RNA干扰对大鲵蛙病毒主要功能基因表达及其增殖的影响

采用q PCR与病毒滴度测定观察RNA干扰(RNA interference,RNAi)对大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)主要衣壳蛋白(major capsid protein,MCP)、甲基转移酶(methyltransferases,MTases)、DNA多聚酶(DNA polymerase)基因表达与病毒增殖的影响。结果表明,小干扰RNA(small interfering RNA,siRNA)能推迟鲤鱼上皮瘤细胞(epithelioma papillosum cyprini,EPC)出现病变,且病变程度也较对照组轻。在各功能基因表达量上,NC-FAM对照组与阴性对照组差异不显著(P0.05);而干扰组的干扰率极显著高于阴性对照组,其中siR-DP-1组siRNA对MCP基因的干扰率为79%,极显著高于其余组(P0.01);siR-MT-1、siR-MT-2组siRNA对MTases基因的干扰率为77%,极显著高于其余组(P0.01);siR-DP-1组siRNA对DNA polymerase基因的干扰率为79%,极显著高于其余组(P0.01)。干扰实验组病毒滴度与NC-FAM对照组和阴性对照组相比也有不同程度的降低。阴性对照组lg TCID50为8.362,NC-FAM对照组lg TCID50为7.848,siR-MCP、siR-MT、siR-DP实验组最低lg TCID50分别为5.764、5.317、5.362。证实RNAi能够抑制CGSRV主要功能基因的表达并影响病毒的复制。     

一株湖北源大口黑鲈蛙病毒的分离鉴定

采用电子显微镜观察和细胞培养等技术, 从湖北黄陂某养殖场患病大口黑鲈(Micropterus salmoides)体内发现并分离到一株蛙病毒。患病大口黑鲈的临床症状主要表现为体表出血、溃疡, 肝脏发白。将病鱼内脏组织匀浆超微滤液接种鳜脑细胞系(mandarin fish brain, MFB)细胞能产生典型细胞病变效应(cytopathic effect, CPE), 病毒滴度达到 10^8.36±0.15 TCID₅₀/mL。细胞培养病毒的超薄切片电镜观察结果显示, 细胞质中存在大量直径约为 150 nm 左右的正六边形病毒粒子, 呈晶格排列。细胞培养病毒的人工感染大口黑鲈试验结果显示, 7 d 内试验鱼死亡率高达 100%, 其临床症状与自然发病鱼相似。采用大口黑鲈病毒(largemouth bass virus, LMBV)的特异性 PCR 检测方法对患病鲈组织样品和细胞培养病毒样品进行检测, 均能扩增出 241 bp 的单一目的条带。进一步根据 GenBank 中 LMBV 主衣壳蛋白(major capsid protein, MCP)基因序列设计特异性引物, 均能从上述样品中扩增出 1392 bp 的 MCP 基因开放阅读框(open reading frame, ORF)全长。将 MCP 氨基酸全序列进行比对, 结果显示其与 Santee-Cooper 蛙病毒、孔雀鱼病毒 6 型及大口黑鲈溃疡综合征病毒的 MCP 氨基酸序列同源性高达 100%。系统进化结果显示, 与感染鱼类的虹彩病毒科蛙病毒属病毒, 如鳜鱼蛙病毒、Santee-Cooper 蛙病毒、孔雀鱼病毒 6 型和大口黑鲈溃疡综合征病毒等聚成一支。这些结果证明, 该分离株为虹彩病毒科蛙病毒属的成员, 暂命名为大口黑鲈蛙病毒(largemouth bass ranavirus, LMBRaV)湖北株 LMBRaV-HB001。病毒敏感细胞系筛选试验结果表明, 病毒 LMBRaV-HB001 感染鲤上皮瘤细胞(epithelioma papulosum cyprinid, EPC)、草鱼性腺细胞(grass carp ovary, GCO)、大鲵肌肉细胞(giant salamander muscle, GSM)和鲫脑组织细胞(gibel carp brain, GiCB)均能产生典型 CPE, 病毒滴度可达 10^8.0 TCID₅₀/mL 以上。本研究首次在湖北省养殖大口黑鲈体内分离与鉴定了 LMBRaV 病毒, 建立了病毒的细胞培养方法, 为进一步研究该病毒的传播、诊断和防控技术提供了重要参考。     

