First detection of spring viraemia of carp virus in common carp (Cyprinus carpio L.) affected by a septicaemic disease in Mexico

Spring viraemia of carp (SVC) is an infectious disease responsible for severe economic losses for various cyprinid species, particularly common carp (Cyprinus carpio carpio). The causative agent is the SVC virus (SVCV), a member of the Sprivivirus genus, Rhabdoviridae family, and a List 1 pathogen notifiable by the World Organization for Animal Health. This study describes the diagnosis of an SVCV pathogen isolated in October 2015 from wild common carp inhabiting a natural lagoon in central Mexico. While neither an epidemic nor fish mortalities were reported, the collected killed specimens exhibited clinical signs of disease (e.g., exopthalmia, moderate abdominal distension and haemorrhaging, as well as internal haemorrhages and adhesions). Histological results of injuries were consistent with the pathology caused by SVCV. This finding was supported by the isolation of a virus in EPC and BF‐2 cells and subsequent RT‐PCR confirmation of SVCV. The phylogenetic analyses of partial SVCV glycoprotein gene sequences classified the isolates into the Ia genogroup. These findings make this the first report of SVCV detection in Mexico, extending the southern geographical range of SVCV within North America. However, since this pathogen was detected in fish inhabiting a natural body of water without tributaries or effluents, it is difficult to estimate the risk of SVCV for other wild/feral cohabitating cyprinid species in the lagoon. The status of this virus is also unknown for other bodies of water within this region.     

鲤Rhbdd3蛋白的抗病毒功能

Rhbdd3(Rhomboid domain-containing protein 3)蛋白在哺乳动物天然免疫中发挥了重要作用,但水生动物中rhbdd3基因的确定序列及Rhbdd3蛋白的功能均尚未见报道。为研究鲤(Cyprinus carpio)的Rhbdd3蛋白在鱼类细胞中的功能,探讨其过表达对鱼类病毒感染的影响,本研究通过PCR扩增得到了鲤rhbdd3基因的编码序列,并将其克隆至pCI-neo载体上,构建了真核表达质粒pCI-rhbdd3。pCI-rhbdd3转染鲤上皮瘤细胞EPC(epithelioma papulosum cyprinid)和鲑囊胚细胞CHSE-214(chinook salmon embryo)后利用制备的特异性抗体进行Western blot,检测Rhbdd3蛋白的表达情况,并利用CCK-8试剂检测其过表达对细胞增殖的影响。转染后分别进行鲤春病毒血症病毒(SVCV)和传染性胰腺坏死病毒(IPNV)的感染实验,并利用间接免疫荧光、Western blot和RT-qPCR方法检测Rhbdd3过表达对SVCV和IPNV增殖的影响。结果显示,pCI-rhbdd3转染后Rhbdd3蛋白在EPC和CHSE-214细胞中得到了过表达,且Rhbdd3蛋白的过表达能显著抑制SVCV和IPNV的复制,但不影响两种细胞的正常活性。本研究为鱼类广谱抗病毒药物的开发提供了新的实验依据,也为鱼类抗病毒新品种的培育奠定了重要基础。     

黑龙江地区鲤春病毒血症病毒的分离与基因型分析

对2015—2016年黑龙江不同地区的40个养殖场送检的鲤(Cyprinus carpio)进行鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的细胞培养分离、PCR鉴定、病毒滴度测定、病毒表面糖蛋白(glycoprotein,G)氨基酸序列聚类分析及基因分型研究。细胞培养结果显示,来自4个不同养殖场的鲤组织样本能够感染鲤上皮细胞(epithelioma papulosum cyprini,EPC)产生典型细胞病变(cytopathic effect,CPE),收集病毒悬液分别称为Shlj1~Shlj4。PCR鉴定结果表明,该4株病毒均为SVCV。病毒滴度实验测算出SVCV Shlj 1~Shlj 4的滴度分别为10~(6.28)、10~(6.88)、10~(7.57)和106.38 TCID50/mL。Shlj的糖蛋白基因核苷酸序列的聚类分析和遗传进化分析结果显示,Shlj 1~Shlj 4与Gen Bank收录的中国参考株A2、BJ0505-2和美国参考株USA、212364聚为一簇,同源性为98.4%~99.8%;Shlj 1~Shlj 4毒株之间的糖蛋白核苷酸序列相似性在98.6%~99.8%,其中Shlj 3与美国SCVC毒株USA、212364具有最高的核苷酸相似性(99.8%),Shlj 2与英国参考毒株880163具有最低的相似性(88.0%)。糖蛋白氨基酸序列比对结果显示,Shlj4中氨基酸突变最多,与另3个毒株差异较大。基因型分析结果显示,Shlj 1~Shlj 4均为基因Ia型。本研究结果表明,黑龙江地区2015—2016年间的SVCV检出率约为10%,并且来源于不同养殖场的病毒分离株的核酸序列呈现不同程度的差异,该结果进一步证明SVCV毒株在中国不同的鲤养殖环境中正在不断地进化。     

