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RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus
Infection, Genetics and Evolution 80 (2020) 104206
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Research paper
RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus
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Xuexing Zhenga, , Yelei Zhua,c, Zhongxin Zhaoa, Lina Yana, Tong Xua, Xianwei Wangd, ⁎ Hongbin Hee, Xianzhu Xiaf, Wenwen Zhenga, Xianghong Xueb, a
Department of Virology, School of Public Health, Shandong University, Jinan 250012, China Division of Infectious Diseases of Special Animal, Institute of Special Animal and Plant Sciences, The Chinese Academy of Agricultural Sciences, Changchun 130122, China c Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China d College of Life Sciences, Shandong University, Qingdao 266237, China e College of Life Sciences, Shandong Normal University, Jinan 250014, China f Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Canine distemper virus Antiviral responses Macrophages Transcriptomic Differentially expression genes
Virulent morbillivirus infections, including Meals Virus (MeV) and Canine Distemper Virus (CDV), caused severe immune suppression and leukopenia, while attenuated vaccine strains developed protective host immune responses. However, the detailed molecular foundations of host antiviral responses were poorly characterized. In order to better understand the interactions between attenuated vaccine and host antiviral responses, the global gene expression changes in CDV-11-infected DH82 cells, a macrophage-derived cell line from canine, were investigated by transcriptomic analysis, and portions of results were confirmed with quantitative RT-PCR. The results exhibited that 372 genes significantly up-regulated (p < .01) and 119 genes were significantly downregulated (p < .01) in CDV-infected macrophages DH82 at 48 h p.i.. The enriched functions of the significantly up-regulated (p < .01) genes were closely associated with interferon stimulated genes (ISGs), chemokine genes and pro-inflammatory factor genes. Gene ontology and pathway analysis of differentially expressed genes (DEGs) revealed that the most significantly involved pathways in CDV-infected DH82 cells were NF-κB and TNF signaling pathway, cytokine-cytokine receptor interaction, and pathogen associated molecular patterns (PAMPs), such as Toll-like, RIG-I-like and NOD-like receptor signalings. Thus, the findings indicated that pattern recognition receptors (PRRs) possibly mediated host innate and protective antiviral immune responses in CDV-11 infected DH82 cells.
1. Introduction Canine distemper (CD), a highly contagious viral disease, caused by the agent of canine morbillivirus (formerly known as canine distemper virus, CDV) which belonged to the Morbillivirus genus within the Paramyxoviridae family and infected a broad host range of carnivores, including the Canidae, Procyonidae, Felidae, Mustelidae, Mephitidae, Ailuridae, Viverridae, Hyaenidae and Phocidae (Barrett, 1999; de Swart et al., 1995; Harder and Osterhaus, 1997; Ohashi et al., 2001; Summers and Appel, 2010) with multi-systemic disorders and severe immune suppression before clinical signs. Recently, it has been reported that CDV caused lethal outbreaks in nonhuman primates (Qiu et al., 2011; Sun et al., 2010) and endangered wildlife (Na et al., 2016). Persistent infection and immunosuppression have increased the host susceptibility
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to opportunistic infections, which mainly contributes to canine morbillivirus-associated morbidity and mortality (Pillet and von Messling, 2009; Xue et al., 2019). Primary replication of CDV in ferret and dog took place in the lymphatic tissue of the respiratory tract. Virulent CDV infected macrophages rapidly and massively, circulating B and T lymphocytes (Ferreira et al., 2010; Lemon et al., 2011) based on the bindings to signaling lymphocyte activation molecule (SLAM) with its hemagglutinin (H) protein (Pratakpiriya et al., 2012; Tatsuo et al., 2000; von Messling et al., 2004), and then developed severe immune suppression by damaging secondary lymphatic organs including spleen, lymph nodes, and gut-associated and mucosal lymphoid tissues (Sawatsky et al., 2018). However, an avirulent CDV infection caused transitory immune suppression in ferrets and dogs, and developed robust protective immune responses (von Messling et al., 2004). Not only
Corresponding authors. E-mail addresses: [email protected] (X. Zheng), [email protected] (X. Xue).
https://doi.org/10.1016/j.meegid.2020.104206 Received 15 October 2019; Received in revised form 18 January 2020; Accepted 22 January 2020 Available online 23 January 2020 1567-1348/ © 2020 Published by Elsevier B.V.
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receptor choice but also post entry host cell control and immune evasion mechanisms determined morbillivirus tropism (von Messling et al., 2004). Macrophages are cellular components of the innate immune system that reside in virtually all tissue and contribute to immunity, repair, and homeostasis (Tugal et al., 2013). The present work demonstrated that the attenuated vaccine strain CDV-11 replicated massively without the typical syncytia in DH82 cells. Thus, macrophages might play a crucial role during CDV early proliferation and infection in vitro. In this study, we employed RNA-sequencing and transcriptomic analyses to find out the comprehensive host antiviral responses at transcriptional levels in DH82, a cell line of macrophages, infected with CDV-11 strain. The results demonstrated that CDV infection in DH82 cells caused a strong induction of genes involved in host innate immune responses and inflammatory responses, which may provide new heights of understanding the host antiviral responses of CDV and finding out the potential therapeutic targets.
cells/well) were infected with CDV-11 at an MOI of 0.1 and grew at 37 °C in a 5% CO2 humidified cell incubator. After virus infection, the cells and supernatants were collected at 24 h, 48 h, 72 h, 96 h and 120 h for determination of virus titers as described in Section 2.3.
2. Materials and methods
Purified RNA samples were submitted to Aksomics Bio-tech Group. Inc. (Shanghai, China) for transcriptomic sequencing of mRNA with three independent biological replicates. A detailed description of the Aksomics Bio-tech company RNA transcriptomic procedures can be found at http://www.aksomics.com/. We enriched RNA from intact total RNA by oligo (dT) and removed the rRNA of the degraded total RNA from DH82 cells infected with or without CDV-11. Random primers were used to synthetize the first strand cDNA from RNA fragments. Based on dUTP, the second strand cDNAs were synthesized. Double-strand cDNA were added dATP to the ends and then Illumina adaptor ligation and PCR amplification for library data. Finally, the library data were detected by Agilent 2100 for quality control and quantified with qPCR. The library was sequenced with Illumina Hiseq 4000 and the raw data were obtained for further analysis.
2.5. Total RNA preparation Total RNA samples of the mock-infected and CDV-11-infected DH82 cells were lysed using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Each sample was prepared for three replications. The RNA integrity and gDNA contamination were tested by Denaturing Agarose Gel Electrophoresis, and the total RNA quantification and quality assurance by spectrophotometer NanoDrop ND1000. 2.6. Transcriptomic sequencing
2.1. Cell culture and virus Vero cells (ATCC-81) and DH82 cells (ATCC-CRL-10389), a cell line from canine macrophages, were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA), 1000 U/mL penicillin (Gibco, CA, USA) and 100 μg/mL streptomycin (Gibco, CA, USA). The attenuated live vaccine CDV-11 strain was gifted by the QiLu animal health products limited company in China. The CDV-11 strain was propagated in Vero and DH82 cells following six passages. The virus titers, determined in Vero and DH82 cells, were calculated by the method of Reed & Muench based on cytopathic effects (CPEs) or indirect immunofluorescent assay (IFA).
2.7. Transcriptomic data analysis
2.2. Indirect immunofluorescent assay (IFA)
The raw data of mRNA sequencings from CDV-11-infected and mock-infected DH82 cells with replicates were averaged. The data size of the RNA-sequencing was 6G of paired-end sequencing. The reference genome was CanFam3.1. The alignments between rata data and the reference genome were used with Hisat2 Software (v2.0.4) (Kim et al., 2015). The transcription abundance of the rata data were estimated using StringTie Software (v1.2.3) (Pertea et al., 2015) in comparison of the genomes of reference annotation information in official database. The FPKM (Fragments Per Kilobase of gene/transcript model per Million mapped fragments) (Mortazavi et al., 2008) values of genes and transcription levels were calculated with Ballgown Software (Frazee et al., 2015), and the differentially expressed genes and transcriptions were screened between samples or groups. The differentially expression genes from inter-groups of the mock- and CDV-infected samples were filtered out by mean FPKM > 0.5. Finally, the Clustering, Gene Ontology (GO) (The Gene Ontology, 2019) and pathway enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Huang da et al., 2009) (http://david.abcc. ncifcrf.gov/) to annotate the functions of differentially expression genes (DEGs) with KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa et al., 2019) pathways. A cutoff of p ≤ .05 for KEGG pathway was analyzed (http://www.geneontology/).
CDV in cultured cells were detected by IFA as described previously (Wang et al., 2012). Briefly, Vero and DH82 cells were infected with CDV-11 at an MOI of 0.1 in DMEM supplement with 2% FBS at 37 °C for 72 h (h). Discarding the supernatant, the cells were fixed with chilled 80% acetone for 30 min (min) at room temperature, and washed for 3 times with PBS. The cells were incubated with murine anti-CDV-N monoclonal antibody (VRMB, WA, USA) at a dilution of 1:500 for 1 h at 37 °C, and then stained with FITC-labeled goat anti-mouse lgG (TransGen, Beijing, China) for 1 h. Finally, the cells were examined under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The percentages of N-positive cells were calculated with reference to the total number of cells. 2.3. Titration of CDV-11 Virus Titers were performed in 96-well plates. Monolayer of Vero or DH82 cells were infected with 50 μL of viral suspensions at serial 10fold dilutions (from 10−1 to 10−8) for six replicates and incubated at 37 °C for 72 h. Discarding the supernatant, the DH82 cells were incubated with anti-CDV-N monoclonal antibody at 37 °C for 1 h and stained with FITC-labeled goat anti-mouse antibody at 37 °C for 1 h. Finally, the results were examined under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The results of virus titers of CDV-11 in Vero cells were determined by CPEs. The 50% tissue cultures infection dose (TCID50) of CDV-11 was calculated according to the method of Reed and Muench [15].
