流行性造血器官坏死病

<正>流行性造血器官坏死病(Epizootic haematopoietic necrosis,E H N)是由一种虹彩病毒感染所引起的全身性疾病,主要感染赤鲈(Perca fluviatilis,俗名河鲈)     

Epizootic haematopoietic necrosis virus: detection by ELISA, immunohistochemistry and immunoelectron-microscopy

Abstract. Epizootic haematopoietic necrosis virus (EHNV) was isolated from cultured rainbow trout, Oncorhynchus mykiss (Walbaum). Antibodies to the virus and to associated capsid subunits have been produced and used in immunohistochemistry, immunoelectron-microscopy and an antigcn-capture-immunosorbent assay (ELISA). The results show that both antibodies can be used by various immuno-procedures to detect both redfin perch, Perca ftuviatilis L., and rainbow trout isolates of EHNV. The procedures described provide for the first time rapid and specific tests for the detection of EHNV in cultured and clinical material.     

鱼类传染性造血器官坏死病临床诊断及检测

<正>鱼传染性造血器官坏死病(Infectious haematopoietic necrosis of fish,IHN)。是一种毒力很强的弹状病毒所引起的的急性、全身性的严重传染病。该病主要侵害虹鳟,包括硬头鳟、大鳞大马哈鱼、红大马哈鱼和大西洋大马哈鱼,银大马哈鱼(银鳟)在自然条件下对IHN病毒有抵抗力。     

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。     

Inactivated infectious haematopoietic necrosis virus (IHNV) vaccines

The inactivation dynamics of infectious haematopoietic necrosis virus (IHNV) by b-propiolactone (BPL), binary ethylenimine (BEI), formaldehyde or heat and the antigenic and immunogenic properties of the inactivated vaccines were evaluated. Chemical treatment of IHNV with 2.7 mm BPL, 1.5 mm BEI or 50 mm formaldehyde abolished virus infectivity within 48 h whereas heat treatment at 50 or 100 degrees C rendered the virus innocuous within 30 min. The inactivated IHNV vaccines were recognized by rainbow trout, Oncorhynchus mykiss, IHNV-specific antibodies and were differentially recognized by antigenic site I or antigenic site II IHNV glycoprotein-specific neutralizing monoclonal antibodies. The BPL inactivated whole virus vaccine was highly efficacious in vaccinated rainbow trout challenged by waterborne exposure to IHNV 7, 28, 42 or 56 days (15 degrees C) after immunization. The formaldehyde inactivated whole virus vaccine was efficacious 7 or 11 days after vaccination of rainbow trout but performed inconsistently when tested at later time points. The other vaccines tested were not efficacious.     

Infectious haematopoietic necrosis virus from salmonids cultured in Korea

Abstract. Four isolates of infectious haematopoietic necrosis virus (IHNV) were obtained from rainbow trout, Oncorhynchus mykiss (Walbaum), and masu salmon, Oncorhynchus masou (Walbaum), fry during epizootics at hatcheries in Korea. The four isolates of IHNV were compared with three from salmonids in the USA (SRCV, OSV and RB-76) by analysis of virion proteins in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and neutralization tests, with two monoclonal antibodies raised against SRCV (MAb SRCV/A4) and RB-76 (MAb RB/B5). Based on the antigenicity and the size of the structural proteins, one Korean isolate from masu salmon (SCS) is similar to RB-76 and is an electropherotype 1. The other three isolates from rainbow trout (PRT, YRT and MRT) were identical to each other in the mobilities of their virion proteins in SDS-PAGE, and although their nucleocapsid (N) proteins comigrated with that of the RB-76 isolate, they differed from RB-76 in the smaller matrix 2 (M2) protein they contained. In addition, the three Korean isolates (PRT, YRT and MRT) could be divided into two groups by reactivity with MAb RB/B5. While the YRT isolate was neutralized by this MAb, the PRT and MRT isolates were not, suggesting that there are at least two neutralizing antigenic variants of IHNV in Korea.     