中华鲟IFNe2的抗病毒天然免疫作用研究

为探究中华鲟干扰素e2（AsIFNe2）的免疫调控作用,本研究通过原核表达获得了重组中华鲟干扰素e2蛋白（rAsIFNe2）,并分析了它对抗病毒相关基因的影响及其抗病毒活性。实时荧光定量PCR（RT-qPCR）结果显示,rAsIFNe2能显著激活中华鲟鳍细胞中干扰素刺激基因（IFN-stimulated genes,ISGs）的表达,如Mx、PKR、Viperin和ADAR4 。此外,rAsIFNe2还能通过激活IRFs和IFNes基因的表达,从而帮助宿主细胞迅速建立抗病毒状态。在鲤春病毒血症病毒（spring viremia of carp virus,SVCV）感染鲤鱼上皮细胞（EPC）模型中,rAsIFNe2能够诱导EPC细胞中Mx、PKR和Viperin的表达,并降低EPC细胞中SVCV病毒G、N和P基因的表达,减少病变效应的产生。上述结果表明AsIFNe2在宿主抗病毒天然免疫反应中发挥作用,为深入了解中华鲟的干扰素免疫系统以及治疗病毒性疾病提供了理论依据。     

Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish

Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full‐length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G‐gene to make the rIHNV‐Gsvcv. The chimeric rIHNV‐Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild‐type SVCV infection was tested. The rIHNV‐Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV‐Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV‐Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV‐Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.     

黑头软口鲦上皮瘤细胞ifn-1基因的表达及抗病毒活性

为了体外表达黑头软口鲦上皮瘤细胞(EPC)I型干扰素(IFN-1),本实验通过RTPCR从EPC中扩增ifn-1基因,构建重组表达质粒pET-32a-IFN-1,并转化到感受态细胞Transetta(DE3),体外纯化后检测其抗病毒活性。结果显示,ifn-1编码区大小为552 bp,编码184个氨基酸,与草鱼干扰素1(CiIFN1)亲缘关系最近。通过SDS-PAGE分析,重组表达质粒pET-32a-IFN-1在宿主菌中可明显表达约35 ku的融合蛋白条带,且部分呈可溶性表达,进而通过亲和纯化可溶性重组IFN-1(rIFN-1),免疫新西兰大白兔获得效价较高的抗IFN-1多克隆抗体,可用于检测细胞内源性的IFN-1。定量PCR显示rIFN-1与EPC细胞孵育可以诱导抗病毒蛋白Mx1的表达,并抑制鲤春病毒血症病毒(SVCV)引起的细胞病变(CPE)及SVCV的复制,表明rIFN-1具有抗病毒活性。     

鲤春病毒血症病毒糖蛋白的截短表达及其免疫原性分析

为了揭示鲤春病毒血症病毒(spring viremia of carp virus,SVCV)糖蛋白编码基因的主要免疫原性决定区域,本研究对SVCV的糖蛋白基因进行截短表达,并用纯化的重组蛋白作为免疫原制备了兔抗血清以分析其免疫原性。通过SOSUI以及DNAStar 6.0软件对SVCV糖蛋白基因的跨膜区、亲水性以及抗原表位进行分析后,采用RT-PCR对该基因主要抗原决定区域编码片段进行扩增,并构建重组表达质粒p GEX-Gtr,对其进行诱导表达后获得截短的SVCV糖蛋白的重组蛋白。将表达的重组蛋白进行纯化复性后,作为免疫原制备兔抗血清,采用ELISA法检测其效价,采用免疫印迹以及间接免疫荧光技术检测该重组蛋白的免疫原性。研究结果显示,截短表达的糖蛋白编码基因长1317 bp,编码439个氨基酸(29~467),推测分子量为49.6 k D。利用该重组蛋白制备的兔抗血清,其与重组蛋白的反应效价为1∶64000,。免疫印迹及间接免疫荧光结果显示,该兔抗血清与SVCV-HN株能发生特异性反应,表明该重组蛋白与天然的病毒表面糖蛋白的免疫原性无差异。上述研究结果表明,截短表达的重组蛋白具有很好的免疫原性,可应用于免疫诊断技术研发与基因工程疫苗的研制。     

