大口黑鲈病毒性溃疡病病原的分离和鉴定

对广东省佛山地区2008年夏季高温时期暴发的大口黑鲈溃疡病的病原进行分离，从患病鱼的病灶肌肉组织中分离到5株嗜水气单胞菌,人工感染健康大口黑鲈，均未出现溃疡暴发病的典型症状病鱼体表大片溃烂，裸露肌肉坏死并有出血，尾鳍、胸鳍和背鳍基部红肿溃烂。制备病灶肌肉组织的除菌过滤液，背部肌肉注射感染健康大口黑鲈，7 d后出现典型的溃疡病症状。取自然发病鱼和人工感染的患病鱼病灶肌肉组织制作超薄切片，经电镜观察，均发现组织中有大量病毒颗粒，病毒粒子有囊膜，呈六角形，为正20面体对称结构，大小约为145.5 nm。根据已知虹彩病毒主要衣壳蛋白（MCP）基因序列设计特异引物，提取人工感染发病鱼病灶肌肉组织的DNA进行PCR扩增，将扩增片段进行序列测定与分析，结果表明该序列与已报道的虹彩病毒MCP基因有着较高同源性（39.5％～100％）。从电镜观察和MCP基因测序分析结果确认该病毒为虹彩病毒科蛙病毒属中的一种病毒，与国外报道的大口黑鲈病毒（LMBV）在分子特性和引起的疾病特征上有一定差异。研究结果表明引起大口黑鲈溃疡性暴发病的病原是虹彩病毒，将该病暂命名为大口黑鲈病毒性溃疡病。     

大口黑鲈蛙病毒分子流行病学及组织病理分析

为探讨大口黑鲈蛙病毒（LMBV）的分子流行规律及在感染后的组织病理变化，实验对2019—2021年采集的723份患病大口黑鲈样品进行荧光定量PCR （qPCR）检测。结果显示，LMBV的阳性率为63.62%，挑选阳性样品接种鳜脑细胞（Chinese perch brain cells,CPB），共获得93株LMBV。通过对分离株MCP、ATP酶、DNA聚合酶和甲基转移酶基因进行PCR扩增与测序分析，发现上述基因在LMBV分离株中均保守，其中MCP、ATP酶基因一致性均为100.0%、甲基转移酶基因一致性为99.7%～100.0%、DNA聚合酶基因一致性为99.8%～100.0%。选取1株LMBV毒株感染健康大口黑鲈，采用qPCR方法对不同组织中的病毒载量进行检测，结果显示，大口黑鲈蛙病毒在心脏、肝脏、脾脏、肾脏、胃、肠和脑等组织中均有分布，人工感染LMBV后的前5天病毒载量逐步升高；感染后第5天，心脏中病毒载量最高（9.5×10⁵个/mg拷贝），脑组织中病毒载量最低（2.9×10²个/mg拷贝）。组织病理结果表明，LMBV可导致大口黑鲈多种组...     

大口黑鲈肝脾肿大病病原研究

为了查明2009年10月广东省佛山地区养殖大口黑鲈(Micropterus salmoides)中暴发的传染性疾病的病原,对病鱼的肝脏、脾脏和腹隔膜进行切片电镜观察,发现细胞质中有大量病毒颗粒,切面为六角形,直径约145～150 nm,病毒为二十面体对称结构、无囊膜、似虹彩病毒的病毒粒子.用除菌的病鱼组织滤液感染健康大口黑鲈,被感染鱼死亡率达90%以上.根据已知虹彩病毒主要衣壳蛋白(MCP)基因序列设计特异引物,提取人工感染发病鱼的肝脏、脾脏、肾脏组织的DNA进行PCR扩增,将扩增片段进行序列测定与分析,结果表明该序列与已报道的鳜(Siniperca chuatsi)传染性脾肾坏死病毒(Infectious Spleen and Kidney Necrosis Virus,ISKNV)MCP基因同源性为98%.电镜观察和MCP基因测序分析结果显示,该病毒的分类地位为虹彩病毒科(Iridoviridae)细胞肿大病毒属(Megalocytivirus).     

