Introduction
Materials and methods
Study design
Animals
Inoculation
Observation and scoring
Leucocyte count
Post-mortem sampling
Isolation of total RNA
Microarray analysis
Microarray comparisons
Real-time PCR for CSFV
Real-time PCR for gene expression
Gene | Acc. number | Forward primer | Reverse primer | Tm (°C) | Reference |
---|---|---|---|---|---|
OAS2 | AY288913 | ACAGTCTTGAGGGGCAACTCTGA | GCTGGCTTTCATCATATCCAAGGA | 60 | |
CXC10 | AY789646 | TGCCCACATGTTGAGATCAT | CGGCCCATCCTTATCAGTAG | 60 | |
TRAIL | NM_001024696 | CAACAAGGCATTCCTCACCT | CCAGCTCTCCATTCCTCAAG | 60 | |
IFN alpha | NM_214393 | TACTCAGCTGCAATGCCATC | TCTGTGCTGAAGAGCTGGAA | 58 | |
18S RNA | AF102857 | GTTCAAAGCAGGCCCGAG | CGCCGCCGCATCGCCA | 57 | De groot et al. (2005) |
Beta actine | AY550069.1 | GGCATCCTGACCCTCAAGTA | GGGTCATCTTCTCACGGTTG | 58 | |
C4-bp | NM_213942 | CTCTGGTTGGAGAGGACAGA | GCTCACAGTCTTCGGGGTA | 60 |
Results
Clinical parameters after infection with CSFV strains
Gene expression analysis
ID (#) | Fold changea
| Blastn or tblastxb
| Tentative function (functional cluster)c
| ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Spleen | Lymph node | Tonsil | |||||||||||
LV | MV | HV | LV | MV | HV | LV | MV | HV | Gene name or tentative annotation (T[H]C) | Acc. number |
E-value (nt) | ||
Higher in infected | |||||||||||||
CSF-1 | – | – | – | 4.0 | – | – | – | – | – | Allograft inflammatory factor 1 (AIF1) |
XM_001490247
| 4E-154 (474) | Growth-suppressing (IFN-γ) |
CSF-2 | – | – | – | 3.8 | – | – | – | – | – | IFN-gamma-inducible protein 16, transcript variant 1 (IFI16), mRNA |
ref|XM-001170179.1|
| 4E-47 (656) | Growth-suppressing (IFN-γ) |
CSF-3 (2) | – | – | – | 4.2 | – | – | – | – | – | Guanylate binding protein 2 (interferon-inducible) (GBP2), mRNA |
ref|NM-004120.
| 1E-123 (711) | Signal transduction/antiviral effect (IFN-γ) |
CSF-4 | – | – | – | 3.5 | 4.6 | – | – | – | – | Similar to dendritic cell-derived IFNG-induced monocyte protein 5 (MOP5) |
pig|TC265382
| 2E-54 (432) | Metal dependent phosphohydrolase (IFN-γ) |
CSF-5 | – | – | – | 3.5 | – | 4.8 | – | – | – | Polyubiquitin, transcript variant 12 (UBC), mRNA |
XM_874550.3
| 0 (790) | Component proteasome (UMP) |
CSF-6 | – | 5.3 | 5.0 | – | – | – | – | – | – | Proteasome (prosome, macropain) subunit alpha type, 6 (Psma6) mRNA |
NM_002791
| 0 (658) | Component proteasome (UMP) |
CSF-7 (3) | 5.6 | 5.7 | 6.1 | 5.3 | 5.1 | – | 5.3 | 5.4 | 6.0 | HECT domain and RCC1-like domain-containing protein 6 (HERC6) |
Pig|TC273610
| 2.9E-121 (715) | Potential ubiquitin ligase (UMP) |
CSF-8 (2) | 4.6 | 5.7 | 5.9 | 4.3 | 5.7 | – | 3.9 | 5.4 | 4.6 | Similar to poly (ADP-ribose) polymerase family, member 14, mRNA (BAL2) |
BC120371
| 5E-179 (626) | DNA-damage induced/cell survival (UMP/Apopt.) |
CSF-9 (2) | – | 5.5 | 6.0 | – | – | – | – | 5.7 | 5.1 | Similar to HUMAN (Q9H0J9) Poly [ADP-ribose] polymerase 12 (PARP12) |
pig|TC260164
| 2E-101(752) | DNA-damage induced/cell survival (UMP/Apopt.) |
CSF-10 | – | 4.7 | 4.9 | – | – | – | – | – | – | Poly (ADP-ribose) polymerase family, member 9 (PARP9)(BAL1), mRNA |
NM_001076828.
