is set. Within the Tool Palette the options include Image Adjust, Corrections, Image Information, and ROI Tools.</li>
 <li>Utilize ROI shapes to identify specific signal strengths based upon location.</li>
 <li>Reconstruct the surface topography to highlight the mouse body, initialize 3D DLIT reconstruction, and use linear fit to set the organ map to the topographic reconstruction. </li>
 </ol>
 </li>
 <li>Further viral titration analysis can be completed through collection of blood and organs. Titration in this study was completed through homogenization of brain tissue in modified eagles medium (MEM). The homogenate was serially diluted and we infected a 6 well plate of Vero cells for 1 hr. The cells were covered with a 1.5% agarose/2xMEM overlay and incubated for 2 days at 37 degrees. Cells were fixed with 10% formalin for 30 min and stained with crystal violet.</li>
</ol>

<ol>
	<li>Steele, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Neurovirulence+and+Tissue+Tropism+of+Wild-type+and+Attenuated+Strains+of+Venezuelan+Equine+Encephalitis+Virus+Administered+by+Aerosol+in+C3H/HeN+and+BALB/c+Mice.">Comparative Neurovirulence and Tissue Tropism of Wild-type and Attenuated Strains of Venezuelan Equine Encephalitis Virus Administered by Aerosol in C3H/HeN and BALB/c Mice.</a> Veterinary Pathology Online. 35, 386-397 (1998).</li><li>Ludwig, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+neurovirulence+of+attenuated+and+non-attenuated+strains+of+Venezuelan+equine+encephalitis+virus+in+mice.">Comparative neurovirulence of attenuated and non-attenuated strains of Venezuelan equine encephalitis virus in mice.</a> Am. J. Trop. Med. Hyg. 64, 49-55 (2001).</li><li>Charles, P. C., Walters, E., Margolis, F., Johnston, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+Neuroinvasion+of+Venezuelan+Equine+Encephalitis+Virus+in+the+Mouse.">Mechanism of Neuroinvasion of Venezuelan Equine Encephalitis Virus in the Mouse.</a> Virology. , 208-662 (1995).</li><li>Volkova, E., Gorchakov, R., Frolov, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficient+packaging+of+Venezuelan+equine+encephalitis+virus-specific+RNAs+into+viral+particles+is+determined+by+nsP1-3+synthesis.">The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1-3 synthesis.</a> Virology. 344, 315-327 (2006).</li><li>Patterson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+non-invasive+imaging+of+alphaviral+brain+infection:+Reducing+animal+numbers+and+morbidity+to+identify+efficacy+of+potential+vaccines+and+antivirals.">Rapid, non-invasive imaging of alphaviral brain infection: Reducing animal numbers and morbidity to identify efficacy of potential vaccines and antivirals.</a> Vaccine. 29, 9345-9351 (2011).</li><li>Cook, S. H., Griffin, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luciferase+Imaging+of+a+Neurotropic+Viral+Infection+in+Intact+Animals.">Luciferase Imaging of a Neurotropic Viral Infection in Intact Animals.</a> J. Virol. 77, 5333-5338 (2003).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nat. Med. 4, 245-247 (1998).</li><li>Osorio, J. E., Iams, K. P., Meteyer, C. U., Rocke, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Monkeypox+Viruses+Pathogenesis+in+Mice+by+In+Vivo+Imaging.">Comparison of Monkeypox Viruses Pathogenesis in Mice by In Vivo Imaging.</a> PLoS ONE. 4, e6592 (2009).</li><li>Luker, G. D., Prior, J. L., Song, J., Pica, C. M., Leib, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+Imaging+Reveals+Systemic+Dissemination+of+Herpes+Simplex+Virus+Type+1+in+the+Absence+of+Interferon+Receptors.">Bioluminescence Imaging Reveals Systemic Dissemination of Herpes Simplex Virus Type 1 in the Absence of Interferon Receptors.</a> J. Virol. 77, 11082-11093 (2003).</li><li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Principles of Humane Experimental Technique. , (1959).</li><li>Kuehne, R. W., Pannier, W. L., Stephen, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+mouse+model+for+the+evaluation+of+potential+antiviral+compounds:+results+with+Venezuelan+equine+encephalomyelitis+virus.">Indirect mouse model for the evaluation of potential antiviral compounds: results with Venezuelan equine encephalomyelitis virus.</a> Antimicrob. Agents Chemother. 11, (1977).</li><li>Lukaszewski, R. A., Brooks, T. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pegylated+Alpha+Interferon+Is+an+Effective+Treatment+for+Virulent+Venezuelan+Equine+Encephalitis+Virus+and+Has+Profound+Effects+on+the+Host+Immune+Response+to+Infection.">Pegylated Alpha Interferon Is an Effective Treatment for Virulent Venezuelan Equine Encephalitis Virus and Has Profound Effects on the Host Immune Response to Infection.</a> J. Virol. 74, 5006-5015 (2000).</li></ol>