2.8. Statistical analysis Results were presented as mean ± standard deviation (SD). Similar results were obtained from at least three independent experiments. Statistical significance were analyzed by one-way ANOVA (Dunnett's ttest) and considered at p < .05. * means p < .01, ** means p < .0001.
2.4. Growth curves Monolayer of Vero and DH82 cells in 6-well culture plates (5 × 105 2
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Fig. 1. Growth characteristics of CDV-11 in Vero and DH82 cells. (A, B, E, F) Light fields of negative Vero (A) and DH82 (E) cells, and the CDV-11-infected Vero (B) and DH82 (F) cells. (C, D, G and H) Indirect Immunofluorescence assay of the Vero (C and D) and DH82 (G and H) cells incubated with CDV-11 virus (D and H) or mock 2% DMEM (C and G). The cells were fixed with 4% paraformaldehyde and stained with primary antibody of anti-CDV N protein (1:500 dilution) and FITC labeled goat anti-mouse IgG (1:100 dilution) at 72 h p.i.. (I) One-step growth curves of CDV-11 in Vero and DH82, respectively. Cells, infected with CDV-11, were collected at the indicated time points, and the viral productions were titrated with TCID50 assay. These data were averaged from three independent experiments, and the error bars indicate standard deviations.
3. Results
cells, monolayer of Vero and DH82 cells were inoculated with CDV-11 at an MOI of 0.1 for 1 h at 37 °C, and then cultured for 96 h with fresh DMEM supplement with 2% FBS. Typical syncytia were observed in CDV-11 infected Vero cells. Surprisingly, the DH82 cells infected with CDV-11 were no cytopathy or syncytia (Fig. 1F) in comparison of the
3.1. Growth characteristics of CDV-11 in Vero and DH82 cells To examine the growth characters of CDV-11 in Vero and DH82 3
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Fig. 2. Gene expression pattern in DH82 infected with CDV-11 at 48 h. (A) The Heatmap of correlation values among the total RNA samples from infected with CDV11 group or mock group. The dark blue of the Heatmap represented the high correlation and the light blue represented the low correlation. (B) Cluster analysis of differentially expressed genes between CDV-11 infection and mock group in DH82 at 48 h post infection were exhibited in a heat map. Each row showed the relative expression level for a single gene and each column showed the expression level of a single sample. The genes were required to satisfy a cutoff p < .01 in direct comparison. Red represented genes with increased expression and green represented genes with decreased expression. (C) Volcano Plot of differentially expressed genes in CDV-11 infected DH82 cells compared to mock group. The x-axis meant log 2 of fold changes, and the y-axis meant log 10 of p-values. The two green vertical lines were up-regulated (right) and down-regulated (left) respectively. The green parallel line corresponded to the threshold of p-value. Green dots represented significantly downregulated expression genes, red dots represented significantly up-regulated expression genes, and black dots represented no significantly difference expressed genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
mock-infected DH82 cells (Fig. 1E). The specific CDV N protein in CDV11-infected DH82 and Vero cells were examined with CDV anti-N monoclonal antibodies (MAbs) at 72 h p.i., which demonstrated strong
reaction signals with the anti-N MAbs in both cells (Fig. 1D and H), and no signals in mock-infected cells (Fig. 1C for Vero cells, Fig. 1G for DH82 cells). To assess growth curves of CDV-11 in both cells, 4
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Table 1 The top 50 up-regulated expressed genes in CDV-11 infection DH82 cells compared to the mock controls. GenBank accession
Gene symbol
Full name
Log2(fold change)
p-Value
NM_001003134.1 XM_003639053.4 XM_860510.5 XM_005636024.3 XM_005622027.3 NM_001003133.1 XM_545847.6 XM_537104.6 XM_843271.5 NM_001003010.2 XM_005626701.3 NM_001010949.1 XM_014116565.2 XM_548077.6 NM_001048091.1 NM_001135787.1 XM_014116241.2 XM_022413453.1 XM_005618758.3 XM_014117383.2 NM_001253787.1 XM_005635291.3 XM_845787.5 XM_846183.4 NM_001197095.1 XM_022404348.1 XM_855170.5 NM_001010960.1 XM_022426251.1 XM_532793.6 XM_545493.5 NM_001005250.1 XM_005616331.3 XM_005623417.3 XM_535344.6 NM_001130836.1 NM_001003297.1 XM_014122423.2 XM_005624038.3 XM_022413781.1 XM_022415661.1 XM_845924.4 XM_539422.6 XM_537413.6 XM_005636026.3 NM_001003200.1 XM_014112321.2 XM_005637591.3 XM_022407392.1 NM_001314132.1
MX1 ISG15 BST2 OAS1 IFI44 MX2 ISG20 IFI44L IFIT1 CCL5 DDX58 CXCL10 DHX58 IFI35 OAS3 IFNB1 RNF213 HERC6 IFIT2 APOL5 Oasl1 HELZ2 PARP14 RSAD2 SERPINE1 EIF2AK2 IRF9 CCL7 MB21D1 IDO1 IFIH1 CCL4 PPP1R15A GCH1 IFI6 TNFSF10 CCL2 IL18BP LGALS3BP DTX3L SRPX2 EHD4 SAMD9L NFKBIA OAS2 CXCL8 TNFAIP3 UBTD1 TMEM236 SIX1
MX dynamin like GTPase 1 ISG15 ubiquitin-like modifier Bone marrow stromal cell antigen 2 2′-5′-oligoadenylate synthetase 1 Interferon induced protein 44 MX dynamin like GTPase 2 Interferon stimulated exonuclease gene 20 Interferon induced protein 44 like interferon-induced protein with tetratricopeptide repeats 1 C-C motif chemokine ligand 5 DExD/H-box helicase 58 C-X-C motif chemokine ligand 10 DExH-box helicase 58 Interferon induced protein 35 2′-5′-oligoadenylate synthetase 3 Interferon beta 1 Ring finger protein 213 HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 Interferon induced protein with tetratricopeptide repeats 2 apolipoprotein L5 2′-5′-oligoadenylate synthetase like helicase with zinc finger 2 Poly(ADP-ribose) polymerase family member 14 Radical S-adenosy l methionine domain containing 2 Serpin family E member 1 Eukaryotic translation initiation factor 2 alpha kinase 2 Interferon regulatory factor 9 C-C motif chemokine ligand 7 Mab-21 domain containing 1 indoleamine 2 Interferon induced with helicase C domain 1 Chemokine (CeC motif) ligand 4 Protein phosphatase 1 regulatory subunit 15A GTP cyclohydrolase 1 Interferon alpha inducible protein 6 TNF superfamily member 10 C-C motif chemokine ligand 2 Interleukin 18 binding protein Calcium activated nucleotidase 1 Deltex E3 ubiquitin ligase 3 L Sushi repeat containing protein EH domain containing 4 Sterile alpha motif domain containing 9 like NFKB inhibitor alpha 2′-5′-oligoadenylate synthetase 2 C-X-C motif chemokine ligand 8 TNF alpha induced protein 3 Ubiquitin domain containing 1 Transmembrane protein 236 SIX homeobox 1
7.22 6.92 5.30 5.20 5.17 5.17 5.06 5.02 4.81 4.45 4.36 4.30 4.27 4.06 3.87 3.81 3.75 3.44 3.37 3.32 3.25 2.99 2.95 2.94 2.78 2.67 2.63 2.58 2.51 2.48 2.45 2.41 2.36 2.35 2.32 2.24 2.23 2.23 2.23 2.21 2.17 2.16 2.15 2.14 2.13 2.12 2.09 2.09 2.05 2.02
4.25E-06 8.26E-07 1.56E-06 3.77E-05 1.88E-08 1.12E-07 1.89E-05 5.70E-06 6.35E-07 4.95E-03 5.47E-07 9.29E-06 9.639E-06 5.66E-05 1.04E-07 7.53E-07 2.03E-08 4.15E-06 6.53E-07 1.51E-05 1.73E-05 3.64E-07 1.68E-06 9.03E-08 2.44E-06 5.27E-07 1.24E-05 1.40E-05 4.93E-05 8.59E-06 2.89E-06 1.89E-04 8.00E-07 5.51E-05 8.20E-04 7.36E-04 3.07E-06 9.80E-05 6.66E-08 3.48E-06 2.72E-05 4.13E-02 3.62E-08 4.42E-06 5.67E-08 4.75E-05 1.15E-05 1.47E-04 3.53 E-02 4.53E-06
were about 0.99, and the correlation between mock and CDV-11-infected groups was more than 0.94 (Fig. 2A). After the filtration of the signals below the threshold level, genes showing at least a 1.5log 2 fold changes in expression with a cutoff of p ≤ .05, were considered to be significant differentially expressed. In summary, 491 differentially expressed genes were identified in CDV-11-infected groups at 48 h p.i. in comparison of the mock group, including 372 up-regulated and 119 down-regulated expression genes (Fig. 2B). The number of up-regulated genes was much more than that of down-regulated genes. The top 50 of up-regulated genes and top 20 down-regulated genes are presented in Tables 1 and 2. The 50 most up-regulated genes from CDV-11-infected macrophages in Table 1 were contained the interferon-stimulated genes (ISGs), such as ISG15, ISG20, OAS1, OAS3, IFIT1, IFI35, IFIT1, IRF9, IFI44L, IFNB1 and IFIT2; chemokine factor genes (CCL5, CXCL10, CCL7, CCL4) and pro-inflammatory cytokines (IL-18BP and TNFSF10). These data also demonstrated that the most up-regulated genes changed 148 folds in CDV-11-infected DH82 cells, while the most down-regulated genes decreased less than 1 fold. The changed folds of up-regulated genes were much higher than those of down-regulated genes.
