Abstract

   The influenza A virus is an acute contagious pathogen that affects the
   human respiratory system and can cause severe lung disease and even
   death. Lariciresinol-4-β-D-glucopyranoside is a lignan that is
   extracted from Isatis indigotica, which is a medicinal herb plant that
   was commonly applied to treat infections, the common cold, fever and
   inflammatory diseases. Our previous study demonstrated that
   lariciresinol-4-β-D-glucopyranoside possesses anti-viral and
   anti-inflammatory properties. However, the comprehensive and detailed
   mechanisms that underlie the effect of
   lariciresinol-4-β-D-glucopyranoside interventions against influenza
   virus infection remain to be elucidated. In this study, we employed
   high-throughput RNA sequencing (RNA-seq) to investigate the
   transcriptomic responses of influenza A virus-infected lung epithelial
   (A549) cells with lariciresinol-4-β-D-glucopyranoside treatment. The
   transcriptome data show that infection with influenza A virus prompted
   the activation of 368 genes involved in RIG-I signalling, the
   inflammatory response, interferon α/β signalling and gene expression
   that was not affected by lariciresinol-4-β-D-glucopyranoside treatment.
   Lariciresinol-4-β-D-glucopyranoside exerted its pharmacological actions
   on the immune system, signal transduction, cell cycle and metabolism,
   which may be an underlying defense mechanism against influenza virus
   infection. In addition, 166 differentially expressed genes (DEGs) were
   uniquely expressed in lariciresinol-4-β-D-glucopyranoside-treated
   cells, which were concentrated in the cell cycle, DNA repair, chromatin
   organization, gene expression and biosynthesis domains. Among them, six
   telomere-associated genes were up-regulated by
   lariciresinol-4-β-D-glucopyranoside treatment, which have been
   implicated in telomere regulation and stability. Collectively, we
   employed RNA-seq analysis to provide comprehensive insight into the
   mechanism of lariciresinol-4-β-D-glucopyranoside against influenza
   virus infection.

Introduction

   The ongoing battle against infectious diseases such as influenza is an
   enduring process. Type A influenza virus causes epidemics each year and
   has contributed to hundreds of thousands of deaths worldwide. Due to
   the viruses’ gene mutations and genome reassortments, the viruses’
   variants have easily acquired resistance to current antiviral
   medications [[32]1, [33]2]. Several studies have documented the recent
   emergence of neuraminidase (NA) inhibitor-resistant avian-origin
   influenza A (H7N9) strains [[34]3, [35]4].

   Influenza A virus infection in the lung is associated with a robust
   host inflammatory response that is characterized by abundant leukocyte
   infiltration (e.g., T cells, macrophages, and monocytes) and sustained
   elevated levels of inflammatory immune mediators, including tumour
   necrosis factor-α (TNF-α), IL-6, and interferon gamma-induced protein
   10 (IP-10) [[36]5–[37]7]. Low or high virulent influenza virus
   infection alters the expression of thousands of host cell genes, such
   as genes related to cell cycle progression, apoptosis, inflammatory
   response, and metabolism [[38]8–[39]11]. In general, the complex
   interactions between the virulence factors of the pathogen and the host
   immune defence were associated with the infectious disease outcome
   [[40]12]. Therefore, the analysis of the global changes in the host
   gene expression profile during influenza A virus infection may
   contribute to the identification of potential novel therapeutic targets
   that are urgently required for preventing influenza-associated
   diseases.

   Chinese herbal medicine (CHM) has served a critical role in preventing
   and controlling influenza infection disease in China since ancient
   times [[41]13]. Current medical plants contain numerous components that
   serve as a source for the development of antiviral agents. Basic
   research of medicinal herbs primarily focuses on bioactive compound
   discovery and the utilization of cell culture or animal models of
   influenza infection to study their pharmacological mechanisms of
   action. The investigation of how these bioactive compounds exert their
   therapeutic effect and restore body system homeostasis is substantially
   based on the investigators’ deductive assumptions, which may result in
   numerous biases and inaccuracies. These unsystematic and biased
   findings from research may pose drug safety concerns and even harm to
   patients’ health in the future. For instance, shikimic acid from
   Illicium verum is a primary precursor for the synthesis of oseltamivir;
   however, oseltamivir has been reported to cause neuropsychiatric side
   effects [[42]14]. Yang et al. has suggested the application of modern
   technologies, such as transcriptomic technologies, to study the
   bioactive compounds from traditional Chinese medicine (TCM) [[43]15].

   RNA-sequencing (RNA-seq), which is a genome-wide analytical technology,
   has been extensively utilized to analyse the transcriptome of various
   infectious diseases, including influenza A virus infection [[44]16,
   [45]17]. RNA-seq analysis of the host response to human or avian
   influenza virus infection has been performed in the mouse, crow,
   chicken and swine and has provided insights into influenza disease
   pathogenesis [[46]18–[47]21]. RNA-seq offers a systematic approach for
   delineating gene expression changes at a given point in time [[48]22],
   which may identify the key molecular events that are linked to the
   pathogenesis of influenza virus infection and provide a comprehensive
   analysis of the pharmacological effects of herbal components.

   The herb Isatis indigotica (Ban-Lan-Gen) is a common Chinese herb that
   is used as an important and popular herbal remedy for the clinical
   treatment of the common cold, fever and influenza [[49]23]. A number of
   active compounds have been identified in I. indigotica, including
   indole alkaloids, flavonoids, and lignans [[50]24, [51]25]. Our
   previous studies have demonstrated that a lignan from I. indigotica,
   lariciresinol-4-β-D-glucopyranoside, exhibited potent inhibitory
   activity against influenza virus A/PR/8/34(H1N1), with 50% inhibitory
   concentrations (IC[50]) of 50 μg/mL and selectively inedx (SI) more
   than 4, while it showed low cytotoxic potency towards MDCK cells for
   which the CC[50] exceeded 200 μg/mL [[52]26]. Furthermore,
   lariciresinol-4-β-D-glucopyranoside suppressed influenza A (H1N1)
   virus-infected or TNF-α-stimulated activation of NF-κB in a NF-κB
   luciferase reporter stable HEK293 cell line. Meanwhile, the increased
   level of pro-inflammatory genes in influenza A H1N1 or H9N2
   viruses-infected cells, such as TNF-α, IL-6, IP-10 and IL-8, were
   inhibted by lariciresinol-4-β-D-glucopyranoside treatment [[53]26].
   Therefore, our previous results suggested that
   lariciresinol-4-β-D-glucopyranoside impaired influenza virus
   propagation and suppressed the human or avian influenza virus-induced
   host inflammatory response via the regulation of NF-κB activation
   [[54]26]. In this study, we used RNA-seq technology to systematically
   assess the transcriptome profile of influenza A virus-infected lung
   epithelial (A549) cells following lariciresinol-4-β-D-glucopyranoside
   treatment, which may result in gaining a comprehensive understanding
   the mechanism of lariciresinol-4-β-D-glucopyranoside against influenza
   A virus infection.