病毒感染过程中青鳉irf2基因过表达的双面性

为了探究干扰素调节因子2 (interferon regulatory factor,IRF2)如何通过调控干扰素(IFN)表达影响鱼类的免疫,实验从青鳉中克隆了irf2 (Olirf2),发现该基因在青鳉各个组织中均有表达;将构建的真核表达载体pTol2/CMV-IRF2/IE1-pr转染到胖头鱥肌肉细胞系(FHM)后,发现瞬时过表达Olirf2能够显著促进鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的复制,并抑制抗病毒相关基因mx1、ifn和irf3的表达。进一步通过双荧光素报告系统发现,Olirf2能够显著抑制NF-κB和ISRE的活性,说明Olirf2可能通过抑制细胞的天然免疫应答进而促进病毒的增殖。然而持续过表达Olirf2则增强了细胞的抗病毒能力,同时促进干扰素相关基因mx1、ifn和irf3的表达。因此,Olirf2基于表达的持续时间不同而具有抗病毒或者促病毒的双面效果。实验通过研究Olirf2在抗病毒信号通路中发挥的作用,为通过基因编辑或者转基因手段来构建抗病毒的鱼类提供了理论基础。     

Establishment of a cell line from the kidney of black carp and its susceptibility to spring viremia of carp virus

In this study, we established and characterized a cell line derived from the kidney of black carp (Mylopharyngodon piceus), which is an important freshwater aquaculture species. The cell line was designated as MPK and subcultured for more than 70 passages in DMEM medium containing 10% fetal bovine serum (FBS) at 28°C. MPK had a modal diploid chromosome number of 48. Moreover, a transient MPK transfection efficiency was up to 18% using a green fluorescent protein plasmid by a modified electroporation. In addition, the MPK cells showed susceptibility to spring viremia of carp virus (SVCV), as demonstrated by the presence of severe cytopathic effects (CPEs) and increased viral RNA. Unexpectedly, the MPK cells expressed pluripotency‐associated genes such as nanog, oct4 and vasa, indicating that these are possibly adult stem cells. Taken together, we have established a stable cell line from kidney that may potentially be utilized as an in vitro platform for genetic modifications and host–pathogen analysis in black carp.     

鲤春病毒血症病毒核蛋白、磷蛋白与基质蛋白的表达、抗体制备及免疫原性比较

鲤春病毒血症病毒(SVCV)能够引起鲤科鱼类大量死亡,被世界动物卫生组织(OIE)列为必须申报的重要疫病,也是我国唯一被列为一类疫病的鱼类传染病。为建立SVCV的快速免疫学诊断方法,研究其主要结构蛋白间的免疫原性差异,实验首先采用原核表达系统克隆并诱导表达,纯化SVCV的核蛋白(N)、磷蛋白(P)和基质蛋白(M),并进一步免疫新西兰白兔制备抗血清,抗血清经Protein A柱进一步纯化获得3种蛋白的多克隆抗体。利用间接酶联免疫吸附测定(ELISA)和免疫印迹实验(Western blot)对抗体效价和特异性进行分析验证。结果发现,SVCV的N、P和M重组蛋白均在原核表达系统获得大量表达,且表达的蛋白经纯化后免疫实验动物产生了相应的多克隆抗体。Western blot结果显示,3种蛋白抗体均与SVCV重组蛋白及天然蛋白发生特异性的免疫反应。ELISA结果显示,针对P蛋白制备的抗体效价最高,可达409 600;针对N和M蛋白制备的抗体效价也均大于204 800。同时,特异性检测实验结果显示,制备的3种蛋白抗体均仅与SVCV发生特异性免疫反应,而与SVCV宿主其他易感病毒均不发生交叉反应。实验结果将对SVCV的快速诊断及疫苗开发提供新的手段和思路。     

Establishment and characterization of a heart-derived cell line from goldfish (<Emphasis Type="Italic">Carassius auratus</Emphasis>)

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10–20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 10^5.0, 10^4.5, and 10^5.0 TCID₅₀/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.     

