Skip to content
1887

Journal of General Virology header logo Journal of General Virology

Volume 104, Issue 11

Establishment of pseudorabies virus latency and reactivation model in mice dorsal root ganglia culture No Access

Abstract

Pseudorabies virus (PRV) belongs to the alpha herpesvirus family and is responsible for Aujeszky’s disease in pigs. Similar to other alpha herpesviruses, PRV establishes a lifelong latent infection in trigeminal ganglion. These latently infected pigs serve as a reservoir for recurrent infections when reactivation is triggered, making the eradication of PRV a challenging task. However, the molecular mechanism underlying PRV latency and reactivation in neurons is still poorly understood due to limitations in the model. To establish a pseudorabies virus latency and reactivation model in primary neuron cultures, we isolated dorsal root ganglion (DRG) from newborn Kunming mice using a method named epineurium-pulling for DRG collection (EPDC) and cultured primary neurons . A dual-colour recombinant PRV BAC mRuby-VP16 was constructed and 0.5 multiplicity of infection (MOI) was found as an appropriate dose in the presence of aciclovir to establish latency. Reactivation was induced using UV-inactivated herpesviruses or a series of chemical inhibitors. Interestingly, we found that not only UV-PRV, but also UV-HSV-1 and UV-BHoV-5 were able to induce rapid PRV reactivation. The efficiency of reactivation for LY294002, forskolin, etoposide, dexamethasone, and acetylcholine was found to be dependent on their concentration. In conclusion, we developed a valuable model of PRV latency and reactivation, which provides a basis for future mechanism research.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): dorsal root ganglion , latency , mice , pseudorabies virus and reactivation
Funding
This study was supported by the:
  • National Natural Science Foundation of China (Award 32170155)
    • Principle Award Recipient: Zheng-FeiLiu
© 2023 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001921
2023-11-22
2025-01-25
Loading full text...

Full text loading...