Materials and methods

Compounds, cell culture, virus infection and sample preparation

   The compound lariciresinol-4-β-D-glucopyranoside was prepared as
   previously described, dissolved in dimethyl sulfoxide (DMSO) (Sigma) as
   a stock solution of 50 mg/ml and stored at -20°C until use. A549 human
   lung adenocarcinoma cells that were purchased from ATCC were cultured
   in MDEM/DF12 (1:1, V/V), with 10% foetal bovine serum (FBS), 100 U/ml
   penicillin, and 100 μg/ml streptomycin under standard conditions at
   37°C in 5% CO2 humidified air. The A549 cells were grown in a monolayer
   to 80% to 90% confluency and detached from the flask using 10 mM EDTA
   (pH 7.4) and 0.25% trypsin. The cells were harvested, and 5 × 10^5 A549
   cells were seeded in 6-well tissue culture plates. The following day,
   the cells were washed twice with PBS and infected with A/PR/8/34 (H1N1)
   at multiplicities of infection (MOIs) of 0.1 using serum-free medium
   for 2 hours at 37°C. The inoculum was removed, and the cells were
   treated with or without lariciresinol-4-β-D-glucopyranoside (400 μg/ml
   to 800 μg/ml). After 24 h, the cells were lysed in TRIzol reagent (Life
   technologies) and stored at -80°C.

RNA isolation, cDNA library construction and sequencing

   Total RNA extracts from each sample were obtained following the
   manufacturer’s instructions (Life technologies). The total RNA quality
   was assessed by 1.5% agarose gel electrophoresis. The A260/A280 ratio
   was determined using a N0061noDrop ND-200 spectrophotometer (NanoDrop
   Technologies, USA). RNA integrity was assessed by Agilent 2200
   TapeStation analysis (Agilent Technologies, Santa Clara, CA, USA). A
   A260/A280 ratio between 1.8 to 2.0 and RIN > 7 were considered
   acceptable. RNA sequencing was performed on an Illumina HiSeq-2000
   RNA-seq sequence production system. RNA-seq data of this study have
   been submitted to the NCBI Gene Expression Omnibus with the accession
   number [55]GSE93999 ([56]https://www.ncbi.nlm.nih.gov/geo/).

Pathway analysis of differentially expressed genes

   We performed Gene Ontology (GO) and pathway enrichment analysis using
   the Database for Annotation, Visualization, and Integrated Discovery
   (DAVID) to obtain a list of differentially expressed genes. We
   identified GO terms and the Kyoto Encyclopaedia of Genes and Genomes
   (KEGG) pathways that were significantly enriched on our list of altered
   genes (P < 0.05). Additionally, the integrated pathways with
   statistical values were computed from our list of DEGs using a reactome
   pathway analysis ([57]http://www.reactome.org/).

Results

Sequencing and alignment

   To understand the molecular mechanism of
   lariciresinol-4-β-D-glucopyranoside action against influenza A virus
   infection, we performed RNA-seq of influenza A virus-infected human
   lung epithelial (A549) cells in the absence or presence of different
   concentrations of lariciresinol-4-β-D-glucopyranoside treatment using
   an Illumina HiSeq 2000 platform, and then, the sequences were aligned
   against human gene sequences (hg19, [58]Table 1). There were four
   experimental conditions, including lung epithelial (A549) cells without
   infection (C), A549 cells infected with A/PR8/34/(H1N1) (CV), A549
   cells infected with A/PR8/34/(H1N1) at the low concentration of
   lariciresinol-4-β-D-glucopyranoside treatment (400 μg/ml) (CVL[L] × 2),
   and A549 cells infected with A/PR8/34/(H1N1) at a high concentration of
   lariciresinol-4-β-D-glucopyranoside treatment (800 μg/ml) (CVL[H] × 2)
   ([59]Fig 1). Then, 24 hours after influenza virus infection, samples
   were harvested and subjected to library construction and sequencing.
   More than 13 million raw reads for each sample were generated from
   constructed RNA-seq libraries using the Illumina HiSeq 2000 platform.
   After stringent data filtering, 12.29 M (93.04%, C), 12.94 M (92.17%,
   CV), 12.35 M (91.50%, CVL[L]) and 12.56 M (90.71%, CVL[H]) sequencing
   reads can be unambiguously mapped against the reference genome
   ([60]Table 1), which suggests the high quality of the sequencing of
   these samples and a satisfactory variable for additional analysis.

Table 1. Statistics of RNA-seq data and mapped reads obtained by an RNA-Seq
analysis of influenza A virus (H1N1)-infected lung epithelial (A549) cells
with or without lariciresinol-4-β-D-glucopyranoside treatment.

   Sample type C CV CVL[L] CVL[H] CVL[L] CVL[H]
   Total reads 13,216,449 (100%) 14,035,807 (100%) 13,497,899 (100%)
   13,841,371 (100%) 13,551,998 (100%) 13,279,470 (100%)
   Total mapped (%) 12,961,879 (98.07%) 13,652,450 (97.27%) 13,045,031
   (96.64%) 13,274,612 (95.91%) 13,034,539 (96.18%) 12,914,700 (97.25%)
   Multiple mapped (%) 665,860 (5.04%) 715,719 (5.10%) 694,903 (5.15%)
   719,288 (5.20%) 723,168 (5.34%) 646,251 (4.87%)
   Uniquely mapped (%) 12,296,019 (93.04%) 12,936,731 (92.17%) 12,350,128
   (91.50%) 12,555,324 (90.71%) 12,311,371 (90.85%) 12,268,449 (92.39%)
   Reads map to “+” 6,311,551 (47.76%) 6,657,196 (47.43%) 6,343,777
   (47.00%) 6,455,222 (46.64%) 6,336,575 (46.76%) 6,298,126 (47.43%)
   Reads map to “-” 6,650,328 (50. 32%) 6,995,254 (49.84%) 6,701,254
   (49.65%) 6,819,390 (49.27%) 6,697,964 (49.42%) 6,616,574 (49.83%)
   [61]Open in a new tab

Fig 1. Experimental design and RNA-seq.

   [62]Fig 1
   [63]Open in a new tab

   The study groups were as follows: one group lung epithelial (A549)
   cells without infection (C), one group of A549 cells infected with
   A/PR8/34/(H1N1) (CV), two groups of A549 cells infected with
   A/PR8/34/(H1N1) at the low concentration of
   lariciresinol-4-β-D-glucopyranoside treatment (400 μg/ml) (CVL[L]), and
   two groups of A549 cells infected with A/PR8/34/(H1N1) at the high
   concentration of lariciresinol-4-β-D-glucopyranoside treatment (800
   μg/ml) (CVL[H]).

