Introduction
Materials and methods
Animal experiment
Isolation of RNA and DNA
Real-time PCR
Microarray analysis
Northern blot analysis
Results
Infection of germ-free piglets with rotavirus
Piglet | Slaugther hpi. | Facesa
| WBCb
| Jejunumc
| ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
% Dry matter/rota virus ELISA | (109 per l) % Lympho/mon. and gran. | Villus μm heigth | Crypt μm dept | V/C ratio | RNA | |||||||
0 hpi | 12 hpi | 18 hpi | 0 hpi | 12 hpi | 18 hpi | Yield (mg/g) | 28S/18S Ratio | |||||
62 Mock | 0 | 20.4/− | NA | NA | (4.8) 71/29 | NA | NA | 654 | 85 | 7.7 | 0.78 | 5.1 |
78 Mock | 0 | 19.2/− | NA | NA | (3.1) 71/29 | NA | NA | 485 | 75 | 6.5 | 0.84 | 4.0 |
85 Mock | 0 | 21.4/− | NA | NA | (3.9) 56/44 | NA | NA | 531 | 79 | 6.7 | 0.84 | 4.4 |
63 Rota | 12 | 17.3/− | 17.9/− | NA | (8.5) 75/25 | (2.8) 61/39 | NA | 416 | 77 | 5.4 | 0.67 | 3.9 |
64 Rota | 12 | 21.6/− | 20.4/+ | NA | (5.5) 67/33 | (3.1) 58/42 | NA | 482 | 88 | 5.5 | 0.65 | 4.1 |
65 Rota | 18 | 18.9/− | 20.6/− | 16.5/+ | (9.1) 64/36 | (8.3) 70/30 | (3.1) 23/77 | 355 | 78 | 4.6 | 0.90 | 4.2 |
67 Rota | 18 | 18.8/− | 19.8/− | 12.4/+ | (7.5) 65/35 | (5.8) 62/38 | (1.8) 11/89 | 354 | 78 | 4.5 | 1.09 | 3.7 |
Microarray analysis
EST nr. | FCa
| FDR (%)b
| Blastn or blastxc
| Tentative function | |||
---|---|---|---|---|---|---|---|
12 h | 18 h | 12 h/18 h | Gene name or tentative annotation (T[H]C) | Acc. number |
E value (nt) | ||
Lower in rotavirus infected | |||||||
R1 | 0.16 | 0.12 | 4.6/0.35 | Cytochrome P450 2C49 (CYP2C49), mRNA | gi:47523893 | 0 (714) | Arachidonic acid metabolism |
R2 | 0.21 | – | 4.6/− | Thioredoxin (TXN) mRNA | gi: 73853748 | 0 (614) | Sulfate-redox metabolism/apoptosis |
R3 | 0.23 | – | 4.6/− | Fyn-related kinase (FRK), mRNA | gi:31657133 | 0 (602) | Signal transduction/cell-proliferation |
R4 | – | 0.13 | −/0.35 | (blastx) NADH dehydrogenase subunit 5 | gi:5835873 | 3E-55 (467) | Fatty acid metabolism |
R5 | – | 0.16 | −/0.35 | Acyl-CoA synthetase long-chain family member three mRNA (ACSL3) | gi:42794753 | 0 (632) | Fatty acid/arachidonic acid metabolism |
R6 | – | 0.17 | −/0.35 | Similar to meprin A beta-subunit precursor | pig|TC275453 | 4.4E-109 (505) | Food digestion and absorption |
R7 | – | 0.18 | −/0.35 | Similar to UDP-glucuronosyltransferase 2B5 precursor (UDPGT) (M-1) | pig|TC129860 | 3E-44 (491) | Androgen and estrogen metabolism |
R8 | – | 0.19 | −/0.35 | Similar to Ig J-chain | pig|TC262280 | 2E-77 (516) | Immune system |
R9 | – | 0.19 | −/0.35 | Similar to transmembrane 4 L six family member 20 (TM4SF20) (L6 membrane protein domain) | pig|TC159234 | 5.2E-119 (585) | Signal transduction/cell-proliferation |
R10 | – | 0.21 | −/0.35 | Human DNA sequence from clone RP11–413P11 | gi:14346089 | 7E-05 (569) | Unknown |