大鲵细胞与大鲵虹彩病毒的微载体规模化培养工艺及优化

利用Cytodex 3微载体悬浮培养系统规模化培养大鲵肌肉细胞(GSM)和大鲵虹彩病毒(GSIV),研究了微载体培养GSM细胞的形态和增殖特性,同时测定了病毒在培养系统中的增殖动态相关指标。结果显示,在Cytodex 3微载体培养系统中,将GSM细胞在贴壁期以转速30 r/min,每静置40 min搅拌2 min的方式间歇搅拌,10 h后贴壁率可达95%,培养基中最适血清浓度为10%,最适微载体浓度为2 g/L,最适细胞初始接种密度为1.2×10~5 cells/mL;增殖期以25 r/min的连续搅拌方式可以达到最佳的细胞生长效能。倒置显微镜与扫描电镜观察结果显示,GSM细胞呈长梭形,紧密贴附在Cytodex 3微载体上,生长良好。采用优化的工艺条件培养GSM细胞,以感染复数(MOI)为0.5的剂量接种GSIV至规模化培养的GSM细胞,48 h后GSM细胞出现典型的细胞病变效应,72 h病毒滴度达到最高TCID_(50)=10~(–8.50±0.20)/mL。本研究为大鲵虹彩病毒病疫苗的规模化生产工艺研究奠定了前期基础。     

一株似鲇高原鳅源蛙病毒的分离与鉴定

从四川乐山某养殖场自然发病的似鲇高原鳅(Triplophysa siluroides)体内分离到一株病毒FYL140220。病毒感染鲤上皮瘤细胞系(epithelima popuasum cuprini,EPC)后,细胞呈现圆缩、坏死、脱落、明显空斑的病变特征。将自然发病鱼组织过滤除菌液和细胞培养病毒液分别接种健康似鲇高原鳅,试验鱼出现与自然发病鱼相同的症状,死亡率分别为30%和40%,而对照组无异常。对经FYL140220感染出现典型细胞病变(CPE)的EPC细胞制备超薄切片进行电镜观察,发现病毒呈正六边形,对称20面体,对角线直径约(103±7)nm;提取细胞培养病毒液、自然发病鱼和人工感染发病鱼的内脏器官总DNA进行蛙病毒主要衣壳蛋白(maior capsid protein,MCP)基因保守区域的PCR扩增,均能扩增出预期大小约500 bp的特异性条带。MCP基因同源性与遗传进化分析表明,分离株FYL140220与蛙病毒属成员聚为一支,尤其与大鲵蛙病毒与沼泽绿蛙病毒同源性最高,分别达99.8%和99.6%。结合PCR检测、电镜观察和系统发育分析,确认分离病毒FYL140220为蛙病毒,这是首次报道蛙病毒可自然感染似鲇高原鳅并致死。     

一个与大鲵蛙病毒 (ADRV) 感染相关基因 —6R的克隆、原核表达及其产物分离

在新近报道大鲵蛙病毒(Andriasda vidianus ranaviurs,ADRV)基因组的基础上,我们对ADRV 6R进行了克隆、原核表达及其产物的分离,这是一个经生物信息分析表明与病毒感染相关的功能基因。先经PCR扩增长到711 bp的ADRV6R,构建含扩增片段的克隆质粒p MD18-T-6R。再构建原核表达质粒p ET-32a-6R,进行测序,并与水生动物蛙病毒相关基因同源序列进行比对,该扩增片段与预期一致,其编码分子量约为27 k D、含236个氨基酸的蛋白,与水产动物病毒US22蛋白家族(US22 family protein)成员有很高的同源性。然后对p ET-32a-6R进行转化、诱导表达,并利用Ni-NTA His-Bind亲和层析及不同浓度咪唑洗脱液对表达产物进行分离、电泳分析,结果显示,获得与预测分子量相符的45 k D融合蛋白(27 k D目的蛋白与18 k D His标签)。这为后续制备抗体、并开展大鲵蛙病毒与宿主的相互作用研究提供了有用的实验材料。     