Identification of differentially expressed genes (DEGs)

   Human lung epithelial (A549) cells with influenza A virus infection
   resulted in the greatest degree of differential expression (CV/C). A
   total of 458 genes (368 genes up-regulated, 90 genes down-regulated)
   were detected to be significantly altered (≥ 1.5-fold change, P < 0.05)
   ([64]Fig 2A). Additionally, 368 (243 up-regulated and 125
   down-regulated) of 458 genes were identified in all
   lariciresinol-4-β-D-glucopyranoside treatments ([65]S1 Table and
   [66]Fig 3A–3D), which reveals that the expression of these genes was
   triggered by host-pathogen interaction genes during influenza virus
   infection but was not regulated by lariciresinol-4-β-D-glucopyranoside
   treatments. To obtain a detailed understanding of the DEGs regulated by
   lariciresinol-4-β-D-glucopyranoside, we compared all treatments (CVL[L]
   and CVL[H]) to CV. We identified 645 significantly altered DEGs (487
   genes up-regulated and 158 genes down-regulated, P < 0.05) in the
   CVL[H]/CV comparison; however, the CVL[L]/CV comparison indicated that
   relatively few genes were significantly changed (8 up-regulated and 16
   down-regulated, P < 0.05) ([67]Fig 2A), which suggests that
   lariciresinol-4-β-D-glucopyranoside treatment exerts its effect against
   influenza virus infection in a dose-dependent manner. In addition, 13
   genes were commonly overlapped and regulated in both pairwise
   comparison C/CV and CV/CVL[L] ([68]Fig 2B). Similarly, a comparison of
   the pairwise comparisons C/CV and CV/CVL[H], 11 overlapping DEGs
   strongly revealed that lariciresinol-4-β-D-glucopyranoside treatment
   against virus infection does not regulate the large components of host
   genes that drive expression by viruses ([69]Fig 2B). Interestingly, 243
   DEGs were solely affected by the high concentration of
   lariciresinol-4-β-D-glucopyranoside treatment (CV/CVL[H]). A detailed
   analysis of the up-regulated and down-regulated genes among the three
   comparisons was performed ([70]Fig 2C–2E). Based on the high
   concentration of lariciresinol-4-β-D-glucopyranoside treatment, 83
   genes and 108 genes in the C/CV and CV/CVL[H] comparisons were
   up-regulated and down-regulated, respectively ([71]Fig 2E). These
   findings indicate that the effects of
   lariciresinol-4-β-D-glucopyranoside against virus infection were not
   attributed to regulation of the genes induced by viruses.

Fig 2. Global overview of the RNA-seq data of influenza A virus-infected lung
epithelial (A549) cells with or without lariciresinol-4-β-D-glucopyranoside
treatment.

   [72]Fig 2
   [73]Open in a new tab

   (a) Summary of the number of more than 1.5 fold up- or down-regulated
   differentially expressed genes (DEGs) as determined by RNA-Seq in all
   four experimental conditions. For example, 368 DEGs were up-regulated
   in CV/C, and 90 DEGs were down-regulated in CV/C. (b) The Venn diagram
   shows the total number of overlapping DEG profiles for three
   comparisons (C/CV, CV/CVL[L], CV/CVL[H]). (c-e) Detailed analysis of
   up-regulated and down-regulated genes among the three comparisons,
   e.g., comparisons of C/CV and CV/CVL[H], as shown in [74]Fig 1E, the
   single up arrow (315↑) or single down arrow (81↓) denotes 315
   up-regulated DEGs or 81 down-regulated DEGs, respectively. Downwards
   paired arrows (12↓↓) or upwards paired arrows (9↑↑) in an overlapping
   area denote 12 common up-regulated DEGs or nine common down-regulated
   DEGs between the comparisons. An upwards arrow leftwards of a downwards
   arrow (31↑↓) denotes 31 up-regulated DEGs in C/CV but down-regulated in
   CV/CVL[H]. Similarly, a downwards arrow leftwards of an upwards arrow
   (0↓↑) denotes no up-regulated DEGs in C/CV but down-regulated DEGs in
   CV/CVL[H].

Fig 3. DEGs by host defence.

   [75]Fig 3
   [76]Open in a new tab

   a) Comparison of 125 down-regulated genes induced by influenza virus
   infection among five samples. b) Comparison of 243 up-regulated genes
   induced by influenza virus infection among five samples. c) Heatmap
   showing 125 down-regulated genes induced by influenza virus infection
   but not altered by lariciresinol-4-β-D-glucopyranoside treatment (high
   expression: yellow; low expression: green). d) Heatmap showing 243
   up-regulated genes induced by influenza virus infection but not altered
   by lariciresinol-4-β-D-glucopyranoside treatment among five samples
   (high expression: yellow; low expression: green). e) Enrichment
   analysis results of 327 up-regulated genes induced by influenza virus
   infection but not altered by lariciresinol-4-β-D-glucopyranoside
   treatment using the reactome database. f) Heatmap showing 16 DEGs in
   the RIG-I-MDA5-mediated induction of IFN-alpha-beta pathway (high
   expression: yellow; low expression: green).

DEGs in lung epithelial (A549) cells with influenza A virus infection

   As previously mentioned, 369 DEGs were triggered by influenza virus
   infection but were not affected by lariciresinol-4-β-D-glucopyranoside
   treatment ([77]Fig 3A–3D and [78]S1 Table). To better understand the
   host cell response to influenza virus infection at 24 hours, we
   characterized the 368 DEGs data by performing the enrichment analysis
   using the DAVID and reactome pathway database. GO enrichment analysis
   suggested that these DEGs were related to the immune system, such as
   the response to wounding (GO:0009611), defence responses (GO:0006952),
   inflammatory response (GO:0006954), response to virus (GO:0009615) and
   immune response (GO:0006955) ([79]Fig 3E and [80]S2 Table).

