牙鲆淋巴囊肿病毒一抗原蛋白的确认及定位

以牙鲆淋巴囊肿病毒（LCDV）为抗原免疫Balb/c小鼠,而后将小鼠脾细胞与P3U1骨髓瘤细胞融合,以囊肿组织冰冻切片的免疫荧光染色筛选杂交瘤细胞,阳性结果显示特异性块状荧光信号集中在囊肿细胞的细胞质边缘部分,且多个荧光信号相连呈现链圈状,有限稀释 法克隆阳性杂交瘤细胞,三次克隆后获得4株稳定产生抗LCDV抗体的单克隆杂交瘤细胞株（1A8、1D7、2B6、2D11）。应用Western-blotting法分析单抗识别蛋白的分子量,结果显示,单抗1D7 和2B6均能特异性结合一条分子量116 kD病毒多肽;应用免疫电镜技术定位单抗识别的抗原决定簇,结果发现胶体金颗粒集中吸附在病毒粒子衣壳周围,且背景清洁,无散在的金颗粒或其他污染物。实验结果说明分子量约为116 kD的蛋白多肽为LCDV病毒衣壳蛋白,且具有线性抗原决定簇。     

病毒铺膜印迹技术分离牙鲆鳃细胞上的淋巴囊肿病毒结合蛋白

利用非变性电泳与病毒铺膜印迹技术(VOPBA)分离了牙鲆(Paralichthys olivaceus)鳃细胞(FG)上淋巴囊肿病毒结合蛋白,结果显示在FG细胞膜上有分子量为135 kD的蛋白与淋巴囊肿病毒特异结合;对该蛋白切胶回收后进行SDS-PAGE与双向电泳,发现135 kD蛋白由3个蛋白组成,分子量分别为58.3 kD、44.6 kD及37.6 kD;135 kD蛋白SDS-PAGE的VOPBA显示,仅出现37.6 kD的蛋白带,而58.3 kD、44.6 kD蛋白皆不与淋巴囊肿病毒结合。结果表明牙鲆FG细胞上135 kD蛋白是淋巴囊肿病毒的结合蛋白,其37.6 kD蛋白具有病毒结合活性。     

鱼肠道弧菌外膜蛋白抗原性分析

采用十二烷基肌氨酸钠法和苯甲基磺酰氟法提取鱼肠道弧菌外膜蛋白.SDS-PAGE图谱显示,PMSF提取的鱼肠道弧菌有19条带,Sarkosyl法提取的鱼肠道弧菌有14条带,其中111、105、87、78、61、58、53、48、45、40、36、33、32、31 kD蛋白带为2种方法共同条带,101、55、42、27、25 kD为PMSF法特有条带.此外,以制备的兔抗鱼肠道弧菌全菌血清为第一抗体,应用Western-blotting技术分析了鱼肠道弧菌外膜蛋白的抗原性,结果显示分子量为106、87、61、58、55、42、36、32 kD的8条蛋白带发生了免疫反应.     

淋巴囊肿病毒27.8 ku受体蛋白在牙鲆组织中的分布

应用抗牙鲆淋巴囊肿病毒(lymphocystis disease virus,LCDV)受体蛋白(27.8 ku)的单克隆抗体(2G11和3D9)定位LCDV受体蛋白在牙鲆组织中的分布。通过对牙鲆外周血、白细胞、鳃、胃、肠、表皮、肝脏、头肾、体肾、脾、性腺、脑、心脏等进行LCDV受体蛋白的间接免疫荧光与免疫组织化学定位观察,发现在牙鲆外周血白细胞的细胞膜、鳃上皮细胞、表皮、胃黏膜上皮细胞顶端、肠上皮细胞、肝细胞、脾表层结缔组织细胞及头肾后端的肾小管上皮细胞内均有较强的阳性信号,表明这些部位分布有LCDV的27.8 ku受体蛋白,但在体肾、性腺、脑、心脏及外周血红细胞中未观察到阳性信号。推测LCDV通过与鳃、表皮及消化道上皮的受体结合进入牙鲆体内,通过与外周血白细胞上的受体结合侵染白细胞而进入血液循环,进而感染肝脏、脾脏、头肾等器官。     

