Abstract
Background
Pseudorabies virus (PRV) causes substantial losses in the swine industry worldwide. Attenuated PRV strains with deletions of immunomodulatory genes glycoprotein E (gE), glycoprotein I (gI) and thymidine kinase (TK) are candidate vaccines. However, the effects of gE/gI/TK deletions on PRV-host interactions are not well understood.
Methods
To characterize the impact of gE/gI/TK deletions on host cells, we analyzed and compared the transcriptomes of PK15 cells infected with wild-type PRV (SD2017), PRV with gE/gI/TK deletions (SD2017gE/gI/TK) using RNA-sequencing.
Results
The attenuated SD2017gE/gI/TK strain showed increased expression of inflammatory cytokines and pathways related to immunity compared to wild-type PRV. Cell cycle regulation and metabolic pathways were also perturbed.
Conclusions
Deletion of immunomodulatory genes altered PRV interactions with host cells and immune responses. This study provides insights into PRV vaccine design.
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Introduction
Pseudorabies virus (PRV), also known as Suid herpesvirus 1 (SuHV1), belongs to the Herpesviridae family and is the causative agent of pseudorabies (PR) or Aujeszky’s disease (AD). Pigs are the natural host of PRV infection and the only animals that can survive PRV infection. PRV causes a highly contagious disease that severely threatens the pig industry, leading to reproductive failure, respiratory and neurological symptoms, and high mortality rates [1]. Infection of other animals with PRV results in acute, fatal disease with intense pruritus. Currently, attenuated live vaccines are the primary means of preventing and controlling pseudorabies [2].
The PRV genome can accommodate large foreign genes without compromising replicative ability, making it an ideal vector for expressing heterologous antigens. [3]. PRV vectors can be used to construct multivalent or broad-spectrum attenuated live vaccines to concurrently prevent PRV and infections by other important animal pathogens [4]. The PRV genome encodes 16 envelope glycoproteins that function in viral entry, egress and cell-to-cell spread. The gE glycoprotein is the major virulence protein enabling PRV to invade the host nervous system [5]. Deletion of the gE gene significantly decreases virulence and prevents invasion of the trigeminal and olfactory nerve terminals [6]. The gI-gE complex, together with gC, mediates viral release and impacts replication and virulence. Ablation of gI and gE functions dramatically affects PRV gene expression during infection. The gE glycoprotein can recruit the microtubule motor protein KIF1A to mediate retrograde axonal transport of PRV particles in neurons [6, 7]. gG is an immunomodulatory envelope protein that induces host cell secretion of interleukin-8 (IL-8) to attract neutrophil and monocyte migration and increase PRV infectivity [8]. gH is an envelope protein with fusion activity that can form a heterodimer with gL and, together with gB and gD, mediate PRV fusion with host cells [9]. gI is an envelope protein that facilitates cell-to-cell spread and retrograde neuronal spread, forming a heterodimer with gE and interacting with the gM/gN complex to impact intercellular PRV diffusion [10]. gK is an envelope protein that regulates viral budding and virulence, forming a heterodimer with UL20 and interacting with gB and gH/gL to impact intracellular transport and egress of PRV [11, 12]. gK can also affect PRV infection and virulence in the eyes, nose and throat [12].gL is an envelope protein with fusion activity that forms a heterodimer with gH and, together with gB and gD, mediates PRV fusion with host cells [9]. gM is an envelope protein that regulates viral budding and cell-to-cell spread, forming a heterodimer with gN and interacting with the gE/gI complex to impact intracellular transport and budding of PRV [13]. gN is an envelope protein that regulates viral budding and cell-to-cell spread, forming a heterodimer with gM and interacting with the gE/gI complex to impact intracellular transport and egress of PRV [13, 14]. The TK gene encodes a nonstructural protein with enzymatic activity to phosphorylate deoxynucleosides, participating in PRV DNA replication and transcription. TK impacts PRV latent infection and virulence [15]. TK-deleted or mutant PRV cannot establish latent infection in ganglia and exhibits attenuated virulence in mice and pigs [16]. TK utilizes host cell nucleotide metabolic pathways to provide necessary deoxyribonucleoside triphosphates (dNTPs) for PRV but can also convert certain antivirals like acyclovir (ACV) into active metabolites to inhibit PRV replication. Numerous PRV proteins are being continually explored for biological functions [17, 18]. Therefore,The glycoproteins gE and gI, along with the thymidine kinase (TK) gene, are major virulence determinants of PRV. gE enables neuroinvasion while TK impacts latent infection and virulence. Deletion of gE/gI/TK genes leads to dramatic attenuation of PRV.