monolayer of Vero and DH82 cells were infected with CDV-11 and the virus titrations were determined at 24 h p.i., 48 h p.i., 72 h p.i. and 96 h p.i., respectively (Fig. 1I). In DH82, CDV-11 exhibited a slightly decline in virus titers, finally reaching 105.5 TCID50/mL at 96 h p.i.. In contrast, virus titers peaked at 106.5 TCID50/mL at 72 h p.i. in Vero cells, and maintained a platform between 72 h p.i. and 96 h p.i., indicating that CDV-11 was sensitive to macrophages DH82 cells with a good duplication ability and the virus production were not significantly impaired. 3.2. Gene expression profiles in CDV-11-infected macrophages In order to understand molecular mechanisms of antiviral responses to CDV vaccine strain in DH82 at transcriptional levels, the total RNA profiles were analyzed with high throughput transcriptomic sequencing (Aksomics Bio-tech Group. Inc.). The total RNA samples were extracted from mock- and CDV-infected DH82 cells at 48 h p.i., and the homogeneity of three replications each group were evaluated based on correlation of gene expression levels and the DEGs between groups. The correlation of the repeated samples in mock- or CDV-11-infected group 5
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Table 2 The top 20 down-regulated expressed genes in CDV-11 infection DH82 cells compared to the mock controls. GenBank accession
Gene symbol
Full name
Log2(fold change)
p-Value
XM_539938.6 XM_849220.5 NM_001205114.1 XM_022403248.1 XM_548978.6 XM_022418589.1 NM_001177734.2 XM_014109177.1 XM_003432297.4 XM_532363.5 XM_005627946.3 XM_533158.6 XM_022417145.1 XM_005616779.3 NM_001002944.1 XM_547049.6 XM_014116110.2 XM_005636328.3 XM_845218.5 XM_014107229.2
VAMP5 RETN HAVCR1 ALX1 CFP MYOCD CD36 NDRG4 LRRC17 HYKK TMEM71 HIPK3 FAM184B DPY19L3 ADORA2B ITGAX STON2 ACACB HPSE2 LZTS3
Vesicle associated membrane protein 5 Resistin Hepatitis A virus cellular receptor 1 ALX homeobox 1 Complement factor properdin Myocardin CD36 molecule NDRG family member 4 Leucine rich repeat containing 17 Hydroxylysine kinase Transmembrane protein 71 Homeodomain interacting protein kinase 3 Family with sequence similarity 184 member B dpy-19 like C-mannosyltransferase 3 Adenosine A2b receptor Integrin subunit alpha X Stonin 2 Acetyl-CoA carboxylase beta Heparanase 2 (inactive) Leucine zipper tumor suppressor family member 3
−1.66 −1.33 −1.23 −1.17 −1.13 −1.07 −1.07 −1.02 −1.01 −1.01 −1.00 −0.94 −0.99 −0.96 −0.91 −0.89 −0.89 −0.88 −0.88 −0.86
4.39E-02 1.21E-03 1.87E-02 4.64E-02 2.57E-04 7.92E-02 3.48E-05 8.09E-06 3.42E-03 6.80E-05 1.12E-02 4.18E-02 3.95E-04 1.58E-05 1.02E-02 3.84E-03 2.93E-05 1.45E-03 1.62E-05 6.40E-04
dendritic cells (DC) in the airways preceded infection of lymphocytes in lymphatic organs in vivo (Ferreira et al., 2010). In the present study, the DH82, a cell line from canine macrophages, supported the attenuated CDV vaccine strain replication with no observed cytopathic effects. RNA transcriptomics, in combination with bioinformatics, had proved to be an efficient and high-throughput tool to establish gene expression patterns, to complete functional clustering, canonical pathways enrichment of the DEGs. Macrophages were infected by multiple viruses, such as human immunodeficiency virus (HIV) (DiNapoli et al., 2016), porcine reproductive and respiratory syndrome virus (PRRSV) (Kim et al., 2010; Zhang et al., 2018), influenza virus (Marvin et al., 2017), respiratory syncytial virus (RSV) (Borchers et al., 2013). CDV replications located primarily in monocytes and macrophages during the early stage of infection in vivo (von Messling et al., 2004). However, it was poorly addressed that the global gene expression profiles in macrophages responses to CDV infection. To better understand the interactions of CDV and macrophages at transcriptional levels, a genome wide gene expression profiles in DH82 cells were performed using transcriptomic analysis. Firstly, the growth characteristics of CDV-11 were examined in Vero and DH82 cells (Fig. 1). The CDV-11 exhibited a perfect replication and propagation in DH82, a cell line from canine macrophages, and viral yield in DH82 cells slightly descended in comparison of its in Vero cells. Then, an optimal moment of 48 h p.i. to perform transcriptomic analyses were determined by monitoring one-step growth curves of CDV-11 in DH82 cells. The results revealed that virus loading maintained a platform between 24 h p.i. and 72 h p.i.. Thus, the total RNA samples for transcriptomic analyses were extracted at 48 h p.i. for adequate data. In totally, 491 differentially expression genes were identified between mock- and CDV-infected DH82 cells at 48 h p.i., including 372 up-regulated and 119 down-regulated expression genes. The significantly up-regulated genes in DH82 responding to CDV-11 infection were ISGs (Mx1, ISG15, BST2, MX2, ISG20, OAS1, IFNB1, IFIT1, IFIT2, OAS3, Oasl1 and IRF9), chemokines (CCL5, CXCL10, CCL7, CCL4) and the pro-inflammatory factors (IL18BP, TNFSF10 and TNFAIP3). All these data were consistent with previous findings that genes involved in innate immunity, pro-inflammatory responses. Activation of IL-1β, TNF-α and CCL5 transcriptions were observed at high levels in RSV persistently infected macrophages. ISG15 modulates the antiviral response during replication of pseudorabies virus (Liu et al., 2018), hepatitis E virus (Sooryanarain et al., 2017), dengue and west nile virus (Dai et al., 2011). Mx and the Mx family of genes encoding large GTPases related to dynamin, showed antiviral activity against a wide
Unsupportively, clustering analysis of the expression profiles revealed a distinct gene signature between CDV- and mock-infected DH82 cells. The DEGs could be grouped into at least 9 subsets based on their modes of expression, which showed that the differentially expressed genes in CDV-11-infected DH82 cells changed greatly compared to the mockinfected DH82 cells at 48 h p.i. (Fig. 2C). 3.3. Confirmation of transcriptome data by qRT-PCR To validate the data from transcriptome sequencing, seven genes chosen randomly (BST2, CCL5, IRF9, TNFSF10, IL18BP, HPSE2) in the DEGs list were subjected to qRT-PCR. The results from qRT-PCR of increased or decreased gene expression levels for the tested genes showed no significant differences with transcriptomic analyses (Supplement Table 1, S3). 3.4. Functional groups and canonical pathways modulated by CDV-11 infection in DH82 cells To clarify the biological functions of the DEGs involved in CDV-11infected DH82 cells, the mostly associated gene ontology (GO) and pathways with DEGs were assessed. The top 10 function groups involving in biological processes, cellular component and molecular function, respectively, showed in Fig. 3A, B and C, such as immune system process, immune responses, negative regulation of viral process and viral life cycle, responses to external biotic stimulus, cytokine receptor binding, chemokine activity, transcriptional factor activity. These results indicated that the transcriptional alterations in CDV-infected macrophages mainly involved in biological processes closely related to antiviral and immune responses, and cellular function and maintenance, respectively. The top 10 pathways involved in responses to CDV-11 infection in DH82 cells were found out with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. The main pathways modulated during infection were those related to NF-κB signaling pathway, NOD-like receptor, RIG-I-like receptor and Toll-like receptor signal pathways, cytokine-cytokine receptor interaction and TNF signaling pathway (Fig. 3D). The DEGs involved in those pathways were listed respectively, and most of these genes were up-regulated (Table 3 and Fig. 4). 4. Discussion Morbillivirus infection of alveolar macrophages and subepithelial 6
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Fig. 3. Enrichment functional gene ontology (GO) and pathways of differentially expressed genes upon CDV-11 infection in macrophages DH82. (A-C) The top 10 significantly relevant GO class articles of Biological Process (BP) (A), Cellular Component (CC) (B) and Molecular Function (C) for differentially expressed genes in CDV-11 or mock-infected DH82 cells were categorized into functional groups. (D) The top 10 significantly involved pathways had been enriched upon CDV-11 infection in DH82. The x-axis (down) meant the enrichment scores, and the x-axis (up) meant number of genes. The y-axis showed GO class articles or signal pathways.