No conflicts of interest declared.

While this protocol covers the imaging aspects for in vivo analysis, it is important to recognize the bioluminescent vector as a key factor for future studies. Our utilization of TC83, an attenuated vaccine strain of VEEV, as a vector for expression of luciferase ensures that large quantities of the enzyme are being produced due to the high replication rate of the virus in the CNS as previously described1-4. While the addition of a second subgenomic promoter and the luciferase gene results in further ...

<table border="1">
<tbody>
 <tr>
 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>D-Luciferin</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Xenogen IVIS System (Spectrum)</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>XGI-8-gas Anesthesia System</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>XIC-3 Containment Box</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>LivingImage 4.0 Software</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Telemetry/identification chips</td>
 <td>Bio Medic Data Systems</td>
 <td>IPTT-300</td>
 <td>Animal ID and Temperature</td>
 </tr>
 <tr>
 <td>BD Integra 1ml TB syringe with 26 g 			x 3/8&#x201D; needle</td>
 <td>Fisher Scientific</td>
 <td>305279</td>
 <td></td>
 </tr>
 <tr>
 <td>Vet Bond tissue adhesive</td>
 <td>Fisher Scientific</td>
 <td>NC9259532 </td>
 <td></td>
 </tr>
 <tr>
 <td>Vetropolycin 			Ophthalmic Ointment </td>
 <td>Webster Veterinary Products</td>
 <td>78444656 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate Buffered 			Saline 1X</td>
 <td>Invitrogen</td>
 <td>14190-144</td>
 <td></td>
 </tr>
 <tr>
 <td>BMDS Chip Reader</td>
 <td>Bio Medic Data Systems</td>
 <td>DAS-7007S</td>
 <td></td>
 </tr>
 <tr>
 <td>DAS-HOST Software</td>
 <td>Bio Medic Data Systems</td>
 <td></td>
 <td>Used to download probe information</td>
 </tr>
 </tbody>
</table>

in vivo imaging systems (ivis) detection of a neuro-invasive encephalitic virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

With a genetically modified virus, TC83-Luciferase, we saw an increase in bioluminescent signal strength as the virus replication moves from the nasal region into the central CNS (Figure 1). Due to the high viral replication rate, we expect to see high levels of bioluminescent signal (Figure 2A) dependent upon the vector and the animal immune response to the vector. We expect this signal increase to continue to a peak, between days 5-7 post infection, in combination with viral replicatio...

Watch this Scientific Journal Video about In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus at JoVE.com

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

in-vivo-imaging-systems-ivis-detection-neuro-invasive-encephalitic

Research

JoVE Journal

Immunology and Infection

20.6K vistas.  University of Texas Medical Branch. Utilizando luciferasa y los sistemas de formación de imágenes in vivo (IVIS) como un nuevo medio para identificar las variables de enfermedad antes de los desarrollos clínicos ocurrir. IVIS nos ha permitido visualizar en tiempo real la invasión de los virus de encefalitis durante varios días, ofreciendo un modelo de enfermedad más preciso para el estudio futuro. También ha permitido identificar las características potenciales de protección de antivirales y vacuna...

Video: In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...

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 ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...