传染性造血器官坏死

传染性造血器官坏死(Infectious haematopoietic necrosis,IHN)是由弹状病毒引起的鱼类急性、全身性传染病,主要发生于鳟和太平洋大马哈鱼鱼苗和种负,病鱼表现为狂游和造血器官坏死,世界动物卫生组织(OIE)将其列为必须申报的动物疫病,为我国的二类疫病.     

Epizootic haematopoietic necrosis, a new viral disease in redfin perch, Perca fluviatilis L., in Australia

Abstract. An epizootic disease in redfin perch, Perca fluviatilis L., in north-east Victoria, Australia, was found to be caused by a recently described iridovirus. The acute disease is characterized by necrosis of the renal haematopoietic tissue, liver and spleen, with variable involvement of the pancreas. Transmission studies confirmed that the iridovirus is the causative agent of the disease. High mortalities occurred in 0+ juveniles in early summer and appear to be a recurrent seasonal event, whereas more sporadic outbreaks occurred in older age classes. Carrier fish were detected at low prevalence. The disease is described as epizootic haematopoietic necrosis (EHN) and is the only viral disease of fish known to occur in Australia.     

A comparison of detection methods for infectious haematopoietic necrosis virus

Abstract. A comparative study of immunological methods for detecting infectious haematopoietic necrosis virus (IHNV) was made. The anti–IHNV antibody titre was measured by solid phase direct binding assays with'¹²⁵iodinated Protein A from Staphylococcus aureus and with immunoperoxidase staining. The binding antibody titre was much higher than that obtained in the virus neutralization assay. The high binding antibody titre of rabbit anti–IHNV sera made the development of two immunological tests for IHNV possible. Virus–specific proteins were detected on nitrocellulose membranes after transfer from denaturing polyacrylamide gels. The immunological methods were highly specific, sensitive lo less than 10 ng of virus protein, and were useful in characterizing the different strains of IHNV.     

An isolate and sequence database of infectious haematopoietic necrosis virus (IHNV)

In the field of fish diseases, the amount of relevant information available is enormous. Internet‐based databases are an excellent tool for keeping track of the available knowledge in the field. Fishpathogens.eu was launched in June 2009 with the aim of collecting, storing and sorting data on fish pathogens. The first pathogen to be included was the rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Here, we present an extension of the database to also include infectious haematopoietic necrosis virus (IHNV). The database is developed, maintained and managed by the European Community Reference Laboratory for Fish Diseases and collaborators. It is available at http://www.fishpathogens.eu/ihnv .     

Detection of infectious haematopoietic necrosis virus in river water and demonstration of waterborne transmission

Abstract. In a study of the possible role of waterborne infectious haematopoietic necrosis virus in transmission of the disease among spawning sockeye salmon, Oncorhynchus nerka (Walbaum), both infection rates and virus titres were higher in fish held at high density in a side channel than in fish in the adjacent river. Virus was never isolated from river water, but was found in water from the side channel at levels ranging from 32.5 to 1600 plaque-forming units (p.f.u.)/ml. Uninfected yearling sockeye salmon held in a box in the side channel developed localized gill infections with IHN virus. The disease did not progress to the viscera until a threshold titre of about 10⁵ p.f.u./g was reached in the gill. The effectiveness of the gill as a barrier limiting development of systemic infections means that waterborne IHN virus probably does not greatly increase the infection rate in a sockeye salmon population during spawning.     

Inhibition of infectious haematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers

Delivery of phosphorodiamidate morpholino oligomers (PMO) into fish cells in vitro and tissues in vivo was examined. Uptake was evaluated by fluorescence microscopy and flow cytometry after treating cultured cells or live rainbow trout with 3' fluorescein-tagged PMO. Arginine-rich peptide conjugated to the 5' end of the PMO markedly enhanced cellular uptake in culture by 8- to 20-fold compared with non-peptide-conjugated PMO as determined by flow cytometry. Enhanced uptake of PMO conjugated to peptide was also observed in tissues of fish treated by immersion. The efficacy of PMO as inhibitors of infectious haematopoietic necrosis virus (IHNV) replication was determined in vitro. Peptide-conjugated PMOs targeting sequences within the IHNV genomic RNA (negative polarity) or antigenomic RNA (positive polarity) significantly inhibited replication in a dose-dependent and sequence-specific manner. A PMO complementary to sequence near the 5' end of IHNV genomic RNA was the most effective, diminishing titre by 97%, as measured by plaque assay and Western blot. These data demonstrate that replication of a negative-stranded non-segmented RNA virus can be inhibited by antisense compounds that target positive polarity viral RNA, or by a compound that targets negative polarity viral RNA.     