against various viruses (Sedewitz and Quann, 2015; Zhang et al., 2015; Zhang et al., 2013) and could also be the candidate effectors to inhibit CDV replication. CDV might make a similar attempt to control their ubiquitination, thus disturbing their antiviral functions. In addition, compared to the mock-infected DH82, the bone marrow stromal cell antigen 2 (BST2), which nonspecifically inhibited retrovirus (such as HIV, SIV and EIAV) (Neil et al., 2008; Yin et al., 2014) and lipid enveloped viruses (such as Ebola, Marburg, Lassa) (Andrey Tokarev et al., 2009) release and could be broadly expressed on IFN-α induction (Blasius et al., 2006), dramatically rose 39 times and accompanied by 5 times up-regulated of IFI6 (Interferon alpha inducible protein 6) in CDV-11 infected DH82 cells. The battle of viral replication and host immune responses was conclusive for animal's clinical outcome of viral infections. Host immune cells, recognizing viral components by pattern-recognition
range of RNA viruses (Karaulov et al., 2017; Randall and Goodbourn, 2008). Mx1 and ISG20 were strongly up-regulated in Influenza A virus infected macrophages but not in epithelial cells, suggesting cell-specific differences in induction of antiviral factors. Transcriptions of ISGs were triggered by nuclear localization of ISGF3 which was formed by the crucial DNA-binding protein IRF9 jointing with heterodimer of phosphorylated STAT1 and STAT2, finally resulting in the establishment of antiviral state in cells (Platanias, 2005; Randall and Goodbourn, 2008). It was widely accepted that the morbillivirus V protein interferes with type I IFN signals (Chinnakannan et al., 2013). This work proved that the type I IFN signals played crucial roles in host antiviral responses with the significantly up-regulation of the ISG15, ISG20 and IRF9 in CDV-11 infection macrophages. Mice lacking the ISG15 gene were more susceptible to infection by influenza, herpes, and sindbis virus (Lenschow et al., 2007). Mx1 and OAS1 exerted antiviral activity 7
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receptors (PRRs), initiated a rapid antiviral response to prevent the spread of the infection, while viruses had evolved the abilities to evade the host immune responses and develop the disease (Zhao et al., 2013). Gene Ontology (GO) and pathway enrichment analyses indicated that most of the up-regulated genes involved in NF-κB signaling pathway, NLRs, RLRs, TLRs signal pathways and cytokine-cytokine receptors interaction in CDV-11-infected DH82. CDV (Onderstepoort strain) induced human osteoclast genesis by activation of NF-κB pathways (Selby et al., 2006). PRRSV inoculation could activate NF-κB by means of I-κB degradation (Lee and Kleiboeker, 2005), leading the production of some inflammatory molecules, such as IL-6, IL-8 and IL-1β (Zhang et al., 2018). HBV induced autophagy to promote viral replication in vitro and in vivo through inhibiting NF-κB signaling pathway (Wang et al., 2018). Hundreds of ISGs, with functions of antiviral, immunomodulatory, cell growth regulatory and metabolic regulatory actions, were induced by NF-κB, and created an antiviral state, as well as a hub of cellular signal transduction pathways involved in host immune responses to virus challenges (Moynagh, 2005; Tong et al., 2017). Therefore, the findings indicated that host innate immune responses against CDV mainly developed the antiviral effects through the NF-κB signaling pathway. Sendai virus activated RIG-I, and was a virus model to stimulate robust type I interferon responses through RIG-I activation. VSV had been demonstrated to trigger specifically RIG-I innate immune signaling (Furr et al., 2010). RIG-I, as well as MDA5, was proved to induce IFN following infection of human cells with measles virus(Ikegame et al., 2010). The viral leader transcript of the measles virus was identified as a potential PAMP ligands of RIG-I (Plumet et al., 2013). In the present work, TNF-α, RIG-I, NF-κB and MyD88 were all significantly up-regulated in CDV-11-infected DH82s. The MyD88 was a major adaptor molecule downstream of several surface and cytosolic PRRs (Kader et al., 2017; Trinchieri and Sher, 2007). RLRs interacting with multi-component proteins, and TLRs dependent or independent on MyD88, both activated IRF3 and NF-κB to induce transcription of a variety of innate immune response genes, including IFNs, direct antiviral genes, and pro-inflammatory genes (Tong et al., 2017; Wilden et al., 2009). The inflammasome NLRP3, known as the member in NODlike-receptor-family, pyrin domain containing 3, was reported to activate the NLRP3 inflammasome, resulting in the caspase-1-mediated maturation of IL-1β (Zilliox et al., 2007). The NF-κB-induced activation of NLRP3 and pro-IL-1β gene expression was requisite for activating caspase-1 to further regulate the secretion of the inflammatory cytokines IL-1β and IL-18 (Motta et al., 2015). The increased TNF-α expression were associated with depletion of Foxp3+ Treg in acutely CDV-infected dogs (Qeska et al., 2013). In this study, nine genes involved in NF-κB signaling pathway, PRRs and downstream cytokines, including CCL4, CXCL8, DDX58, ICAM1, IL1R1, MYD88, NFKBIA, TNF, TNFaIP3 and TRIM25, were significantly up-regulated, and the NF-κB complex inhibitory protein IκB-β was obviously down-regulated. Lethal influenza A virus preferentially activated TLR3 and triggered a severe inflammatory response (Pothlichet et al., 2013). Constitutive TLR3dependent immunity protected cortical neurons from HSV-1 infection. TLR3 recognized double-stranded RNA and was involved in the inflammatory response to RSV infection. Therefore, further investigations needed to be explored how the PRRs worked the functions of antiviral and inflammatory responses during the early stage of CDV infection in lymphocytes.
0.07 0.08 0.07 0.08 3.45E-09 4.68E-09 5.12E-08 1.84E-07
5. Conclusion In this study, our data demonstrated a comprehensive knowledge of gene expression changes in DH82, a cell line from canine macrophages, infected with CDV-11 vaccine strain. The DEGs were indicated to involve in innate immune responses, such as NF-κB signaling pathway, NLRs, RLRs, TLRs signal pathways and cytokine-cytokine receptor interaction, which may provide clues for understanding the host antiviral
cfa04657 cfa04620 cfa04668 cfa05162
0.07 0.12 8.93E-10 1.44E-09
RIG-I-like receptor signaling pathway Cytokine-cytokine receptor interaction IL-17 signaling pathway Toll-like receptor signaling pathway TNF signaling pathway Measles cfa04622 cfa04060
0.11 1.35E-10 NOD-like receptor signaling pathway cfa04621
0.12 6.88E-11 Herpes simplex infection cfa05168
0.09 6.70E-13 NF-κB signaling pathway cfa04064
0.12 1.41E-13 Influenza A cfa05164
ADAR//CCL2//CCL5//CXCL10//CXCL8//DDX58//EIF2AK2//ICAM1//IFIH1//IFNB1//IRF9//MX1//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1// OAS2//OAS3//PML//RNASEL//RSAD2//TICAM1//TLR3//TNF//TNFSF10//TRIM25 BIRC3//CCL4//CD40//CFLAR//CXCL8//DDX58//ICAM1//IL1R1//MYD88//NFKB1//NFKB2//NFKBIA//PLAU//RELB//TICAM1//TNF//TNFAIP3// TNFRSF11A//TNFSF13B//TRIM25 CASP8//CCL2//CCL5//CFP//DDX58//DLA-64//EIF2AK2//FOS//IFIH1//IFNB1//IRF9//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1//OAS2//OAS3// PML//RNASEL//TAP1//TICAM1//TLR3//TNF//TNFRSF14 BIRC3//CASP8//CCL2//CCL5//CXCL8//GBP5//GSDMD//IFNB1//IRF9//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1//OAS2//OAS3//P2RX7// PANX1//RNASEL//TANK//TICAM1//TNF//TNFAIP3 AZI2//CASP8//CXCL10//CXCL8//DDX58//DHX58//IFIH1//IFNB1//ISG15//NFKB1//NFKBIA//NFKBIB//TANK//TNF//TRIM25 CCL17//CCL2//CCL4//CCL5//CCL7//CD40//CSF1//CXCL10//CXCL14//CXCL8//CXCR4//HGF//IFNB1//IL15RA//IL18RAP//IL1R1//OSMR// PDGFB//PDGFRB//TNF//TNFRSF11A//TNFRSF12A//TNFRSF14//TNFSF10//TNFSF13B//TNFSF18//VEGFA CASP8//CCL17//CCL2//CCL7//CEBPB//CXCL10//CXCL8//FOS//FOSL1//MMP1//MMP9//NFKB1//NFKBIA//TNF//TNFAIP3//USP25 CASP8//CCL4//CCL5//CD40//CD80//CXCL10//CXCL8//FOS//IFNB1//IRF5//MYD88//NFKB1//NFKBIA//SPP1//TICAM1//TLR3//TNF BCL3//BIRC3//CASP8//CCL2//CCL5//CEBPB//CFLAR//CSF1//CXCL10//FOS//ICAM1//MMP9//NFKB1//NFKBIA//TNF//TNFAIP3 ADAR//CCND3//DDX58//EIF2AK2//IFIH1//IFNB1//IRF9//MX1//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1//OAS2//OAS3//TNFAIP3//TNFSF10
Genes GeneRatio Fisher-p value Definition Pathway ID
Table 3 Significant pathways of the differential expressed genes in DH82 cells infected with CDV.
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Fig. 4. The simplified models of signal pathways of the DEGs involved in immune response process. Significantly up-regulation or down-regulation genes were represented in red or green, respectively, and the gray meant no changes. The dotted lines showed omitted signal pathways between the signal transfers, and the solid lines showed direct signal transfers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
responses during CDV infection and finding out the targets of therapy. However, further functional elucidations remained to be further investigated for revealing the molecule mechanisms of pathogenic and immune responses during CDV infection.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2020.104206. References
Declaration of Competing Interest von Messling, Veronika, Milosevic, Dragana, Cattaneo, R., 2004. Tropism illuminated: lymphocyte-based pathways blazed by lethal morbillivirus through the host immune system. Proc. Natl. Acad. Sci. U. S. A. 101, 14216–14221. Andrey Tokarev, M.S., Fitzpatrick, K., Guatelli, J., 2009. Antiviral activity of the interferon-induced cellular protein BST-2/Tetherin. AIDS Res. Hum. Retrovir. 25, 1197–1211. Barrett, T., 1999. Morbillivirus infections, with special emphasis on morbilliviruses of carnivores. Vet. Microbiol. 69, 3. Blasius, A.L., Giurisato, E., Cella, M., Schreiber, R.D., Shaw, A.S., Colonna, M., 2006. Bone marrow stromal cell antigen 2 is a specific marker of type I IFN-producing cells in the naive mouse, but a promiscuous cell surface antigen following IFN stimulation. J. Immunol. 177, 3260–3265. Borchers, A.T., Chang, C., Gershwin, M.E., Gershwin, L.J., 2013. Respiratory syncytial virus–a comprehensive review. Clin. Rev. Allergy Immunol. 45, 331–379. Chinnakannan, S.K., Nanda, S.K., Baron, M.D., 2013. Morbillivirus v proteins exhibit multiple mechanisms to block type 1 and type 2 interferon signalling pathways. PLoS One 8, e57063.
