Porcine Reproductive and Respiratory Syndrome Virus Interferes with Swine Influenza A Virus Infection of Epithelial Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Newborn Pig Tracheal Epithelial Cell Line
2.2. Alveolar Macrophages
2.3. Virus Propagation, Purification and Titration
2.4. Real-Time Monitoring of SwIAV Cytopathic Effects Using Real-Time Cell Analysis
2.5. Virus Inactivation
2.6. Precision-Cut Lung Slices
2.7. Virus Infection and Stimulation of Newborn Pig Tracheal Epithelial Cells and Precision-Cut Lung Slices
2.8. Immune Gene Expression Analysis and Virus Detection by Quantitative Real-Time PCR
2.9. Western Blotting
2.10. Immunofluorescence Analyses
2.11. Statistical Analyses
3. Results
3.1. Simultaneous PRRSV-1 Contact with NPTr Epithelial Cells Strongly Decreases swIAV H1N2 Replication and Immune Gene Expression
3.2. The Effect of PRRSV-1 Is till Observed if PRRSV-1 Is Added Shortly before swIAV H1N2 to NPTr Cells
3.3. Inactivation of Non-Internalized Viruses by Acid Wash Abrogates PRRSV-1 Effect on swIAV H1N2 Infection in NPTr Cells
3.4. Inactivated PRRSV-1 still Impacts swIAV H1N2 Infection of Tracheal Epithelial Cells
3.5. PRRSV-1 Impacts swIAV H1N2 Infection in Primary Tissue Lung Slices
3.6. PRRSV-1 and swIAV H1N2-Induced Signaling Pathways in NPTr Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Abbreviation and Full Name | Primer Sequences: Sense (S) and Anti-Sense (AS) | Amplicon Size (bp) | Annealing Temperature (°C) | Accession Number or PMIDs |
---|---|---|---|---|
Viruses | ||||
SwIAV H1N2 M-encoding gene | (S) AGATGAGTCTTCTAACCGAGGTCG (AS) TGCAAAAACATCTTCAAGTCTCTG (P) (6FAM)-TCAGGCCCCCTCAAAGCCGA-(TAM) | 100 | 60 | 15460317 |
PRRSV Finistère ORF5 | (S) AGAACCAGCGCCAATTCAGA (AS) TCTTTTTCGCCTGTCCTCCC (P) (HEX)-AAACACAGCTCCAATGGGGAATGGC-(TAM) | 135 | 60 | 28241868 |
ADV gB-encoding gene | (S) GCGGGTACGTGTACTACGAG (AS) GAGGCCCTGGAAGAAGTTGG (P) (6FAM)-ACTACAGCTACGTGCGCATGGTGGAG-(TAM) | 287 | 63 | NC_006151 |
Reference genes | ||||
B2MI Beta-2-microgobulin | (S) CAAGATAGTTAAGTGGGATCGAGAC (AS) TGGTAACATCAATACGATTTCTGA | 161 | 58 | 17697375 |
HPRT1 Hypoxanthine phosphoribosyltransferase 1 | (S) GGACTTGAATCATGTTTGTG (AS) CAGATGTTTCCAAACTCAAC | 91 | 60 | 17697375 |
GAPDH Glyceraldehyde 3-phosphate dehydrogenase | (S) CTTCACGACCATGGAGAAGG (AS) CCAAGCAGTTGGTGGTACAG | 170 | 63 | AF017079 |
Pattern Recognition Receptors | ||||
MDA5 Melanoma differentiation-associated protein 5 | (S) AGCCCACCATCTGATTGGAG (AS) TTCTTCTGCCACCGTGGTAG | 133 | 60 | MF358967.1 |
RIGI Retinoic acid-inducible gene I | (S) CGACATTGCTCAGTGCAATC (AS) TCAGCGTTAGCAGTCAGAAG | 126 | 60 | NM_213804 |
TLR2 Toll-like receptor 2 | (S) ACGGACTGTGGTGCATGAAG (AS) GGACACGAAAGCGTCATAGC | 101 | 62 | NM_213761.1 |
TLR3 Toll-like receptor 3 | (S) GACCTCCCGGCAAATATAAC (AS) GGGAGACTTTGGCACAATTC | 155 | 60 | NM_001097444 |
TLR4 Toll-like receptor 4 | (S) TGTGCGTGTGAACACCAGAC (AS) AGGTGGCGTTCCTGAAACTC | 136 | 62 | NM_001293316.1 |
TLR6 Toll-like receptor 6 | (S) TCCCAGGATCAAGGTTCTTG (AS) GAGCAGAGTCCCCTTATAAC | 370 | 60 | NM_213760.2 |
TLR7 Toll-like receptor 7 | (S) CGGTGTTTGTGATGACAGAC (AS) AACTCCCACAGAGCCTCTTC | 174 | 62 | NM_001097434.1 |
