博士 / 國立臺灣大學 / 動物學研究所 / 96 / The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (≦1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line.
We obtained a full-length cDNA clone for the Mx gene of barramundi (Lates calcarifer), using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from a barramundi brain cell line cBB. The Mx cDNA of 2.2 kb contains an open reading frame (ORF) of 1875 nucleotides encoding a protein of 624 amino acids. The predicted barramundi Mx protein is 71.4 kDa and contains a tripartite guanosinetriphosphate (GTP)-binding motif at the amino terminal and a leucine zipper at the carboxyl terminal, characteristic of all known Mx proteins. Poly I:C-transfection induced the expression of Mx gene in cBB cells, and the induction level at 28 ℃ was higher than that at 20 ℃. Moreover, Mx gene expression was also induced by viral infection, including fish nodavirus, birnavirus, and iridovirus. Among these, nodavirus was a stronger inducer than the other two viruses. Using an antiviral activity assay, we revealed that poly I:C-transfected cBB cells had antiviral activity against fish nodavirus and birnavirus, but not iridovirus. Furthermore, the replication of nodavirus and birnavirus could be restored after the expression of Mx gene was down-regulated by siRNA. Therefore, these results indicated that the expression of barramundi Mx gene was able to inhibit the proliferation of fish nodavirus and birnavirus.
The expression levels of NNV RNA-dependent RNA polymerase (RdRp) and capsid protein were found to increase in NNV-infected cBB cells within 24 h post-infection (hpi), but later decrease as soon as the expression of barramundi Mx protein. Moreover, NNV capsid protein, RNA2, and viral titer all elevated in the cBB cells when barramundi Mx protein expression was down-regulated by siRNA. Barramundi Mx protein was observed to colocalize with NNV RdRp at the perinuclear area in NNV-infected cBB cells by immunofluorescence staining, and was co-precipitated with NNV RdRp by immunoprecipitation assay. Furthermore, the complexes of barramundi Mx protein and NNV RdRp could colocalize with lysosomes, and then NNV RdRp would be degragded by the lysosome system. Therefore, it was suggested that barramundi Mx protein suppressed NNV RNA synthesis by sequestration of NNV RdRp to the perinuclear area for the following degradation in the lysosomes.
| Identifer | oai:union.ndltd.org:TW/096NTU05312030 |
| Date | January 2008 |
| Creators | Yu-Chi Wu, 吳育騏 |
| Contributors | 齊肖琪 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | en_US |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 129 |
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