Publications by authors named "Tong Ou"

15 Publications

  • Page 1 of 1

Rapid Genetic Diagnosis of Citrin Deficiency by Multicolor Melting Curve Analysis.

Front Pediatr 2021 5;9:654527. Epub 2021 May 5.

Prenatal Diagnosis Center and Medical Laboratory, The Third Affiliated Hospital of Shenzhen University (Luohu Hospital Group), Shenzhen, China.

Citrin deficiency caused by genetic mutations is an autosomal recessive disease, and four prevalent mutations including c.851_854del, c.1638_1660dup, IVS6+5G>A, and IVS16ins3kb make up >80% of total pathogenic mutations within the Chinese population. However, suitable assays for detection of these mutations have not yet been developed for use in routine clinical practice. In the current study, a real-time PCR-based multicolor melting curve analysis (MMCA) was developed to detect the four prevalent mutations in one closed-tube reaction. The analytical and clinical performances were evaluated using artificial templates and clinical samples. All four mutations in the test samples were accurately genotyped their labeling fluorophores and values, and the standard deviations of values were indicated to be <0.2°C. The limit of detection was estimated to be 500 diploid human genomes per reaction. The MMCA assay of 5,332 healthy newborns from southern China identified a total of 107 -mutation carriers, indicating a carrier rate of 2%. The genotypes of 107 carriers and 112 random non-carriers were validated using direct sequencing and Long-range PCR with 100% concordance. In conclusion, the assay developed in this study may potentially serve as a rapid genetic diagnostic tool for citrin deficiency.
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http://dx.doi.org/10.3389/fped.2021.654527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8133314PMC
May 2021

HSP47 contributes to angiogenesis by induction of CCL2 in bladder cancer.

Cell Signal 2021 May 14;85:110044. Epub 2021 May 14.

Institute of Urology, The Third Affiliated Hospital of Shenzhen University (Luohu Hospital Group), Shenzhen 518000, China; Shenzhen Following Precision Medical Research Institute, Luohu Hospital Group, Shenzhen 518000, China; Teaching Center of Shenzhen Luohu Hospital, Shantou University Medical College, Shenzhen 518000, China. Electronic address:

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and is involved in tumor progression by promoting angiogenesis. However, the regulatory network of HSP47 in angiogenesis remains elusive. In this study, we report a novel mechanism of HSP47-induced angiogenesis in bladder cancer (BC). We find that HSP47 is abnormally overexpressed in BC and is correlated with poor prognosis. HSP47 down-regulation suppresses angiogenesis in BC cells. Mechanistically, activation of the ERK pathway and induction of C-C Motif Chemokine Ligand 2 (CCL2) are responsible for HSP47-induced angiogenesis. The correlation between HSP47 with CCL2 and angiogenesis is further confirmed in BC clinical samples. Taken together, our findings suggest that HSP47 contributes to BC angiogenesis by induction of CCL2 and provide a potential anti-angiogenesis target for BC therapy.
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http://dx.doi.org/10.1016/j.cellsig.2021.110044DOI Listing
May 2021

Nitazoxanide impairs mitophagy flux through ROS-mediated mitophagy initiation and lysosomal dysfunction in bladder cancer.

Biochem Pharmacol 2021 May 4;190:114588. Epub 2021 May 4.

Institute of Urology, The Third Affiliated Hospital of Shenzhen University (Luohu Hospital Group), Shenzhen 518000, China; Shenzhen Following Precision Medical Research Institute, Luohu Hospital Group, Shenzhen 518000, China; Teaching Center of Shenzhen Luohu Hospital, Shantou University Medical College, Shenzhen 518000, China; Department of Urology and Guangdong Key Laboratory of Urology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510000, China. Electronic address:

Bladder cancer is one of the most common malignancy in the urinary tract with high recurrence and drug resistance in clinics. Alternative treatments from existing drugs might be a promising strategy. Nitazoxanide (NTZ), an FDA-approved antiprotozoal drug, has got increasingly noticed because of its favorable safety profile and antitumor potential, yet the effects in bladder cancer and underlying mechanisms remain poorly understood. Herein, we find that NTZ induces mitochondrial damage and mitophagy initiation through PINK1-generated phospho-ubiquitin(pS65-Ub) and autophagy receptor-mediated pathway even in the absence of Atg5/Beclin1. Meanwhile, NTZ inhibits lysosomal degradation activity, leading to mitophagy flux impairment at late stage. Mitochondrial reactive oxygen species (ROS) production is critical in this process, as eliminating ROS with N-acetylcysteine (NAC) efficiently inhibits PINK1 signaling-mediated mitophagy initiation and alleviates lysosomal dysfunction. Co-treatment with NTZ and autophagy inhibitor Chloroquine (CQ) to aggravate mitophagy flux impairment promotes NTZ-induced apoptosis, while alleviation of mitophagy flux impairment with ROS scavenger reduces cell death. Moreover, we also discover a similar signaling response in the 3D bladder tumor spheroid after NTZ exposure. In vivo study reveals a significant inhibition of orthotopic bladder tumors with no obvious systemic toxicity. Together, our results uncover the anti-tumor activities of NTZ with the involvement of ROS-mediated mitophagy modulation at different stages and demonstrate it as a potential drug candidate for fighting against bladder tumors.
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http://dx.doi.org/10.1016/j.bcp.2021.114588DOI Listing
May 2021

LvHemB1, a novel cationic antimicrobial peptide derived from the hemocyanin of Litopenaeus vannamei, induces cancer cell death by targeting mitochondrial voltage-dependent anion channel 1.

Cell Biol Toxicol 2021 Feb 25. Epub 2021 Feb 25.

Institute of Marine Sciences and Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, 515063, China.

Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.
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http://dx.doi.org/10.1007/s10565-021-09588-yDOI Listing
February 2021

Development of a novel prognostic signature for predicting the overall survival of bladder cancer patients.

Biosci Rep 2020 06;40(6)

Medical Laboratory of the Third Affiliated Hospital of Shenzhen University, Shenzhen 518001, China.

Background: Bladder cancer is one of the most common malignancies. So far, no effective biomarker for bladder cancer prognosis has been identified. Aberrant DNA methylation is frequently observed in the bladder cancer and holds considerable promise as a biomarker for predicting the overall survival (OS) of patients.

Materials And Methods: We downloaded the DNA methylation and transcriptome data for bladder cancer from The Cancer Genome Atlas (TCGA), a public database, screened hypo-methylated and up-regulated genes, similarly, hyper-methylation with low expression genes, then retrieved the relevant methylation sites. Cox regression analysis was used to identify a nine-methylation site signature of a training group. Predictive ability was validated in a test group by receiver operating characteristic (ROC) analysis.

Results: We identified nine bladder cancer-specific methylation sites as potential prognostic biomarkers and established a risk score system based on the methylation site signature to evaluate the OS. The performance of the signature was accurate, with area under curve was 0.73 in the training group and 0.71 in the test group. Taking clinical features into consideration, we constructed a nomogram consisting of the nine-methylation site signature and patients' clinical variables, and found that the signature was an independent risk factor.

Conclusions: Overall, the significant nine methylation sites could be novel prediction biomarkers, which could aid in treatment and also predict the overall survival likelihoods of bladder cancer patients.
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http://dx.doi.org/10.1042/BSR20194432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286875PMC
June 2020

Whole-genome sequencing identifies ADGRG6 enhancer mutations and FRS2 duplications as angiogenesis-related drivers in bladder cancer.

Nat Commun 2019 02 12;10(1):720. Epub 2019 Feb 12.

Urology Institute of Shenzhen University, The Third Affiliated Hospital of Shenzhen University, Shenzhen University, Shenzhen, 518000, China.

Bladder cancer is one of the most common and highly vascularized cancers. To better understand its genomic structure and underlying etiology, we conduct whole-genome and targeted sequencing in urothelial bladder carcinomas (UBCs, the most common type of bladder cancer). Recurrent mutations in noncoding regions affecting gene regulatory elements and structural variations (SVs) leading to gene disruptions are prevalent. Notably, we find recurrent ADGRG6 enhancer mutations and FRS2 duplications which are associated with higher protein expression in the tumor and poor prognosis. Functional assays demonstrate that depletion of ADGRG6 or FRS2 expression in UBC cells compromise their abilities to recruit endothelial cells and induce tube formation. Moreover, pathway assessment reveals recurrent alterations in multiple angiogenesis-related genes. These results illustrate a multidimensional genomic landscape that highlights noncoding mutations and SVs in UBC tumorigenesis, and suggest ADGRG6 and FRS2 as novel pathological angiogenesis regulators that would facilitate vascular-targeted therapies for UBC.
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http://dx.doi.org/10.1038/s41467-019-08576-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372626PMC
February 2019

Genome analysis and gene nblA identification of Microcystis aeruginosa myovirus (MaMV-DC) reveal the evidence for horizontal gene transfer events between cyanomyovirus and host.