大鲵蛙病毒感染大鲵的动态病理损伤及病原的组织分布

本研究旨在观察大鲵在大鲵蛙病毒(Chinese giant salamander ranavirus,CGSRV)感染过程中组织的动态病理损伤,同时定量检测CGSRV在组织中的动态分布。对大鲵腹腔注射1.0×106.5 TCID50/m L的CGSRV进行人工感染,在感染的0d,3d,5d,7d,9d,13d,16d随机采集3尾,取肝、肾、脾、肺、肠、皮肤、肌肉、脑、心脏和胃等组织,石蜡切片和HE染色对CGSRV感染大鲵的病理损伤过程进行观察,采用SYBR Green q PCR技术对病毒在组织中的动态分布进行定量研究。组织病理结果表明,CGSRV感染导致大鲵多组织器官损伤,其中肝、脾、肾和皮肤肌肉病变严重,为损伤靶器官,且在一些损伤的细胞内见嗜碱性或嗜酸性包涵体。感染后3d肝细胞与肾小管上皮细胞变性,肾间以及肝小叶中央静脉周围淋巴细胞和嗜酸性粒细胞浸润;感染后5~7d实质性器官变性、坏死,炎症加重,胃肠道呈卡他性炎。感染后9d表皮细胞坏死、脱落,肌纤维变性、坏死。感染后13~16d肝出现广泛变性、坏死与炎症,脾淋巴细胞数量显著减少,肾小球渗出性-坏死性炎,皮肤肌肉呈出血性坏死性炎和实质性心肌炎。SYBR Green qPCR结果显示,整个感染进程中各组织CGSRV含量呈上升趋势,不同组织中病毒量为2.36×103~1.84×109 copy/mg组织,其中肺、肠、肝、脾、肾和皮肤肌肉含量高,表明CGSRV具有广泛的组织分布特征,但肝、脾、肾、皮肤肌肉为其复制和损伤的靶器官,且病毒分布量与病理损伤程度呈正相关。     

大鲵虹彩病毒理化及生物学特性研究

对大鲵虹彩病毒(Giant salamander iridovirus,GSIV)的理化特性及生物学特性进行了研究。结果表明:GSIV对热处理敏感,56℃和65℃处理30 min均可彻底灭活病毒;GSIV经酸(pH3)和碱(pH10)处理,病毒滴度(TCID50)与对照组相比较分别下降了8.58、9.04个对数级,差异极显著(P<0.01);GSIV经有机溶剂氯仿、乙醚以及胰蛋白酶处理,TCID50与对照组相比较分别下降了9.33、7.83、6.49个对数级,差异极显著(P<0.01)。冻融次数对GSIV滴度的影响不显著(P>0.05)。GSIV对细胞培养物的感染性试验结果表明,GSIV可在鲤上皮瘤细胞系(Epithelioma papilloma cyprini,EPC)、斑点叉尾鮰肾脏细胞系(Channel catfish kidney,CCK)、虹鳟鱼性腺细胞系(Rainbow trout gonadal,RTG-2)等细胞中增殖,但在EPC、CCK细胞中增殖速度快,TCID50高;GSIV在EPC细胞中的最适生长温度是25℃。GSIV在EPC细胞中增殖动态试验结果表明,GSIV感染细胞6 h后TCID50开始快速上升,进入对数增长期,72 h时TCID50达到最大值,以后趋于稳定。GSIV感染EPC细胞超薄切片透射电镜观察结果显示,在EPC细胞质中可见大量虹彩病毒样颗粒,呈晶格状排列,直径约140 nm。     

