Search Results (5)

Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species. Full article

Glycosylphosphatidylinositol mannosyltransferase I (GPI-MT-I) is an essential glycosyltransferase of glycosylphosphatidylinositol-anchor proteins (GPI-APs) that transfers the first of the four mannoses in GPI-AP precursors, which have multiple functions, including immune response and signal transduction. In this study, the GPI-MT-I gene that regulates GPI-AP biosynthesis in Andrias davidianus (AdGPI-MT-I) was characterized for the first time. The open reading frame (ORF) of AdGPI-MT-I is 1293 bp and encodes a protein of 430 amino acids that contains a conserved PMT2 superfamily domain. AdGPI-MT-I mRNA was widely expressed in the tissues of the Chinese giant salamander. The mRNA expression level of AdGPI-MT-I in the spleen, kidney, and muscle cell line (GSM cells) was significantly upregulated post Chinese giant salamander iridovirus (GSIV) infection. The mRNA expression of the virus major capsid protein (MCP) in AdGPI-MT-I-overexpressed cells was significantly reduced. Moreover, a lower level of virus MCP synthesis and gene copying in AdGPI-MT-I-overexpressed cells was confirmed by western blot and ddPCR. These results collectively suggest that GSIV replication in GSM cells was significantly reduced by the overexpression of the AdGPI-MT-I protein, which may contribute to a better understanding of the antiviral mechanism against iridovirus infection. Full article

Chinese giant salamander iridovirus (GSIV) infection could lead to mitochondrial apoptosis in this animal, a process that involves B-cell lymphoma-2 (BCL-2) superfamily molecules. The mRNA expression level of Bcl-xL, a crucial antiapoptotic molecule in the BCL-2 family, was reduced in early infection and increased in late infection. However, the molecular mechanism remains unknown. In this study, the function and regulatory mechanisms of Chinese giant salamander (Andrias davidianus) Bcl-xL (AdBcl-xL) during GSIV infection were investigated. Western blotting assays revealed that the level of Bcl-xL protein was downregulated markedly as the infection progressed. Plasmids expressing AdBcl-xL or AdBcl-xL short interfering RNAs were separately constructed and transfected into Chinese giant salamander muscle cells. Confocal microscopy showed that overexpressed AdBcl-xL was translocated to the mitochondria after infection with GSIV. Additionally, flow cytometry analysis demonstrated that apoptotic progress was reduced in both AdBcl-xL-overexpressing cells compared with those in the control, while apoptotic progress was enhanced in cells silenced for AdBcl-xL. A lower number of copies of virus major capsid protein genes and a reduced protein synthesis were confirmed in AdBcl-xL-overexpressing cells. Moreover, AdBcl-xL could bind directly to the proapoptotic molecule AdBak with or without GSIV infection. In addition, the p53 level was inhibited and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-xL-overexpressing cells during GSIV infection. These results suggest that AdBcl-xL plays negative roles in GSIV-induced mitochondrial apoptosis and virus replication by binding to AdBak and inhibiting p53 activation. Full article

Mx, Myxovirus resistance is an important interferon-stimulated protein that mediates antiviral responses. In this study, the expression and activities of Chinese giant salamander, Andrias davidianus Mx gene, AdMx, were investigated. The AdMx cDNA sequence contains an open reading frame (ORF) of 2112 nucleotides, encoding a putative protein of 703 aa. Meanwhile, AdMx possesses the conserved tripartite GTP binding motif and a dynamin family signature. qRT-PCR analysis revealed a broad expression of AdMx in vivo, with the highest expression levels in brain, kidney and spleen. The AdMx expression level in kidney, spleen and muscle significantly increased at 6 h after Chinese giant salamander iridovirus (GSIV) infection and peaked at 48 h, while that in muscle cell line (GSM) was not noticeably up-regulated until 72 h post infection. Additionally, a plasmid expressing AdMx was constructed and transfected into the Chinese giant salamander GSM cells. The virus load and gene copies in AdMx over-expressed cells were significantly reduced compared with those in the control cells. Moreover, compared to the control cells, a lower level of virus major capsid protein (MCP) synthesis in AdMx over-expressed cells was confirmed by Western blot. These results collectively suggest that Mx plays an important antiviral role in the immune responses against GSIV in Chinese giant salamander. Full article

Chinese giant salamander iridovirus (GSIV) is the causative pathogen of Chinese giant salamander (Andrias davidianus) iridovirosis, leading to severe infectious disease and huge economic losses. However, the infection mechanism by GSIV is far from clear. In this study, a Chinese giant salamander muscle (GSM) cell line is used to investigate the mechanism of cell death during GSIV infection. Microscopy observation and DNA ladder analysis revealed that DNA fragmentation happens during GSIV infection. Flow cytometry analysis showed that apoptotic cells in GSIV-infected cells were significantly higher than that in control cells. Caspase 8, 9, and 3 were activated in GSIV-infected cells compared with the uninfected cells. Consistently, mitochondria membrane potential (MMP) was significantly reduced, and cytochrome c was released into cytosol during GSIV infection. p53 expression increased at an early stage of GSIV infection and then slightly decreased late in infection. Furthermore, mRNA expression levels of pro-apoptotic genes participating in the extrinsic and intrinsic pathway were significantly up-regulated during GSIV infection, while those of anti-apoptotic genes were restrained in early infection and then rose in late infection. These results collectively indicate that GSIV induces GSM apoptotic cell death involving mitochondrial damage, caspases activation, p53 expression, and pro-apoptotic molecules up-regulation. Full article