J Gen Virol 2015 Dec;96(12):3681-3697

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, PR China.

The genome sequence, genetic characterization and nblA gene function of Microcystis aeruginosa myovirus isolated from Lake Dianchi in China (MaMV-DC) have been analysed. The genome DNA is 169 223 bp long, with 170 predicted protein-coding genes (001L–170L) and a tRNA gene. About one-sixth of these genes have homologues in the host cyanobacteria M. aeruginosa. The genome carries a gene homologous to host nblA, which encodes a protein involved in the degradation of cyanobacterial phycobilisome. Its expression during MaMV-DC infection was confirmed by reverse transcriptase PCR and Western blot detection and abundant expression was companied by the significant decline of phycocyanin content and massive release of progeny MaMV-DC. In addition, expressing MaMV-DC nblA reduced the phycocyanin peak and the phycocyanin to chlorophyll ratio in model cyanobacteria. These results confirm that horizontal gene transfer events have occurred between cyanobacterial host and cyanomyovirus and suggest that MaMV-DC carrying host-derived genes (such as 005L, that codes for NblA) is responsible for more efficient expression of cyanophage genes and release of progeny cyanophage. This study provides novel insight into the horizontal gene transfer in cyanophage and the interactions between cyanophage and their host.
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http://dx.doi.org/10.1099/jgv.0.000290DOI Listing
December 2015

Unraveling the genome structure of cyanobacterial podovirus A-4L with long direct terminal repeats.

Virus Res 2015 May 30;203:4-9. Epub 2015 Mar 30.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Wuhan 430072, China. Electronic address:

The freshwater cyanobacterial virus (cyanophage) A-4L, a podovirus, can infect the model cyanobacterium Anabaena sp. strain PCC 7120 resulting in a high burst size and forming concentric plaques on its lawns. The complete genome sequence of A-4L was determined by the combination of high-throughput sequencing, terminal transferase-mediated polymerase chain reaction and restriction mapping. It contains 41,750 bp with 810 bp direct terminal repeats and 38 potential open reading frames. As compared with other cyanobacterial podoviruses in diverse ecosystems, the A-4L has the longest terminal repeat and shares similar genome organizations with freshwater members. Furthermore, phylogenetic analysis based on concatenated sequences of eight core proteins indicated that freshwater cyanobacterial podoviruses were clustered together and distinct from marine counterparts, suggesting a clear divergence in the cyanobacterial podovirus lineage between freshwater and marine ecosystems. Our findings uncover the unique genome structure of A-4L which contains long direct terminal repeats, and create the first model system to address knowledge gaps in understanding cyanobacterial virus-host interactions at the molecular level.
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http://dx.doi.org/10.1016/j.virusres.2015.03.012DOI Listing
May 2015

[Main reason for concentric rings plaque formation of virus infecting cyanobacteria (A-4L) in lawns of Anabaena variabilis].

Wei Sheng Wu Xue Bao 2014 Feb;54(2):191-9

Objective: To clarify an important biological characteristic of virus infecting cyanobacteria (A-4L) and to isolate, identify new bloom-forming cyanobacteria viruses, we studied A-4L concentric rings plaque formation in Anabaena sp. PCC7120.

Methods: One step growth curve was designed to estimate the latent period and burst size of A4L. The initial titer of A-4L was about 2.8 x 10(10) PFU/mL. The appropriate titer suspension of A-4L was inoculated onto the lawns of Anabaena sp. PCC 7120 which have been cultivated at different time. Pathological change of lawns was observed and recorded daily. To investigate the effect of lighting on the concentric rings plaque formation, plates were cultivated and infected under continuous lighting (L: D = 24 h: 0 h), periodic lighting (L:D = 14 h:10 h) or 3 days continuous lighting after periodic lighting for 3 days. The ultra-morphology of purified A-4L was observed by negative staining electron microscopy.