鳜脑组织细胞系的建立及其病毒敏感性研究

鱼类细胞是开展鱼类病毒分离鉴定、功能基因分析以及生物制品制备等研究的重要物质基础。鳜(Siniperca chuatsi)是深受养殖者和消费者欢迎的养殖品种。随着鳜鱼养殖产量的逐年增加,其病害问题尤其是病毒病问题也日趋严重,但是,可用于鳜鱼病毒分离和基因功能分析的鳜细胞系缺乏。本研究采用组织块消化法,对来源鳜脑组织的细胞进行原代培养,建立了鳜脑组织细胞系,命名为MFB。MFB细胞在28℃含10%胎牛血清的L-15中已稳定传代超过70次,第25代鳜脑组织细胞的染色体众数为56。采用免疫荧光细胞化学技术(β-tubulin和Neu-N)鉴定MFB细胞的神经元纯度,结果显示,培养的MFB细胞为神经元类细胞。病毒敏感性实验结果显示,鳜蛙虹彩病毒(MFRaIV)、大口黑鲈蛙虹彩病毒(LMBRaIV)和大鲵虹彩病毒(GSIV)均可在MFB细胞中产生典型细胞病变效应,病毒滴度分别为108.68±0.12、108.36±0.15、1010.15±1.85 TCID50/mL。使用脂质体Lipofectamine®2000将pEGFP-N1转入MFB细胞,转染效率可达20%。本研究建立的鳜脑组织细胞系不仅对多种蛙虹彩病毒敏感,而且转染质粒效率较高,为鳜病毒性病原的分离及基因功能研究奠定了前期基础。     

大鲵感染蛙病毒的病理特征及预防措施

近年来随着大鲵人工养殖的迅速发展,病害发生日渐严重,已成为大鲵升级养殖的瓶颈,自2010年在陕西汉中、四川绵阳等地发生了大面积的大鲵感染蛙病毒事件,给大鲵养殖户造成了巨大的损失。现将大鲵感染蛙病毒的病理特征及预防措施分析、归纳和总结如下,供同行参考。一、大鲵感染蛙病毒的特征大鲵感染蛙病毒的特征主要表现在:大鲵摄食量减少,食欲降低甚至完全没有食欲,某些患病大鲵还发生吐食的情况,体表分泌大量的黏液,活动减少,躺卧在池底,并在患病后的5～10天内死亡。感染蛙     

大鲵虹彩病毒的形态结构及其包涵体特征

运用电镜和免疫荧光技术,对纯化的大鲵(Andrias davidianus)虹彩病毒粒子、感染病毒的EPC细胞以及确诊感染病毒的病鲵组织样品进行观察和分析。结果表明,纯化的大鲵虹彩病毒负染后电镜下显示球形结构,具囊膜,直径约150 nm;感染EPC细胞中的病毒颗粒呈典型的正二十面体结构,由核衣壳和核心构成,核衣壳呈正六边形,对角直径为(150±5)nm(N=30),核衣壳厚度约5 nm,核心直径为(98±18)nm(N=27)。在病鲵病变的肺和肾组织中发现存在大量聚集或分散的病毒颗粒,其形态特征和感染EPC细胞超薄切片观察的结果一致。免疫荧光电镜观察结果显示,感染病毒的细胞可观察到明显的红色荧光信号,且呈斑块状分布,大小不等。综合大鲵虹彩病毒初步的形态发生和免疫荧光观察结果,认为病毒感染细胞后在不同的发生时期会形成不同类型的病毒包涵体。     

Characterization and virus susceptibility of a skin cell line from red-spotted grouper (Epinephelus akaara)

A red-spotted grouper Epinephelus akaara skin (RGS) cell line was established and characterized. RGS cells had a normal diploid chromosome number of 2n = 48, the morphology of which was fibroblastic-like in 3 days and epithelial-like over 5 after 16 passages. The cells multiplied well in Dulbecco’s modified Eagle’s medium supplemented with 10% of fetal bovine serum at 25°C. Susceptibilities of RGS and grass carp ovary (GCO) cells to two viruses were tested, and the results showed that the titer of an iridovirus Rana grylio virus (RGV) in RGS cells was 10^3.5 TCID₅₀ ml^?1, which was much higher than a rhabdovirus spring viremia of carp virus (SVCV) in the cells (10^0.5 TCID₅₀ ml^?1). The titers of RGV and SVCV in GCO were 10^6.0 TCID₅₀ ml^?1 and 10^8.0 TCID₅₀ ml^?1, respectively, which were higher than those in RGS cells. The data may imply that RGS cells could be selectively resistible to some viruses during infection. RT-PCR analysis of RGV-infected RGS cells showed that RGV could replicate in RGS cells. Further study of virus replications in RGS cells was conducted by electron microscopy and immunofluorescence microscopy has shown that virus particles scattered in the cytoplasm and virus protein appeared in both the cytoplasm and nucleus. The results suggested that RGS cells could be used as a potential in vitro model to study the cutaneous barrier function against virus infection.     

