We thank the reviewers for their thoughtful suggestions that helped to improve this manuscript. This work was supported by grants to MVC (NS21072, HD23315), ACW (GM61139, S10RR017970) and IM (AI073898, GM056927) from the NIH. MK was supported in part by an NIH training grant (5T32 AI007180).

1. Isolation and Culture of SCG Neurons from Rat Embryos
To provide a useful context for understanding this protocol, and for a comprehensive discussion of earlier literature that established methods of SCG neuron culture, including the basis for SCG in vitro culture, plate-coating substrates, and the components of serum-free media, the reader is referred to references2-4.
<ol>
 <li>The use of rats as a source for SCG neurons was conducted in accordance with NIH ...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+latency+in+vitro+by+the+herpes+virus+type+1+mutant+in1918.">Establishment of latency in vitro by the herpes virus type 1 mutant in1918.</a> J. Gen. Virol. 72, 907-913 (1991).</li><li>Wagner, E. K., Bloom, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+investigation+of+herpes+simplex+virus+latency.">Experimental investigation of herpes simplex virus latency.</a> Clin. Microbiol. Rev. 10, 419-443 (1997).</li><li>Strelow, L. I., Laycock, K. A., Jun, P. Y., Rader, K. A., Brady, R. H., Miller, J. K., Pepose, J. S., Leib, D. 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This primary neuron culture and infection system provides a simple and effective method to explore the molecular mechanisms underlying HSV-1 latency and reactivation. The system faithfully recapitulates the accepted hallmarks of latency defined in both human infections and in live-animal models. When the virus is latent in the SCG cultures, infectious particles and viral lytic gene products cannot be detected. The only viral gene product detected in latently-infected neurons is the non-coding LAT trans...

<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Catalog#</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>70&mu;m nylon filter( cell strainer)</td>
 <td>BD Biosciences</td>
 <td>352350</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1x Hank's Balanced Salt Solution (HBSS-/-) </td>
 <td>Invitrogen</td>
 <td>14175</td>
 <td>w/o CaCl2 and MgCl2</td>
 </tr>
 <tr>
 <td>1x Minimum Essential Media (MEM) </td>
 <td>Invitrogen</td>
 <td>11095-080</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5-Fluoro-2'-deoxyuridine</td>
 <td>Sigma</td>
 <td>F0503</td>
 <td>prepare 20 mM stock in 1x MEM; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>96-well flat well bottom TC plates</td>
 <td>Corning</td>
 <td>3599</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Acyclovir </td>
 <td>Calbiochem</td>
 <td>114798 </td>
 <td>prepare 31 mM stock in DMSO; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Aphidicolin </td>
 <td>Calbiochem</td>
 <td>178273 </td>
 <td>prepare 10 mM stock in DMSO; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>B-27 Supplement </td>
 <td>Invitrogen</td>
 <td>17504-44</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Collagenase </td>
 <td>Sigma </td>
 <td>C2674</td>
 <td>prepare 10 mg/ml stock in HBSS-/-; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>D-(+)-Glucose </td>
 <td>Sigma</td>
 <td>G6152</td>
 <td>prepare 40% stock in H2O; filter sterilize &amp; store at 4&deg;C</td>
 </tr>
 <tr>
 <td>L-Glutamine </td>
 <td>Invitrogen</td>
 <td>25030-081</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laminin </td>
 <td>Sigma</td>
 <td>L2020</td>
 <td>prepare 1 mg/ml stock in H2O; quick-freeze 20 &mu;l aliquats &amp; store at -80&deg;C; dilute to 2 &mu;g/ml working conc. in sterile H2O</td>
 </tr>
 <tr>
 <td>Leibovit'z L-15 media </td>
 <td>Invitrogen</td>
 <td>11415</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nerve Growth Factor </td>
 <td>Harlan Laboratories</td>
 <td>BT.5017</td>
 <td>prepare 50 &mu;g/ml stock in HBSS-/-; store at -80&deg;C</td>
 </tr>
 <tr>
 <td>Neurobasal medium </td>
 <td>Invitrogen</td>
 <td>12348</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphonoacetic acid (PAA) </td>
 <td>Sigma</td>
 <td>P6909 </td>
 <td>prepare 75 mg/ml stock in H2O; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide </td>
 <td>Sigma</td>
 <td>P0899 </td>
 <td>prepare 20 mg/ml stock in H2O; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Rat-tail collagen </td>
 <td>Millipore</td>
 <td>08-115</td>
 <td>Concentration varies with supply lot; store at 4&deg;C and dilute to 0.66 mg/ml working conc. in sterile H2O</td>
 </tr>
 <tr>
 <td>Trichostatin A </td>
 <td>Sigma</td>
 <td>T8552</td>
 <td>prepare 1 mM stock in DMSO; store at -20&deg;C</td>
 </tr>
 <tr>
 <td>Trypsin 2.5% </td>
 <td>Invitrogen</td>
 <td>15090-04</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a primary neuron culture system for the study of herpes simplex virus latency and reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

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Immunology and Infection

17.2K visualizações.  New York University School of Medicine. O protocolo descreve um sistema de modelo eficiente e reprodutível para estudar vírus herpes simplex tipo 1 (HSV-1) a latência e reactivação. O ensaio emprega homogéneos culturas de neurónios simpáticos e permite a dissecção molecular do vírus de neurónios-interacções usando uma variedade de ferramentas, incluindo a interferência de RNA e de expressão de proteínas recombinantes.