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。     

罗非鱼暴发性流行病病毒病原的电镜观察初报

利用电子显微镜技术,首次在国内从患暴发性流行病罗非鱼体内发现病毒病原。电镜观察结果显示,在患病罗非鱼脾脏、心脏以及鳃组织细胞质中观察到大量病毒颗粒。病毒颗粒呈球形,无囊膜,直径约30~40 nm,与鱼类神经坏死病毒相近。     

鲤疱疹病毒Ⅱ型的理化及生物学特性和超微形态发生

为了查明鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,Cy HV-2)的理化与生物学特性以及病毒在细胞内的超微形态发生过程,利用新建立的对Cy HV-2敏感的异育银鲫脑组织细胞系(Gi CB),对Cy HV-2的理化及生物学特性进行了详细研究,比较了不同来源鱼类细胞系对Cy HV-2感染的敏感性,并对体外培养细胞中Cy HV-2病毒粒子及其超微形态发生过程进行了电镜观察。结果显示,Cy HV-2对热、酸、碱、有机溶剂和冻融敏感;常用鱼类细胞系EPC、RTG-2、Koi-Fin、CIK、CCK、PF-Fin对Cy HV-2的感染不敏感,特异性巢式PCR检测盲传至第7代Cy HV-2细胞培养物,结果均为阴性;Cy HV-2在Gi CB细胞中的增殖动态研究结果表明:病毒感染细胞经过12 h的隐晦期,24 h开始进入对数生长期,96 h病毒滴度达到最高值(107.52±0.26 TCID50/m L),然后进入平台期;透射电子显微镜观察结果显示,Cy HV-2感染细胞可分为吸附与侵入、复制与装配、成熟与释放3个主要过程,病毒进入对数生长期后,被感染细胞内可见形态典型的疱疹病毒颗粒。     

暗纹东方鱼屯的一种病毒性疾病

发病暗纹东方鱼屯以体表和内脏出血为主要症状,死亡率可达80%以上。显微镜检查体表、鳃丝及肠腔未见寄生虫。病鱼的肝脏和血液中未能分离到病菌。经电镜检查,在肝脏组织中发现大量球形或多角形病毒颗粒,核衣壳直径约85 nm,双层衣壳,病毒核心直径50~60 nm,无囊膜,未见包涵体。病毒颗粒常聚集成团,呈晶格状排列,聚集团外有包膜,有的病毒颗粒分散于细胞中。病毒的形态结构及大小与呼肠孤病毒相似。对该病的防治方法作了讨论。     

草鱼呼肠孤病毒JX-0902株的分离和鉴定

收集江西南昌地区患典型草鱼出血病的病鱼组织,进行超薄切片电镜观察,结果显示,在脾、肾样品中发现大量病毒颗粒,病毒无囊膜,近球形,直径约70 nm,形态与已报道的草鱼呼肠孤病毒(grass carp reovirus,GCRV)相似。取病鱼肌肉、内脏研磨,组织液经离心、过滤除菌后进行鱼体人工感染试验和细胞感染实验,结果发现,人工感染试验鱼出现死亡,且具有典型出血症状;组织滤液感染草鱼肾细胞系(CIK)细胞后,盲传6代未观察到典型细胞病变效应,但感染细胞固定后经超薄切片电镜观察,细胞质内有大量病毒存在,与病鱼组织切片观察到的病毒形态一致。SDS-PAGE结果进一步揭示,新分离病毒株(暂命名JX-0902)基因组由11条dsRNA组成,呈现水生呼肠孤病毒基因组典型特征,但基因组带型与GCRV 873株存在差异,而与HZ08株接近。根据GenBank上已提交的草鱼呼肠孤病毒S6序列(GQ896337)设计特异引物,以JX-0902株的cDNA作为模板,可扩增出特异性条带。基于S6序列的聚类分析结果显示,JX-0902与GCRV HZ08株、GD108株S6同源性高达98%、99%,该分离株应为草鱼呼肠孤病毒。     

栉孔扇贝大规模死亡致病病原的研究

对2000-2002年山东沿海6个疫区患病栉孔扇贝进行电镜观察，在消化腺、外套膜、肾和肠的结缔组织细胞和间质细胞中发现一种球形病毒．并引起相直的病理学变化。该病毒具囊膜，直径为130～170nm，核衣壳直径为90～140nm。病毒分离纯化后．观察到的病毒囊膜表面覆有长20nm的纤突。病毒在细胞质中的囊泡样结构内完成装配，其内未发现包涵体存在。从发病疫区栉孔扇贝组织中分离出病毒毒种对键康扇贝进行人工感染。结果显示，各感染实验组发病扇贝表现出与自然海区发病扇贝相同的临床症状。病毒注射组死r二率为?5％，病毒浸浴组死亡率为68．7％．灭活病毒注射组和空白对照组死亡率皆为12．5％。病毒注射、浸浴组与灭活病毒注射组、空白对照组死亡率差异显著。电镜复检结果显示，各感染实验组发病扇贝组织中分布有大量病毒粒子，与自然海区发病扇贝组织所观察到的病毒粒子在形态特征和病理学特征上完全一致。以上结果证明，病毒是导致栉孔扇贝大规模死亡的直接病原。     