Results: The latent period of A-4 (L) was 0.5 h-2 h and the burst size was about 247 infectious units per cell. Under periodic lighting, concentric rings plaques were observed in the plate after infection 3 days to 4 days and the distance between two rings was about 3 mm. Statistic analysis showed that there was a correlation between the number of concentric rings in plaques and infection days, which was "n -1". Compared with the periodic lighting, the plaques without concentric rings were observed under continuous lighting. However, the concentric rings formed under periodic lighting disappeared gradually after turning to continuous lighting, which demonstrated that the formation of concentric rings plaques depended on the periodic lighting. Negative staining electron microscopy showed that the A-4L particle had a spheroidal head with diameter about 50 nm and a tail with length about 10 nm which was similar to the characteristic morphology of cyanobacterial podoviruses.

Conclusion: A-4L is a virus infecting cyanobacteria which can form concentric rings plaque. And periodic lighting is the key conditions for the concentric rings plaque formation of A4L.
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February 2014

Development of an Ussuri catfish Pseudobagrus ussuriensis skin cell line displaying differential cytopathic effects to three aquatic animal viruses.

Virus Res 2014 Aug 30;189:56-62. Epub 2014 Apr 30.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Electronic address:

An Ussuri catfish Pseudobagrus ussuriensis skin (UCS) cell line was developed and subcultured for more than 60 passages. UCS cells consisted of mostly epithelial-like cells and multiplied well in TC199 medium supplemented with 10% fetal bovine serum at 25°C. Chromosome analysis revealed that most UCS cells had a normal diploid karyotype with 2n=52. UCS cells showed differential cytopathic effects (CPEs) after inoculation of spring viremia of carp virus (SVCV, a negative-strand RNA virus), grass carp reovirus (GCRV, a multi-segmented double-stranded RNA virus) and Rana grylio virus (RGV, a large double-stranded DNA virus), and were indicative of high sensitivities to these three aquatic animal viruses by a virus titration study. The CPE caused by SVCV appeared as rounded and granular cells, grape-like clusters and small lytic plaques. Characteristic CPE containing plaque-like syncytia was induced by GCRV. RGV-infected cells produced typical CPE characterized by cells shrinkage and aggregation, formation of clear plaques and cell sheet detachment. Furthermore, significant fluorescent signals were observed after UCS cells were transfected with green fluorescent protein reporter plasmids, and the development of CPE induced by a recombinant RGV, ΔTK-RGV, in UCS cells was illustrated using a combination of light and fluorescence microscopy. The data from this study suggested that UCS cell line can potentially serve as a useful tool for the comparison study of different aquatic animal viruses and the isolation of some newly emerging viruses in Ussuri catfish farming.
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http://dx.doi.org/10.1016/j.virusres.2014.04.013DOI Listing
August 2014

Isolation and identification of a lethal rhabdovirus from farmed rice field eels Monopterus albus.

Dis Aquat Organ 2013 Nov;106(3):197-206

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate University of Chinese Academy of Sciences, Wuhan 430072, PR China.

We provide the first description of a virus responsible for a systemic hemorrhagic disease causing high mortality in farmed rice field eels Monopterus albus in China. Typical signs exhibited by the diseased fish were extensive hemorrhages in the skin and viscera and some neurological signs, such as loss of equilibrium and disorganized swimming. Histopathological examination revealed various degrees of necrosis within the spleen and liver. Virus isolation was attempted from visceral tissues of diseased fish by inoculation on 6 fish cell lines. Typical cytopathic effects (CPE) were produced in bluegill fry (BF2) cells, so this cell line was chosen for further isolation and propagation of the virus. Electron microscopy observation showed that the negative stained viral particles had the characteristic bullet shape of rhabdoviruses and an estimated size of 60 × 120 nm. We therefore tentatively refer to this virus as Monopterus albus rhabdovirus (MoARV). Molecular characterization of MoARV, including sequence analysis of the nucleoprotein (N), phosphoprotein (P), and glycoprotein (G) genes, revealed 94.5 to 97.3% amino acid similarity to that of Siniperca chuatsi rhabdovirus. Phylogenetic analysis based on the amino acid sequences of N and G proteins indicated that MoARV should be a member of the genus Vesiculovirus. Koch's postulates were fulfilled by infecting healthy rice field eels with MoARV, which produced an acute infection. RT-PCR analysis demonstrated that MoARV RNA could be detected in both naturally and experimentally infected fish. The data suggest that MoARV was the causative pathogen of the disease.
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http://dx.doi.org/10.3354/dao02660DOI Listing
November 2013

Two virus-like particles that cause lytic infections in freshwater cyanobacteria.