大菱鲆致病性溶藻弧菌SR1的外膜蛋白及其抗原性分析

用十二烷基肌氨酸钠(Sarkosyl)抽提结合超速离心的方法提取了一株大菱鲆致病性溶藻弧菌(Vibrio alginolyticus)SR1和其他7株弧菌的外膜蛋白。通过SDS-PAGE图谱分析比较了这8株弧菌外膜蛋白的组成,结果表明,8株弧菌的外膜蛋白电泳一般可得到6-12条条带,其分子量多集中在65-106 kD和28-48 kD,其中36 kD的蛋白带为8株弧菌所共有。用兔抗SR1全菌血清进行Western-blot印迹显示,菌株SR1的外膜蛋白条带中有6条发生了阳性反应,其分子量分别为73 kD、48 kD4、5 kD3、9 kD、36 kD和32 kD。而其他7株弧菌的外膜蛋白与兔抗SR1血清也发生程度不等的阳性反应,这些阳性反应条带的分子量集中在65-73 kD、45-48 kD和36-41 kD之间,其中36 kD的外膜蛋白在8株弧菌中均出现明显的阳性反应,说明36 kD的外膜蛋白是这8株弧菌共有的特异性抗原。     

牙鲆秦皇岛弧菌HQ010712-1外膜蛋白及其抗原性分析

采用十二烷基肌氨酸钠(Sarkosyl)和苯甲基磺酰氟(PMSF)2种方法提取秦皇岛弧菌HQ010712-1(Vibrio qinhuangdaora sp.nov.)外膜蛋白,结果显示 Sarkosyl法提取效果较好,且所提取的主要外膜蛋白分子量为102kD、45 kD、39 kD、36 kD、30 kD、28 kD、24 kD、22 kD;为比较该菌株与弧菌属其他细菌外膜蛋白组分及抗原性异同,以鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、溶藻胶弧菌(Vibrio alginolyticus)为对照,电泳图谱显示4种弧菌外膜蛋白的分子量主要集中在22～48 kD之间;利用抗秦皇岛弧菌HQ010712-1血清的免疫印迹表明菌株HQ010712-1外膜蛋白中分子量为45 kD、36 kD的蛋白条带呈现阳性反应,其他3种弧菌外膜蛋白中均有与该抗血清反应的条带,且分子量为36 kD的反应带为菌株HQ010712-1、副溶血弧菌、溶藻胶弧菌共有.本研究旨在为进一步筛选和研究致病性弧菌的共同保护性抗原提供参考.     

人工感染淋巴囊肿病毒牙鲆的免疫球蛋白水平变化

用患淋巴囊肿病牙鲆体表肿瘤纯化的淋巴囊肿病毒对健康牙鲆进行人工感染,应用抗牙鲆免疫球蛋白单克隆抗体通过间接酶联免疫法（ELISA）检测感染之后的牙鲆血清总抗体水平和抗LCDV特异性抗体水平的变化规律。结果表明,感染7d后,牙鲆血清中特异性抗体和总抗体水平都显著升高。抗LCDV特异性抗体水平在405nm处的OD值从对照的0.129上升至0.238;随着感染时间延长抗体水平持续升高（第14天0.247,第21天0.410）,在第28天达到最高值0.436,然后开始缓慢降低（第35天0.385,第98天0.357）;总抗体水平在405nm处的OD值从对照的0.135上升至0.250;随着感染时间延长抗体水平持续升高（第14天0.266,第21天0.561）,在第28天达到最高值0.613,然后开始缓慢降低（第35天0.480,第98天0.475）。通过间接免疫荧光技术（IFAT）在感染第14天以后的牙鲆的肠、胃中检测到病毒。     

牙鲆淋巴囊肿病毒双抗体夹心ELISA检测方法的建立

以牙鲆淋巴囊肿病毒(Lymphocystis disease virus,LCDV)特异性单克隆抗体为捕获抗体,纯化的兔抗LCDV血清为检测抗体,建立了LCDV病毒双抗夹心间接ELISA检测方法.该方法最佳反应条件为:单克隆抗体包被质量浓度为2 μg/ml,兔抗血清工作浓度为1∶1200稀释,以3%牛血清白蛋白作为封闭液.应用本方法对体表具有明显发病症状的牙鲆、与发病鱼同池但体表无症状的牙鲆及健康牙鲆的肝、脾、肾、胃、肠、心脏、鳃等7种组织进行检测,在前两种鱼的胃、肠、鳃等组织检测到病毒,而健康牙鲆各组织器官反应结果均为阴性,试验结果表明,所建立的ELISA检测方法具有特异性强、可靠性高的特点,可用于养殖牙鲆LCDV的检测.     