蛙病毒PCR检测方法的建立与比较分析

为了进一步丰富蛙虹彩病毒检测方法，实验针对蛙病毒3型(frog virus 3,FV3)核衣壳蛋白(major capsid protein,MCP)基因保守区设计一对特异性引物，并选取另外2种蛙病毒PCR检测方法进行对比实验，通过反应体系的优化、反应特异性和敏感性实验，建立了一种针对FV3的PCR检测方法。结果显示，该方法最低检测限可达1.2个拷贝数的病毒粒子，与神经坏死病毒、虾血细胞虹彩病毒、大口黑鲈虹彩病毒、锦鲤疱疹病毒、鲤浮肿病毒、传染性脾肾坏死病毒、加州鲈弹状病毒等常见水产动物致病毒株无交叉反应。临床样品检测表明，该方法所获得的检测结果与另外2种方法一致，结果可靠。本研究所建立的FV3 PCR检测方法，具有简便、快速、敏感度好、特异性高、低成本等特点，可用于FV3蛙病毒的快速诊断和分子流行病学调研。     

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。     

虎纹蛙病毒主要衣壳蛋白基因的克隆及其序列

从新分离感染虎纹蛙的病毒培养细胞中提取病毒DNA作模板，用分别对应于蛙病毒－3型（FV3）主要衣壳蛋白（Major Capsid Protein,MCP）基因读码框两侧的寡核苷酸片段作引物进行PCR扩增，得到预期大小基因片段，进一步将此基因片段插入到pGEM-T载体中，进行全长片段的序列测定和分析。结果表明，编码虎纹蛙病毒的MCP基因的读码框核苷酸数为1392bp，编码463个氨基酸；基因的核苷酸序列与其他脊椎动物虹彩病毒的MCP基因序列比较结果显示，该病毒与蛙病毒属的FV3的同源性（98％)明显高于囊肿病毒属的FLDV－1（52％），并且与虹彩病毒科其他成员的MCP基因序列均有所不同，说明该病毒株是虹彩病毒科蛙病毒属的新成员。     

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。     

大口黑鲈溃疡综合征病毒MCP基因序列分析及PCR快速检测方法的建立

为了早期快速诊断近年来流行于广东省养殖大口黑鲈(Micropterussalmoides)中的病毒性溃疡综合征,本研究用基因组步移的方法获得了大口黑鲈溃疡综合征病毒(Largemouthbassulcerativesyndromevirus,LBUSV)主要衣壳蛋白(MCP)基因,该基因编码区全长1392bp。通过序列比较分析,在MCP基因内确定了一段241bp的特异性较强的片段作为靶序列,设计并合成引物,经过优化PCR反应条件,建立了可以快速检测大口黑鲈溃疡综合征病毒的PCR方法。实验表明,在PCR进行到30个循环反应时可以检测到的质粒最小浓度是104拷贝数/μL,相当于104个病毒粒子。利用该方法,从天然感染LBUSV的大口黑鲈脾脏组织DNA可扩增出241bp的片段,而健康大口黑鲈和感染了传染性脾肾坏死病毒样病毒的大口黑鲈脾脏组织则没有扩增条带。本研究建立的PCR检测方法具有检测快速、成本低、准确性高的特点,适用于大范围早期病害诊断的推广应用。     

一株大黄鱼虹彩病毒的分离鉴定及多克隆抗体的制备

2021年7月,浙江省象山某养殖场养殖的大黄鱼（Larimichthys crocea）出现类似大黄鱼虹彩病毒引起的疾病。采用鲤上皮瘤细胞培养和病毒主要衣壳蛋白测序分析的方法,从患病的大黄鱼中分离到一株病毒。该病毒接种到鲤上皮瘤细胞（EPC）后出现空斑、脱落的细胞病变症状。根据虹彩病毒MCP和ATPase基因保守序列设计特异性引物对病毒组织样本进行PCR扩增,得到分别为1 367 bp和740 bp的目的基因片段。将MCP基因扩增片段测序,经BLAST对比及系统发育树聚类分析,确定该分离的病毒属虹彩病毒科细胞肿大病毒属。通过蔗糖密度梯度离心纯化,用透射电镜观察该病毒粒子呈正六边形,直径为120～150 nm。用纯化病毒作为抗原免疫小鼠获得抗大黄鱼虹彩病毒的多克隆抗体,效价为1∶7 000;通过SDS-PAGE和Western blotting初步确定3个免疫蛋白。本研究为大黄鱼虹彩病毒纯化提供一种新方法,并初步分离出免疫蛋白,为该病毒相关分子生物学研究、蛋白研究以及疫苗制备等提供理论依据。     

养殖银鲳虹彩病毒感染的组织病理学分析及分子检测

2018年7月,宁波象山某养殖场内银鲳出现集中死亡现象,累积死亡率高达80%以上,为探究此次疾病暴发的原因,本研究通过对患病银鲳进行组织病理学分析、透射电镜观察、病原的分子生物学检测及分析来对其病原进行鉴定,以便制定合理的防控措施。患病银鲳临床表现为厌食、身体失衡、脾脏肿大、肝脏颜色异常等病征。组织病理学观察发现,患病银鲳肝脏、脾脏和肾脏均出现直径10～15μm、细胞质嗜碱性的肿大细胞。进一步的透射电镜观察则在脾脏、肾脏等组织细胞内发现了病毒包涵体结构以及大量的病毒粒子,直径为140～160 nm。通过虹彩病毒特异性PCR检测发现,患病组织样品为虹彩病毒阳性。此外,通过病毒主要衣壳蛋白基因(MCP)序列分析,发现银鲳源病毒与大黄鱼虹彩病毒(GenBank登录号:AY779031.1)MCP同源性最高为99.76%,认为此分离病毒属于虹彩病毒科、肿大细胞病毒属、真鲷虹彩病毒(Red sea bream iridovirus, RSIV)类群。本实验首次报道了全人工养殖环境下银鲳感染虹彩病毒的病例,该研究将为养殖银鲳虹彩病毒病的诊断和防治提供重要的参考依据。     