   Results from the enrichment analysis of the reactome indicated that 243
   up-regulated genes were also significantly enriched in 60 pathways
   related to the immune system, disease, cell cycle and programmed cell
   death (P < 0.01, FDR < 0.05), such as antiviral defense, innate
   immunity, type I interferon signaling pathway and cytokine signaling in
   immune system ([81]Fig 3E and [82]S2 Table). There were 16 DEGs that
   were enriched in the RIG-I-like receptor signalling pathway ([83]Fig 3F
   and [84]S2 Table), which activates signalling cascades to produce type
   I interferon (IFN-α/β) and mediate the anti-viral response. These DEGs,
   which mainly included HERC5, DDX58, IRF1, IFR7, and ISG15, were
   reported to be involved in initiating the antiviral programme. DDX58
   (RIG-I) recognized intracellular virus-derived RNA molecules and
   activated host defence responses in RNA-virus-infected cells [[85]27].
   Activation of the RIG-I signalling pathway triggers the signalling
   pathway that leads to the activation of the transcription factors NF-κB
   and IRF3/7, which transcribe cytokines and chemokines such as IFN-β,
   IL-8, TNF-α, IFN-λ1/2 and RANTES [[86]28]. Robust type I interferon
   production by virus infection requires the IRF7-RIG-I amplification
   loop and the stability of RIG-I via post-translational modification by
   type I IFN [[87]29, [88]30]. Interferon signalling is essential for the
   production of anti-viral mediators (such as OAS1, Mx1, SP100, and
   TRIM22) to defend against virus infection [[89]31, [90]32]. The
   IFN-stimulated gene TRIM22 exerts its antiviral activity via
   polyubiquitination and degradation of viral nucleoprotein (NP)
   [[91]32], whereas the viruses have evolved numerous strategies to
   counteract the interferon-mediated anti-viral response [[92]33].

   125 down-regulated genes that are significantly enriched in 12 pathways
   are involved in metabolism and gene expression (P < 0.01, FDR < 0.05),
   such as L13a-mediated translational silencing of ceruloplasmin
   expression, eukaryotic translation initiation and cap-dependent
   translation initiation ([93]S1 Fig and [94]S3 Table). The generation of
   a capped RNA primer from the host cell pre-mRNAs by the endonuclease
   activity of PB2 polymerase subunit is required for initiating viral
   mRNA transcription and causes the degradation of cellular mRNA
   [[95]34]. Influenza virus protein translation is completely dependent
   on the host cell protein synthesis machinery, whereas the host’s
   cellular protein synthesis is shut down during the infection [[96]35].

Pattern against viral infections by lariciresinol-4-β-D-glucopyranoside

   We determined that the regulation of gene expression effective against
   viral infections by lariciresinol-4-β-D-glucopyranoside has been
   demonstrated in two different ways: (i) direct regulation of the DEGs
   induced by influenza A virus infection, and (ii) regulation of the
   expression of other genes that are not affected by viruses to exert its
   effects.

Influenza A virus-induced DEGs directly regulated by
lariciresinol-4-β-D-glucopyranoside treatment

   Using a criteria for data analysis (fold change ≥ ± 1.5 and p-value <
   0.05), 146 DEGs (included 29 genes up-regulated and 117 genes
   down-regulated) have been identified significant difference between two
   comparison (CV/CVL[L], CV/CVL[H]) ([97]Fig 4A–4D and [98]S4 Table). We
   performed the enrichment analysis on the DEGs that were directly
   regulated by lariciresinol-4-β-D-glucopyranoside. The GO enrichment
   analysis revealed that the 146 DEGs were significantly enriched in the
   immune response, cell cycle, metablism, cellular responses to stress
   and cell-cell communication ([99]Fig 4E), such as negative regulation
   of viral genome replication (GO:0045071), chemokine interleukin-8-like
   domain (IPR001811), GO terms related to chemotaxis (GO:0006935,
   GO:0060326 and GO:0008009) and defense response to virus (GO:0051607)
   ([100]S5 Table). We discovered that the expression of genes involved in
   the inflammatory response was down-regulated after
   lariciresinol-4-β-D-glucopyranoside treatment, such as CXCL1, CXCL2,
   CXCL3, CXCL5, IL-8, CCL2 and CX3CL1. Interestingly, GBP4 gene
   expression (gene name, guanylate binding protein 4; fold change = 2.76;
   p value = 3.22E-05) was up-regulated after treatment with
   lariciresinol-4-β-D-glucopyranoside ([101]Fig 4D and [102]S4 Table).
   GBPs are a group of IFN-induced GTPases that are essential for the host
   antiviral defence. GBP1 has been extensively investigated and has been
   shown to act against many viruses, including vesicular stomatitis virus
   (VSV), encephalomyocarditis virus (ECMV) and hepatitis C virus (HCV)
   [[103]36, [104]37]. In addition to proinflammatory cytokine genes,
   influenza virus infection also elicits the transcription of
   gene-encoding lipid mediators (including COX, LTA4H and PGES)
   [[105]38–[106]40]. The lariciresinol-4-β-D-glucopyranoside treatment
   down-regulated influenza virus-induced expression of PTGES (gene name,
   prostaglandin E-synthase; fold change = 3.33; p value = 1.73E-06).
   Increases in PTGES expression cause the generation of its metabolic
   product, prostaglandin E2 (PGE2), which has been shown to be involved
   in increasing viral replication and inhibiting type I IFN secretion
   [[107]38]. KEGG pathway enrichment analysis showed that the
   down-regulated genes are enriched in the chemokine signaling pathway,
   metabolic pathways and toll-like receptor signaling pathway ([108]S5
   Table and [109]S6 Table), such as interleukin-7 signalling, prolactin
   receptor signalling and NOTCH2 activation and the transmission of
   signals to the nucleus.

Fig 4. Bioinformatics analysis of the DEGs regulated by
lariciresinol-4-β-D-glucopyranoside treatment.

   [110]Fig 4
   [111]Open in a new tab

   a) Comparison of 117 down-regulated genes regulated by
   lariciresinol-4-β-D-glucopyranoside treatment among five samples. b)
   Heatmap showing 23 DEGs regulated by
   lariciresinol-4-β-D-glucopyranoside treatment among five samples (high
   expression: yellow; low expression: green). c) Comparison of 29
   up-regulated genes regulated by lariciresinol-4-β-D-glucopyranoside
   treatment among five samples. d) Heatmap showing 121 DEGs regulated by
   lariciresinol-4-β-D-glucopyranoside treatment among five samples (high
   expression: yellow; low expression: green). e) Enrichment analysis
   results of 146 DEGs regulated by lariciresinol-4-β-D-glucopyranoside
   treatment using the reactome database.

Emerging DEGs by lariciresinol-4-β-D-glucopyranoside treatment

   Genes expression that was up-regulated or down-regulated at least
   1.5-fold (fold change ≥ ± 1.5, p-value less than 0.05), but not in
   accordance with this criteria in virus-infected group (CV), which were
   defined as emerging DEGs by lariciresinol-4-β-D-glucopyranoside
   treatment. A total of 166 DEGs (70 up-regulated genes and 96
   down-regulated genes) belong to the emerging DEGs that are considered
   to be indirectly effective genes that are not induced by viruses
   ([112]Fig 5A and 5B and [113]S7 Table). For the up-regulated genes,
   they are significantly enriched in the response to extracellular
   stimuli (GO:000999) and the response to nutrient (GO:0031667,
   GO:0007584) in the DAVID results ([114]S8 Table). Pathway analysis
   using KEGG revealed that these DEGs were primarily concentrated in the
   cell cycle, DNA repair, chromatin organization and gene expression
   ([115]Fig 5C) including 56 pathways (P<0.01, FDR < 0.05), such as the
   packaging of telomere ends, HDACs deacetylation of histones and RNA
   polymerase I promoter opening ([116]Table 2).