References

  1. Lee BH, Tebaldi G, Pritchard SM, Nicola AV. Host cell neddylation facilitates alphaherpesvirus entry in a virus-specific and cell-dependent manner. Microbiol Spectr 2022; 10:e0311422 [View Article] [PubMed]
     [Google Scholar]
  2. Connolly SA, Jardetzky TS, Longnecker R. The structural basis of herpesvirus entry. Nat Rev Microbiol 2021; 19:110–121 [View Article] [PubMed]
     [Google Scholar]
  3. Wang X, Wu C-X, Song X-R, Chen H-C, Liu Z-F. Comparison of pseudorabies virus China reference strain with emerging variants reveals independent virus evolution within specific geographic regions. Virology 2017; 506:92–98 [View Article] [PubMed]
     [Google Scholar]
  4. Pomeranz LE, Reynolds AE, Hengartner CJ. Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 2005; 69:462–500 [View Article] [PubMed]
     [Google Scholar]
  5. Sun Y, Luo Y, Wang C-H, Yuan J, Li N et al. Control of swine pseudorabies in China: opportunities and limitations. Vet Microbiol 2016; 183:119–124 [View Article] [PubMed]
     [Google Scholar]
  6. Chen J, Li G, Wan C, Li Y, Peng L et al. A comparison of pseudorabies virus latency to other α-herpesvirinae subfamily Mmembers. Viruses 2022; 14:1386 [View Article] [PubMed]
     [Google Scholar]
  7. Wilson AC. Impact of cultured neuron models on α-herpesvirus latency research. Viruses 2022; 14:1209 [View Article] [PubMed]
     [Google Scholar]
  8. Laval K, Enquist LW. The neuropathic itch caused by pseudorabies virus. Pathogens 2020; 9:254 [View Article] [PubMed]
     [Google Scholar]
  9. Tanaka S, Imamura T, Sakaguchi M, Mannen K. Acetylcholine reactivates latent pseudorabies virus in mice. J Virol Methods 1998; 70:103–106 [View Article] [PubMed]
     [Google Scholar]
  10. Wang H-H, Liu J, Li L-T, Chen H-C, Zhang W-P et al. Typical gene expression profile of pseudorabies virus reactivation from latency in swine trigeminal ganglion. J Neurovirol 2020; 26:687–695 [View Article] [PubMed]
     [Google Scholar]
  11. Camarena V, Kobayashi M, Kim JY, Roehm P, Perez R et al. Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons. Cell Host Microbe 2010; 8:320–330 [View Article] [PubMed]
     [Google Scholar]
  12. Cliffe AR, Arbuckle JH, Vogel JL, Geden MJ, Rothbart SB et al. Neuronal stress pathway mediating a histone Methyl/Phospho switch is required for herpes simplex virus reactivation. Cell Host Microbe 2015; 18:649–658 [View Article] [PubMed]
     [Google Scholar]
  13. Arbuckle JH, Turner A-M, Kristie TM. Analysis of HSV viral reactivation in explants of sensory neurons. Curr Protoc Microbiol 2014; 35:14e [View Article] [PubMed]
     [Google Scholar]
  14. Kuhn MA, Nayak S, Camarena V, Gardner J, Wilson A et al. A cell culture model of facial palsy resulting from reactivation of latent herpes simplex type 1. Otol Neurotol 2012; 33:87–92 [View Article] [PubMed]
     [Google Scholar]
  15. Roehm PC, Camarena V, Nayak S, Gardner JB, Wilson A et al. Cultured vestibular ganglion neurons demonstrate latent HSV1 reactivation. Laryngoscope 2011; 121:2268–2275 [View Article] [PubMed]
     [Google Scholar]
  16. Wilcox CL, Johnson EM. Nerve growth factor deprivation results in the reactivation of latent herpes simplex virus in vitro. J Virol 1987; 61:2311–2315 [View Article] [PubMed]
     [Google Scholar]
  17. Goswami P, Ives AM, Abbott ARN, Bertke AS. Stress hormones epinephrine and corticosterone selectively reactivate HSV-1 and HSV-2 in sympathetic and sensory neurons. Viruses 2022; 14:1115 [View Article] [PubMed]
     [Google Scholar]
  18. Koyuncu OO, MacGibeny MA, Hogue IB, Enquist LW. Compartmented neuronal cultures reveal two distinct mechanisms for alpha herpesvirus escape from genome silencing. PLoS Pathog 2017; 13:e1006608 [View Article] [PubMed]
     [Google Scholar]
  19. Zheng K, Jiang F-F, Su L, Wang X, Chen Y-X et al. Highly efficient base editing in viral genome based on bacterial artificial chromosome using a cas9-cytidine deaminase fused protein. Virol Sin 2020; 35:191–199 [View Article] [PubMed]
