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

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Abstract

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.

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Acknowledgments

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 (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.

Funding

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.

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Authors and Affiliations

  1. State Key Laboratory of Agricultural Microbiology and Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, 430070, China

    Xiang Guan, Jie Liu, Hui Jiang, Chang-Xian Wu, Huan-Chun Chen & Zheng-Fei Liu

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  1. Xiang Guan
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Correspondence to Zheng-Fei Liu.

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Electronic supplementary material

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)

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)

Supplementary Table 1

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Supplementary Table 2

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Guan, X., Liu, J., Jiang, H. et al. Expression of pseudorabies virus-encoded long noncoding RNAs in epithelial cells and neurons. J. Neurovirol. 24, 597–605 (2018). https://doi.org/10.1007/s13365-018-0651-3

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