No conflict of interest.
Acknowledgements We gratefully acknowledge the infrastructure support of the college of public health in Shandong University. This work was supported by The National Key Research and Development of China (2016YFD0501000), The National Nature Science Foundation of China (31902275, 31902308), and The Fundamental Research Funds of Shandong University (2018JC050).
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Research paper
RNA sequencing analyses of gene expressions in a canine macrophages cell line DH82 infected with canine distemper virus
T
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Xuexing Zhenga, , Yelei Zhua,c, Zhongxin Zhaoa, Lina Yana, Tong Xua, Xianwei Wangd, ⁎ Hongbin Hee, Xianzhu Xiaf, Wenwen Zhenga, Xianghong Xueb, a
Department of Virology, School of Public Health, Shandong University, Jinan 250012, China Division of Infectious Diseases of Special Animal, Institute of Special Animal and Plant Sciences, The Chinese Academy of Agricultural Sciences, Changchun 130122, China c Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China d College of Life Sciences, Shandong University, Qingdao 266237, China e College of Life Sciences, Shandong Normal University, Jinan 250014, China f Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130122, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Canine distemper virus Antiviral responses Macrophages Transcriptomic Differentially expression genes
Virulent morbillivirus infections, including Meals Virus (MeV) and Canine Distemper Virus (CDV), caused severe immune suppression and leukopenia, while attenuated vaccine strains developed protective host immune responses. However, the detailed molecular foundations of host antiviral responses were poorly characterized. In order to better understand the interactions between attenuated vaccine and host antiviral responses, the global gene expression changes in CDV-11-infected DH82 cells, a macrophage-derived cell line from canine, were investigated by transcriptomic analysis, and portions of results were confirmed with quantitative RT-PCR. The results exhibited that 372 genes significantly up-regulated (p < .01) and 119 genes were significantly downregulated (p < .01) in CDV-infected macrophages DH82 at 48 h p.i.. The enriched functions of the significantly up-regulated (p < .01) genes were closely associated with interferon stimulated genes (ISGs), chemokine genes and pro-inflammatory factor genes. Gene ontology and pathway analysis of differentially expressed genes (DEGs) revealed that the most significantly involved pathways in CDV-infected DH82 cells were NF-κB and TNF signaling pathway, cytokine-cytokine receptor interaction, and pathogen associated molecular patterns (PAMPs), such as Toll-like, RIG-I-like and NOD-like receptor signalings. Thus, the findings indicated that pattern recognition receptors (PRRs) possibly mediated host innate and protective antiviral immune responses in CDV-11 infected DH82 cells.
1. Introduction Canine distemper (CD), a highly contagious viral disease, caused by the agent of canine morbillivirus (formerly known as canine distemper virus, CDV) which belonged to the Morbillivirus genus within the Paramyxoviridae family and infected a broad host range of carnivores, including the Canidae, Procyonidae, Felidae, Mustelidae, Mephitidae, Ailuridae, Viverridae, Hyaenidae and Phocidae (Barrett, 1999; de Swart et al., 1995; Harder and Osterhaus, 1997; Ohashi et al., 2001; Summers and Appel, 2010) with multi-systemic disorders and severe immune suppression before clinical signs. Recently, it has been reported that CDV caused lethal outbreaks in nonhuman primates (Qiu et al., 2011; Sun et al., 2010) and endangered wildlife (Na et al., 2016). Persistent infection and immunosuppression have increased the host susceptibility
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to opportunistic infections, which mainly contributes to canine morbillivirus-associated morbidity and mortality (Pillet and von Messling, 2009; Xue et al., 2019). Primary replication of CDV in ferret and dog took place in the lymphatic tissue of the respiratory tract. Virulent CDV infected macrophages rapidly and massively, circulating B and T lymphocytes (Ferreira et al., 2010; Lemon et al., 2011) based on the bindings to signaling lymphocyte activation molecule (SLAM) with its hemagglutinin (H) protein (Pratakpiriya et al., 2012; Tatsuo et al., 2000; von Messling et al., 2004), and then developed severe immune suppression by damaging secondary lymphatic organs including spleen, lymph nodes, and gut-associated and mucosal lymphoid tissues (Sawatsky et al., 2018). However, an avirulent CDV infection caused transitory immune suppression in ferrets and dogs, and developed robust protective immune responses (von Messling et al., 2004). Not only
Corresponding authors. E-mail addresses: [email protected] (X. Zheng), [email protected] (X. Xue).
https://doi.org/10.1016/j.meegid.2020.104206 Received 15 October 2019; Received in revised form 18 January 2020; Accepted 22 January 2020 Available online 23 January 2020 1567-1348/ © 2020 Published by Elsevier B.V.
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receptor choice but also post entry host cell control and immune evasion mechanisms determined morbillivirus tropism (von Messling et al., 2004). Macrophages are cellular components of the innate immune system that reside in virtually all tissue and contribute to immunity, repair, and homeostasis (Tugal et al., 2013). The present work demonstrated that the attenuated vaccine strain CDV-11 replicated massively without the typical syncytia in DH82 cells. Thus, macrophages might play a crucial role during CDV early proliferation and infection in vitro. In this study, we employed RNA-sequencing and transcriptomic analyses to find out the comprehensive host antiviral responses at transcriptional levels in DH82, a cell line of macrophages, infected with CDV-11 strain. The results demonstrated that CDV infection in DH82 cells caused a strong induction of genes involved in host innate immune responses and inflammatory responses, which may provide new heights of understanding the host antiviral responses of CDV and finding out the potential therapeutic targets.
cells/well) were infected with CDV-11 at an MOI of 0.1 and grew at 37 °C in a 5% CO2 humidified cell incubator. After virus infection, the cells and supernatants were collected at 24 h, 48 h, 72 h, 96 h and 120 h for determination of virus titers as described in Section 2.3.
2. Materials and methods
Purified RNA samples were submitted to Aksomics Bio-tech Group. Inc. (Shanghai, China) for transcriptomic sequencing of mRNA with three independent biological replicates. A detailed description of the Aksomics Bio-tech company RNA transcriptomic procedures can be found at http://www.aksomics.com/. We enriched RNA from intact total RNA by oligo (dT) and removed the rRNA of the degraded total RNA from DH82 cells infected with or without CDV-11. Random primers were used to synthetize the first strand cDNA from RNA fragments. Based on dUTP, the second strand cDNAs were synthesized. Double-strand cDNA were added dATP to the ends and then Illumina adaptor ligation and PCR amplification for library data. Finally, the library data were detected by Agilent 2100 for quality control and quantified with qPCR. The library was sequenced with Illumina Hiseq 4000 and the raw data were obtained for further analysis.
2.5. Total RNA preparation Total RNA samples of the mock-infected and CDV-11-infected DH82 cells were lysed using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. Each sample was prepared for three replications. The RNA integrity and gDNA contamination were tested by Denaturing Agarose Gel Electrophoresis, and the total RNA quantification and quality assurance by spectrophotometer NanoDrop ND1000. 2.6. Transcriptomic sequencing
2.1. Cell culture and virus Vero cells (ATCC-81) and DH82 cells (ATCC-CRL-10389), a cell line from canine macrophages, were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, CA, USA), 1000 U/mL penicillin (Gibco, CA, USA) and 100 μg/mL streptomycin (Gibco, CA, USA). The attenuated live vaccine CDV-11 strain was gifted by the QiLu animal health products limited company in China. The CDV-11 strain was propagated in Vero and DH82 cells following six passages. The virus titers, determined in Vero and DH82 cells, were calculated by the method of Reed & Muench based on cytopathic effects (CPEs) or indirect immunofluorescent assay (IFA).
2.7. Transcriptomic data analysis
2.2. Indirect immunofluorescent assay (IFA)
The raw data of mRNA sequencings from CDV-11-infected and mock-infected DH82 cells with replicates were averaged. The data size of the RNA-sequencing was 6G of paired-end sequencing. The reference genome was CanFam3.1. The alignments between rata data and the reference genome were used with Hisat2 Software (v2.0.4) (Kim et al., 2015). The transcription abundance of the rata data were estimated using StringTie Software (v1.2.3) (Pertea et al., 2015) in comparison of the genomes of reference annotation information in official database. The FPKM (Fragments Per Kilobase of gene/transcript model per Million mapped fragments) (Mortazavi et al., 2008) values of genes and transcription levels were calculated with Ballgown Software (Frazee et al., 2015), and the differentially expressed genes and transcriptions were screened between samples or groups. The differentially expression genes from inter-groups of the mock- and CDV-infected samples were filtered out by mean FPKM > 0.5. Finally, the Clustering, Gene Ontology (GO) (The Gene Ontology, 2019) and pathway enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (Huang da et al., 2009) (http://david.abcc. ncifcrf.gov/) to annotate the functions of differentially expression genes (DEGs) with KEGG (Kyoto Encyclopedia of Genes and Genomes) (Kanehisa et al., 2019) pathways. A cutoff of p ≤ .05 for KEGG pathway was analyzed (http://www.geneontology/).