Establishment and characterization of a new cell line from koi brain (Cyprinus carpio L.)

A novel permanently growing brain cell line from koi (Cyprinus carpio L.) (KB cell line) was established, and its suitability for detection of koi herpesvirus (KHV) was demonstrated in this study. The KB cell line was optimally maintained at 27°C in Leibovitz's L‐15 medium supplemented with 10% foetal bovine serum (FBS). It was subcultured more than 100 times, and chromosome analysis revealed that 51.54% of KB cells at passage 80 maintained the abnormal diploid chromosome number 2n = 96 while the modal chromosome number was 2n = 100. The cell line was cryopreserved in liquid nitrogen at ?196°C and was recovered from storage after 1 year with good cell viability and vitality. The results of virus isolation demonstrated that KB cells were susceptible to KHV, which was shown by the presence of an obvious cytopathic effect and abundant virus particles. The viral titres of KHV in KB reached 10^5.73TCID₅₀/0.1 ml within 7 days. Immunofluorescence and Western blot assays confirmed that KB replicated KHV. The newly established KB cell line will serve as a useful tool to elucidate KHV disease (KHVD) pathogenesis.     

In vitro cultivation model for Heterosporis saurida (Microsporidia) isolated from lizardfish,Saurida undosquamis (Richardson)

Heterosporis saurida is a microsporidian that infects lizardfish, Saurida undosquamis (Richardson, 1848), in the Arabian Sea. Spores were isolated from infected lizardfish and used to infect derived fish cell lines: common carp brain (CCB), epithelioma papulosum cyprinid (EPC), fathead minnow epithelial (FHM), rainbow trout gonad (RTG), bluegill fry (BF‐2) and chinook salmon embryo (CHSE). Non‐fish cell lines were also tested that include: insect (SF‐9), rabbit (RK‐13) and African green monkey (Vero E6). No growth of H. saurida was observed in any fish cell line, SF‐9 or Vero E6 cell lines. H. saurida spores grew only in RK‐13 cell line and were detected by immunofluorescence. Developmental stages of H. saurida were seen in RK‐13 cells by light and transmission electron microscopy, and species identification was confirmed by sequencing. This study demonstrated that H. saurida was able to proliferate in the mammalian RK‐13 cell line, which thus represents an in vitro model for conducting molecular genetics and cell–pathogen interaction studies of Heterosporis.     

一株传染性造血器官坏死病病毒的致病性研究

为了对分离于山东某虹鳟养殖场的一株传染性造血器官坏死病毒株(IHNV-Sn1203)进行致病性检测与研究,将该IHNV-Sn1203毒株进行虹鳟鱼苗人工回接感染实验。结果显示,8d内人工感染实验鱼累计死亡率高达100%。收集大批濒死的病鱼样本,制备病理组织切片;利用鲤上皮细胞(EPC)进行细胞感染实验、病毒电镜观察、空斑实验、病毒滴度检测和聚类分析。病理组织切片显示,该病毒可造成虹鳟造血器官广泛性坏死;细胞感染实验结果显示,接种24 h后EPC细胞出现葡萄串状典型细胞病变(cytopathic effect,CPE),72 h后大部分细胞崩解脱落形成网状孔洞;电镜下清晰可见弹状病毒粒子大量存在于细胞质内,其在EPC细胞上的滴度为108.36TCID50/mL,并能形成2~4 mm空斑。对病毒核蛋白氨基酸序列的聚类分析结果显示,该病毒与标准毒株RB-1和WRAC的同源性分别为97%和93%,与国内报道的zyx株具有最高的同源性(99%)。研究表明,IHNV-Sn1203毒株能够在鱼体及敏感细胞中稳定繁殖,产生典型病变,具有较高的病毒滴度,对虹鳟鱼苗有很高的感染性和致死性。     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Isolation of a novel polyomavirus,related to Japanese eel endothelial cell‐infecting virus,from marbled eels,Anguilla marmorata (Quoy & Gaimard)