In this study, we analyzed the transcriptomic changes in PK15 cells infected with the previously isolated virulent C strain, the attenuated SD2017gE/gI/TK strain with deletions of the major virulence determinants gE, gI and TK generated through homologous recombination using RNA-sequencing with Illumina platform. The goal was to elucidate the effects of key PRV virulence gene deletions on host cells, gain further insights into PRV pathogenesis, and establish a basis for novel attenuated live vaccine development.
Materials and methods
Virus and cell line
The highly virulent wild-type PRV mutant SD2017 strain was isolated in 2017 from the brains of PRV-infected piglets in Linyi, China [11]. SD2017gE/gI/TK was constructed in Shandong key laboratory of preventive veterinary medicine using homologous recombination to delete the gI, gE and TK genes, as described previously. PK15 (Sus scrofa epithelial kidney) cells used for PRV culture were obtained from the American Type Culture Collection (Manassas, VA, USA).
Cell culture and virus infection
PK15 porcine kidney cells were cultured in high-glucose DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. PRV SD2017 and PRV 2017gE/gI/TK were added at an MOI of 0.1 for 1 h, and the cells were then washed followed by the addition of 2% FBS / DMEM. PBS was used for mock infected control. Cells were harvested at 24 h post infection (hpi) in 3 independent biological replicates. RNA samples were extracted and stored at -80℃. the integrity, degradation and contamination of RNA were analyzed by agarose gel electrophoresis. The purity of RNA (OD260/280 and OD260/230 ratio) was detected by NanoDrop ND-1000 spectrophotometer (Nano Drop Inc., Wilmington, DE, USA). Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) was used to accurately detect 28 S/18 or 23 S/16S and RIN values, and accurately detect RNA integrity.
Library construction and transcriptome sequencing
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations, and index codes were added to attribute sequences to each sample [19].This kit was used to prepare sequencing libraries from total RNA. In order to select cDNA fragments preferably 250 ~ 300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Considering shorter fragments contain less sequencing information, we optimized conditions to obtain ideal 300–400 bp fragments, which allows richer sequencing information while ensuring quality. Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °F before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on the Agilent Bioanalyzer 2100 system. We ensured all sample libraries met requirements for subsequent sequencing [20, 21].
The index-coded libraries were pooled and clustering was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform generating 150 bp paired-end reads. Downstream quality control and information analysis were carried out to ensure accuracy of analysis. [21, qPCR was used to assess the intracellular PRV replication levels in samples infected for the same duration by both the PRV 2017gE/gI/TK and PRV SD2017 strains. The original virus strain exhibited significantly higher replication levels compared to the virulence gene-deleted strain, with its highest CT value at 14.408 and the lowest CT value for the deletion strain at 15.620 (Fig. 1A). RNA was extracted from PK15 cells infected with PRV 2017gE/gI/TK and control groups. The RNA integrity was assessed by measuring the ratio of 28 S to 18 S rRNA and the RIN value. As shown in Fig. 1B, the ratio of 28 S to 18 S was greater than 1.5 for all samples except PK15 03, which had a ratio of 0.8. However, the RIN value of PK15 03 was 9, indicating high RNA quality. The other samples also had RIN values above, which is generally considered acceptable for downstream experiments. The data has been submitted to the SRA database, with the accession number PRJNA1001590. To screen for differentially expressed genes (DEGs) in PK15 cells after infection with PRV 2017gE/gI/TK And PRV SD2017 we used the DESeq2 software package to perform differential expression analysis of the transcriptome se-quencing data and plotted the results. Principal component analysis showed the PRV 2017gE/gI/TK deletion mu-tant, wild-type PRV, and PK15 groups were closely clustered (Figure 2A). Therefore, this study focused on the dif-ferences between the attenuated PRV 2017gE/gI/TK strain and the wild-type PRV SD2017 strain, as well as the PK15 control group (Figure 2B). Venn diagrams displayed the DEGs between the 3 groups (Figure 2C). We set the screen-ing criteria for differential expression as |log2 fold change | > 1 and adjusted p-value < 0.005. According to these criteria, we screened the PRV 2017gE/gI/TK vs PRV SD2017 comparison and identified 93 up-regulated and 188 downregulated genes, the PRV 2017gE/gI/TK vs PK15 comparison showed 262 upregulated and 1021 downregulated genes, and the PRV SD2017 vs PK15 comparison showed 836 upregulated and 1299 downreg-ulated genes. Volcano plots were used to show the number and distribution of DEGs between groups (Figure 2D). The GO analysis mainly focused on functional annotation differences between the attenuated PRV 2017gE/gI/TK strain and the virulent PRV SD2017 strain, primarily by comparing the top 30 significant