CDV in cultured cells were detected by IFA as described previously (Wang et al., 2012). Briefly, Vero and DH82 cells were infected with CDV-11 at an MOI of 0.1 in DMEM supplement with 2% FBS at 37 °C for 72 h (h). Discarding the supernatant, the cells were fixed with chilled 80% acetone for 30 min (min) at room temperature, and washed for 3 times with PBS. The cells were incubated with murine anti-CDV-N monoclonal antibody (VRMB, WA, USA) at a dilution of 1:500 for 1 h at 37 °C, and then stained with FITC-labeled goat anti-mouse lgG (TransGen, Beijing, China) for 1 h. Finally, the cells were examined under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The percentages of N-positive cells were calculated with reference to the total number of cells. 2.3. Titration of CDV-11 Virus Titers were performed in 96-well plates. Monolayer of Vero or DH82 cells were infected with 50 μL of viral suspensions at serial 10fold dilutions (from 10−1 to 10−8) for six replicates and incubated at 37 °C for 72 h. Discarding the supernatant, the DH82 cells were incubated with anti-CDV-N monoclonal antibody at 37 °C for 1 h and stained with FITC-labeled goat anti-mouse antibody at 37 °C for 1 h. Finally, the results were examined under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). The results of virus titers of CDV-11 in Vero cells were determined by CPEs. The 50% tissue cultures infection dose (TCID50) of CDV-11 was calculated according to the method of Reed and Muench [15].
2.8. Statistical analysis Results were presented as mean ± standard deviation (SD). Similar results were obtained from at least three independent experiments. Statistical significance were analyzed by one-way ANOVA (Dunnett's ttest) and considered at p < .05. * means p < .01, ** means p < .0001.
2.4. Growth curves Monolayer of Vero and DH82 cells in 6-well culture plates (5 × 105 2
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Fig. 1. Growth characteristics of CDV-11 in Vero and DH82 cells. (A, B, E, F) Light fields of negative Vero (A) and DH82 (E) cells, and the CDV-11-infected Vero (B) and DH82 (F) cells. (C, D, G and H) Indirect Immunofluorescence assay of the Vero (C and D) and DH82 (G and H) cells incubated with CDV-11 virus (D and H) or mock 2% DMEM (C and G). The cells were fixed with 4% paraformaldehyde and stained with primary antibody of anti-CDV N protein (1:500 dilution) and FITC labeled goat anti-mouse IgG (1:100 dilution) at 72 h p.i.. (I) One-step growth curves of CDV-11 in Vero and DH82, respectively. Cells, infected with CDV-11, were collected at the indicated time points, and the viral productions were titrated with TCID50 assay. These data were averaged from three independent experiments, and the error bars indicate standard deviations.
3. Results
cells, monolayer of Vero and DH82 cells were inoculated with CDV-11 at an MOI of 0.1 for 1 h at 37 °C, and then cultured for 96 h with fresh DMEM supplement with 2% FBS. Typical syncytia were observed in CDV-11 infected Vero cells. Surprisingly, the DH82 cells infected with CDV-11 were no cytopathy or syncytia (Fig. 1F) in comparison of the
3.1. Growth characteristics of CDV-11 in Vero and DH82 cells To examine the growth characters of CDV-11 in Vero and DH82 3
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Fig. 2. Gene expression pattern in DH82 infected with CDV-11 at 48 h. (A) The Heatmap of correlation values among the total RNA samples from infected with CDV11 group or mock group. The dark blue of the Heatmap represented the high correlation and the light blue represented the low correlation. (B) Cluster analysis of differentially expressed genes between CDV-11 infection and mock group in DH82 at 48 h post infection were exhibited in a heat map. Each row showed the relative expression level for a single gene and each column showed the expression level of a single sample. The genes were required to satisfy a cutoff p < .01 in direct comparison. Red represented genes with increased expression and green represented genes with decreased expression. (C) Volcano Plot of differentially expressed genes in CDV-11 infected DH82 cells compared to mock group. The x-axis meant log 2 of fold changes, and the y-axis meant log 10 of p-values. The two green vertical lines were up-regulated (right) and down-regulated (left) respectively. The green parallel line corresponded to the threshold of p-value. Green dots represented significantly downregulated expression genes, red dots represented significantly up-regulated expression genes, and black dots represented no significantly difference expressed genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
mock-infected DH82 cells (Fig. 1E). The specific CDV N protein in CDV11-infected DH82 and Vero cells were examined with CDV anti-N monoclonal antibodies (MAbs) at 72 h p.i., which demonstrated strong
reaction signals with the anti-N MAbs in both cells (Fig. 1D and H), and no signals in mock-infected cells (Fig. 1C for Vero cells, Fig. 1G for DH82 cells). To assess growth curves of CDV-11 in both cells, 4
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Table 1 The top 50 up-regulated expressed genes in CDV-11 infection DH82 cells compared to the mock controls. GenBank accession
Gene symbol
Full name
Log2(fold change)
p-Value
NM_001003134.1 XM_003639053.4 XM_860510.5 XM_005636024.3 XM_005622027.3 NM_001003133.1 XM_545847.6 XM_537104.6 XM_843271.5 NM_001003010.2 XM_005626701.3 NM_001010949.1 XM_014116565.2 XM_548077.6 NM_001048091.1 NM_001135787.1 XM_014116241.2 XM_022413453.1 XM_005618758.3 XM_014117383.2 NM_001253787.1 XM_005635291.3 XM_845787.5 XM_846183.4 NM_001197095.1 XM_022404348.1 XM_855170.5 NM_001010960.1 XM_022426251.1 XM_532793.6 XM_545493.5 NM_001005250.1 XM_005616331.3 XM_005623417.3 XM_535344.6 NM_001130836.1 NM_001003297.1 XM_014122423.2 XM_005624038.3 XM_022413781.1 XM_022415661.1 XM_845924.4 XM_539422.6 XM_537413.6 XM_005636026.3 NM_001003200.1 XM_014112321.2 XM_005637591.3 XM_022407392.1 NM_001314132.1
MX1 ISG15 BST2 OAS1 IFI44 MX2 ISG20 IFI44L IFIT1 CCL5 DDX58 CXCL10 DHX58 IFI35 OAS3 IFNB1 RNF213 HERC6 IFIT2 APOL5 Oasl1 HELZ2 PARP14 RSAD2 SERPINE1 EIF2AK2 IRF9 CCL7 MB21D1 IDO1 IFIH1 CCL4 PPP1R15A GCH1 IFI6 TNFSF10 CCL2 IL18BP LGALS3BP DTX3L SRPX2 EHD4 SAMD9L NFKBIA OAS2 CXCL8 TNFAIP3 UBTD1 TMEM236 SIX1
MX dynamin like GTPase 1 ISG15 ubiquitin-like modifier Bone marrow stromal cell antigen 2 2′-5′-oligoadenylate synthetase 1 Interferon induced protein 44 MX dynamin like GTPase 2 Interferon stimulated exonuclease gene 20 Interferon induced protein 44 like interferon-induced protein with tetratricopeptide repeats 1 C-C motif chemokine ligand 5 DExD/H-box helicase 58 C-X-C motif chemokine ligand 10 DExH-box helicase 58 Interferon induced protein 35 2′-5′-oligoadenylate synthetase 3 Interferon beta 1 Ring finger protein 213 HECT and RLD domain containing E3 ubiquitin protein ligase family member 6 Interferon induced protein with tetratricopeptide repeats 2 apolipoprotein L5 2′-5′-oligoadenylate synthetase like helicase with zinc finger 2 Poly(ADP-ribose) polymerase family member 14 Radical S-adenosy l methionine domain containing 2 Serpin family E member 1 Eukaryotic translation initiation factor 2 alpha kinase 2 Interferon regulatory factor 9 C-C motif chemokine ligand 7 Mab-21 domain containing 1 indoleamine 2 Interferon induced with helicase C domain 1 Chemokine (CeC motif) ligand 4 Protein phosphatase 1 regulatory subunit 15A GTP cyclohydrolase 1 Interferon alpha inducible protein 6 TNF superfamily member 10 C-C motif chemokine ligand 2 Interleukin 18 binding protein Calcium activated nucleotidase 1 Deltex E3 ubiquitin ligase 3 L Sushi repeat containing protein EH domain containing 4 Sterile alpha motif domain containing 9 like NFKB inhibitor alpha 2′-5′-oligoadenylate synthetase 2 C-X-C motif chemokine ligand 8 TNF alpha induced protein 3 Ubiquitin domain containing 1 Transmembrane protein 236 SIX homeobox 1
7.22 6.92 5.30 5.20 5.17 5.17 5.06 5.02 4.81 4.45 4.36 4.30 4.27 4.06 3.87 3.81 3.75 3.44 3.37 3.32 3.25 2.99 2.95 2.94 2.78 2.67 2.63 2.58 2.51 2.48 2.45 2.41 2.36 2.35 2.32 2.24 2.23 2.23 2.23 2.21 2.17 2.16 2.15 2.14 2.13 2.12 2.09 2.09 2.05 2.02
4.25E-06 8.26E-07 1.56E-06 3.77E-05 1.88E-08 1.12E-07 1.89E-05 5.70E-06 6.35E-07 4.95E-03 5.47E-07 9.29E-06 9.639E-06 5.66E-05 1.04E-07 7.53E-07 2.03E-08 4.15E-06 6.53E-07 1.51E-05 1.73E-05 3.64E-07 1.68E-06 9.03E-08 2.44E-06 5.27E-07 1.24E-05 1.40E-05 4.93E-05 8.59E-06 2.89E-06 1.89E-04 8.00E-07 5.51E-05 8.20E-04 7.36E-04 3.07E-06 9.80E-05 6.66E-08 3.48E-06 2.72E-05 4.13E-02 3.62E-08 4.42E-06 5.67E-08 4.75E-05 1.15E-05 1.47E-04 3.53 E-02 4.53E-06
were about 0.99, and the correlation between mock and CDV-11-infected groups was more than 0.94 (Fig. 2A). After the filtration of the signals below the threshold level, genes showing at least a 1.5log 2 fold changes in expression with a cutoff of p ≤ .05, were considered to be significant differentially expressed. In summary, 491 differentially expressed genes were identified in CDV-11-infected groups at 48 h p.i. in comparison of the mock group, including 372 up-regulated and 119 down-regulated expression genes (Fig. 2B). The number of up-regulated genes was much more than that of down-regulated genes. The top 50 of up-regulated genes and top 20 down-regulated genes are presented in Tables 1 and 2. The 50 most up-regulated genes from CDV-11-infected macrophages in Table 1 were contained the interferon-stimulated genes (ISGs), such as ISG15, ISG20, OAS1, OAS3, IFIT1, IFI35, IFIT1, IRF9, IFI44L, IFNB1 and IFIT2; chemokine factor genes (CCL5, CXCL10, CCL7, CCL4) and pro-inflammatory cytokines (IL-18BP and TNFSF10). These data also demonstrated that the most up-regulated genes changed 148 folds in CDV-11-infected DH82 cells, while the most down-regulated genes decreased less than 1 fold. The changed folds of up-regulated genes were much higher than those of down-regulated genes.
