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. 2022 May 27;14(6):1158.
doi: 10.3390/v14061158.

Metabolomics Analysis of PK-15 Cells with Pseudorabies Virus Infection Based on UHPLC-QE-MS

Panrao Liu  1 , Danhe Hu  1 , Lili Yuan  1 , Zhengmin Lian  1 , Xiaohui Yao  1 , Zhenbang Zhu  1 , Xiangdong Li  1   2
Affiliations

Metabolomics Analysis of PK-15 Cells with Pseudorabies Virus Infection Based on UHPLC-QE-MS

Panrao Liu et al. Viruses. .

Abstract

Viruses depend on the metabolic mechanisms of the host to support viral replication. We utilize an approach based on ultra-high-performance liquid chromatography/Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass (UHPLC-QE-MS) to analyze the metabolic changes in PK-15 cells induced by the infections of the pseudorabies virus (PRV) variant strain and Bartha K61 strain. Infections with PRV markedly changed lots of metabolites, when compared to the uninfected cell group. Additionally, most of the differentially expressed metabolites belonged to glycerophospholipid metabolism, sphingolipid metabolism, purine metabolism, and pyrimidine metabolism. Lipid metabolites account for the highest proportion (around 35%). The results suggest that those alterations may be in favor of virion formation and genome amplification to promote PRV replication. Different PRV strains showed similar results. An understanding of PRV-induced metabolic reprogramming will provide valuable information for further studies on PRV pathogenesis and the development of antiviral therapy strategies.

Keywords: PK-15 cells; UHPLC-QE-MS; metabolomic analysis; pseudorabies virus.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Infections of different PRV strains in PK-15 cells. (A,B) The cells were infected with PRV variant strain (abbreviation of PRV-G) and PRV Bartha K61 strain (abbreviation of PRV-K) at MOI = 1, and cell samples were collected at 6, 12, and 24 h for immunoblotting detection. (C,D) PK-15 cells were infected with PRV-G and PRV-K at MOI = 1 for 6, 12, and 24 h at 37 °C with 5% CO2. The expression levels of PRV-gB protein detected by IFA. Scale bars = 200 μm.
Figure 2
Figure 2
Score scatter plots of PCA and OPLS-DA of PRV-infected and uninfected cells. (A,B) Score scatter plot of the PCA model for the different infection groups versus mock group. Electrospray ionization served as the source of UHPLC-QE-MS, including positive and negative ion modes (POS and NEG). (A) was derived from POS and (B) from NEG. The lines denote 95% confidence interval Hotelling’s ellipses. (C) OPLS-DA model for the different PRV-strain infection group versus mock group.
Figure 3
Figure 3
Analysis of differentially expressed metabolites in PK-15 cells infected with different PRV strains. (A,B) Venn diagrams between PRV-infected groups (6H, 12H, 24H) and mock group. (C,D) Numbers of differentially expressed metabolites upregulated (red) and downregulated (blue) in infected groups. (E–H) Pie charts and the histogram graphs showing proportions of different categories among differentially expressed metabolites in PRV-infected PK-15 cells.
Figure 4
Figure 4
Heatmap analysis of 103 and 136 metabolites among PRV-G, PRV-K, and mock groups. Rows: metabolites; columns: samples. The color of each rectangle represents the relative level of the differential metabolites. Red: upregulated; blue: downregulated. (A) PRV-G; (B) PRV-K.
Figure 5
Figure 5
The KEGG-enrichment pathway analysis of differentially expressed metabolites for PK-15 cells infected with different PRV strains in different time courses. (A–C) PRV-G (6H, 12H, 24H); (D–F) PRV-K (6H, 12H, 24H).
Figure 6
Figure 6
Schematic overview of altered metabolic pathways in PK-15 cells infected with different PRV strains. The metabolites were shown in different colors according to their changes. Black: unchanged; red: upregulated; blue: downregulated.
Figure 7
Figure 7
The mRNA levels of sphingolipid- and glycerophospholipid-metabolism-related enzymes after different PRV-strain infections. PK-15 cells were harvested with different MOI (1, 5, 10) at 24 h.p.i after PRV (PRV-G or PRV-K) infection, while non-infected cells were used as control. The mRNA levels of different enzymes were determined by qRT-PCR. β-actin was used as an internal reference gene. (A,B) PRV-G; (C,D) PRV-K. The significance in the figure was indicated as follows: *, p < 0.05; **, p < 0.01.
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Grants and funding

This work was funded by the National Natural Science Foundation of China (Nos. 32102637 and 32172823) and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).