Fig 5. Bioinformatics analysis of the emerging DEGs that regulated by
lariciresinol-4-β-D-glucopyranoside treatment.

   [117]Fig 5
   [118]Open in a new tab

   a) Comparison of 70 up-regulated genes that regulated by
   lariciresinol-4-β-D-glucopyranoside treatment among five samples but no
   significant change by influenza virus infection. b) Comparison of 70
   down-regulated genes that regulated by
   lariciresinol-4-β-D-glucopyranoside treatment among five samples but no
   significant change by influenza virus infection. c) Enrichment analysis
   results of 70 up-regulated genes regulated by
   lariciresinol-4-β-D-glucopyranoside treatment using the reactome
   database. d) Enrichment analysis results of 96 down-regulated genes
   regulated by lariciresinol-4-β-D-glucopyranoside treatment using the
   reactome database.

Table 2. Enriched KEGG pathway of DEGs in lariciresinol-4-β-D-glucopyranoside
treatment.

   Pathway name Entities found Entities total Entities P-Value Entities
   FDR
   Packaging of telomere ends 10 32 3.14E-13 5.79E-11
   HDACs deacetylate histones 12 63 3.83E-13 5.79E-11
   RNA polymerase I promoter opening 9 32 1.27E-11 1.06E-09
   HATs acetylate histones 13 110 1.41E-11 1.06E-09
   DNA methylation 9 35 2.79E-11 1.47E-09
   DNA damage/telomere stress-induced senescence 11 70 2.94E-11 1.47E-09
   Meiotic synapsis 10 60 1.39E-10 5.97E-09
   PRC2 methylates histones and DNA 9 43 1.68E-10 6.23E-09
   SIRT1 negatively regulates rRNA expression 9 44 2.06E-10 6.79E-09
   Formation of the beta-catenin: TCF transactivating complex 10 64
   2.58E-10 7.73E-09
   Activated PKN1 stimulates transcription of AR (androgen receptor)
   regulated genes KLK2 and KLK3 9 47 3.65E-10 9.84E-09
   RMTs methylate histone arginines 9 49 5.23E-10 1.31E-08
   Telomere maintenance 10 73 9.02E-10 1.86E-08
   Condensation of prophase chromosomes 9 53 1.03E-09 1.86E-08
   Nucleosome assembly 9 53 1.03E-09 1.86E-08
   Deposition of new CENPA-containing nucleosomes at the centromere 9 53
   1.03E-09 1.86E-08
   Transcriptional regulation by small RNAs 10 77 1.50E-09 2.54E-08
   Meiotic recombination 9 58 2.24E-09 3.59E-08
   RNA polymerase I chain elongation 9 59 2.60E-09 3.89E-08
   meiosis 10 91 7.21E-09 1.08E-07
   Chromosome maintenance 10 101 1.91E-08 2.55E-07
   Chromatin modifying enzymes 14 241 1.96E-08 2.55E-07
   Chromatin organization 14 241 1.96E-08 2.55E-07
   RHO GTPases activate PKNs 9 77 2.52E-08 3.02E-07
   NoRC negatively regulates rRNA expression 9 79 3.13E-08 3.75E-07
   RNA polymerase I promoter clearance 9 81 3.86E-08 4.25E-07
   Gene silencing by RNA 10 111 4.58E-08 5.04E-07
   RNA polymerase I transcription 9 84 5.25E-08 5.25E-07
   Senescence-associated secretory phenotype (SASP) 9 85 5.79E-08 5.79E-07
   Negative epigenetic regulation of rRNA expression 9 88 7.75E-08
   7.75E-07
   Oxidative stress-induced senescence 9 107 3.95E-07 3.55E-06
   Epigenetic regulation of gene expression 9 114 6.65E-07 5.99E-06
   Cellular senescence 11 190 7.54E-07 6.79E-06
   PERK regulates gene expression 6 38 1.07E-06 8.57E-06
   RNA polymerase I, RNA polymerase III, and mitochondrial transcription 9
   121 1.08E-06 8.67E-06
   Mitotic prophase 9 128 1.71E-06 1.37E-05
   ATF4 activates genes 5 32 9.26E-06 7.41E-05
   TCF-dependent signalling in response to WNT 10 212 1.47E-05 1.03E-04
   Cellular responses to stress 13 455 1.35E-04 9.45E-04
   Nonhomologous end-joining (NHEJ) 5 57 1.40E-04 9.83E-04
   G2/M DNA damage checkpoint 5 57 1.40E-04 9.83E-04
   Recruitment and ATM-mediated phosphorylation of repair and signalling
   proteins at DNA double strand breaks 5 65 2.57E-04 0.001796963
   ATF6-alpha activates chaperone genes 3 15 3.11E-04 0.002176276
   DNA double-strand break response 5 73 4.35E-04 0.002608677
   ATF6-alpha activates chaperones 3 17 4.47E-04 0.002682118
   Signalling by Wnt 10 326 4.90E-04 0.00294052
   RHO GTPase effectors 9 295 9.84E-04 0.005904968
   Processing of DNA double-strand break ends 5 90 0.001106 0.006633812
   G2/M checkpoints 5 92 0.001218 0.007306391
   M Phase 9 306 0.001268 0.007606081
   Unfolded protein response (UPR) 6 149 0.001829 0.009145478
   Amino acid synthesis and interconversion (transamination) 4 61 0.001947
   0.009737152
   HDR through homologous recombination (HR) or single strand annealing
   (SSA) 5 122 0.004091 0.020454387
   Homology directed repair 5 128 0.004998 0.02499001
   Signalling by Rho GTPases 9 408 0.008327 0.041636644
   Serine biosynthesis 2 16 0.008486 0.042427933
   [119]Open in a new tab

   Six DEGs were located in the packaging of telomere ends pathway,
   including HIST1H2AC, HIST1H2BC, HIST1H2BJ, MID1IP1 and TERF2IP (S5
   Fig). Histones (HIST1H2AC, HIST1H2BC, and HIST1H2BJ) have been
   identified as essential for transcription regulation, DNA repair, DNA
   replication and chromosomal stability. Telomeres serve a critical role
   in chromosome end-protection and genomic stability. Telomeric Repeat
   Binding Factor 2, Interacting Protein (TERF2IP) functions as a
   regulator of telomeres and transcription and is involved in the
   regulation of telomere length and protection [[120]41]. TERF2IP is
   essential for negatively regulating telomere recombination and
   repressing homology-directed repair (HDR), which can affect telomere
   length [[121]41].