Virol Sin 2013 Oct 12;28(5):303-5. Epub 2013 Sep 12.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.

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http://dx.doi.org/10.1007/s12250-013-3339-0DOI Listing
October 2013

Cultivation and characterization of the MaMV-DC cyanophage that infects bloom-forming cyanobacterium Microcystis aeruginosa.

Virol Sin 2013 Oct 26;28(5):266-71. Epub 2013 Aug 26.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China.

The MaMV-DC cyanophage, which infects the bloom-forming cyanobacterium Microcystis aeruginosa, was isolated from Lake Dianchi, Kunming, China. Twenty-one cyanobacterial strains were used to detect the host range of MaMV-DC. Microcystic aeruginosa FACHB-524 and plaque purification were used to isolate individual cyanophages, and culturing MaMV-DC with cyanobacteria allowed us to prepare purified cyanophages for further analysis. Electron microscopy demonstrated that the negatively stained viral particles are tadpole-shaped with an icosahedral head approximately 70 nm in diameter and a contractile tail approximately 160 nm in length. Using one-step growth experiments, the latent period and burst size of MaMV-DC were estimated to be 24-48 hours and approximately 80 infectious units per cell, respectively. Restriction endonuclease digestion and agarose gel electrophoresis were performed using purified MaMV-DC genomic DNA, and the genome size was estimated to be approximately 160 kb. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed four major structural proteins. These results support the growing interest in using freshwater cyanophages to control bloom-forming cyanobacterium.
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http://dx.doi.org/10.1007/s12250-013-3340-7DOI Listing
October 2013

Rana grylio virus (RGV) 50L is associated with viral matrix and exhibited two distribution patterns.

PLoS One 2012 13;7(8):e43033. Epub 2012 Aug 13.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan, China.

Background: The complete genome of Rana grylio virus (RGV) was sequenced and analyzed recently, which revealed that RGV 50 L had homologues in many iridoviruses with different identities; however, the characteristics and functions of 50 L have not been studied yet.

Methodology/principal Findings: We cloned and characterized RGV50L, and revealed 50 L functions in virus assembly and gene regulation. 50 L encoded a 499-amino acid structural protein of about 85 kDa in molecular weight and contained a nuclear localization signal (NLS) and a helix- extension-helix motif. Drug inhibition assay demonstrated that 50 L was an immediate-early (IE) gene. Immuno-fluorescence assay revealed that 50 L appeared early and persisted in RGV-infected cells following two distribution patterns. One pattern was that 50 L exhibited a cytoplasm-nucleus- viromatrix distribution pattern, and mutagenesis of the NLS motif revealed that localization of 50 L in the nucleus was NLS-dependent; the other was that 50 L co-localized with viral matrix which plays important roles in virus assembly and the life circle of viruses.

Conclusions/significance: RGV 50L is a novel iridovirus IE gene encoded structural protein which plays important roles in virus assembly.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043033PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418244PMC
May 2013

Sequencing and analysis of the complete genome of Rana grylio virus (RGV).

Arch Virol 2012 Aug 28;157(8):1559-64. Epub 2012 Apr 28.

State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Wuhan 430072, China.

Infection with Rana grylio virus (RGV), an iridovirus isolated in China in 1995, resulted in a high mortality rate in frogs. The complete genome sequence of RGV was determined and analyzed. The genomic DNA was 105,791 bp long, with 106 open reading frames (ORFs). Dot plot analysis showed that the gene order of RGV shared colinearity with three completely sequenced ranaviruses. A phylogenetic tree was constructed based on concatenated sequences of iridovirus 26 core-gene-encoded proteins, and the result showed high bootstrap support for RGV being a member of the genus Ranavirus and that iridoviruses of other genera also clustered closely. A microRNA (miRNA) prediction revealed that RGV could encode 18 mature miRNAs, many of which were located near genes associated with virus replication. Thirty-three repeated sequences were found in the RGV genome. These results provide insight into the genetic nature of RGV and are useful for laboratory diagnosis for vertebrate iridoviruses.
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http://dx.doi.org/10.1007/s00705-012-1316-9DOI Listing
August 2012