TLR8 Toll-like receptor 8 | (S) CACATTTGCCCGGTATCAAG (AS) TGTGTCACTCCTGCTATTCG | 145 | 58 | NM_214187.1 |
TLR9 Toll-like receptor 9 | (S) GGCCTTCAGCTTCACCTTGG (AS) GGTCAGCGGCACAAACTGAG | 151 | 64 | NM_213958.1 |
TLR10 Toll-like receptor 10 | (S) CTTTGATCTGCCCTGGTATCTCA (AS) CATGTCCGTGCCCACTGAC | 51 | 60 | AB_208699,1 |
Interferons | ||||
IFNα Interferon alpha | (S) GGCTCTGGTGCATGAGATGC (AS) CAGCCAGGATGGAGTCCTCC | 197 | 62 | JQ839262.1 |
IFNβ Interferon beta | (S) AGTTGCCTGGGACTCCTCAA (AS) CCTCAGGGACCTCAAAGTTCAT | 70 | 60 | 21645029 |
IFNλ1 Swine interferon lamba 1 | (S) GAGGCTGAGCTAGACTTGAC (AS) CCTGAAGTTCGACGTGGATG | 115 | 60 | NM_001142837 |
IFNλ3 Swine interferon lamba 3 | (S) CCTGGAAGCCTCTGTCATGT (AS) TCTCCACTGGCGACACATT | 72 | 60 | 29677213 |
Interferon-stimulated genes | ||||
PKR Protein kinase R | (S) CACATCGGCTTCAGAGTCAG (AS) GGGCGAGGTAAATGTAGGTG | 166 | 61 | NM_214319 |
OAS1 2’-5’-Oligoadenylate Synthetase 1 | (S) CCCTGTTCGCGTCTCCAAAG (AS) GCGGGCAGGACATCAAACTC | 303 | 64 | NM_214303 |
MX1 Myxovirus resistance protein 1 | (S) AGTGTCGGCTGTTTACCAAG (AS) TTCACAAACCCTGGCAACTC | 151 | 60 | NM_214061 |
MX2 Myxovirus resistance protein 2 | (S) CCGACTTCAGTTCAGGATGG (AS) ACAGGAGACGGTCCGTTTAC | 156 | 62 | AB258432 |
ISG12 Interferon-stimulated gene 12 | (S) GTACTTCCTCCCTGATAGG (AS) ACAGCTACAGAAGCCTTG | 76 | 54 | NM_001198921.1 |
ISG15 Interferon stimulated gene 15 | (S) GATCGGTGTGCCTGCCTTC (AS) CGTTGCTGCGACCCTTGT | 176 | 58 | NM_001128469.3 |
Targeted Protein | Specific Antibody |
---|---|
-Phospho-AKT | -Rabbit polyclonal anti-phospho-AKT (Ser473) #9271 (Ozyme) |
-AKT | -Rabbit monoclonal anti-AKT (11E7) #4685 (Ozyme) |
-Phospho-AMPK -AMPK | -Rabbit monoclonal anti-phospho-AMPK alpha (Thr172) (40H9) #2535L (Ozyme) -Rabbit polyclonal anti-Total AMPK #2532L (Ozyme) |
-Phospho-ERK1/2 -ERK2 | -Rabbit monoclonal anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) #4370 (Ozyme) -Rabbit polyclonal anti-ERK2 (GTX27948) (Tebu-bio) |
-Phospho-JAK2 -JAK2 | -Rabbit polyclonal anti-phospho-JAK2 (Tyr1007/1008) #3771 (Ozyme) -Rabbit monoclonal anti-JAK2 (D2E12) #3230 (Ozyme) |
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Saade, G.; Ménard, D.; Hervet, C.; Renson, P.; Hue, E.; Zhu, J.; Dubreil, L.; Paillot, R.; Pronost, S.; Bourry, O.; et al. Porcine Reproductive and Respiratory Syndrome Virus Interferes with Swine Influenza A Virus Infection of Epithelial Cells. Vaccines 2020, 8, 508. https://doi.org/10.3390/vaccines8030508
Saade G, Ménard D, Hervet C, Renson P, Hue E, Zhu J, Dubreil L, Paillot R, Pronost S, Bourry O, et al. Porcine Reproductive and Respiratory Syndrome Virus Interferes with Swine Influenza A Virus Infection of Epithelial Cells. Vaccines. 2020; 8(3):508. https://doi.org/10.3390/vaccines8030508
Chicago/Turabian StyleSaade, Georges, Déborah Ménard, Caroline Hervet, Patricia Renson, Erika Hue, Jianzhong Zhu, Laurence Dubreil, Romain Paillot, Stéphane Pronost, Olivier Bourry, and et al. 2020. "Porcine Reproductive and Respiratory Syndrome Virus Interferes with Swine Influenza A Virus Infection of Epithelial Cells" Vaccines 8, no. 3: 508. https://doi.org/10.3390/vaccines8030508