   For the down-regulated genes, they were primarily enriched in the
   biosynthetic processes ([122]S8 Table), such as lipid (GO:0008610),
   cholesterol, steroid and sterol biosynthesis in the DAVID results. In
   the reactome results, they were primarily enriched in the domains of
   metabolism, haemostasis and DNA replication ([123]Fig 5D) including 45
   pathways, such as the regulation of cholesterol biosynthesis by SREBP
   (SREBF) and cholesterol biosynthesis ([124]S9 Table). The key G1-phase
   cell-cycle regulator CCND3 (gene name, cyclin D3; fold change = 4.59; p
   value = 1.61E-16) was down-regulated in
   lariciresinol-4-β-D-glucopyranoside-treated cells.

Discussion

   Traditional Chinese medicine (TCM) may be an alternative approach for
   the treatment of influenza virus infection. Although the underlying
   mechanism remains elusive, substantial clinical evidence that clearly
   demonstrates the beneficial effect of TCM on influenza diseases is
   available. Due to the complex interactions network between TCM
   compounds and their anti-influenza targets, the transcriptome
   sequencing (RNA-seq) technology offers a systematic analysis for the
   study of TCM pharmacology. To gain insight into the molecular mechanism
   of lariciresinol-4-β-D-glucopyranoside action against influenza virus
   infection, our present study for the first time employed RNA-seq
   technology to analyse the global gene expression profiling of human
   alveolar epithelial (A549) cells that relate to the host antiviral
   response.

   Host gene expression profiling analysis revealed association of
   activated inflammatory response and defence responses in influenza A
   virus-infected cells ([125]S2 Table and [126]Fig 3E). Moreover, our
   results also proved that the expression of RIG-I signalling or
   antiviral response-related genes, such as DDX58, NLRC5, IRF7, ISG15 and
   TNFAIP3 elevated significantly at 24 h after influenza virus infection
   ([127]Fig 3F and [128]S2 Table). All these data are consistent with
   previous findings that genes involved in innate immunity and
   pro-inflammatory responses were elicited by challenge with human or
   avian influenza A virus [[129]20, [130]42, [131]43].

   Go enrichment analysis showed that the DEGs directly regulated by
   lariciresinol-4-β-D-glucopyranoside treatment were mainly located in
   immune system and defense response to virus (GO:0051607) ([132]Fig 4E
   and [133]S5 Table). We observed that influenza A virus infection
   induced up-regulated expression of cytokines/chemokines, such as IL-6,
   IL8, CCL2, CX3CL1, CCL3 and CXCL3, were all decreased upon
   lariciresinol-4-β-D-glucopyranoside treatment ([134]Fig 4B, [135]Fig 4D
   and [136]S4 Table). IKBKE (also known as IKK-i), an IKK-related kinase,
   had a 4.94-fold decrease in lariciresinol-4-β-D-glucopyranoside
   treatment group ([137]Fig 4D and [138]S4 Table), which stimulates NF-κB
   signal transduction via activation of p65/RelA and trigger inflammatory
   responses [[139]44, [140]45]. Previous study had demonstrated that
   IKBKE knockout attenuated inflammation and had a protective effective
   against diet-induced obesity [[141]46]. We also noted that
   lariciresinol-4-β-D-glucopyranoside treated decreased the
   NF-κB-regulated genes encoding for the acute phase marker SAA1
   [[142]47] and inflammatory cytokine IL-6 were down-regulated (8.84 and
   2.41-fold decrease, respectively) ([143]Fig 4D and [144]S4 Table),
   which reflects the systemic inflammatory response and key factor in
   symptom formation in influenza. These data are consistent with our
   previous finding that lariciresinol-4-β-D-glucopyranoside inhibited
   influenza virus-induced pro-inflammatory reaction via down-regulated
   the activation of NF-κB.

   Moreover, TRIM25 is a key component of the RIG-I signaling pathway and
   subsequently initiate host antiviral immunity to RNA viruses infection
   [[145]48], which was up-regulated 2.23-fold by
   lariciresinol-4-β-D-glucopyranoside treatment ([146]Fig 4D and [147]S4
   Table). The vital role of TRIM25 in the IFN induction and
   RIG-I-dependent restriction of multiple RNA viruses has emerged from
   TRIM25-deficient study [[148]48, [149]49]. The up-regulation of TRIM25
   suggests that the transcript levels of the RIG-I signalling downstream
   genes type I/III interferons may also be elevated in infected cells
   with lariciresinol-4-β-D-glucopyranoside treatment. Accordingly, our
   results also clearly demonstrated that the expression of type III
   interferons (IFNL2 and IFNL3) were increased (1.93 and 1.90-fold
   increase, respectively) by lariciresinol-4-β-D-glucopyranoside
   treatment ([150]Fig 4D and [151]S4 Table). Interferons exerts their
   antiviral activity by inducing expression thousands of IFN-inducible
   antiviral effectors, such as MX1, OAS, IGS15, viperin and IFITM3
   [[152]50]. Interestingly, the expression of the IFN-inducible antiviral
   effector RSAD2 (radical S-adenosyl methionine domain containing 2, also
   known as viperin), belonging to the radical SAM superfamily that
   inhibits influenza virus release by disrupting lipid raft formation
   [[153]51], was increased 3.17-fold as compared with that of
   viral-infected group ([154]Fig 4D and [155]S4 Table). But the
   expression of other the antiviral effector TRIM22, IFITM1, IFI27, BST2
   (PDCA-1) and IFITM3 were not further up-regulated by
   lariciresinol-4-β-D-glucopyranoside treatment ([156]Fig 4D and [157]S4
   Table). Increased expression of antiviral defence-related genes
   (TRIM25, IFNL2, IFNL3 and RSAD2) has provided some understanding into
   the mechanism of the antiviral effect of
   lariciresinol-4-β-D-glucopyranoside reported in our previous study,
   that’s lariciresinol-4-β-D-glucopyranoside treatment may enhance the
   host intrinsic antiviral immunity against viral infection.

