Journal of NeuroVirology

, Volume 24, Issue 5, pp 597–605 | Cite as

Expression of pseudorabies virus-encoded long noncoding RNAs in epithelial cells and neurons

  • Xiang Guan
  • Jie Liu
  • Hui Jiang
  • Chang-Xian Wu
  • Huan-Chun Chen
  • Zheng-Fei LiuEmail author


Long noncoding RNAs (lncRNAs) play important roles in regulating eukaryotic genome replication and gene expression in diverse biological systems. Here, we identified lncRNAs transcribed from pseudorabies virus (PRV)-infected PK-15 cells. Based on high-throughput sequencing data, we obtained 87,263,926 and 93,947,628 clean reads from mock-infected and PRV-infected PK-15 cells, respectively. Through a normalized analytic protocol, we identified three novel viral lncRNAs. According to an analysis of differential expression between the mock-infected and PRV-infected cells, 4151 host lncRNAs were significantly upregulated and 2327 host lncRNAs were significantly downregulated in the latter group. Viral lncRNAs and several host lncRNAs were verified by northern blotting and real-time PCR. The findings showed that the viral lncRNA LDI might regulate the expression of IE180, a potent transcriptional activator of viral genes. Furthermore, we characterized the expression of viral lncRNAs in a culture of infected primary chicken dorsal root ganglia (DRG). Collectively, the obtained data suggest that PRV generates lncRNAs in both epithelial cells and chick DRG neurons.


lncRNA Pseudorabies virus Epithelial cell Chick DRG neuron 



We thank Bin Wu (Huazhong Agricultural University) for the generous gift of the PRV SMX new variant strain. We also thank Liwen Bianji, Edanz Group China (, for editing the English text of a draft of this manuscript.

Funding information

This work was supported by the Natural Science Foundation of China (31770191, 31470259) and National Key Research and Development Program (2016YFD0500105) to Z.F. Liu.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

13365_2018_651_MOESM1_ESM.jpg (172 kb)
Supplementary Figure 1 Conservation of PRV lncRNAs in different PRV strains. (A) PRV Bartha vaccine strain. (B) Emerging variant HNX. (C) Emerging variant SMX. (JPG 171 kb)
13365_2018_651_MOESM2_ESM.pptx (1.4 mb)
Supplementary Figure 2 Indirect immunofluorescence of chick DRG neurons. The primary DRG neurons were cultivated in plates coated with laminin and supplied with plating medium. After 2 days, the medium was replaced with fresh plating medium supplemented with cytosine β-D-arabinofuranoside. Neurons were cultured for 9-12 days until use. Fresh explant culture (undifferentiated) and differentiated neurons were fixed with 4% paraformaldehyde and permeabilized with 0.2% TritonX-100. Then cells were stained with Anti-68 kDa neurofilament antibody (1:500), followed by the secondary antibody goat anti-chicken IgG/Y (H&L) (1:1000). (A) Fresh explant culture (undifferentiated) was stained with 4′6-diamidino-2-phenylindole (DAPI). (B) Fresh explant culture was stained anti-68 kDa neurofilament antibody, followed by secondary antibody Goat anti-Chicken IgG/Y (H&L). (C) Differented chicken neurons were stained with DAPI. (D) Differented chicken neurons were stained anti-68 kDa neurofilament antibody, followed by secondary antibody Goat anti-Chicken IgG/Y (H&L). (PPTX 1438 kb)
13365_2018_651_MOESM3_ESM.docx (19 kb)
Supplementary Table 1 (DOCX 19 kb)
13365_2018_651_MOESM4_ESM.docx (19 kb)
Supplementary Table 2 (DOCX 18 kb)