牙鲆淋巴囊肿病毒纯化及不同来源病毒免疫特性比较

比较了3种不同的牙鲆淋巴囊肿病毒纯化方法,优化后的方法如下：剥离囊肿表面薄膜,收集内容物,匀浆后再用超声波细胞破碎仪破碎,反复冻融,650×g、1800×g差速离心,30%（W/W）蔗糖垫底超速离心（78500×g）浓缩病毒,最后蔗糖密度梯度超速离心（78500×g）纯化病毒。电镜观察发现,出现在47%～52%蔗糖密度区域的病毒带含有多量、纯净和结构一致的病毒粒子。此外,利用制备的兔抗血清对不同地区的病毒进行了免疫特性分析,Western blotting检测显示来自威海、青岛及秦皇岛3个地区的淋巴囊肿病毒反应结果是一致的,均有3条蛋白带发生反应,其分子量分别为125、66和55kDa。     

牙鲆淋巴囊肿病毒灭活疫苗制备及应用

淋巴囊肿病毒（Lymphocystis virus）属于虹彩病毒科（I ridoviridae）,病毒粒子含DNA正二十面体,直径一般200-250nm,该病毒的感染性在18～20℃水中能保持5d以上,是感染高峰期。淋巴囊肿病是一种慢性皮肤瘤。牙鲆淋巴囊肿主要症状表现在鱼类的皮肤上、鳍上和眼球等处出现小水泡状的肿状物,这些肿状物多呈白色、淡灰色、灰黄色、个别囊肿物因寄生部位充血而略显红色,囊肿大小1～10mm并紧密相连呈桑椹状。淋巴囊肿病全年可见,     

Phylogenetic analysis and molecular methods for the detection of lymphocystis disease virus from yellow perch, Perca flavescens (Mitchell)

Lymphocystis disease is a prevalent, non-fatal disease that affects many teleost fish and is caused by the DNA virus lymphocystis disease virus (LCDV). Lymphocystis-like lesions have been observed in yellow perch, Perca flavescens (Mitchell), in lakes in northern Alberta, Canada. In an effort to confirm the identity of the virus causing these lesions, DNA was extracted from these lesions and PCR with genotype generic LCDV primers specific to the major capsid protein (MCP) gene was performed. A 1357-base pair nucleotide sequence corresponding to a peptide length of 452 amino acids of the MCP gene was sequenced, confirming the lesions as being lymphocystis disease lesions. Phylogenetic analysis of the generated amino acid sequence revealed the perch LCDV isolate to be a distinct and novel genotype. From the obtained sequence, a real-time PCR identification method was developed using fluorgenic LUX primers. The identification method was used to detect the presence/absence of LCDV in yellow perch from two lakes, one where lymphocystis disease was observed to occur and the other where the disease had not been observed. All samples of fin, spleen and liver tested negative for LCDV in the lake where lymphocystis disease had not been observed. The second lake had a 2.6% incidence of LCD, and virus was detected in tissue samples from all individuals tested regardless of whether they were expressing the disease or not. However, estimated viral copy number in spleen and liver of symptomatic perch was four orders of magnitude higher than that in asymptomatic perch.     

Identification and characterization of a novel lymphocystis disease virus isolate from cultured grouper in China

Grouper Epinephelus spp. is one of the most important mariculture fish species in China and South-East Asian countries. The emerging viral diseases, evoked by iridovirus which belongs to genus Megalocytivirus and Ranavirus, have been well characterized in recent years. To date, few data on lymphocystis disease in grouper which caused by lymphocystis disease virus (LCDV) were described. Here, a novel LCDV isolate was identified and characterized. Based on the sequence of LCDV major capsid protein (MCP) and DNA polymerase gene, we found that the causative agents from different species of diseased groupers were the same one and herein were uniformly defined as grouper LCDV (GLCDV). Furthermore, H&E staining revealed that the nodules on the skin were composed of giant cells that contained inclusion bodies in the cytoplasm. Numerous virus particles with >210 nm in diameter and with hexagonal profiles were observed in the cytoplasm. In addition, phylogenetic analysis based on four iridovirus core genes, MCP, DNA polymerase, myristoylated membrane protein (MMP) and ribonucleotide reductase (RNR), consistently showed that GLCDV was mostly related to LCDV-C, followed by LCDV-1. Taken together, our data firstly provided the molecular evidence that GLCDV was a novel emerging iridovirus pathogen in grouper culture.     