Experimental oral transmission of largemouth bass virus

Largemouth bass virus (LMBV) is a recently discovered iridovirus that causes a fatal disease of largemouth bass, Micropterus salmoides (Lacepède). Fish can become infected by waterborne LMBV, but oral transmission of this virus has not been demonstrated previously. Largemouth bass were gavaged with guppies, Poecilia reticulata Peters, which had been injected with LMBV, and then sampled periodically during a 7‐week observation period. The dose of LMBV averaged 10^5.6 tissue culture infectious doses – 50% cytopathic endpoint (TCID₅₀) per largemouth bass. Five of 24 largemouth bass exposed to LMBV became infected with the virus, but none of the fish had clinical signs typical of LMBV disease. Virus titres in largemouth bass were highest in swim bladder (10^5.5–9.5 TCID₅₀ g^?1) and were 10^5.2 TCID₅₀ g^?1 or lower in cutaneous mucus, head kidney, trunk kidney, spleen, gonad and intestine. These results indicate that LMBV can be transmitted orally to largemouth bass, but further study is needed to determine the factors affecting pathogenicity of the virus.     

Outbreaks of ulcerative disease associated with ranavirus infection in barcoo grunter,Scortum barcoo (McCulloch & Waite)

In 2013, an outbreak of ulcerative disease associated with ranavirus infection occurred in barcoo grunter, Scortum barcoo (McCulloch & Waite), farms in Thailand. Affected fish exhibited extensive haemorrhage and ulceration on skin and muscle. Microscopically, the widespread haemorrhagic ulceration and necrosis were noted in gill, spleen and kidney with the presence of intracytoplasmic eosinophilic inclusion bodies. When healthy barcoo grunter were experimentally challenged via intraperitoneal and oral modes with homogenized tissue of naturally infected fish, gross and microscopic lesions occurred with a cumulative mortality of 70–90%. Both naturally and experimentally infected fish yielded positive results to the ranavirus‐specific PCR. The full‐length nucleotide sequences of major capsid protein gene of ranaviral isolates were similar to largemouth bass virus (LMBV) and identical to largemouth bass ulcerative syndrome virus (LBUSV), previously reported in farmed largemouth bass (Micropterus salmoides L.), which also produced lethal ulcerative skin lesions. To the best of our knowledge, this is the first report of a LMBV‐like infection associated with skin lesions in barcoo grunter, adding to the known examples of ranavirus infection associated with skin ulceration in fish.     

Establishment and characterization of a liver cell line,ALL, derived from yellowfin sea bream,Acanthopagrus latus,and its application to fish virology

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%–20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus–yellowfin sea bream interaction.     

Isolation and characterization of a ranavirus from koi,Cyprinus carpio L., experiencing mass mortalities in India

We investigated mass mortalities of koi, Cyprinus carpio Linnaeus, 1758, experienced in South Indian fish farms by virus isolation, electron microscopy, PCR detection, sequencing of capsid protein gene and transmission studies. Samples of moribund koi brought to the laboratory suffered continuous mortality exhibiting swimming abnormalities, intermittent surfacing and skin darkening. Irido-like virus was isolated from the infected fish in the indigenous snakehead kidney cell line (SNKD2a). Icosahedral virus particles of 100 to 120 nm were observed in the infected cell cultures, budding from the cell membrane. Virus transmission and pathogenicity studies revealed that horizontal transmission occurred associated with mortality. PCR analysis of infected fish and cell cultures confirmed the presence of Ranavirus capsid protein sequences. Sequence analysis of the major capsid protein gene showed an identity of 99.9% to that of largemouth bass virus isolated from North America. Detection and successful isolation of this viral agent becomes the first record of isolation of a virus resembling Santee–Cooper Ranavirus from a koi and from India. We propose the name koi ranavirus to this agent.     