| 2E-165 (817) | DNA-damage induced/cell survival (UMP/Apopt.) |
CSF-11 | 7.2 | 6.8 | 9.9 | 6.3 | – | – | – | – | – | Similar to HCV-associated microtubular aggregate protein p44 (IFI44) |
THC2463234
| 2.9E-23 (534) | Antiproliferative activity (IFN-α/β) |
CSF-12 | 39 | 49 | 38 | 40 | 39 | 41 | 27 | 45 | 38 | CXC-like protein gene, complete cds (CXCL10/IP10) |
AY894692.
| 0 (533) | Pleiotropic cytokine (IFN-α/β) |
CSF-13 | 21 | 23 | 18 | 14 | 13 | 13 | 18 | 25 | 24 | 2′–5′ oligoadenylate synthetase 2 (OAS2) |
ref|NM-001031796.1|
| 2E-136 (309) | dsRNA mediated antiviral response (IFN-α/β) |
CSF-14 | 6.0 | 7.7 | 7.1 | 3.7 | 5.2 | – | – | 5.8 | 4.6 | TNF-related apoptosis-inducing ligand (TRAIL) mRNA |
ref|NM-001024696.1|
| 0 (1004) | Cytokine TNF ligand family (Apopt.) |
CSF-15 | – | 5.8 | 6.5 | – | 6.0 | 10.2 | – | 7.2 | 4.4 | EST weakly similar to HUMAN (Q7Z5U7) cathepsin C (CTSC) |
human|THC2686463
| 0.22 (276) | Lysosomal cysteine proteinase/requires Cl− (Apopt.) |
CSF-16 | – | 4.0 | 6.0 | 4.4 | – | – | – | – | – | Similar to NADH-ubiquinone oxidoreductase chain 1 (NADH1) |
ref|XM-001113286.1|
| 6e-81(374) | Subunit mitochondrial complex I (Apopt.) |
CSF-17 | – | – | – | 4.4 | 4.0 | – | – | – | – | Myeloid cell leukemia sequence 1 (MCL1)(BCL2-related) |
ref|NM-021960.3|
| 3E-136 (671) | Cell survival/anti-apoptotic (Apopt.) |
CSF-18 | 12 | 18 | 13 | 12 | 9.4 | – | 11 | 15 | 14 | Glutamate-cysteine ligase, modifier subunit (GCLM), mRNA |
ref|NM-199845.1|
| 5E-29 (244) | Key enzyme glutathion metabolism (Apopt.) |
CSF-19 (3) | – | 5.6 | 6.3 | 4.0 | 5.8 | 8.7 | – | 6.4 | 5.0 | Glutaredoxin (thioltransferase) (GLRX), mRNA |
ref|NM-001037604.1|
| 7e-51 (611) | Cell redox homeostasis/deglutathionylation (Apopt.) |
CSF-20 | – | 5.1 | 6.6 | 5.9 | 5.9 | – | – | – | – | Calbindin D-9 k mRNA |
NM_214140.
| 1E-174 (516) | Calcium binding and transport activity (Apopt.) |
CSF-21 | 3.6 | – | – | – | – | – | – | – | – | Sus scrofa MHC class I antigen 1 (SLA-1), mRNA |
ref|NM-001097431.1|
| 1E-86 (255) | Antigen presentation MHC I (Innate immune AP) |
CSF-22 | – | – | – | – | – | – | – | 3.5 | – | Beta-2-microglobulin (B2 M), mRNA |
ref|NM-004048.2|
| 1E-26 (476) | Antigen presentation MHC I (Innate immune AP) |
CSF-23 | – | – | – | – | – | – | – | 3.9 | – | Fc fragment of IgG, low affinity IIIb, receptor (CD16b) |
ref|NM-214391.1|
| 0 (794) | Natural killer cell mediated cytotoxicity (Innate Immune–AP) |
CSF-24 (7) | – | – | – | 4.5 | 4.2 | – | – | – | – | Immunoglobulin J chain |
ref|XM-001105782
| 8e-98 (348) | Joining IgM and IgA monomers (Adapt. Immune–AP) |
CSF-25 (2) | – | – | – | 3.8 | 3.7 | – | – | – | – | Immunoglobulin lambda-chain (CD179b antigen) |
ref|XM-850728.1|
| 2e-96 (677) | Pre- B or T cell receptor (Adapt. Immune–AP) |
CSF-26 (5) | – | – | – | 3.9 | 4.2 | – | – | – | – | Kappa light chain mRNA |
ref|NM-001093783.1|
| 1E-77 (494) | Pre- B or T cell receptor (Adapt. Immune–AP) |
CSF-27 (2) | – | – | – | 3.7 | – | – | – | – | – | Immunoglobulin mu heavy chain constant region, mRNA |
gb|S42881.1|
| 0 (661) | Pre- B or T cell receptor (Adapt. immune–AP) |
CSF-28 (2) | – | 3.8 | – | – | – | – | – | – | – | Placenta-specific 8 (PLAC8), mRNA |
NM_016619.