Marbled eels, Anguilla marmorata (Quoy & Gaimard), cultured in Taiwan exhibited haemorrhage and mortality in January 2012. The severely diseased eels bled from the gills and showed congestion of the central venous sinus of the gill filaments and haemorrhage throughout the body similar to viral endothelial cell necrosis of eel. In this study, a novel polyomavirus (AmPyV) was isolated from the diseased eels using the AMPF cell line established from the pectoral fin of healthy marbled eels. AmPyV was found to encode a long T‐antigen orthologous gene. Phylogenetic analysis showed that AmPyV was closely related to Japanese eel endothelial cell‐infecting virus. PCR assays revealed AmPyV infection throughout the systemic organs. AmPyV proliferated in the AMPF, EK‐1 and EO‐2 cells at temperatures 25–30 °C, and the progeny virus yields were 10^7.0, 10^7.4 and 10^7.7 TCID₅₀ mL^?1, respectively. The purified virions were icosahedral particles, 70–80 nm in diameter. No clinical signs or mortality was observed among the eels injected with the virus; however, the virus was reisolated from the brain, eyes, kidneys, fins and gills of infected eels 2 month after injection. Our results suggest that AmPyV exhibits a latent infection. Pathogen of the disease needs to study further.     

Polyunsaturated fatty acid metabolism in a cell culture model of essential fatty acid deficiency in a freshwater fish, carp (Cyprinus carpio)

Proliferation of an essential fatty acid deficient cell line from carp (EPC-EFAD; epithelioma papillosum carp-essential fatty acid deficient) is stimulated by supplementing the cells with C₂₀, but not C₁₈ polyunsaturated fatty acids (PUFA). It is hypothesized that the differential ability of the PUFA to stimulate proliferation of the EPC-EFAD cells may be related to the extent of the cells' ability to desaturate and elongate C₁₈ PUFA. In the present study, the metabolism of ¹⁴C-labeled C₁₈ and C₂₀ PUFA was investigated in EPC-EFAD cells in comparison with normal EPC cells. The incorporation of all the PUFA was significantly greater in EPC-EFAD cells but the rank order, 20:5n-3 > 18:3n-3 = 18:2n-6 >20:4n-6 was the same in both cell lines. The proportion of radioactivity from all labeled PUFA recovered in phosphatidylethanolamine and total polar lipids was significantly lower in EPC-EFAD cells compared to EPC cells, whereas the proportion of radioactivity recovered in all the other phospholipid classes and total neutral lipid was greater in EPC-EFAD cells. Both cell lines desaturated[1-¹⁴C]18:3n-3 and [1-¹⁴C]20:5n-3 to a greater extent than the corresponding (n-6) substrates but the desaturation of all the ¹⁴ C-labeled PUFA was significantly greater in EPC-EFAD cells compared to EPC cells. The results showed that, although essential fatty acid deficiency had several significant effects on PUFA metabolism in EPC cells, the fatty acid desaturation/elongation pathway was not impaired in EPC-EFAD cells and so they can desaturate 18:3n-3 to 20:5n-3 and 22:6n-3, and 18:2n-6 to 20:4n-6. However, 20:4n-3 and 20:3n-6, and not 20:4n-6 and 20:5n-3, were the predominant C₂₀ PUFA produced by the elongation and desaturation of [1-¹⁴C]18:3n-3 and [1-¹⁴C]18:2n-6, respectively. Therefore, the previously reported inability of 18:3n-3 and 18:2n-6, compared to 20:5n-3 and 20:4n-6, to stimulate proliferation of the cells is apparently not due to a general deficiency in the fatty acid desaturation pathway in EPC-EFAD cells but may be related to potential differences in eicosanoid profiles in cells supplemented with C₁₈ PUFA compared to C₂₀ PUFA.     

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.