  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402CrossRefGoogle Scholar
  2. Ben-Porat T, Kaplan AS (1985) Molecular biology of pseudorabiesvirus. Plenum Press, New YorkGoogle Scholar
  3. Brown JA, Valenstein ML, Yario TA, Tycowski KT, Steitz JA (2012) Formation of triple-helical structures by the 3′-end sequences of MALAT1 and MENβ noncoding RNAs. Proc Natl Acad Sci U S A 109:19202–19207CrossRefGoogle Scholar
  4. Casero D, Sandoval S, Seet CS, Scholes J, Zhu Y, Ha VL, Luong A, Parekh C, Crooks GM (2015) Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages. Nat Immunol 16:1282–1291CrossRefGoogle Scholar
  5. Cheung AK (1989) Detection of pseudorabies virus transcripts in trigeminal ganglia of latently infected swine. J Virol 63:2908–2913PubMedPubMedCentralGoogle Scholar
  6. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S et al (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29:15–21CrossRefGoogle Scholar
  7. Engel EA, Song R, Koyuncu OO, Enquist LW (2015) Investigating the biology of alpha herpesviruses with MS-based proteomics. Proteomics 15:1943–1956CrossRefGoogle Scholar
  8. Gardner EJ, Nizami ZF, Talbot CC Jr, Gall JG (2012) Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis. Genes Dev 26:2550–2559CrossRefGoogle Scholar
  9. Hafezi W, Lorentzen EU, Eing BR, Muller M, King NJ, Klupp B, Mettenleiter TC, Kuhn JE (2012) Entry of herpes simplex virus type 1 (HSV-1) into the distal axons of trigeminal neurons favors the onset of nonproductive, silent infection. PLoS Pathog 8:e1002679CrossRefGoogle Scholar
  10. Hu B, Huo Y, Chen G, Yang L, Wu D, Zhou J (2016) Functional prediction of differentially expressed lncRNAs in HSV-1 infected human foreskin fibroblasts. Virol J 13:137CrossRefGoogle Scholar
  11. Huang J, Ma G, Fu L, Jia H, Zhu M, Li X, Zhao S (2014) Pseudorabies viral replication is inhibited by a novel target of miR-21. Virology 456-457:319–328CrossRefGoogle Scholar
  12. Iyer MK, Niknafs YS, Malik R, Singhal U, Sahu A, Hosono Y, Barrette TR, Prensner JR, Evans JR, Zhao S, Poliakov A, Cao X, Dhanasekaran SM, Wu YM, Robinson DR, Beer DG, Feng FY, Iyer HK, Chinnaiyan AM (2015) The landscape of long noncoding RNAs in the human transcriptome. Nat Genet 47:199–208CrossRefGoogle Scholar
  13. Jin L, Scherba G (1999) Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells. J Virol 73:9781–9788PubMedPubMedCentralGoogle Scholar
  14. Klupp BG, Hengartner CJ, Mettenleiter TC, Enquist LW (2003) Complete, annotated sequence of the pseudorabies virus genome. J Virol 78:424–440CrossRefGoogle Scholar
  15. Kulesza CA, Shenk T (2004) Human cytomegalovirus 5-kilobase immediate-early RNA is a stable intron. J Virol 78:13182–13189CrossRefGoogle Scholar
  16. Kulesza CA, Shenk T (2006) Murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse. Proc Natl Acad Sci U S A 103:18302–18307CrossRefGoogle Scholar
  17. Li C, Fitzgerald ME, Del Mar N, Cuthbertson-Coates S, LeDoux MS, Gong S, Ryan JP, Reiner A (2015) The identification and neurochemical characterization of central neurons that target parasympathetic preganglionic neurons involved in the regulation of choroidal blood flow in the rat eye using pseudorabies virus, immunolabeling and conventional pathway tracing methods. Front Neuroanat 9:65PubMedPubMedCentralGoogle Scholar
  18. Li Y, Zheng G, Y Zhang, Yang X, Liu H, Chang H, Wang X, Zhao J, Wang C, Chen L (2017). MicroRNA analysis in mouse neuro-2a cells after pseudorabies virus infection. J NeuroVirol 1–11Google Scholar
  19. Liu F, Zheng H, Tong W, Li G-X, Tian Q, Liang C, Li L-W, Zheng X-C, Tong G-Z (2016) Identification and analysis of novel viral and host dysregulated microRNAs in variant pseudorabies virus-infected PK15 cells. PLoS One 11:e0151546CrossRefGoogle Scholar
  20. Mahjoub N, Dhorne-Pollet S, Fuchs W, Endale Ahanda ML, Lange E, Klupp B, Arya A, Loveland JE, Lefevre F, Mettenleiter TC, Giuffra E (2015) A 2.5-kilobase deletion containing a cluster of nine microRNAs in the latency-associated-transcript locus of the pseudorabies virus affects the host response of porcine trigeminal ganglia during established latency. J Virol 89:428–442CrossRefGoogle Scholar
  21. Marquitz AR, Mathur A, Edwards RH, Raab-Traub N (2015) Host gene expression is regulated by two types of noncoding RNAs transcribed from the Epstein-Barr virus BamHI A rightward transcript region. J Virol 89:11256–11268CrossRefGoogle Scholar
  22. Mettenleiter TC (2000) Aujeszky’s disease (pseudorabies) virus: the virus and molecular pathogenesis—state of the art, June 1999. Vet Res 31:99–115PubMedGoogle Scholar
  23. Musacchia F, Basu S, Petrosino G, Salvemini M, Sanges R (2015) Annocript: a flexible pipeline for the annotation of transcriptomes able to identify putative long noncoding RNAs. Bioinformatics 33:2199–2201CrossRefGoogle Scholar
