Project description:The DNAJ family, a class of chaperone proteins involved in protein folding, assembly, and transport, plays an essential role in viral infections. However, the role of DNAJA4 (DnaJ Heat Shock Protein Family (Hsp40) Member A4) in the ranavirus infection has not been reported. This study demonstrates the function of the epithelial papilloma of carp (EPC) DNAJA4 in Chinese giant salamander (Andrias davidianus) iridovirus (CGSIV) replication. DNAJA4 consists of 1479 base pairs and encodes a 492 amino acid polypeptide. Sequence analysis has shown that EPC DNAJA4 contains a conserved J domain and shares 84% homology with Danio rerio DNAJA4 and 68% homology with Homo sapiens DNAJA4. EPC DNAJA4 was localized in the cytoplasm, and its expression was significantly upregulated after CGSIV infection. Overexpression of EPC DNAJA4 promotes CGSIV replication and CGSIV DNA replication. siRNA knockdown of DNAJA4 expression attenuates CGSIV replication and viral DNA replication. Overexpression and interference experiments have proved that EPC DNAJA4 is a pro-viral factor. Co-IP, GST-pulldown, and immunofluorescence confirmed the interaction between EPC DNAJA4 and CGSIV proliferating cell nuclear antigen (PCNA). Our results demonstrate for the first time that EPC DNAJA4 is involved in viral infection by promoting viral DNA replication and interacting with proteins associated with viral replication.
Project description:The emergence of an infectious viral disease caused by the Chinese giant salamander iridovirus (GSIV) has led to substantial economic losses. However, no more molecular information is available for the understanding of the mechanisms associated with virus-host interaction. In this study, de novo sequencing was used to obtain abundant high-quality ESTs and investigate differentially-expressed genes in the spleen of Chinese giant salamanders that were either infected or mock infected with GSIV. Comparative expression analysis indicated that 293 genes were down-regulated and 220 genes were up-regulated. Further enrichment analysis showed that the most enriched pathway is "complement and coagulation cascades", and significantly enriched diseases include "inherited thrombophilia", "immune system diseases", "primary immunodeficiency", "complement regulatory protein defects", and "disorders of nucleotide excision repair". Additionally, 30 678 simple sequence repeats (SSRs) from all spleen samples, 26 355 single nucleotide polymorphisms (SNPs) from the spleens of uninfected animals and 36 070 SNPs from the spleens of infected animals were detected. The large amount of variation was specific for the Chinese giant salamanders that were infected with GSIV. The results reported herein provided significant and new EST information that could contribute greatly in investigations into the molecular functions of immune genes in the Chinese giant salamander.
Project description: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.
Project description:Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), which is extensively expressed in vertebrates, is a deubiquitinating enzymes that inhibits the degradation of proteins by reversing ubiquitination modification. Herein, a 1087-bp sequence encoding UCHL1 was identified from the Chinese giant salamander (CGS; Andrias davidianus). The coding sequences (CDS) of UCHL1 encoded a putative poly peptide of 222 amino acids. The CGS UCHL1 isoforms were more related to their human and mouse counterparts. The phylogenic tree of vertebrate UCHL1 indicated that CGS UCHL1 has the closest relationship with human UCHL1 (up to 73.99 %). Before the gonads of male CGSs matured, the peak level of UCHL1 expression in testes appeared in 3-year-old CGSs according to RT-qPCR and western blot. In adult testes, the level of UCHL1 protein was lower in the breeding period than in the post-breeding period, whereas the level of UCHL1 protein in interstitial fluid of adult CGS testes was higher during the breeding period than during the post-breeding period. In testicular seminiferous lobules in the developmental stage of CGSs, immunohistochemistry displayed three kinds of localizing patterns of UCHL1, including nuclear localization at half year old, cytoplasmic localization from one year to three years old, and extracellular localization in adult. In testicular seminiferous lobules of adult CGS, the different developmental germ cells were separated by cysts containing UCHL1 protein, but UCHL1 did not localize on the mature sperm. The results showed that extracellular UCHL1 loaded on exosomes, as a component of the homogeneous germ cell cysts, could regulate the synchronous development of sperm in testes of adult CGS.
Project description: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.