     [Google Scholar]
  20. Liu J, Song X-R, Zheng K, Zhang W-J, Chen H-C et al. Feedback inhibition of bovine herpesvirus 5 replication by dual-copy bhv5-miR-B10-3p. J Gen Virol 2020; 101:290–298 [View Article] [PubMed]
     [Google Scholar]
  21. Yan K, Liu J, Guan X, Yin Y-X, Peng H et al. The carboxyl terminus of tegument protein pUL21 contributes to pseudorabies virus neuroinvasion. J Virol 2019; 93:e02052-18 [View Article] [PubMed]
     [Google Scholar]
  22. Van Cleemput J, Koyuncu OO, Laval K, Engel EA, Enquist LW. CRISPR/Cas9-constructed pseudorabies virus mutants reveal the importance of UL13 in alphaherpesvirus escape from genome silencing. J Virol 2021; 95:e02286-20 [View Article] [PubMed]
     [Google Scholar]
  23. Perner C, Sokol CL. Protocol for dissection and culture of murine dorsal root ganglia neurons to study neuropeptide release. STAR Protoc 2021; 2:100333 [View Article] [PubMed]
     [Google Scholar]
  24. Fornaro M, Sharthiya H, Tiwari V. Adult mouse DRG explant and dissociated cell models to investigate neuroplasticity and responses to environmental insults including viral infection. J Vis Exp 201856757 [View Article] [PubMed]
     [Google Scholar]
  25. Albuquerque C, Joseph DJ, Choudhury P, MacDermott AB. Dissection, plating, and maintenance of dorsal root ganglion neurons for monoculture and for coculture with dorsal horn neurons. Cold Spring Harb Protoc 2009; 2009:pdb.prot5275 [View Article] [PubMed]
     [Google Scholar]
  26. Hermes T de A, Jarry V de M, Reis F, Minatel E. Anatomy and imaging of the spinal cord: an overview. Semin Ultrasound CT MR 2023; 44:400–407 [View Article] [PubMed]
     [Google Scholar]
  27. Ramo NL, Troyer KL, Puttlitz CM. Viscoelasticity of spinal cord and meningeal tissues. Acta Biomater 2018; 75:253–262 [View Article] [PubMed]
     [Google Scholar]
  28. Anderson MB, Das S, Miller KE. Subcellular localization of neuronal nuclei (NeuN) antigen in size and calcitonin gene-related peptide (CGRP) populations of dorsal root ganglion (DRG) neurons during acute peripheral inflammation. Neurosci Lett 2021; 760:135974 [View Article] [PubMed]
     [Google Scholar]
  29. Koyuncu OO, Song R, Greco TM, Cristea IM, Enquist LW. The number of alphaherpesvirus particles infecting axons and the axonal protein repertoire determines the outcome of neuronal infection. mBio 2015; 6:e00276-15 [View Article] [PubMed]
     [Google Scholar]
  30. Goswami SC, Mishra SK, Maric D, Kaszas K, Gonnella GL et al. Molecular signatures of mouse TRPV1-lineage neurons revealed by RNA-Seq transcriptome analysis. J Pain 2014; 15:1338–1359 [View Article] [PubMed]
     [Google Scholar]
  31. Malin SA, Davis BM, Molliver DC. Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity. Nat Protoc 2007; 2:152–160 [View Article] [PubMed]
     [Google Scholar]
  32. Owen DE, Egerton J. Culture of dissociated sensory neurons from dorsal root ganglia of postnatal and adult rats. Methods Mol Biol 2012; 846:179–187 [View Article] [PubMed]
     [Google Scholar]
  33. de Luca AC, Faroni A, Reid AJ. Dorsal root ganglia neurons and differentiated adipose-derived stem cells: an in vitro co-culture model to study peripheral nerve regeneration. J Vis Exp 201552543 [View Article] [PubMed]
     [Google Scholar]
  34. Shen H, Gan M, Yang H, Zou J. An integrated cell isolation and purification method for rat dorsal root ganglion neurons. J Int Med Res 2019; 47:3253–3260 [View Article] [PubMed]
     [Google Scholar]
  35. Valtcheva MV, Copits BA, Davidson S, Sheahan TD, Pullen MY et al. Surgical extraction of human dorsal root ganglia from organ donors and preparation of primary sensory neuron cultures. Nat Protoc 2016; 11:1877–1888 [View Article] [PubMed]
     [Google Scholar]
  36. Rafael D, Tomer E, Kobiler O. A single herpes simplex virus 1 genome reactivates from individual cells. Microbiol Spectr 2022; 10:e0114422 [View Article] [PubMed]