differences in PRV 2017gE/gI/TK vs. PRV SD2017 and PRV 2017gE/gI/TK vs. PK15, and PRV SD2017 vs. PK15 (Fig. 3). The GO analysis revealed that both infections significantly enriched processes highly relevant to DNA replication and damage repair, like cell cycle checkpoint (GO:0000075) and DNA repair (GO:0006281). These results indicate the viral infection jeopardized the genome integrity of host cells. Additionally, altered RNA splicing and processing (GO:0008380) and cytoskeleton organization and dynamics (GO:0000226) were observed, suggesting the viruses likely hijacked host RNA processing and intracellular trafficking systems. Some immune and inflammatory processes (GO:0006955) were also enriched, especially in the virulent strain infection, reflecting the immune responses elicited by the viruses. Moreover, modulated signal transduction pathways (GO:0007173) and protein degradation pathways (GO:0030163) manifested the extensive effects of viral infections on host cells. In summary, the GO analysis portrayed how the virulent strain intricately manipulated host immunity, genome stability, signal transduction, etc., inflicting more severe infection and damage to host cells. The loss of virulence genes in the attenuated strain may contribute to these observations. KEGG analysis mainly focused on the pathway annotation differences between PRV 2017gE/gI/TK weak strain and PRV SD2017 by analyzing the significant differences in top 20 pathways between PRV 2017gE/gI/TK vs. PRV SD2017 and PRV 2017gE/gI/TK vs. PK15, PRV SD2017 vs. PK15 as shown in (Fig. 4). For virulent strain vs. blank, cell cycle pathway was significantly enriched, with genes GADD45B, CDC7, CCNB3. Focal adhesion pathway was also significantly enriched, with genes SNAI1, TGFBR1, CTNND1. Serine protease inhibitor aging pathway was significantly enriched with gene GADD45B. For attenuated strain vs. virulent strain, IL-17 signaling pathway was significantly enriched, with inflammatory genes CCL20, CXCL2, CXCL8 significantly upregulated. TNF signaling pathway was significantly enriched, with inflammatory genes CCL20, CXCL2, TNF significantly upregulated. Chemokine signaling pathway was significantly enriched, with inflammatory genes CCL20, CXCL8, CXCL10 significantly upregulated. Enriched pathways also included rheumatoid arthritis, pathogen recognition receptor pathways and other immune-related pathways. For attenuated strain vs. blank PK15 cells, significantly enriched pathways included: Drug resistance pathway (ko01524), with genes MSH2, BIRC3 upregulated; Cellular senescence (ko04218), with genes SIRT1, NBN upregulated; cAMP signaling pathway (ko04024), with genes FOS, PDE4D upregulated. Analysis of virulent strain vs. blank showed virulent strain disrupted host basic survival functions, which may be due to higher replication efficiency and more damage to host cells. The enrichment of immune and inflammatory pathways and significant upregulation of inflammatory genes like CCL20, CXCL2, CXCL8 in attenuated strain can serve as evidence for easier recognition and clearance of attenuated strain by host (Fig. 4). To further validate the transcriptome analysis results, we performed a RT-qPCR analysis to determine the reproducibility of the differential gene expression. GAPDH mRNA was amplified as the endogenous control. Four down-regulated genes (STAT1, CD80, CD40, FGFR2) and three up-regulated genes (IL6, ISG15, CCL20) identified in RNA-seq were selected for RT-qPCR verification. The RT-qPCR results showed that the expression trends of these 7 genes were consistent with the RNA-seq data, though the extent of up/down-regulation varied. 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J Virol. 2023;97:e0041223. https://doi.org/10.1128/jvi.00412-23. Wang J, Lu SF, Wan B, Ming SL, Li GL, Su BQ, Liu JY, Wei YS, Yang GY, Chu BB. Maintenance of cyclic GMP-AMP homeostasis by ENPP1 is involved in pseudorabies virus Infection. Mol Immunol. 2018;95:56–63. https://doi.org/10.1016/j.molimm.2018.01.008. At the same time, we would like to thank the professor Yongjun Wen for the technical support. This research was funded by the Qingdao science and technology benefit the people demonstration project (NO. 23-2-8-xdny-14-nsh), the China Agriculture Research System of MOF and MARA. **aoli Wang: Conceptualization, Methodology, Writing-Review & Editing. Yingguang Li: Formal analysis, Writing-Original Draft. Shaoming Dong: Investigation, Data Curation, Writing-Review & Editing. Cong Wang: Validation, Data Curation. Yongming Wang: Formal analysis, Resources, Supervision. Hongliang Zhang: Project administration, Funding acquisition. 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Group Comments Supplementary RSD2017vsPK15_all_GOenrich Supplementary RSD2017vsPK15_all_KEGGenrich Supplementary RSD2017vsSD2017_all_GOenrich Supplementary RSD2017vsSD2017_all_KEGGenrich Supplementary SD2017vsPK15_all_GOenrich Supplementary SD2017vsPK15_all_KEGGenrich Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. 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Virol J 20, 303 (2023). https://doi.org/10.1186/s12985-023-02265-y Received: Accepted: Published: DOI: https://doi.org/10.1186/s12985-023-02265-yResults
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