Induction of apoptosis in a flounder gill cell line by lymphocystis disease virus infection

Lymphocystis disease virus (LCDV), a large icosahedral DNA virus classified to the iridovirus family, is the causative agent of lymphocystis, a disease which occurs in marine and freshwater fish species and is characterized by formation of papilloma-like lesions on the surface of the skin. In vitro, LCDV infection causes flounder gill cells, an adherent cell line, to exhibit an obvious cytopathic effect (CPE). In order to test whether apoptosis is responsible for the observed CPE, cells infected with LCDV at a multiplicity of infection (m.o.i.) of 5 PFU per cell were examined at various time intervals for the appearance of apoptotic signs. Nuclear fragmentation, DNA laddering and caspase activation were observed in the infected cells at the time (i.e. 10 days post-infection) when an intensive CPE was observed. These findings demonstrate that LCDV is capable of inducing apoptosis in vitro, which is different from the result of LCDV infection in vivo, and consequently suggest an intricate LCDV-host interaction.     

Isolation of lymphocystis disease virus from sole, Solea senegalensis Kaup, and blackspot sea bream, Pagellus bogaraveo (Brunnich)

Two viruses were isolated from cultured sole, Solea senegalensis, and wild blackspot sea bream, Pagellus bogaraveo, and preliminarily characterized as lymphocystis disease viruses (LCDVs). Viral isolates were characterized by morphological, biochemical and biophysical properties. In addition, the susceptibility of four fish cell lines was also tested. LCDV isolates developed cytopathic effects on the SAF-1 cell line at 5 and 6 days post-infection and reached titres of 10(6) TCID50 mL(-1). The antigenic and structural protein analysis of the two new LCDV isolates showed identical profiles to that obtained for LCDV strain Leetown NFH (ATCC VR-342), used as a reference viral strain, and for an LCDV isolate collected from gilt-head sea bream, Sparus aurata, cultured in southern Spain. Molecular confirmation was performed by polymerase chain reaction. Specific primers for LCDV produced a 270-bp DNA fragment, the expected size for LCDV.     

军曹鱼淋巴囊肿的病理学研究

应用病理组织学和电镜方法,对患疑似淋巴囊肿病的军曹鱼(Rachycentron canadum)的各器官进行观察.结果表明,患病军曹鱼的皮肤囊肿组织由一些淋巴囊肿细胞集合体组成,这些囊肿细胞排列紧密,直径为10～150 μm,细胞呈圆形、锥形不规则状;细胞外有一层厚的囊膜;细胞质内散布有大量的嗜碱性包涵体,且多数集中在细胞的边缘部分;电镜观察到囊肿细胞质中有大量二十面体的病毒粒子,病毒颗粒直径220 nm.据此确认该病为病毒性淋巴囊肿病.其他器官主要组织病理学变化有:在心脏、肝脏、脾脏和头肾中也存在囊肿细胞,心肌纤维水肿;肾间质淋巴细胞增生,巨噬中心出现,肾小管上皮细胞变性和坏死;脾淋巴细胞增生,脾髓质出血;肝脂肪变性;鳃上皮肿胀.根据观察结果可以认为,该病毒不仅损伤鱼的皮肤,致使病鱼外观异样而严重影响其商品价值,而且对鱼的内脏和免疫器官也造成严重的致命损伤.     

牙鲆抗淋巴囊肿病家系选育及生长和抗病性能分析

为了选育抗淋巴囊肿的牙鲆(Paralichthys olivaceus)新品种,2015年建立了5个普通家系(C1~C5)、3个雌核发育家系(G1、G2、G3)和1个对照组,在淋巴囊肿病高发养殖场进行自然染毒实验,统计各家系的抗病保护率。同时对各家系120 d、180 d、240 d和300 d时的生长性状进行跟踪测量和比较。结果表明,各家系牙鲆在不同时期有各自不同的生长规律,呈现出家系间生长规律的不一致性。但在所有家系中,家系G2各个时期的生长表现始终排名靠前。390 d统计抗病保护率的结果显示,对照组的抗病保护率只有59.57%,而经选育所有实验组的抗病保护率都在60%以上,最高达97.20%(家系G2)。但总体上,390 d时牙鲆淋巴囊肿抗病和患病个体间体重和体长差异均不显著;体重、体长与抗病保护率为正相关,但相关性均不显著。家系G2抗病个体的平均体重显著高于患病个体(P0.05)。以G2家系为基础,继续进行选育,有望获得牙鲆抗淋巴囊肿速生新品种。