     [Google Scholar]
  37. Thompson RL, Preston CM, Sawtell NM. De novo synthesis of VP16 coordinates the exit from HSV latency in vivo. PLoS Pathog 2009; 5:e1000352 [View Article] [PubMed]
     [Google Scholar]
  38. Kim JY, Mandarino A, Chao MV, Mohr I, Wilson AC. Transient reversal of episome silencing precedes VP16-dependent transcription during reactivation of latent HSV-1 in neurons. PLoS Pathog 2012; 8:e1002540 [View Article] [PubMed]
     [Google Scholar]
  39. Deshmane SL, Fraser NW. During latency, herpes simplex virus type 1 DNA is associated with nucleosomes in a chromatin structure. J Virol 1989; 63:943–947 [View Article] [PubMed]
     [Google Scholar]
  40. Wang Q-Y, Zhou C, Johnson KE, Colgrove RC, Coen DM et al. Herpesviral latency-associated transcript gene promotes assembly of heterochromatin on viral lytic-gene promoters in latent infection. Proc Natl Acad Sci U S A 2005; 102:16055–16059 [View Article] [PubMed]
     [Google Scholar]
  41. Stevens JG, Wagner EK, Devi-Rao GB, Cook ML, Feldman LT. RNA complementary to a herpesvirus alpha gene mRNA is prominent in latently infected neurons. Science 1987; 235:1056–1059 [View Article] [PubMed]
     [Google Scholar]
  42. Guo H, Zhou R, Xi Y, Xiao S, Chen H. Transcriptional suppression of IE180 and TK promoters by the EP0 of pseudorabies virus strains Ea and Fa. Virus Genes 2009; 38:269–275 [View Article] [PubMed]
     [Google Scholar]
  43. Linderman JA, Kobayashi M, Rayannavar V, Fak JJ, Darnell RB et al. Immune escape via a transient gene expression program enables productive replication of a latent pathogen. Cell Rep 2017; 18:1312–1323 [View Article] [PubMed]
     [Google Scholar]
  44. Dochnal S, Merchant HY, Schinlever AR, Babnis A, Depledge DP et al. DLK-dependent biphasic reactivation of herpes simplex virus latency established in the absence of antivirals. J Virol 2022; 96:e0050822 [View Article] [PubMed]
     [Google Scholar]
  45. Hu H-L, Shiflett LA, Kobayashi M, Chao MV, Wilson AC et al. TOP2β-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency. Mol Cell 2019; 74:466–480 [View Article] [PubMed]
     [Google Scholar]
  46. Cuddy SR, Schinlever AR, Dochnal S, Seegren PV, Suzich J et al. Neuronal hyperexcitability is a DLK-dependent trigger of herpes simplex virus reactivation that can be induced by IL-1. eLife 2020; 9:e58037 [View Article] [PubMed]
     [Google Scholar]
  47. Cacioppo JT, Kiecolt-Glaser JK, Malarkey WB, Laskowski BF, Rozlog LA et al. Autonomic and glucocorticoid associations with the steady-state expression of latent epstein-barr virus. Horm Behav 2002; 42:32–41 [View Article] [PubMed]
     [Google Scholar]
  48. Chang P-J, Chen L-W, Chen L-Y, Hung C-H, Shih Y-J et al. Effects of the NEDD8-activating enzyme inhibitor MLN4924 on lytic reactivation of Kaposi’s sarcoma-associated herpesvirus. J Virol 2017; 91:e00505-17 [View Article] [PubMed]
     [Google Scholar]
  49. Huang S-Y, Fang C-Y, Tsai C-H, Chang Y, Takada K et al. N-methyl-N’-nitro-N-nitrosoguanidine induces and cooperates with 12-O-tetradecanoylphorbol-1,3-acetate/sodium butyrate to enhance epstein-barr virus reactivation and genome instability in nasopharyngeal carcinoma cells. Chem Biol Interact 2010; 188:623–634 [View Article] [PubMed]
     [Google Scholar]
  50. Saviola AJ, Zimmermann C, Mariani MP, Signorelli SA, Gerrard DL et al. Chromatin profiles of chromosomally integrated human herpesvirus-6A. Front Microbiol 2019; 10:1408 [View Article] [PubMed]
     [Google Scholar]
/content/journal/jgv/10.1099/jgv.0.001921
Loading
Establishment of pseudorabies virus latency and reactivation model in mice dorsal root ganglia culture
J. Gen. Virol. 104, 001921 (2023); https://doi.org/10.1099/jgv.0.001921
/content/journal/jgv/10.1099/jgv.0.001921
/content/journal/jgv/10.1099/jgv.0.001921
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error