Name: in vivo imaging systems (ivis) detection of a neuro-invasive encephalitic virus
Uploaded: 2012-12-02T14:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus at JoVE.com

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 ani...

<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. 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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 ...

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 <td>Name 			of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
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 <td>D-Luciferin</td>
 <td></td>
 <td></td>
 <td></td>
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 <td>Isoflurane</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
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 <td>Xenogen IVIS System (Spectrum)</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
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 <td>XGI-8-gas Anesthesia System</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
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 <td>XIC-3 Containment Box</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
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 <td>LivingImage 4.0 Software</td>
 <td>Caliper Life Sciences</td>
 <td></td>
 <td></td>
 </tr>
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 <td>Telemetry/identification chips</td>
 <td>Bio Medic Data Systems</td>
 <td>IPTT-300</td>
 <td>Animal ID and Temperature</td>
 </tr>
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 <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>
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 <td>Vet Bond tissue adhesive</td>
 <td>Fisher Scientific</td>
 <td>NC9259532 </td>
 <td></td>
 </tr>
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 <td>Vetropolycin 			Ophthalmic Ointment </td>
 <td>Webster Veterinary Products</td>
 <td>78444656 </td>
 <td></td>
 </tr>
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 <td>Dulbecco's Phosphate Buffered 			Saline 1X</td>
 <td>Invitrogen</td>
 <td>14190-144</td>
 <td></td>
 </tr>
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 <td>BMDS Chip Reader</td>
 <td>Bio Medic Data Systems</td>
 <td>DAS-7007S</td>
 <td></td>
 </tr>
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 <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 ...

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