| Infectious bursal disease virus (IBDV) causes a severe immunosuppression by destroying the precursors of B lymphocytes in the bursa of Fabricius. To investigate effects of virus infection as well as its encoded genes on the IBDV-susceptible cells, Vero cell was used as a model and its mRNA profiles were analyzed by long serial analysis of gene expression (LongSAGE) after infected by IBDV or transformed with various encoded genes of IBDV A-segment.Cloning of the full-length genomic A-segment of IBDV The full-length cDNA of genomic A-segment was amplified from IBDV NB strain by a long accurate reverse-transcription polymerase chain reaction (LA-PCR). The cloned A-segment of 3,259 nucleotides contains two partially overlapping open reading fragments (ORF1 and ORF2) flanked by 5' and 3' noncoding regions (NCR). The deduced amino acid sequence has 81.8-99.9% identities to other IBDV strains in GenBank. Phylogenetic analyses indicated that NB strain is mainly closed to the isolates of JD1, Cu-1, P2 and CEF94, but is distinct from vvIBDV and variant strains isolated from Europe, Hong Kong and Japan. It has the highest identity to JD1 on the polyprotein (VP2/4/3) encoded by ORF1 (99.5%), VP3 (99.2%) and VP4 (100%), to the isolates of JD1, CEf94 and D78 on VP2 (99.8%), and to JD1, HZ2, P2, CEF94, CT, Cu-1 and D78 on VP5 (99.3%).Establishment of Vero cell lines expressing IBDV genes in stable The full-length IBDV A-segment, polyprotein (VP2/4/3), VP3, VP5 and serial truncated VP2s genes were respectively inserted into pCI-neo and pEGFP-C2 vectors resulting in 21 recombinant expression plasmids of pCI-neo-A, pCI-neo-VP2/4/3, pCI-neo-VP3, pCI-neo-VP5 and pCI-neo-pVP2s, as well as pEGFP-VP5 and pEGFP-pVP2s. The recombinant plasmids of pEGFP-VP5 and pEGFP-pVP2s were firstly transfected into Vero cells and expression of the EGFP-fusion proteins were analyzed. The results demonstrated that Vero cells began to express EGFP-VP5 molecules around the plasma membrane of Vero cells at 4 to 5 h after transfected. Full and truncated pVP2 proteins fused with EGFP were observed in the cytoplasm of Vero cell at 9 to 12 h post-transfected. After transfected with pCI-neo-A, pCI-neo-VP2/4/3, pCI-neo-VP5, pCI-neo-VP3, or pCI-neo-pVP2s, Vero cells were selected by antibiotic G418 and cloned by limiting dilution. The A-segment, VP2/4/3, VP5, VP3 or truncated pVP2 genes in the monoclonal G418-resistant cells were detected by PCR/Southern blot, RT-PCR/Northern blot, Indirect Immunofluorescent Antibody (IFA), Immunoperoxidase Monolayer Assay (IPMA) and western blot analyses, respectively. As a result, we established the tranfected Vero cell lines expressing IBDV genes as follow: two monoclonal cells integrated with the A-segment expressing all proteins of VP2, VP4, VP3 and VP5, three monoclonal cells integrated with the VP2/4/3 gene expressing VP2, VP4 and VP3 proteins, Seven monoclonal cells expressing VP5 protein, seventeen monoclonal cells integrated with the truncated pVP2s expressing the corresponding complete or truncated pVP2 proteins, and three monoclonal cells expressing VP3 protein.Construction and analysis of LongSAGE libraries Five LongSAGE libraries were constructed using mRNAs isolated from the IBDV-infected Vero cells, monoclonal cell lines (Vero-A cells, Vero-VP2/4/3 cells, Vero-VP5 cells), and Vero cells, respectively. About 2,000 positive transformants carrying the interested fragments (>500 bp) were selected for sequencing from each library. The expressed tags were extracted, after base-calling and implementation in R project. In total, 96,213 tags were generated from five LongSAGE libraries, including Vero cells (17,663 tags), IBDV-infected cells (22,193 tags), Vero-A cells (22,968 tags), Vero-VP243 cells (19,168 tags) and Vero-VP5 cells (14,221 tags). 96,213 tags represent 24,475 transcripts (25.44%), extracting from Vero cells (8,369 transcripts), IBDV-infected cells (9,187 transcripts), Vero-A cells (10,160 transcripts), Vero-VP243 cells (8,840 transcripts) and Vero-VP5 cells (7,130 transcripts).In Fisher's exact test, there were 37 up-regulated and 84 down-regulated transcripts in IBDV-infected cells, 40 up-regulated and 104 down-regulated transcripts in Vero-A cells, 43 up-regulated and 74 down-regulated transcripts in Vero-VP2/4/3 cells, and 36 up-regulated and 18 down-regulated transcripts in Vero-VP5 cells comparing with the normal Vero cells (P<0.05). Comparing with IBDV-infected cell, 335 transcripts in Vero-A cells, 334 transcripts in Vero-VP2/4/3 cells, and 91 transcripts in Vero-VP5 cells were transcripted differentially (P<0.05). Comparing with Vero-A cells, there were 49 differentially expressed transcripts in Vero-VP2/4/3 cells and 113 differentially expressed transcripts in Vero-VP5 cells. Comparing with Vero-VP2/4/3 cells, 104 transcripts were expressed differentially in Vero-VP5 cells. Virtual northern blot and chromsome location showed that 3,124 assigned genes were mapped to human chromosomes.
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