三种水生动物细胞系对两株蛙病毒敏感性的比较

利用3个不同物种的水生动物细胞系,包括爪蟾肾细胞系(A6)、大鲵胸腺细胞系(GSTC)和鲤上皮瘤细胞系(EPC),分别用沼泽绿牛蛙蛙病毒(RGV)和大鲵蛙病毒(ADRV)感染,进一步研究细胞病变显微形态、病毒滴度、细胞病变与不同感染时间的相关性等。结果显示,在光镜下可见感染病毒的细胞发生病变,A6和EPC细胞肿胀或破裂;GSTC细胞收缩或聚在一起形成多层。同种水产动物细胞系对不同蛙病毒的敏感性不同,在A6、EPC和GSTC细胞中,RGV的滴度分别为10~(3.6)、10~(5.9)和10~(6.6) TCID_(50)/m L;ADRV的滴度分别为10~(4.3)、10~(5.4)和10~(6.1) TCID_(50)/m L,表明GSTC细胞系对两种蛙病毒都更敏感。研究为后续蛙病毒致病机理提供了有用的信息和重要实验材料。     

Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China

Fish rhabdoviruses are a family of viruses responsible for large‐scale fish die‐offs worldwide. Here, we reported the isolation and identification of a member of rhabdoviruses from wild largemouth bass (Micropterus salmoides) in the coastal area of the Pearl River Estuary, China. This virus isolate was identified as viral haemorrhagic septicaemia virus (VHSV) by specific RT‐PCR. Furthermore, the virus (VHSVLB2018) was isolated by cell culture using fathead minnow cells and confirmed by RT‐PCR. Electron microscopy showed the presence of bullet‐shaped viral particles in the cytoplasm of infected cells. The complete sequencing of VHSVLB2018 confirmed that it was genome configuration typical of rhabdoviruses. Phylogenetic analysis based on whole‐genome sequences and G gene nucleotides sequences revealed that VHSVLB2018 was assigned to VHSV genogroup Ⅳa. The pathogenicity of VHSVLB2018 was determined in infection experiments using specific pathogen‐free largemouth bass juveniles. VHSVLB2018‐infected fish showed typical clinical signs of VHSV disease, including darkened skin, petechial haemorrhages and pale enlarged livers, with the cumulative mortalities reached 63.3%–93.3% by 7 days post‐infection. VHSVLB2018 was re‐isolated from dead fish and confirmed by RT‐PCR. Together, this is the first report of isolation and identification of a VHSV isolate from wild largemouth bass in China.     

Comparison of the pathogenicity of Francisella orientalis in Nile tilapia (Oreochromis niloticus), Asian seabass (Lates calcarifer) and largemouth bass (Micropterus salmoides) through experimental intraperitoneal infection

Francisella orientalis is a highly virulent, emerging bacterium that causes mass mortalities in tilapia. This pathogen also affects numerous other warm-water fish species, including three-line grunt, hybrid striped bass and various ornamental fish. This study sheds light on two new species of fish that are susceptible to F. orientalis. Asian seabass and largemouth bass showed variable levels of susceptibility in a bacterial challenge experiment. After intraperitoneally injected with a dose of 10⁶ CFU/fish, a total of 64.28% and 21.42% mortalities were obtained in Asian seabass and largemouth bass, respectively. Meanwhile, Nile tilapia showed acute mortality of 100%. All fish showed typical lesions of francisellosis, including multifocal granulomas in the spleen and head kidney. Immunohistochemical analysis revealed strong positive signals inside the granulomas of all fish. The bacterial recovery in solid media from infected fish was highest in Nile tilapia (85.71%), followed by Asian seabass (35.71%) and largemouth bass (21.42%). PCR results tested 100% positive for Nile tilapia, and 78.57% and 21.42% for Asian seabass and largemouth bass, respectively. In conclusion, Asian seabass and largemouth bass are susceptible to this pathogen, which warrants new management strategies when employing predation polyculture systems of these species with tilapia.     

虎纹蛙病毒甲基转移酶基因的克隆和分析

经PCR扩增得到虎纹蛙病毒DNA甲基转移酶完整基因片段，并将其克隆到pUCm-T载体，测序可知该基因读码框大小为642bp，编码一由214个氨基酸组成的，预期分子量为24.8kD的多肽。RTV的MTase基因与蛙病毒属的蛙病毒-3型、叉尾Hui病毒和Regina病毒的一致性为96%～97%，与淋巴囊肿病毒属的比目鱼病毒的一致性为56%，从而进一步证明RTV蛙病毒的分类地位；与其它脊椎动物的虹彩病毒一样，该基因包含原核生物5′甲基转移酶的前四个高度保守区而缺少第五个区域，可能只是构成甲基转移酶的一个亚基，RTV、裂唇鱼病毒和大口黑鲈病毒之间MTase基因的一致性与衣壳蛋白基因的差异较大，说明同一种类的不同基因甚至同一基因的不同区域间演化速率不同，因此在虹彩病毒的演化研究中选择合适的基因或基因区域极为重要。