鲤科疱疹病毒Ⅱ型射阳株的分离与鉴定

对患"鳃出血"病的异育银鲫(Carassius auratus gibelio)病灶组织进行分离,并通过回归感染试验、细胞分离鉴定、电镜观察以及PCR几种方法结合来鉴定。结果显示:健康异育银鲫注射组织滤液5 d后开始发病,且症状与原发病症状一样,死亡率为80%,对照组没有死亡。对患病鱼内脏组织经超薄切片,电子显微镜观察发现组织内有大量的病毒颗粒,成熟的病毒粒子直径大小170~200 nm,与疱疹病毒Ⅱ型病毒粒子相符。滤液经疱疹病毒Ⅱ型特异性的PCR检测,获得阳性目的片段,同时将无菌处理的病样滤液接种胖头鲤细胞(FHM),培养1 d后出现明显的细胞病变(CPE),单层细胞网状收缩,坏死细胞聚集。收集细胞培养液,进行病毒特异性的PCR检测,并检测到436 bp的阳性片段。结果表明:从江苏射阳某"鳃出血"病异育银鲫养殖鱼塘分离到的病毒为疱疹病毒Ⅱ型(Cy HV-2)病毒。     

暗纹东方鲀的一种病毒性疾病

发病暗纹东方鲀以体表和内脏出血为主要症状,死亡率可达80%以上.显微镜检查体表、鳃丝及肠腔未见寄生虫.病鱼的肝脏和血液中未能分离到病菌.经电镜检查,在肝脏组织中发现大量球形或多角形病毒颗粒,核衣壳直径约85 nm,双层衣壳,病毒核心直径50～60 nm,无囊膜,未见包涵体.病毒颗粒常聚集成团,呈晶格状排列,聚集团外有包膜,有的病毒颗粒分散于细胞中.病毒的形态结构及大小与呼肠孤病毒相似.对该病的防治方法作了讨论.     

栉孔扇贝急性病毒性坏死症(AVND)病理学观察与分析

2000~2002年,应用光镜和电子显微技术对AVND发生期间栉孔扇贝(Chlamys farreri)各主要组织器官病理变化进行了连续观察。光镜观察显示,除生殖腺、闭壳肌外,濒死栉孔扇贝的外套膜、鳃、肾、消化腺和肠组织都有不同程度的组织病理变化。病灶主要出现在上述各器官的结缔组织以及上皮组织,病理变化表现为细胞核肿大、固缩、破裂,核染色质边缘化或空泡化,受感染细胞崩解、脱落,留下大片均质无结构的空白区域,形成凝固性坏死,结缔组织细胞质中有嗜碱性包涵体样颗粒存在。电镜观察显示,在病灶出现的组织细胞内,细胞核染色质异常凝集和边缘化、核膜周隙扩张或溶解。细胞器病理变化明显,内质网扩张,核糖体脱落,有髓鞘样小体出现;线粒体肿大、嵴融解,整个细胞出现解体现象。病变的结缔组织细胞与间质细胞细胞质中有大量直径为130~170nm、具有囊膜的球形病毒粒子存在,负染电镜观察表明,病毒囊膜表面覆有长20nm放射状纤突。病毒的发生基质在细胞质内,并被一膜性结构所包围。病毒在宿主细胞中的繁殖分为3个阶段,即病毒发生期、病毒装配期和病毒释放期。病理学观察证明,病毒感染与病理变化具有明显的相关性。     

杆状病毒性的皮下及造血组织坏死─对虾暴发性流行病的病原和病理学

１９９３年对虾暴发性流行病的患病对虾经光镜组织病理学研究和电镜观察，在对虾的皮下组织（包括前肠上皮、后肠上皮、真皮和鳃等）、结缔组织、造血组织、触角腺及血淋巴细胞等的细胞核均发现核肿大和深的嗜酸性着色。在电镜下，病变组织的细胞核内为一种杆状病毒充满，病毒大小为１２０ｎｍ×３６０ｎｍ，有囊膜，在细胞内不形成核型多角体或颗粒体类包涵体，属于杆状病毒属的无包涵体型杆状病毒亚群，即Ｃ型杆状病毒亚群。根据该对虾暴发性流行病的症状、病毒感染的靶组织和病毒的特征，该病毒被认为是一种新的对虾病毒，暂定名为皮下及造血组织坏死杆状病毒。     