Establishment of a new cell line from the heart of giant grouper,Epinephelus lanceolatus (Bloch), and its application in toxicology and virus susceptibility

A new marine fish cell line, derived from the heart of giant grouper, Epinephelus lanceolatus (Bloch), was established and characterized. The cell line was designated as ELGH and subcultured with more than 60 passages. The ELGH cells were mainly composed of fibroblast-like cells and multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum (FBS) at 28 °C. Chromosome analysis indicated that the modal chromosome number was 48. The fluorescent signals were detected in ELGH when transfected with green fluorescent protein reporter plasmids. The 50% cytotoxic concentration (CC₅₀) of the extracellular products (ECPs) from Streptococcus iniae and Vibrio alginolyticus E333 on ELGH cells was 60.02 and 12.49 μg mL⁻¹, respectively. Moreover, the ELGH cells showed susceptibility to Singapore grouper iridovirus (SGIV), but not to soft-shelled turtle iridovirus (STIV), red-spotted grouper nervous necrosis virus (RGNNV) and spring viremia of carp virus (SVCV), which was demonstrated by the presence of a severe cytopathic effect (CPE) and increased viral titres. In addition, electron microscopy observation showed that abundant virus particles were present in the infected cells. Taken together, our data above provided the potential utility of ELGH cells for transgenic and genetic manipulation, as well as cytotoxicity testing and virus pathogenesis.     

Koi herpesvirus and carp oedema virus: Infections and coinfections during mortality events of wild common carp in the United States

Koi herpesvirus (KHV; cyprinid herpesvirus‐3) and carp oedema virus (CEV) are important viruses of common and koi carp (Cyprinus carpio); however, the distribution of these viruses in wild common carp in North America is largely unknown. During the summers of 2017 and 2018, 27 mass mortalities of common carp were reported from four states in the USA (Minnesota, Iowa, Pennsylvania and Wisconsin), the majority of which were distributed across eight major watersheds in southern Minnesota. Samples from 22 of these mortality events and from five clinically healthy nearby carp populations were screened for KHV, CEV and SVCV using real‐time polymerase chain reaction (qPCR). KHV was confirmed in 13 mortality events, CEV in two mortality events and coinfections of KHV/CEV in four mortality events. Nucleotide sequence analysis revealed that the KHV and CEV detected here are closely related to European lineages of these viruses. While molecular detection alone cannot conclusively link either virus with disease, the cases described here expand the known range of two important viruses. This is also the first reported detection of KHV and CEV coinfections in wild carp populations.     

一株鲈鲤源鲤春病毒血症病毒的分离鉴定及其病理观察

2016年4月,四川某鲈鲤(Percocypris pingi)养殖场流行一种以鳃、鱼鳔和内脏器官出血为临床特征的传染病。组织病理学观察发现,患病鲈鲤全身多组织器官均发生明显的病理损伤,尤其是肝、脾、肾、鳃和肠表现为明显的出血、变性、坏死以及炎症细胞浸润。取病鱼组织匀浆滤液接种鲤上皮瘤细胞(epithelioma papulosum cyprini, EPC),盲传3代出现典型的细胞病变(cytopathic effects, CPE)。将自然发病鱼组织匀浆滤液和细胞培养病毒液分别接种健康鲈鲤,实验鱼出现与自然发病鱼相同的症状,死亡率分别为60%和50%,而对照组未见异常。对经分离毒株ZLP160415感染出现CPE的EPC细胞制备超薄切片进行电镜观察,发现病毒呈弹状,长90～150 nm,宽40～60 nm;对自然发病鱼、人工感染发病鱼内脏组织和细胞培养病毒液进行鲤春病毒血症病毒(springviremiaofcarpvirus,SVCV)的RT-PCR检测,均扩增出目的条带。基于SVCV糖蛋白基因进行系统发育分析,结果显示分离毒株(ZLP160415)属于Ia型。结合本次疾病的流行病学与病理损伤特点、病毒分离鉴定、人工感染试验结果和透射电镜检查,确定此次流行病的病原为SVCV。     

鲤春病毒血症病毒生物学研究进展

近年来,鱼类急性出血性疾病——鲤春病毒血症的暴发屡屡给水产养殖业带来巨大的经济损失.鲤春病毒血症是由鲤春病毒血症病毒引起的,该病毒具有传染性强,发病率高的特点,目前尚无有效治疗药物.文章从流行特点、临床症状、生物学特征、检测技术和疫苗研究等方面总结概述了鲤春病毒血症病毒的生物学研究进展,可为进一步深入研究该病毒提供参考...