| Classical swine fever virus (CSFV) is a small enveloped virus with a positive stranded RNA genome which belongs in the genus Pestivirus, together with two bovine viral diarrhea viruses (BVDV) and border disease virus (BDV), within the family Flaviviridae. The viral genome is approximately 12.5 kb in size and contains a single large open reading frame that encodes a 3,898 amino acid polyprotein. CSFV has a particular tropism for cells of the immune system and is known to cause severe leukopenia, in particular lymphopenia, featuring atrophy of primary lymphoid tissue and bone marrow, and depletion of different subsets of leukocytes (T- and B-lymphocytes, monocytes-macrophages and granulocytes) in infected pigs. All leukocytes are depleted during CSFV infection, but B-lymphocytes are particularly susceptible. Besides the reduction in leukocyte numbers, lymphocyte activation and function during virulent CSFV infection are severely impaired, thereby significantly decreasing antibody production and host defenses. The destruction of leukocytes following CSFV infection is largely associated with apoptosis in thymus, spleen, lymph nodes and bone marrow.DNA microarray technology, in combination with bioinformatics, has proved to be a very efficient high-throughput tool and offers great advantages in the study of genomic expression profiles of cells. The present study provides the first pan-genomic microarray analysis of pig transcriptional responses to CSFV infection, in which the genomic transcriptional levels of porcine PBL prepared from CSFV-inoculated pigs with severe clinical symptoms were analyzed using the Affymetrix porcine GeneChip. Total RNA was extracted from PBL collected before and after infection. Results showed that expression of 2,919, 2,859 and 2,995 genes from the 3 pigs were altered post-infection, of which altered expression of 2,662 genes was identified in two or three animals and subsequently subjected to one-way ANOVA statistical analysis (p<0.05). In all, 1,745 genes (8.64 % of all genes present in the array) were confirmed as having a greater than twofold altered expression, with 877 up-regulated and 868 down-regulated. Of 877 up-regulated genes, 24.3% (213/877) were found in all three animals, 61.2% (537/877) found up-regulated in two animals while not changed in another, 14.5% (127/877) found up-regulated in two animals while down-regulated in another. Of 868 down-regulated genes, 37.2% (323/868) were found in all three animals, 55.6% (483/868) found down-regulated in two animals while not changed in one, 7.2% (62/868) found down-regulated in two animals while up-regulated in the third. The functions of the 1,745 genes were observed by the Gene Ontology tool, available from the Affymetrix web site. Surprisingly, 88.3 % (1,540/1,745) were not functionally annotated, with only 205 genes (11.7 %) showing clear functional annotation. These clustered into 8 functional groups: cell proliferation and cycle (62/1745; 3.6 %), immune response (37/1745; 2.1 %), protein kinase activity (25/1745; 1.4 %) , apoptosis (24/1745; 1.4 %), signal transduction (24/1745; 1.4 %), transcription (13/1745; 0.7 %), receptor activity (13/1745; 0.7 %), cytokine/ chemokine (7/1745; 0.4 %).Viral infections are generally associated with numerous changes in host gene expression that determine the fate of the infected cells and the eventual outcome of the viral infection. Molecular pathogenetic studies of viral infection involve the investigation of these specific changes within the host cell or, more completely, within a specific tissue or organ. The blood samples used in the study were collected at day 7 p.i., the most severe phase of the disease. Real time RT-PCR showed that the numbers of gene copies of CSFV replicating in PBL of the infected pigs were 106.03±526 copies 100/ng total RNA (mean±SD), indicating that a high proportion of the leukocytes were infected. A rapid onset of leukopenia was detected with levels of PBL in all 3 infected animals declining from 20,667±1,379 cells/μl before infection to 12,000±1,646 cells/μl at day 4 p.i. This loss of PBL further progressed until day 10 p.i., reaching levels as low as 7,100±1,176 cells/μl. This result confirms the depletion of PBL in the infected pigs. To investigate further loss of lymphocytes - the main target of CSFV during infection - the T-cell subpopulations of the 3 pigs were analyzed by FACS at days 4, 7 and 10. Results showed that the severest depletion of CD3+CD4+ and CD3+CD8+ T-cell subsets (P <0.05) were observed by day 7 p.i. This is consistent with the findings of a previous study, showing that CD3+CD4+ cells and CD3+CD8+ cytotoxic T-cell subsets were strongly influenced by the viral infection. To explore the factors causing leukopenia apoptosis analysis was applied to PBL using the Annexin V: FITC Apoptosis Detection Kit and FACScan flow cytometry and using the manufacturers'protocols. Results clearly showed that apoptosis was observed in PBL from the same blood samples of the 3 pigs used for microarray with the proportion of apoptotic cells in a total of 10,000 leucocytes per test being significantly increased from 7.03 % before infection to 25.56 % (P<0.05) afterwards, while cell death increased correspondingly, from 1.48 % to 6.46 % (P<0.05). This increase in apoptotic PBL is consistent with the observations of a previous study. Analyzed the 24 apoptotic genes showed that the significant differences were observed in cellular apoptosis genes expression post-infection (p.i.) and the network of apoptosis was concluded. This study provided a valuable information for further exploring the molecular mechanism of apoptosis caused by CSFV infection.In this study we defined Heme oxygenase 1 (HO-1) as a novel CSFV E2 action partner in PK-15 cells. E2 binding was confined to productively infected PK-15. We demonstrate that the CSFV E2 interaction likely occurs at the limiting membranes of late endosomes/multivesicular bodies and that HO-1 depletion is associated with a significant decline in the viral replication and infectivity of released virions; this don't coincided with the increasing of apoptosis occurred in PK-15 inoculated CSFV compared to contrals. Cumulatively, our data suggest that HO-1 is essential for the proper life cycle of CSFV in target cells.In conclusion, the present study has described a complete transcriptional response of pigs to CSFV infection. Microarray analysis has shown that expression of 1,745 genes in PBL were altered following CSFV infection, This work has established a most comprehensive differential transcriptional profile of CSFV-infected pigs, although the application of these data to elucidate the viral pathogenesis is still limited. Further functional investigation of the altered genes may facilitate understanding of the pathogenic mechanisms and molecular responses of host cells to CSFV infection. |
PDF Full Text Request