  24. Nicoll MP, Hann W, Shivkumar M, Harman LE, Connor V, Coleman HM, Proenca JT, Efstathiou S (2016) The HSV-1 latency-associated transcript functions to repress latent phase lytic gene expression and suppress virus reactivation from latently infected neurons. PLoS Pathog 12:e1005539CrossRefGoogle Scholar
  25. Nishitsuji H, Ujino S, Yoshio S, Sugiyama M, Mizokami M, Kanto T, Shimotohno K (2016) Long noncoding RNA# 32 contributes to antiviral responses by controlling interferon-stimulated gene expression. Proc Natl Acad Sci U S A 113:10388–10393CrossRefGoogle Scholar
  26. Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295CrossRefGoogle Scholar
  27. Pomeranz LE, Reynolds AE, Hengartner CJ (2005) Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine. Microbiol Mol Biol Rev 69:462–500CrossRefGoogle Scholar
  28. Powell S, Vinod A, Lemons ML (2014) Isolation and culture of dissociated sensory neurons from chick embryos J Vis Exp 51991Google Scholar
  29. Priola SA, Stevens JG (1991) The 5′ and 3′ limits of transcription in the pseudorabies virus latency associated transcription unit. Virology 182:852–856CrossRefGoogle Scholar
  30. Priola SA, Gustafson DP, Wagner EK, Stevens JG (1990) A major portion of the latent pseudorabies virus genome is transcribed in trigeminal ganglia of pigs. J Virol 64:4755–4760PubMedPubMedCentralGoogle Scholar
  31. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140CrossRefGoogle Scholar
  32. Rock DL, Hagemoser WA, Osorio FA, McAllister HA (1988) Transcription from the pseudorabies virus genome during latent infection. Arch Virol 98:99–106CrossRefGoogle Scholar
  33. Schlackow M, Nojima T, Gomes T, Dhir A, Carmo-Fonseca M, Proudfoot NJ (2017) Distinctive patterns of transcription and RNA processing for human lincRNAs. Mol Cell 65:25–38CrossRefGoogle Scholar
  34. Strang BL, Stow ND (2005) Circularization of the herpes simplex virus type 1 genome upon lytic infection. J Virol 79:12487–12494CrossRefGoogle Scholar
  35. Su YHMM, Ng AK, Lin J, Jordan R, Fraser NW, Block TM (2002) Stability and circularization of herpes simplex virus type 1 genomes in quiescently infected PC12 cultures. J Gen Virol 83:2943–2950CrossRefGoogle Scholar
  36. Sun R, Lin SF, Gradocille L, Miller G (1996) Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus. Proc Natl Acad Sci U S A 93:11883–11888CrossRefGoogle Scholar
  37. Tafer H, Hofacker IL (2008) RNAplex: a fast tool for RNA-RNA interaction search. Bioinformatics 24:2657–2663CrossRefGoogle Scholar
  38. Tombacz D, Csabai Z, Olah P, Havelda Z, Sharon D, Snyder M, Boldogkoi Z (2015) Characterization of novel transcripts in pseudorabies virus. Viruses 7:2727–2744CrossRefGoogle Scholar
  39. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515CrossRefGoogle Scholar
  40. Wang M, Yuan D, Tu L, Gao W, He Y, Hu H, Wang P, Liu N, Lindsey K, Zhang X (2015) Long noncoding RNAs and their proposed functions in fibre development of cotton (Gossypium spp.). New Phytol 207:1181–1197CrossRefGoogle Scholar
  41. Wapinski O, Chang HY (2011) Long noncoding RNAs and human disease. Trends Cell Biol 21:354–361CrossRefGoogle Scholar
  42. Wilusz JE (2016) Long noncoding RNAs: re-writing dogmas of RNA processing and stability. Biochim Biophys Acta 1859:128–138CrossRefGoogle Scholar
  43. Wu Y, Wei B, Liu H, Li T, Rayner S (2011) MiRPara: a SVM-based software tool for prediction of most probable microRNA coding regions in genome scale sequences. BMC Bioinf 12:107CrossRefGoogle Scholar
  44. Wu YQ, Chen DJ, He HB, Chen DS, Chen LL, Chen HC, Liu ZF (2012) Pseudorabies virus infected porcine epithelial cell line generates a diverse set of host microRNAs and a special cluster of viral microRNAs. PLoS One 7:e30988CrossRefGoogle Scholar
  45. Wu H, Yin QF, Luo Z, Yao RW, Zheng CC, Zhang J, Xiang JF, Yang L, Chen LL (2016) Unusual processing generates SPA LncRNAs that sequester multiple RNA binding proteins. Mol Cell 64:534–548CrossRefGoogle Scholar
  46. Yin QF, Yang L, Zhang Y, Xiang JF, Wu YW, Carmichael GG, Chen LL (2012) Long noncoding RNAs with snoRNA ends. Mol Cell 48:219–230CrossRefGoogle Scholar
  47. Zhang Y, Zhang XO, Chen T, Xiang JF, Yin QF, Xing YH, Zhu S, Yang L, Chen LL (2013) Circular intronic long noncoding RNAs. Mol Cell 51:792–806CrossRefGoogle Scholar
  48. Zhou J, Li S, Wang X, Zou M, Gao S (2017) Bartha-k61 vaccine protects growing pigs against challenge with an emerging variant pseudorabies virus. Vaccine 35:1161–1166CrossRefGoogle Scholar

Copyright information

© Journal of NeuroVirology, Inc. 2018

Authors and Affiliations

  • Xiang Guan
    • 1
  • Jie Liu
    • 1
  • Hui Jiang
    • 1
  • Chang-Xian Wu
    • 1
  • Huan-Chun Chen
    • 1
  • Zheng-Fei Liu
    • 1
    Email author
  1. 1.State Key Laboratory of Agricultural Microbiology and Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary MedicineHuazhong Agricultural UniversityWuhanChina

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