monolayer of Vero and DH82 cells were infected with CDV-11 and the virus titrations were determined at 24 h p.i., 48 h p.i., 72 h p.i. and 96 h p.i., respectively (Fig. 1I). In DH82, CDV-11 exhibited a slightly decline in virus titers, finally reaching 105.5 TCID50/mL at 96 h p.i.. In contrast, virus titers peaked at 106.5 TCID50/mL at 72 h p.i. in Vero cells, and maintained a platform between 72 h p.i. and 96 h p.i., indicating that CDV-11 was sensitive to macrophages DH82 cells with a good duplication ability and the virus production were not significantly impaired. 3.2. Gene expression profiles in CDV-11-infected macrophages In order to understand molecular mechanisms of antiviral responses to CDV vaccine strain in DH82 at transcriptional levels, the total RNA profiles were analyzed with high throughput transcriptomic sequencing (Aksomics Bio-tech Group. Inc.). The total RNA samples were extracted from mock- and CDV-infected DH82 cells at 48 h p.i., and the homogeneity of three replications each group were evaluated based on correlation of gene expression levels and the DEGs between groups. The correlation of the repeated samples in mock- or CDV-11-infected group 5
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Table 2 The top 20 down-regulated expressed genes in CDV-11 infection DH82 cells compared to the mock controls. GenBank accession
Gene symbol
Full name
Log2(fold change)
p-Value
XM_539938.6 XM_849220.5 NM_001205114.1 XM_022403248.1 XM_548978.6 XM_022418589.1 NM_001177734.2 XM_014109177.1 XM_003432297.4 XM_532363.5 XM_005627946.3 XM_533158.6 XM_022417145.1 XM_005616779.3 NM_001002944.1 XM_547049.6 XM_014116110.2 XM_005636328.3 XM_845218.5 XM_014107229.2
VAMP5 RETN HAVCR1 ALX1 CFP MYOCD CD36 NDRG4 LRRC17 HYKK TMEM71 HIPK3 FAM184B DPY19L3 ADORA2B ITGAX STON2 ACACB HPSE2 LZTS3
Vesicle associated membrane protein 5 Resistin Hepatitis A virus cellular receptor 1 ALX homeobox 1 Complement factor properdin Myocardin CD36 molecule NDRG family member 4 Leucine rich repeat containing 17 Hydroxylysine kinase Transmembrane protein 71 Homeodomain interacting protein kinase 3 Family with sequence similarity 184 member B dpy-19 like C-mannosyltransferase 3 Adenosine A2b receptor Integrin subunit alpha X Stonin 2 Acetyl-CoA carboxylase beta Heparanase 2 (inactive) Leucine zipper tumor suppressor family member 3
−1.66 −1.33 −1.23 −1.17 −1.13 −1.07 −1.07 −1.02 −1.01 −1.01 −1.00 −0.94 −0.99 −0.96 −0.91 −0.89 −0.89 −0.88 −0.88 −0.86
4.39E-02 1.21E-03 1.87E-02 4.64E-02 2.57E-04 7.92E-02 3.48E-05 8.09E-06 3.42E-03 6.80E-05 1.12E-02 4.18E-02 3.95E-04 1.58E-05 1.02E-02 3.84E-03 2.93E-05 1.45E-03 1.62E-05 6.40E-04
dendritic cells (DC) in the airways preceded infection of lymphocytes in lymphatic organs in vivo (Ferreira et al., 2010). In the present study, the DH82, a cell line from canine macrophages, supported the attenuated CDV vaccine strain replication with no observed cytopathic effects. RNA transcriptomics, in combination with bioinformatics, had proved to be an efficient and high-throughput tool to establish gene expression patterns, to complete functional clustering, canonical pathways enrichment of the DEGs. Macrophages were infected by multiple viruses, such as human immunodeficiency virus (HIV) (DiNapoli et al., 2016), porcine reproductive and respiratory syndrome virus (PRRSV) (Kim et al., 2010; Zhang et al., 2018), influenza virus (Marvin et al., 2017), respiratory syncytial virus (RSV) (Borchers et al., 2013). CDV replications located primarily in monocytes and macrophages during the early stage of infection in vivo (von Messling et al., 2004). However, it was poorly addressed that the global gene expression profiles in macrophages responses to CDV infection. To better understand the interactions of CDV and macrophages at transcriptional levels, a genome wide gene expression profiles in DH82 cells were performed using transcriptomic analysis. Firstly, the growth characteristics of CDV-11 were examined in Vero and DH82 cells (Fig. 1). The CDV-11 exhibited a perfect replication and propagation in DH82, a cell line from canine macrophages, and viral yield in DH82 cells slightly descended in comparison of its in Vero cells. Then, an optimal moment of 48 h p.i. to perform transcriptomic analyses were determined by monitoring one-step growth curves of CDV-11 in DH82 cells. The results revealed that virus loading maintained a platform between 24 h p.i. and 72 h p.i.. Thus, the total RNA samples for transcriptomic analyses were extracted at 48 h p.i. for adequate data. In totally, 491 differentially expression genes were identified between mock- and CDV-infected DH82 cells at 48 h p.i., including 372 up-regulated and 119 down-regulated expression genes. The significantly up-regulated genes in DH82 responding to CDV-11 infection were ISGs (Mx1, ISG15, BST2, MX2, ISG20, OAS1, IFNB1, IFIT1, IFIT2, OAS3, Oasl1 and IRF9), chemokines (CCL5, CXCL10, CCL7, CCL4) and the pro-inflammatory factors (IL18BP, TNFSF10 and TNFAIP3). All these data were consistent with previous findings that genes involved in innate immunity, pro-inflammatory responses. Activation of IL-1β, TNF-α and CCL5 transcriptions were observed at high levels in RSV persistently infected macrophages. ISG15 modulates the antiviral response during replication of pseudorabies virus (Liu et al., 2018), hepatitis E virus (Sooryanarain et al., 2017), dengue and west nile virus (Dai et al., 2011). Mx and the Mx family of genes encoding large GTPases related to dynamin, showed antiviral activity against a wide
Unsupportively, clustering analysis of the expression profiles revealed a distinct gene signature between CDV- and mock-infected DH82 cells. The DEGs could be grouped into at least 9 subsets based on their modes of expression, which showed that the differentially expressed genes in CDV-11-infected DH82 cells changed greatly compared to the mockinfected DH82 cells at 48 h p.i. (Fig. 2C). 3.3. Confirmation of transcriptome data by qRT-PCR To validate the data from transcriptome sequencing, seven genes chosen randomly (BST2, CCL5, IRF9, TNFSF10, IL18BP, HPSE2) in the DEGs list were subjected to qRT-PCR. The results from qRT-PCR of increased or decreased gene expression levels for the tested genes showed no significant differences with transcriptomic analyses (Supplement Table 1, S3). 3.4. Functional groups and canonical pathways modulated by CDV-11 infection in DH82 cells To clarify the biological functions of the DEGs involved in CDV-11infected DH82 cells, the mostly associated gene ontology (GO) and pathways with DEGs were assessed. The top 10 function groups involving in biological processes, cellular component and molecular function, respectively, showed in Fig. 3A, B and C, such as immune system process, immune responses, negative regulation of viral process and viral life cycle, responses to external biotic stimulus, cytokine receptor binding, chemokine activity, transcriptional factor activity. These results indicated that the transcriptional alterations in CDV-infected macrophages mainly involved in biological processes closely related to antiviral and immune responses, and cellular function and maintenance, respectively. The top 10 pathways involved in responses to CDV-11 infection in DH82 cells were found out with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases. The main pathways modulated during infection were those related to NF-κB signaling pathway, NOD-like receptor, RIG-I-like receptor and Toll-like receptor signal pathways, cytokine-cytokine receptor interaction and TNF signaling pathway (Fig. 3D). The DEGs involved in those pathways were listed respectively, and most of these genes were up-regulated (Table 3 and Fig. 4). 4. Discussion Morbillivirus infection of alveolar macrophages and subepithelial 6
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Fig. 3. Enrichment functional gene ontology (GO) and pathways of differentially expressed genes upon CDV-11 infection in macrophages DH82. (A-C) The top 10 significantly relevant GO class articles of Biological Process (BP) (A), Cellular Component (CC) (B) and Molecular Function (C) for differentially expressed genes in CDV-11 or mock-infected DH82 cells were categorized into functional groups. (D) The top 10 significantly involved pathways had been enriched upon CDV-11 infection in DH82. The x-axis (down) meant the enrichment scores, and the x-axis (up) meant number of genes. The y-axis showed GO class articles or signal pathways.