Project description:B-cell lymphoma-2 (BCL-2) superfamily molecules play crucial roles in mitochondrial apoptosis induced by Chinese giant salamander iridovirus (GSIV). As an anti-apoptotic molecule in the BCL-2 family, the molecular mechanism of Bcl-w during GSIV infection remains unknown. In this study, we characterized for the first time an amphibian Bcl-w from Chinese giant salamander Andrias davidianus (AdBcl-w), and its function and regulatory mechanism during GSIV infection were investigated. AdBcl-w possesses the conserved structural features of Bcl-w and shares 35-54% sequence identities with other Bcl-w. mRNA expression of AdBcl-w was most abundant in liver and muscle. The AdBcl-w mRNA expression was regulated during GSIV infection. Western blotting assays revealed that the level of Bcl-w protein was downregulated markedly as the infection progresses. Confocal microscopy showed that overexpressed AdBcl-w was translocated to the mitochondria after infection with GSIV. Flow cytometry analysis demonstrated that compared with control, the apoptotic progress in cells transfected with AdBcl-w was reduced while that in cells transfected with AdBcl-w siRNA was enhanced. The number of virus major capsid protein gene copies was lower and protein synthesis was reduced in AdBcl-w overexpressing cells. In addition, AdBcl-w could bind directly to the pro-apoptotic molecule AdBak, while this interaction was weakened with GSIV infection. Moreover, p53 level was reduced and the mRNA expression levels of crucial regulatory molecules in the p53 pathway were regulated in AdBcl-w overexpressing cells during GSIV infection. These results suggested that AdBcl-w inhibit GSIV replication by regulating the virus induced mitochondrial apoptosis.
Project description: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.
Project description:Understanding animal movement is a key question in ecology and biodiversity conservation, which is particularly important for the success of reintroduction projects. The movement of critically endangered Chinese giant salamander (Andrias davidianus) remains poorly understood due to the rareness of wild individuals of this species. We lack movement details about the full annual cycle after reintroduction, especially the abiotic and biotic influences that affect its movement. We utilized pilot reintroduction projects as opportunities to fill in some knowledge gaps on their movement ecology. We released 31 juvenile captive-reared Chinese giant salamanders of two age groups in two rivers in the Qinling Mountains of central China and monitored their daily movements for 16 months using surgically implanted radio transmitters. We examined the impacts of individual traits (body mass, body condition) and environmental conditions (temperature, precipitation, and moon phase) on their daily movement patterns. Data were analyzed using a mixed-effects logistic regression model to understand the drivers of their movement tendency (i.e., whether they move or not) and a linear mixed-effects model was used to understand the drivers of their movement distance. We found that movement tendency of the older salamander cohort was positively affected by the moon phase, increasing near the Full Moon, whereas the younger cohort of animals were not impacted by the moon phase. For daily distance moved, we found temperature had a strong positive effect on both cohorts, whereas precipitation had moderate but opposite effects on the two cohorts Body mass and body condition did not have any significant impacts on either age classes' movement tendency or distance. This study provides insight into the abiotic factors that impact the temporal and spatial movement ecology of reintroduced giant salamander, which will in turn help with designing best practices for future releases and conservation of this iconic montane aquatic predator.
Project description:We report here the complete genome sequence of a megalocytivirus strain, GSIV-K1, isolated from a farmed giant sea perch (Lates calcarifer) in Kaohsiung, Taiwan. GSIV-K1 causes mortality in farmed marine fish, including giant sea perch and groupers. The genome sequence is nearly identical to the genome of the orange-spotted grouper iridovirus.
Project description:Singapore grouper iridovirus (SGIV), one of the nucleocytoviricota viruses (NCVs), is a highly pathogenic iridovirid. SGIV infection results in massive economic losses to the aquaculture industry and significantly threatens global biodiversity. In recent years, high morbidity and mortality in aquatic animals have been caused by iridovirid infections worldwide. Effective control and prevention strategies are urgently needed. Here, we present a near-atomic architecture of the SGIV capsid and identify eight types of capsid proteins. The viral inner membrane-integrated anchor protein colocalizes with the endoplasmic reticulum (ER), supporting the hypothesis that the biogenesis of the inner membrane is associated with the ER. Additionally, immunofluorescence assays indicate minor capsid proteins (mCPs) could form various building blocks with major capsid proteins (MCPs) before the formation of a viral factory (VF). These results expand our understanding of the capsid assembly of NCVs and provide more targets for vaccine and drug design to fight iridovirid infections.