   There is growing evidencing that metabolism, such as lipid metabolism
   or amino acid (AA) metabolism, plays a potential role in viral
   infection [[158]40, [159]52]. For instance, phospholipase D (PLD)
   catalyses of the hydrolysis phosphatidylcholine (PC), whereas it is
   reported that PLD can facilitate the entry of influenza virus
   [[160]53]. Consistent with these, the present gene expression profiling
   showed some genes related to metabolism were altered influenza virus
   infection, such as LAT4H (leukotriene A4 hydrolase),
   IDO1(indoleamine-2,3-dioxygenase 1), PTGES (prostaglandin E synthase),
   ASS1 (argininosuccinate synthase 1) ([161]Fig 4B, [162]Fig 4D and
   [163]S4 Table). PTGES expression was up-regulated upon influenza virus
   influenza and then contribute to PGE2 generation, which has been shown
   to increase viral replication via inhibition of IRF3-mediated type I
   IFN signaling pathway [[164]38]. Infection with influenza virus has
   also been shown to increase the expression level of IDO1 in vitro and
   in vivo [[165]54, [166]55]. Tryptophan (Trp) is an α-amino acid that
   can be metabolized by IDO1 to produce kynurenine (Kyn). Administration
   of a specific inhibitor of IDO led to elevation of influenza-specific
   CD8^+ T-cells in influenza-infected mice [[167]54]. IDO knockout mice
   were protected from lethal influenza challenge [[168]56]. In our
   transcriptome data, gene expression of PTGES and IDO1 induced by
   influenza virus infection were down-regulated by
   lariciresinol-4-β-D-glucopyranoside treatment ([169]Fig 4B, [170]Fig
   4D, [171]S4 Table and [172]S8 Table). These results suggest that
   lariciresinol-4-β-D-glucopyranoside modulated the host lipid metabolism
   and amino acid (AA) metabolism against influenza infection.

   Functional enrichment analysis suggests that some DEGs regulated by
   lariciresinol-4-β-D-glucopyranoside treatment were involved in cell
   cycle and DNA repair ([173]Fig 4E and [174]Fig 5C). Indeed, it was
   showed that many of the DEGs related to cell cycle (CDKN1A, CCND3,
   GADD45a, GADD45b, MCM5, MCM7, ID1) and DNA damage (ATF3, ATF4, TIPARP,
   BBC3) were observed in the infected-cell with or without
   lariciresinol-4-β-D-glucopyranoside treatment ([175]Fig 4D, [176]Fig 6E
   and [177]Fig 6F). Many viruses manipulate host cell division cycle to
   support their own replication [[178]57–[179]59]. Cell cycle analysis
   demonstrated that the induction of G0/G1 phase cell cycle arrest is
   associated with influenza virus efficient replication [[180]9,
   [181]11]. Compared to cell cycle arrest at G2/M phase, cell cycle G0/G1
   phase arrest is the most optimal condition for influenza virus protein
   synthesis [[182]9]. Our results revealed that
   lariciresinol-4-β-D-glucopyranoside treatment increased expression of
   GADD45 (GADD45A, GADD45B) ([183]Fig 4D, [184]Fig 6E, [185]S4 Table and
   [186]S7 Table). In fact, many studies have found overexpression of
   GADD45, one of p53 target genes in response to DNA damage, causes G2/M
   cell cycle arrest in different human cell lines [[187]60, [188]61].
   Based upon the accumulated evidence and our findings, we have
   reasonable to speculate lariciresinol-4-β-D-glucopyranoside
   up-regulated the expression of GADD45 leading to cell cycle arrest at
   G2/M phase and decreased the synthesis of influenza virus proteins.
   Likewise, overexpression of ID1 inhibited the phosphorylation of
   p16^INK4a and RhoA [[189]62]. It was found that inhibition of
   phosphorylation of p16^INK4a and RhoA by influenza virus NS1 protein
   were correlated with cell cycle G0/G1 phase arrest [[190]9], thus
   establishing favorable conditions for progeny virus production.
   Although the expression of ID1 in the influenza virus-infected cells
   (CV) was comparable to levels in uninfected cells (C),
   lariciresinol-4-β-D-glucopyranoside treatment showed a prominent
   decrease in the expression of ID1 ([191]Fig 6F and [192]S7 Table).
   Thereby, we suppose that the down-regulation of ID1 by
   lariciresinol-4-β-D-glucopyranoside leads to increase the activity of
   p16^INK4a and RhoA, which would inhibit the cell cycle arrest at the
   G0/G1 cell phase for viral replication.

Fig 6. Heatmap depicting the emerging DEGs.

   [193]Fig 6
   [194]Open in a new tab

   E) Heatmap of 70 DEGs that was up-regulated solely by
   lariciresinol-4-β-D-glucopyranoside treatment (high expression: yellow;
   low expression: green). F) Heatmap of 96 DEGs that was down-regulated
   solely by lariciresinol-4-β-D-glucopyranoside treatment (high
   expression: yellow; low expression: green).

   As identifying novel molecular targets involved in
   lariciresinol-4-β-D-glucopyranoside treatment is important, we observed
   the up-regulation of telomere end-packaging genes, such as HIST1H2AC,
   HIST1H2BC, HIST1H2BJ, MID1IP1 and TERF2IP ([195]Fig 6E and [196]S8
   Table). However, the expression of these genes (HIST1H2AC, HIST1H2BC
   and MID1IP1) were only slightly down-regulated in influenza
   virus-infected cells (CV), but did not reach statistical significance
   ([197]S2 Fig). Telomeres are essential for chromosome end-protection
   and genome stability. Dysregulation of telomeres has been implicated in
   many human diseases, including atherosclerosis, cancer, cardiovascular
   and infectious diaseaes [[198]63–[199]66]. Virus infection has been
   reported to alter host cell telomere maintenance and shorten the length
   of telomeres, which lead to DNA damage [[200]66–[201]68]. Vaccinia
   virus (VACV)- and cytomegalovirus (CMV)-specific CD4^+ T cells have
   longer telomere lengths than IAV-specific CD4^+ T cells [[202]68]. A
   previous study reported that HeLa cells with influenza virus/B/Lee/40
   infection significantly decreased the activity of telomerase on day 18
   post-infection [[203]69]. A recent study reveals that influenza
   virus-induced inflammation contribute to cellular oxidative DNA damage,
   which may play a role in the pathogenesis of influenza diseases
   [[204]70]. Down-regulated expression of TERF2IP has been shown to
   trigger telomere dysfunction-induced DNA damage [[205]71, [206]72],
   whereas lariciresinol-4-β-D-glucopyranoside treatment increased
   expression of TERF2IP. Although we did not observe prominent changes in
   the gene expression of the packaging of telomere ends by influenza A
   virus infection. We postulated that maybe the time-point of infection
   do not alter the expression of telomere-associated genes. These results
   suggests that lariciresinol-4-β-D-glucopyranoside may have beneficial
   effect in telomere protection and thus protect from influenza
   virus-induced telomere-related DNA damage.

Conclusions

   Our data provide insight into the transcriptome profile of host
   responses that are involved in the complex interplay of molecular
   pathways of virus-host interactions at the cellular level in lung
   epithelial A549 cells and a systematic understanding of the
   pharmacological mechanism of lariciresinol-4-β-D-glucopyranoside
   against viral infection.