against various viruses (Sedewitz and Quann, 2015; Zhang et al., 2015; Zhang et al., 2013) and could also be the candidate effectors to inhibit CDV replication. CDV might make a similar attempt to control their ubiquitination, thus disturbing their antiviral functions. In addition, compared to the mock-infected DH82, the bone marrow stromal cell antigen 2 (BST2), which nonspecifically inhibited retrovirus (such as HIV, SIV and EIAV) (Neil et al., 2008; Yin et al., 2014) and lipid enveloped viruses (such as Ebola, Marburg, Lassa) (Andrey Tokarev et al., 2009) release and could be broadly expressed on IFN-α induction (Blasius et al., 2006), dramatically rose 39 times and accompanied by 5 times up-regulated of IFI6 (Interferon alpha inducible protein 6) in CDV-11 infected DH82 cells. The battle of viral replication and host immune responses was conclusive for animal's clinical outcome of viral infections. Host immune cells, recognizing viral components by pattern-recognition
range of RNA viruses (Karaulov et al., 2017; Randall and Goodbourn, 2008). Mx1 and ISG20 were strongly up-regulated in Influenza A virus infected macrophages but not in epithelial cells, suggesting cell-specific differences in induction of antiviral factors. Transcriptions of ISGs were triggered by nuclear localization of ISGF3 which was formed by the crucial DNA-binding protein IRF9 jointing with heterodimer of phosphorylated STAT1 and STAT2, finally resulting in the establishment of antiviral state in cells (Platanias, 2005; Randall and Goodbourn, 2008). It was widely accepted that the morbillivirus V protein interferes with type I IFN signals (Chinnakannan et al., 2013). This work proved that the type I IFN signals played crucial roles in host antiviral responses with the significantly up-regulation of the ISG15, ISG20 and IRF9 in CDV-11 infection macrophages. Mice lacking the ISG15 gene were more susceptible to infection by influenza, herpes, and sindbis virus (Lenschow et al., 2007). Mx1 and OAS1 exerted antiviral activity 7
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receptors (PRRs), initiated a rapid antiviral response to prevent the spread of the infection, while viruses had evolved the abilities to evade the host immune responses and develop the disease (Zhao et al., 2013). Gene Ontology (GO) and pathway enrichment analyses indicated that most of the up-regulated genes involved in NF-κB signaling pathway, NLRs, RLRs, TLRs signal pathways and cytokine-cytokine receptors interaction in CDV-11-infected DH82. CDV (Onderstepoort strain) induced human osteoclast genesis by activation of NF-κB pathways (Selby et al., 2006). PRRSV inoculation could activate NF-κB by means of I-κB degradation (Lee and Kleiboeker, 2005), leading the production of some inflammatory molecules, such as IL-6, IL-8 and IL-1β (Zhang et al., 2018). HBV induced autophagy to promote viral replication in vitro and in vivo through inhibiting NF-κB signaling pathway (Wang et al., 2018). Hundreds of ISGs, with functions of antiviral, immunomodulatory, cell growth regulatory and metabolic regulatory actions, were induced by NF-κB, and created an antiviral state, as well as a hub of cellular signal transduction pathways involved in host immune responses to virus challenges (Moynagh, 2005; Tong et al., 2017). Therefore, the findings indicated that host innate immune responses against CDV mainly developed the antiviral effects through the NF-κB signaling pathway. Sendai virus activated RIG-I, and was a virus model to stimulate robust type I interferon responses through RIG-I activation. VSV had been demonstrated to trigger specifically RIG-I innate immune signaling (Furr et al., 2010). RIG-I, as well as MDA5, was proved to induce IFN following infection of human cells with measles virus(Ikegame et al., 2010). The viral leader transcript of the measles virus was identified as a potential PAMP ligands of RIG-I (Plumet et al., 2013). In the present work, TNF-α, RIG-I, NF-κB and MyD88 were all significantly up-regulated in CDV-11-infected DH82s. The MyD88 was a major adaptor molecule downstream of several surface and cytosolic PRRs (Kader et al., 2017; Trinchieri and Sher, 2007). RLRs interacting with multi-component proteins, and TLRs dependent or independent on MyD88, both activated IRF3 and NF-κB to induce transcription of a variety of innate immune response genes, including IFNs, direct antiviral genes, and pro-inflammatory genes (Tong et al., 2017; Wilden et al., 2009). The inflammasome NLRP3, known as the member in NODlike-receptor-family, pyrin domain containing 3, was reported to activate the NLRP3 inflammasome, resulting in the caspase-1-mediated maturation of IL-1β (Zilliox et al., 2007). The NF-κB-induced activation of NLRP3 and pro-IL-1β gene expression was requisite for activating caspase-1 to further regulate the secretion of the inflammatory cytokines IL-1β and IL-18 (Motta et al., 2015). The increased TNF-α expression were associated with depletion of Foxp3+ Treg in acutely CDV-infected dogs (Qeska et al., 2013). In this study, nine genes involved in NF-κB signaling pathway, PRRs and downstream cytokines, including CCL4, CXCL8, DDX58, ICAM1, IL1R1, MYD88, NFKBIA, TNF, TNFaIP3 and TRIM25, were significantly up-regulated, and the NF-κB complex inhibitory protein IκB-β was obviously down-regulated. Lethal influenza A virus preferentially activated TLR3 and triggered a severe inflammatory response (Pothlichet et al., 2013). Constitutive TLR3dependent immunity protected cortical neurons from HSV-1 infection. TLR3 recognized double-stranded RNA and was involved in the inflammatory response to RSV infection. Therefore, further investigations needed to be explored how the PRRs worked the functions of antiviral and inflammatory responses during the early stage of CDV infection in lymphocytes.
0.07 0.08 0.07 0.08 3.45E-09 4.68E-09 5.12E-08 1.84E-07
5. Conclusion In this study, our data demonstrated a comprehensive knowledge of gene expression changes in DH82, a cell line from canine macrophages, infected with CDV-11 vaccine strain. The DEGs were indicated to involve in innate immune responses, such as NF-κB signaling pathway, NLRs, RLRs, TLRs signal pathways and cytokine-cytokine receptor interaction, which may provide clues for understanding the host antiviral
cfa04657 cfa04620 cfa04668 cfa05162
0.07 0.12 8.93E-10 1.44E-09
RIG-I-like receptor signaling pathway Cytokine-cytokine receptor interaction IL-17 signaling pathway Toll-like receptor signaling pathway TNF signaling pathway Measles cfa04622 cfa04060
0.11 1.35E-10 NOD-like receptor signaling pathway cfa04621
0.12 6.88E-11 Herpes simplex infection cfa05168
0.09 6.70E-13 NF-κB signaling pathway cfa04064
0.12 1.41E-13 Influenza A cfa05164
ADAR//CCL2//CCL5//CXCL10//CXCL8//DDX58//EIF2AK2//ICAM1//IFIH1//IFNB1//IRF9//MX1//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1// OAS2//OAS3//PML//RNASEL//RSAD2//TICAM1//TLR3//TNF//TNFSF10//TRIM25 BIRC3//CCL4//CD40//CFLAR//CXCL8//DDX58//ICAM1//IL1R1//MYD88//NFKB1//NFKB2//NFKBIA//PLAU//RELB//TICAM1//TNF//TNFAIP3// TNFRSF11A//TNFSF13B//TRIM25 CASP8//CCL2//CCL5//CFP//DDX58//DLA-64//EIF2AK2//FOS//IFIH1//IFNB1//IRF9//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1//OAS2//OAS3// PML//RNASEL//TAP1//TICAM1//TLR3//TNF//TNFRSF14 BIRC3//CASP8//CCL2//CCL5//CXCL8//GBP5//GSDMD//IFNB1//IRF9//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1//OAS2//OAS3//P2RX7// PANX1//RNASEL//TANK//TICAM1//TNF//TNFAIP3 AZI2//CASP8//CXCL10//CXCL8//DDX58//DHX58//IFIH1//IFNB1//ISG15//NFKB1//NFKBIA//NFKBIB//TANK//TNF//TRIM25 CCL17//CCL2//CCL4//CCL5//CCL7//CD40//CSF1//CXCL10//CXCL14//CXCL8//CXCR4//HGF//IFNB1//IL15RA//IL18RAP//IL1R1//OSMR// PDGFB//PDGFRB//TNF//TNFRSF11A//TNFRSF12A//TNFRSF14//TNFSF10//TNFSF13B//TNFSF18//VEGFA CASP8//CCL17//CCL2//CCL7//CEBPB//CXCL10//CXCL8//FOS//FOSL1//MMP1//MMP9//NFKB1//NFKBIA//TNF//TNFAIP3//USP25 CASP8//CCL4//CCL5//CD40//CD80//CXCL10//CXCL8//FOS//IFNB1//IRF5//MYD88//NFKB1//NFKBIA//SPP1//TICAM1//TLR3//TNF BCL3//BIRC3//CASP8//CCL2//CCL5//CEBPB//CFLAR//CSF1//CXCL10//FOS//ICAM1//MMP9//NFKB1//NFKBIA//TNF//TNFAIP3 ADAR//CCND3//DDX58//EIF2AK2//IFIH1//IFNB1//IRF9//MX1//MYD88//NFKB1//NFKBIA//NFKBIB//OAS1//OAS2//OAS3//TNFAIP3//TNFSF10
Genes GeneRatio Fisher-p value Definition Pathway ID
Table 3 Significant pathways of the differential expressed genes in DH82 cells infected with CDV.
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Fig. 4. The simplified models of signal pathways of the DEGs involved in immune response process. Significantly up-regulation or down-regulation genes were represented in red or green, respectively, and the gray meant no changes. The dotted lines showed omitted signal pathways between the signal transfers, and the solid lines showed direct signal transfers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
responses during CDV infection and finding out the targets of therapy. However, further functional elucidations remained to be further investigated for revealing the molecule mechanisms of pathogenic and immune responses during CDV infection.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.meegid.2020.104206. References
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No conflict of interest.
Acknowledgements We gratefully acknowledge the infrastructure support of the college of public health in Shandong University. This work was supported by The National Key Research and Development of China (2016YFD0501000), The National Nature Science Foundation of China (31902275, 31902308), and The Fundamental Research Funds of Shandong University (2018JC050).
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