| 3e-59 (330) | Expressed in neutrophils/cell survival (Apopt ?) |
CSF-29 | – | 3.8 | – | – | – | – | – | – | – | Sterile alpha motif domain containing 9 (SAMD9) |
XM_001492397
| 2e-170 (595) | Cell proliferation (Apopt. ?) |
CSF-30 | – | – | 4.6 | – | – | – | – | – | – | Similar to frizzled 5 (LOC710796), mRNA (FZD5) |
ref|XR-012291.1|
| 2e-12 (418) | Receptor for Wnt signaling proteins |
CSF-31 | 4.5 | 4.5 | 4.5 | 5.3 | 4.7 | – | – | 5.5 | – | EST weakly similar to myelin expression factor 2 (MyEF2) |
human|THC2606750
| 0.00012 (547) | Myocyte transcription enhancer |
CSF-32 | – | 5.5 | 6.0 | – | 4.9 | – | – | – | – | Ribosomal protein L9 homologue (RPL9) |
XM_001149849
| 7E-160 (790) | Structural component ribosome |
CSF-33 | – | 4.1 | 4.9 | – | – | – | – | – | – | Chloride intracellular channel 5 (CLIC5), transcript variant 2, mRNA |
ref|NM-016929.3|
| 5e-40 (564) | Voltage-gated chloride channel (cytoskeleton) |
Lower in infected | |||||||||||||
CSF-34 | – | 0.16 | 0.18 | – | – | – | – | – | – | Actin, alpha 2, smooth muscle, aorta (ACTA2), mRNA |
ref|NM-001034502.1|
| 9e-76 (237) | Component cytoskeleton (cytoskeleton) |
CSF-35 | – | 0.20 | 0.22 | – | – | – | – | – | – | Tropomyosin, transcript variant 25 (TPM1), mRNA |
ref|XM-860199.1|
| 5e-167 (623) | Component cytoskeleton (cytoskeleton) |
CSF-36 (7) | – | 0.29 | – | 0.12 | 0.13 | 0.16 | – | – | – | Tblastx; mRNA clone:LVRM10070B07 homologue to B. taurus elastin |
dbj|AK233022.1|
| 0 (613) | Extracellular fibre |
CSF-37 (4) | – | – | – | 0.09 | 0.07 | 0.11 | – | – | – | Complement component 4 binding protein, alpha (C4BPA)(APOR), mRNA |
ref|NM-213942.1|
| 0.0 (537) | Complement cascades (innate and adapt immune–AP) |
CSF-38 (6) | – | – | – | 0.14 | 0.17 | – | – | – | – | Beta globin mRNA |
ref|XM-537902.2|
| 5e-172 (565) | Oxygen transport (Ox-P) |
CSF-39 | – | – | – | 0.24 | – | – | – | – | – | Hemoglobin alpha chain |
ref|NM-001077422.2|
| 4e-117 (593) | Oxygen transport (Ox-P) |
CSF-40 (2) | – | – | – | 0.25 | – | – | – | – | – | Solute carrier family 40 member 1 (Ferroportin 1), mRNA |
XM_001250723
| 6e-87 (388) | Cellular iron ion homeostasis (Ox-P) |
CSF-41 (3) | – | 0.23 | 0.21 | – | – | – | – | 0.27 | 0.22 | Cytochrome b (CYTB) |
ref|NP-008646.1
| 1E-54 (434) | Oxidative phosphorylation (Ox-P) |
CSF-42 | – | 0.29 | 0.29 | – | – | – | – | – | – | EST homoloque to NADH dehydrogenase subunit 4L (ND4L) |
pig|TC238572
| 1.0e-18 (468) | Oxidative phosphorylation (Ox-P) |
CSF-43 | – | – | 0.28 | – | – | – | – | – | 0.23 | Blastx; NADH dehydrogenase subunit 6 (ND6) |
ref|NP-008645.1
| 3e-50 (524) | Oxidative phosphorylation (Ox-P) |
CSF-44 | – | – | 0.23 | – | – | – | – | – | – | Ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3/CD203c) |
ref|NM-001075923.
| 0.0 (670) | Hydrolysis of Flavin adenine dinucleotide (Ox-P) |