Supporting information

   S1 Fig. Enrichment analysis of 125 DEGs, which were down-regulated by
   influenza virus infection.

   Enrichment analysis showed that 125 DEGs which were down-regulated by
   influenza virus infection but not altered by
   lariciresinol-4-β-D-glucopyranoside treatment, enriched in the
   metabolism, gene expression and metabolism of proteins.

   (TIF)
   [207]Click here for additional data file.^ (2.6MB, tif)
   S2 Fig. The DEGs located in the packaging of telomere ends pathway in
   lariciresinol-4-β-D-glucopyranoside treatments cells.

   The expression of these genes was altered at least fold change ≥ ± 1.5
   and p-value < 0.05, but not in accordance with the criteria in
   virus-infected group (CV).

   (TIF)
   [208]Click here for additional data file.^ (6.8MB, tif)
   S1 Table. List of influenza A virus-induced DEGs which was not altered
   by lariciresinol-4-β-D-glucopyranoside treatment.

   The expression of these genes was altered at least fold change ≥ ± 1.5
   and p-value < 0.05.

   (XLSX)
   [209]Click here for additional data file.^ (23KB, xlsx)
   S2 Table. Enriched GO categories and KEGG pathway of influenza A
   virus-induced DEGs, which were not altered by
   lariciresinol-4-β-D-glucopyranoside treatment.

   GO enrichment analysis showed that these DEGs induced by influenza
   virus infection were enriched in antiviral defence, type I interferon
   signaling pathway and innate immune response. KEGG pathway enrichment
   analysis showed that these DEGs induced by influenza virus infection
   were enriched in pathways such as TNF signaling pathway, NOD-like
   receptor signaling pathway and chemokine signaling pathway.

   (XLSX)
   [210]Click here for additional data file.^ (79.2KB, xlsx)
   S3 Table. The enriched KEGG pathway of 125 DEGs, which were
   down-regulated by influenza virus infection.

   KEGG pathway enrichment analysis showed that 125 DEGs, which were
   down-regulated by influenza virus infection but not altered by
   lariciresinol-4-β-D-glucopyranoside treatment, enriched in 28 pathways
   such as L13a-mediated translational silencing of Ceruloplasmin
   expression, eukaryotic Translation Initiation and synthesis of bile
   acids and bile salts via 27-hydroxycholesterol.

   (XLSX)
   [211]Click here for additional data file.^ (12.8KB, xlsx)
   S4 Table. List of influenza A virus-induced DEGs, which were directly
   regulated by lariciresinol-4-β-D-glucopyranoside treatment.

   The expression of these genes was altered at least fold change ≥ ± 1.5
   and p-value < 0.05.

   (XLSX)
   [212]Click here for additional data file.^ (19.1KB, xlsx)
   S5 Table. Enriched GO categories and KEGG pathway of influenza A
   virus-induced DEGs, which were directly regulated by
   lariciresinol-4-β-D-glucopyranoside treatment.

   GO enrichment analysis showed these DEGs directly regulated by
   lariciresinol-4-β-D-glucopyranoside were enriched in defence response
   to virus, immune response and antiviral defence. KEGG pathway
   enrichment analysis showed that these DEGs were enriched in pathways
   such as chemokine signaling pathway, cytokine-cytokine receptor
   interaction and T cell receptor signaling pathway.

   (XLSX)
   [213]Click here for additional data file.^ (45.5KB, xlsx)
   S6 Table. The enriched KEGG pathway of 62 DEGs, which were
   down-regulated by lariciresinol-4-β-D-glucopyranoside treatment.

   KEGG pathway enrichment analysis showed that 62 DEGs down-regulated by
   lariciresinol-4-β-D-glucopyranoside treatment were enriched in pathways
   such as interleukin-7 signaling, prolactin receptor signaling and
   signaling by FGFR1 fusion mutants.

   (XLSX)
   [214]Click here for additional data file.^ (9.5KB, xlsx)
   S7 Table. List of emerging DEGs altered solely by
   lariciresinol-4-β-D-glucopyranoside treatment.

   Genes expression altered fold change ≥ ± 1.5 and p-value < 0.05 were
   considered as emerging DEGs, but not in accordance with the criteria in
   virus-infected group (CV).

   (XLSX)
   [215]Click here for additional data file.^ (15.3KB, xlsx)
   S8 Table. Enrichment analysis of biological process GO terms of
   emerging DEGs by lariciresinol-4-β-D-glucopyranoside treatment.

   Genes expression that up-regulated or down-regulated at least fold
   change ≥ ± 1.5 and p-value < 0.05 were considered as emerging DEGs, but
   not in accordance with the criteria in virus-infected group (CV).

   (XLSX)
   [216]Click here for additional data file.^ (56.9KB, xlsx)
   S9 Table. The enriched KEGG pathway of 96 emerging DEGs, which were
   down-regulated solely by lariciresinol-4-β-D-glucopyranoside treatment.

   These emerging DEGs (Genes expression were down-regulated by at least
   1.5-fold change and p-value < 0.05 were considered as emerging DEGs,
   but the expression of these DEGs not in accordance with the criteria in
   virus-infected group (CV)) were enriched in 46 pathways such as
   activation of gene expression by SREBF, cholesterol
   biosynthesis,linoleic acid (LA) metabolism and MAP2K and MAPK
   activation.

   (XLSX)
   [217]Click here for additional data file.^ (17.5KB, xlsx)

Data Availability

   All relevant data are within the paper and its Supporting Information
   files. RNA-seq data have been deposited to the NCBI Gene Expression
   Omnibus (GEO) ([218]https://www.ncbi.nlm.nih.gov/geo/), the accession
   number is GSE93999.

Funding Statement

   This study was supported by the National Natural Science Foundation of
   China (Grant no. U1201227)
   ([219]http://www.nsfc.gov.cn/publish/portal1/); the Science and
   Technology Development Fund in Macao Special Administrative Region
   (Grant no. 019/2013/A1) ([220]http://www.fdct.gov.mo/about); the
   National Natural Science Foundation of China (Grant no. 81428024)
   ([221]http://www.nsfc.gov.cn/publish/portal1/); the Ministry of Science
   and Technology of China (Grant no. 2015DFM30010)
   ([222]http://www.most.gov.cn/index.htm); the Guangzhou Postdoctoral
   Research Foundation, China (Grant no. 2015LJ)
   ([223]http://www.hrssgz.gov.cn/gzbsh/); the Natural Science Foundation
   of Guangdong Province, China (Grant no. 2014A030310115)
   ([224]http://www.gdstc.gov.cn/); and the Scientific Research Project of
   College Directly Under the Guangzhou Education Bureau (Grant no.
   1201430183) ([225]http://www.gzedu.gov.cn/).

References