Molecular characterization of three Rana grylio virus (RGV) isolates and Paralichthys olivaceus lymphocystis disease virus (LCDV-C) in iridoviruses

Three Rana grylio virus (RGV) isolates and lymphocystis disease virus (LCDV-C) were molecularly characterized by antigenicity comparison, Western blot detection of viral polypeptides, restriction fragment length polymorphism analysis of viral genomes, and MCP sequence analysis. Significant antigenicity differences existed among the three RGV isolates and LCDV-C. Western blot detection indicated that the viral polypeptides of three RGV isolates could be recognized by the anti-RGV9807 serum, whereas no bands were observed in the LCDV-C, and significant differences exist among the band patterns of three RGV isolates. Restriction fragment length polymorphism (RFLP) analysis was performed by digesting genomic DNA of the four iridovirus isolates with restriction endonucleases HindIII, KpnI, XbaI and BamHI. On the whole, obvious discrepancies existed between LCDV-C and RGV isolates, and some significant band pattern differences were also revealed between RGV9808 and RGV9506 (or RGV9807) in the profiles of restriction endonucleases XbaI, KpnI and BamHI. PCR amplification and sequence analysis of MCP gene sequence further revealed their phylogenetic relationship among the three RGV isolates, LCDV-C and other iridoviruses. RGV9506, RGV9807 and RGV9808 are clustered together with other ranaviruses, such as FV3, BIV, TFV and ENHV, although the RGV9808 is more close to EHNV than to other ranaviruses. Additionally, LCDV-C is clustered with LCDV-1, the type species of genus Lymphocystisvirus. The current study provides clear evidence that significant genetic difference exists among the three RGV isolates. Therefore, further work on comparative genomic studies will contribute significantly to understanding of their taxonomic position and pathological mechanism.     

Magnitude of the US trade in amphibians and presence of Batrachochytrium dendrobatidis and ranavirus infection in imported North American bullfrogs (Rana catesbeiana)

Amphibians are globally threatened by anthropogenic habitat loss, the wildlife trade and emerging diseases. Previous authors have hypothesized that the spread of the amphibian disease chytridiomycosis (Batrachochytrium dendrobatidis) and amphibian ranaviruses are associated with the international trade in live amphibians. The North American bullfrog (Rana catesbeiana) is thought to be a carrier of these pathogens, is globally traded as a live commodity, and is sold live in US markets. We obtained importation data for all live amphibians, and parts thereof, into three major US ports of entry (Los Angeles, San Francisco and New York) from 2000 to 2005. Importation of live amphibians into these ports totaled almost 28 million individuals over this 6-year period. We collected samples from freshly-imported market frogs and found infection with both pathogens in all three cities and all seasons, with an overall infection prevalence of 62% (306/493) and 8.5% (50/588) for B. dendrobatidis and ranaviruses, respectively, by PCR. This study definitively identifies these two important pathogens in recently imported live market frogs and suggests that the amphibian trade can contribute to introductions of these pathogens into new regions. It provides support for the recent listing of B. dendrobatidis and ranaviral diseases by the OIE, and provides evidence for measures to be taken to eradicate these pathogens from the trade.     

蛙病毒的转录组和蛋白质组分析研究进展

蛙病毒属是一群大型的胞浆型DNA 病毒属虹彩病毒科能导致包括鱼类、两栖类和爬行类等冷血动 物产生严重疾病,蛙病毒分子生物学研究是阐明病毒复制,疾病产生各种相关因子作用机制的基础,微阵列分析技 术,蛋白和DNA 复制抑制下的转录产物分析,质谱蛋白质组学等分子生物学技术已被广泛应用于分析蛙病毒的基 因转录和蛋白表达多种蛙病毒转录组和蛋白质组分析使人们能够了解这一类病毒基因的转录产物转录时相病 毒蛋白表达的整体状态功能结构和可能的调控方式.     

大口黑鲈虹彩病毒双重PCR检测方法的建立

为同时检测患病大口黑鲈中不同属虹彩病毒感染和带毒情况,针对蛙病毒属和肿大细胞病毒属大口黑鲈虹彩病毒主衣壳蛋白（major capsid protein,MCP）基因的序列,分别设计1对特异性引物Rana-mcp F/R和Mega-mcp F/R,扩增产物片段大小分别为475 bp和262 bp。通过PCR反应体系的优化、反应的特异性和灵敏度试验,建立一种可以同时检测大口黑鲈蛙病毒属和肿大细胞病毒属虹彩病毒感染的双重PCR检测方法,最低DNA检测量分别为6.5 pg和14.5 pg。用此方法,对临床获得的15个大口黑鲈样品进行双重PCR检测和序列测定,获得5个蛙病毒属和1个肿大细胞病毒属虹彩病毒检测阳性结果。本研究中建立的基于MCP基因的大口黑鲈虹彩病毒双重PCR检测方法,可用于养殖大口黑鲈虹彩病毒病快速鉴别诊断和分子流行病学调查。     

大口黑鲈虹彩病毒病研究进展

大口黑鲈虹彩病毒(LMBV;又称SCRV)引起的病毒病是迄今为止国内外大口黑鲈病害研究报道最多的病毒性疾病之一.在大口黑鲈原产地美国,LMBV广泛的感染性和致病性引起科研人员和养殖人员的高度关注.论文综述了大口黑鲈虹彩病毒病的来源、地理分布、传播途径、致病性及防控措施等方面的研究进展,并对该病的防控进行了展望,对于国内...     

Investigation of ornamental fish entering the EU for the presence of ranaviruses

A survey was performed on ornamental fish imported into the EU to detect viral agents belonging to the genus Ranavirus. The objective was to gain knowledge of the potential for these systemic iridoviruses to gain entry into the EU via international trade in ornamental fish. A total of 208 pooled samples, representing 753 individual fish, were tested. The samples included 13 orders and 37 families, originating from different countries and continents. Tissues from fish that died during or just after transport were collected and examined by standard virological techniques in epithelioma papulosum cyprini cells, by transmission electron microscopy and by PCR for the detection of the major capsid protein and DNA polymerase gene sequences of ranaviruses. Virus was isolated from nine fish species but ranavirus was not identified in those samples. The results suggest that ranaviruses are not highly prevalent in ornamental fish imported into the EU.     

蛙虹彩病毒巢式 PCR检测方法的建立

蛙虹彩病毒属(Ranavirus)病毒宿主广泛,可以感染爬行类、鱼类和两栖类,大部分病毒对宿主都有较强的致病性和致死性.为建立一种快速高效的蛙虹彩病毒的检测方法,本研究利用中华鳖虹彩病毒(soft-shelled Turtle Iridovirus,STIV)核衣壳蛋白(Major Capsid Protein,MCP)基因保守区设计内引物和外引物,建立了特异性检测流行性造血器官坏死病毒(Epizootic Haematopoietic Necrosis Virus,EHNV)、中华鳖虹彩病毒和虎纹蛙虹彩病毒(Tiger Frog Virus,TFV)的巢式PCR(巢式PCR)检测方法,并制备了重组质粒pGem-T-S作为阳性对照标准品.检测限试验结果显示,该方法可以检测102拷贝的病毒粒子.而且与传染性造血器官坏死病毒、鲤春病毒、病毒性出血性败血症病毒、斑点叉尾(鱼回)病毒、传染性胰脏坏死病毒、真鲷虹彩病毒、牙鲆弹状病毒以及锦鲤疱疹病毒等其他非蛙虹彩病毒无交叉反应.该体系具有简便、快速、敏感、特异性高、低成本等特点,为诊断与预防蛙虹彩病毒提供了一项重要的技术手段.