R11 | – | 0.21 | −/0.35 | Fatty acid binding protein 2, intestinal (IFABp 2) | gi:10938019 | 1E-118 (605) | Lipid transport |
R12 | – | 0.22 | −/0.35 | Homo sapiens uncharacterized bone marrow protein BM041 mRNA (proline rich 13 [PRR13]) | gi:7688976 | 2E-49 (599) | Transcription regulator/apoptosis |
R13 | – | 0.25 | −/0.35 | Na+/glucose cotransporter protein (SGLT1) mRNA, 3′ end | gi:164674 | 0 (473) | Membrane-transport |
Higher in rotavirus infected | |||||||
R14 | 9.4 | 15.0 | 4.6/0.35 | Similar to Homo sapiens transmembrane protein 106B (TMEM106B), mRNA (DUF1356.domain) | pig|TC209451 | 1.9E-34 (328) | Unknown |
R15 | 8.9 | 14.2 | 4.6/0.35 | (blastx) Phospholipase inhibitor (PLA2 inhibitor domain) | gi:37182061 | 1E-39 (779) | Signal transduction/membrane metabolism |
R16 | 7.0 | 14.2 | 4.6/0.35 | Mucus-type core 2 beta-1,6-N-acetylglucosaminyltransferase mRNA (GCNT3) | gi:32396225 | 0 (624) | Glycan/mucus metabolism |
R17 | 6.6 | – | 4.6/− | Weakly similar to oligo/dipeptide transport protein sulfolobus solfataricus (DppC-2) | cattle|TC272801 | 7E-44 (664) | Membrane-transport |
R18 | 6.3 | – | 4.6/− | Similar to HCV-associated microtubular aggregate protein p44 (IFN-induced protein 44/IFI44) | hum|THC2463234 | 2.9E-23 (534) | Cytoskeleton (IFN-induced) |
R19 | 6.3 | 3.7 | 4.6/0.35 | Maltase–glucoamylase (MGAM), mRNA [2.2/Probable alpha-glucosidase] | gi:4758711 | 0 (589) | Food digestion |
R20 | 5.0 | 4.0 | 4.6/0.35 | Keratin 20 (KRT20), mRNA | gi:27894336 | 3E-59 (413) | Cytoskeleton |
R21 | 4.7 | – | 4.6/− | Actin, beta (ACTB), mRNA | gi:57977284 | 3E-38 (235) | Cytoskeleton |
R22 | 4.6 | 4.2 | 4.6/0.35 | THO complex 4 (THOC4), mRNA | gi:55770863 | 1E-40 (280) | Transcriptional activator |
R23 | – | 7.5 | −/0.35 | Guanylate binding protein 2 (GBP-2), interferon-inducible, mRNA [5.1/GBP-1] | gi:18490137 | 1E-123 (711) | Signal transduction (IFN-induced) |
R24 | – | 7.2 | −/0.35 | Spermidine/spermine N-acetyltransferase (SAT), mRNA [6,7] | gi:47523773 | 1E-109 (546) | Polyamine metabolism/immune defence |
R25 | – | 6.4 | −/0.35 | Unannotated pig EST | pig|CN159449 | 2.0E-40 (616) | Unknown |
R26 | – | 5.6 | −/0.35 | Hypoth. prot small intestine (CD20/IgE Fc receptor subunit β domain/MS4A2) | gi:4186144 | 0 (354) | Signal transduction/Ca2+ homeostasis |
R27 | – | 5.3 | −/0.35 | Homo sapiens cDNA: FLJ21643 fis, clone COL08382 (RNA-binding protein) | gi:10437783 | 2E-55 (617) | RNA binding/translation |
R28 | – | 5.2 | −/0.35 | Sus scrofa caspase-3 (CASP3), mRNA | gi:47523065 | 0 (617) | Effector protein apoptosis |
R29 | – | 5.2 | −/0.30 | Similar to pyrophosphatase 1 (PPA1) mRNA, (LOC716720) [2,3] | gi:109089532 | 5E-28 (146) | Phosphate metabolism |
R30 | – | 4.3 | −/0.35 | proteasome (prosome, macropain) subunit alpha type, 6 (Psma6) mRNA | gi:23110943 | 0 (658) | Ubiquitin mediated proteolysis |