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。     

Extra small virus-like particles (XSV) and nodavirus associated with whitish muscle disease in the giant freshwater prawn, Macrobrachium rosenbergii

A disease of Macrobrachium rosenbergii, the giant freshwater prawn, farmed in China was recently recorded in Zhejiang, Jiangsu, Shanghai, Guangxi and Guangdong provinces. The clinical sign of the disease, which develops in post-larvae (PL), is a whitish appearance of the muscles, particularly noticeable in the abdomen. Mortalities may reach 100% in some hatcheries. Investigations by transmission electron microscopy after negative staining of diseased PL homogenates showed the presence of two types of viral particles: one, unenveloped, icosahedral in shape, 26-27 nm in diameter, the second, much smaller, about 14-16 nm in diameter, designated extra small virus particle (XSV). The large virus has a genome with two pieces of ssRNA (RNA-1 and RNA-2), of 3 and 1.2 kb, respectively. Hybridization tests confirmed that this large virus is closely related to M. rosenbergii nodavirus (MrNV) which was isolated from diseased prawns in a hatchery in the French West Indies. Its very small size and hypothesized biochemical and biological characteristics suggest XSV is a new type of crustacean virus. As XSV has always been found associated with the larger virus (nodavirus) and is located in muscle and connective cells of diseased animals, it could be an autonomous virus, a helper-type virus or a satellite-like virus.     

中华绒螯蟹小核糖核酸病毒病及其组织病理学

本文对所谓“抖抖病”的中华绒螯蟹病蟹病因进行了分析研究。经电镜观察,病蟹的心脏、了、鳃、肠和肝脏胰腺中存在着球状病毒粒子,病毒无囊膜,直径为２８－３２ｎｍ左右,分布在细胞浆内,不形成包涵体。从病毒形态和染色反应特性确定该病毒为小ＲＮＡ病毒科病毒。经人工注射病蟹组织浆除菌上清液,被感染的健康蟹出现在自然发病蟹足严重抖动等一样的症状,表明病囊中的小ＲＮＡ病毒为该病原。病蟹病理特征为：细胞肿大,核溶解七     

一种真鲷球形病毒的形态及细胞病理学电子显微镜观察

对１９９５年青岛地区患病真鲷肝脏、肠上皮组织、贤脏、鳃等组织进行电镜观察,发现一种球形病毒,该病毒具有囊腊,直径为８０￣１００ｎｍ,在核和细胞质中均形成不规则形态包涵体;细胞病理变化表现为核内染色质变性,核膜水肿变形且部分溶解,内质网膨胀,胞质内有大量游离核糖体分布,线粒体外膜及内嵴变形且有少量溶解。     

鲈脾肿大症的病原及细胞病理学的初步研究

首次报道了鲈脾肿大症的发病情况。病鱼以脾脏肿大为主要症状，死亡率高达50％以上。对病鱼进行显微镜检查和细菌分离培养，未见寄生虫和病原菌。经电镜检查，在脾、肝等组织中发现大量病毒颗粒。该病毒颗粒为六边形，没有囊膜，衣壳直径180-220nm，认为该病毒屑虹彩病毒(Iridoviridae)。细胞病理变化表现为感染细胞肿胀，细胞超微结构破坏，细胞质出现大面积空泡。血细胞中出现大量异形淋巴细胞，占淋巴细胞总数的50％以上。     

Studies on epizootic iridovirus infection among red sea bream,Pagrus major (Temminck & Schlegel), cultured in Taiwan

Since 1993, an epizootic viral disease has occurred in net-cage cultured red sea bream, Pagrus major (Temminck & Schlegel), in Peng-hu Island located on the south-western coast of Taiwan. The diseased fish exhibited abnormal swimming and were lethargic, but few visible external signs were observed. The cumulative mortality because of the disease sometimes reached 50-90% over 2 months. Histopathogical studies of the affected fish showed enlarged basophilic cells in the gill, kidney, heart, liver and spleen. These necrotic cells were Feulgen-positive and stained blue using Giemsa. Transmission electron microscopy revealed icosahedral virions in the cytoplasm of the necrotic cells. The viral particles consisted of a central nucleocapsid (75-80 nm) and envelope, and were 120-150 nm in diameter. These results suggest that the virus belongs to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570 bp fragments were produced from the viral DNA using as a template 1-F and 1-R primers derived from red seabream iridovirus (RSIV) from red sea bream in Japan. Similar results were also obtained using nested-PCR with different primer sets (1-F, 2-R and 2-F, 1-R). Although the size and some features of epizootics of this virus differed from RSIV in Japan, it shows close genetic affinities with the latter and it is suggested that RSIV has been introduced to Taiwan.     