Herpesviral haematopoietic necrosis virus (CyHV-2) infection: case studies from commercial goldfish farms

Herpesviral haematopoietic necrosis is a disease of goldfish, Carassius auratus , caused by Cyprinid herpesvirus-2 (CyHV-2) infection. Quantitative PCR was carried out on tissue homogenates from healthy goldfish fingerlings, broodfish, eggs and fry directly sampled from commercial farms, from moribund fish submitted to our laboratory for disease diagnosis, and on naturally-infected CyHV-2 carriers subjected to experimental stress treatments. Healthy fish from 14 of 18 farms were positive with copy numbers ranging from tens to 10⁷ copies μg⁻¹ DNA extracted from infected fish. Of 118 pools of broodfish tested, 42 were positive. The CyHV-2 was detected in one lot of fry produced from disinfected eggs. Testing of moribund goldfish, in which we could not detect any other pathogens, produced 12 of 30 cases with 10⁶–10⁸ copies of CyHV-2 μg⁻¹ DNA extracted. Subjecting healthy CyHV-2 carriers to cold shock (22–10 °C) but not heat, ammonia or high pH, increased viral copy numbers from mean copy number (±SE) of 7.3 ± 11 to 394 ± 55 μg⁻¹ DNA extracted after 24 h. CyHV-2 is widespread on commercial goldfish farms and outbreaks apparently occur when healthy carriers are subjected to a sharp temperature drop followed by holding at the permissive temperature for the disease.     

传染性造血器官坏死病毒-Sn1203株的基因型及糖蛋白的生物信息学分析

利用鲤(Cyprinus carpio)上皮细胞(epitheliaoma papulosum cyprini, EPC)培养传染性造血器官坏死病毒- Sn1203分离株(IHNV-Sn1203), 根据GenBank中IHNV G蛋白基因开放阅读框(open reading frame, ORF)的序列设计引物(GenBank序列编号AB288207), 采用RT-PCR的方法克隆得到IHNV-Sn1203株G蛋白全长ORF, 克隆至表达载体pET27b(+)中, 构建了pET27-G重组质粒, 并进行了测序分析。生物信息学分析结果显示, IHNV-Sn1203株G蛋白基因序列长度为1 527 bp, 与韩国株具有最高的核酸同源性(96.86%)和氨基酸同源性(97.05%)。该基因编码508个氨基酸残基, 推导分子量约为56.55 kD, 等电点为6.15; 氨基酸序列分析表明, G蛋白富含丝氨酸、苏氨酸和酪氨酸, 存在28个潜在的磷酸化位点; 存在4个潜在的N-糖基化位点和7个潜在的O-糖基化位点; G蛋白N端含有20个氨基酸的信号肽; 亲水性大于输水性; 位于483~508位氨基酸存在一跨膜区; 抗原表位预测显示抗原性良好; 系统进化树分析显示, IHNV-Sn1203株与日本株和韩国株聚为一簇, 都属于JRt基因型。

     

A single-step multiplex PCR for simultaneous detection of white spot syndrome virus and infectious hypodermal and haematopoietic necrosis virus in penaeid shrimp

White spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic necrosis virus (IHHNV) are the major viral pathogens of penaeid shrimp worldwide (Lightner & Redman 1998). Litopenaeus vannamei was introduced into China from the Americas, and quickly became widely cultured. Following its introduction, both IHHNV and WSSV have become important pathogens of cultured penaeid shrimp and have had a huge impact on the culture industry in China in recent years.