Mass mortalities associated with viral nervous necrosis (VNN) disease in two species of hatchery-reared grouper, Epinephelus fuscogutatus and Epinephelus akaara (Temminck & Schlegel)

Mass mortalities of hatchery-reared juvenile groupers have occurred in southern Taiwan. The diseased fish swam in a darting, corkscrew fashion. Light microscopy revealed vacuolation in the brain tissue. Electron microscopy showed numerous non-enveloped, cytoplasmic viral particles (20–25 nm in diameter) in the brain cells, and many virions were enclosed in the membrane-bound organelles of the cells. Two structural proteins of the purified grouper virus, with molecular weights of 44 and 43 kDa, were revealed by SDS-PAGE. Moreover, the results of RT-PCR and nested PCR diagnosis using primers specific to the T2 and T4 target segments of striped jack nervous necrosis virus (SJNNV) RNA2 genes suggest that this virus is a fish nodavirus, and is designated as GNNV 9410 strain (grouper nervous necrosis virus strain 9410). This is the first case report of viral nervous necrosis among marine fish in Taiwan.     

一株锯缘青蟹呼肠孤病毒的分离纯化及鉴定

在研究锯缘青蟹病害的过程中,从广东某养殖场分离到一株呼肠孤病毒(命名为SsRV)。病毒粒子为球状,直径约45 nm。病毒粒子基因组为12个节段的双链RNA;在1%琼脂糖电泳中SsRV基因组呈1/5/6带型,其分子量分别为3.8,2.5,2.4,2.0,1.9,1.8,1.3,1.2,1.1,1.1,1.0,1.0 kbp。根据病毒的宿主范围、基因组节段数及电泳型,此病毒很可能与地中海滨蟹呼肠孤病毒P和W 2共同构成呼肠孤病毒科一个新的属。     

Presence of infectious salmon anaemia virus (ISAV) in tissues of Atlantic salmon, Salmo salar L., collected during three separate outbreaks of the disease

Abstract. Infectious salmon anaemia (ISA) is a viral disease of farmed Atlantic salmon, Salmo salar L., in Norway. The enveloped virus particles (100nm) believed to be the causative agent of the disease have been observed budding from endothelial cells in heart blood vessels. However, it is not known if the virus propagates in endothelial cells in all tissues/organs, if other target cells exist or if material collected from different salmon farms with natural outbreaks of ISA contain the same virus particles. Salmon smolts from three hatcheries with no history of disease were taken into the laboratory and experimentally challenged with ISA collected from Atlantic salmon during natural outbreaks of the disease in three different fish farms outside Bergen. Norway. Tissues for TEM studies were Collected from: (1) organs that showed clinical signs of ISA (i.e. used in the diagnosis of the disease); (2) tissues believed to be important in transmission of the virus (integument, kidney, urinary bladder, gut and somatic muscle); and (3) hormone-producing tissues (pituitary gland, saccus vasculosus, thymus, thyroid, ultimobranehial gland, gonad, head kidney, heart and ventral aorta). The same virus as that believed to be the causative agent of ISA was found in all tissues examined from the challenged fish, i.e. a multiorgan infection with the same virus present in salmon from all three fish farms. The virus particles are about 100 nm in diameter, consisting of a slightly pleomorphic unit membrane envelope within which are a number of granules about 10–12nm in diameter. The granules seemed to be arranged in two concentric circles (spheres). The virus was seen budding from the surface of endothelial cells in blood vessels/sinus only. However, the virus was found intracellularly in both endothelial cells and in leucocytes.     

Viral-like particles associated with cuticular epithelium necrosis in cultured Litopenaeus vannamei (Decapoda: Crustacea) in Ecuador

A new viral agent was found associated with the endoplasmic reticulum of epithelial cells of the Pacific white shrimp Litopenaeus vannamei (Boone) sampled during mass mortalities. A 40% mortality rate affected nursery and grow‐out ponds during the first 50–60 days of culture, and peak mortality in ponds occurred when shrimp reached 2–4 g. Histopathological changes of affected shrimp showed different grades of necrosis in epithelial cells and, in some cases, other tissues were affected. Transmission electron microscopy (TEM) of columnar cells of the cuticular epithelium showed the accumulation of viral particles, either dispersed in the cytoplasm or in a string‐like or paracrystalline array. These arrays of virions were within membrane‐bound vesicles formed from the endoplasmic reticulum (ER), in orderly arrays on the outer nuclear membrane or along the ER. The virus particles had apparently proliferated in the ER. The virions had an opaque area with an approximate diameter of 20 nm and an electron‐lucent surface layer. The approximate diameter of the non‐enveloped virions was 25 nm. The cytological changes observed are similar to those associated with the Picornaviridae and Nodaviridae families. The histopathology and ultrastructure of a new disease in L. vannamei is associated with the presence of a putative new virus. Until further isolation and characterization is performed, it is recommended to refer to the agent as Litopenaeus vannamei viral‐like particles (LvVLPs).