重组虹鳟IFN-γ2的原核表达及抗IHNV活性分析

为获得虹鳟IFN-γ2(rt IFN-γ2)抗传染性造血器官坏死病毒(IHNV)活性的相关数据,实验根据NCBI已发表序列设计引物,提取经植物血凝素刺激后的虹鳟头肾细胞总RNA,采用RT-PCR方法扩增471 bp的该基因完整开放阅读框。将该基因重组至原核表达载体p ET32a中,并转化大肠杆菌Rosetta,进行诱导表达,SDS-PAGE结果显示,目的蛋白以包涵体形式表达,大小约为38.4 ku。重组蛋白经复性、纯化后在CHSE-214细胞上进行抗IHNV活性分析,结果显示,rt IFN-γ2在CHSE-214细胞上抗IHNV活性为6.63×106U/mg。Real-time PCR结果显示,rt IFN-γ2免疫后,虹鳟头肾、脾、肝中IRF-1、IRF-2、IFN-I、IFN-γ和Mx表达水平均显著提高,总体而言免疫后2天机体抗病毒状态弱于免疫后1天。攻毒保护实验结果显示,免疫后1天进行IHNV攻击时,鱼死亡率为40%,而免疫后2天进行IHNV攻击时,鱼死亡率达到80%。研究表明,原核表达系统制备的重组虹鳟IFN-γ2不仅具有体外抗IHNV活性,更能激发虹鳟的抗病毒状态,从而为虹鳟抵抗IHNV感染提供一定的保护力。     

传染性造血器官坏死病灭活疫苗的制备及免疫保护效果

为了研发用于预防传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)灭活疫苗,实验以不同感染复数(multiplicity of infection,MOI)在胖头鱥上皮细胞(Epithelioma papulosum cyprinid,EPC)上对传染性造血器官坏死病毒(infectious hematopoietic necrosis virus,IHNV)进行连续传代培养,通过测定各代病毒滴度,结合病毒收获时间确定最佳增殖方案;采用不同浓度的β-丙内酯(β-propanolactone,BPL)在24℃下灭活,经安全性实验验证后确定最佳灭活条件。以不同剂量腹腔注射免疫虹鳟,以磷酸盐缓冲溶液(PBS)为阴性对照组,通过检测攻毒后相对免疫保护率(relative percent survival,RPS)、免疫相关因子表达量及血清中和抗体效价来分析该疫苗的保护效果。结果显示,最佳增殖方案为以MOI为0.000 1接种,15℃培养3 d;最佳灭活条件为以终浓度3.0 mmol/L的BPL将IHNV在24℃下灭活24 h;以最佳免疫剂量10μL/尾腹腔注射免疫虹鳟,免疫后7、21、45和60 d的相对免疫保护率分别为91.37%、84.28%、84.15%和47.5%,免疫后60 d时RPS显著低于其他时间点免疫组;免疫相关基因的实时荧光定量PCR(realtime quantitative PCR,RT-qPCR)结果显示,与阴性对照组相比,Mx-1和IFN-γ表达量在免疫后7、15和30 d脾脏和头肾中均显著上调,在第7天时达到最大值(5倍);CD4和IgM表达量在免疫后15 d脾脏和头肾中均显著上调;在免疫后第30、45和60天虹鳟血清IHNV中和抗体效价依次为67.25、43.40和29.78,呈下降趋势且各组间差异显著。研究表明,该灭活疫苗可诱导虹鳟产生特异性免疫和非特异性免疫反应,对虹鳟具有显著的免疫保护作用。本研究为IHNV灭活疫苗研发提供参考。     

传染性造血器官坏死病毒糖蛋白抗血清的制备及应用

应用逆转录聚合酶链式反应(RT-PCR)从传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)感染的细胞悬液克隆病毒的糖蛋白基因,将其亚克隆至原核表达载体pCWori,转化到大肠杆菌DH 5α,通过发酵大肠杆菌制备病毒糖蛋白.经SDS-PAGE分析,诱导表达的重组蛋白主要以包涵体的形式存在,使用Ni-NTA亲和层析柱在变性条件下进行纯化并透析复性,最终得到了较高纯度的可溶性糖蛋白,分子量约为57 kDa.Western-blot分析结果显示,所表达的蛋白能够被IHNV病毒制备的兔抗IHNV血清识别.用复性后的蛋白免疫小鼠制备抗血清,ELISA显示抗体效价可达1∶64 000.经制备的抗血清可以作为一抗建立ELISA检测方法,用于检测细胞悬液的病毒粒子,将抗血清稀释到1∶16 000仍能与IHNV全病毒发生反应.本研究利用重组的IHNV糖蛋白成功制备了高效价的抗血清,并能够与IHNV全病毒发生特异性结合,为IHNV免疫学检测方法建立奠定了基础.     

传染性造血器官坏死病毒微型基因组表达干扰素对病毒的抑制效应

为构建传染性造血器官坏死病毒(IHNV HLJ-09)微型基因组并表达虹鳟IFN,采用RT-PCR扩增IHNV HLJ-09株的N、P、L、G和NV蛋白基因并亚克隆入真核表达载体pCI中,构建辅助质粒pCI-N、pCI-P、pCI-L、pCI-G和pCI-NV;将扩增获得的IHNV基因组两末端序列、增强型绿色荧光蛋白(EGFP)报告基因、虹鳟I型干扰素(IFN)基因克隆到真核表达载体pCI中构建出表达EGFP的IHNV微型基因组pCI-LFGT和表达IFN的IHNV微型基因组pCI-LFIT;将pCI-LFIT质粒转染已接种IHNV HLJ-09毒株的EPC细胞,实时荧光定量PCR法测定细胞中IHNV G基因RNA。结果显示:构建的微型基因组不论与辅助病毒还是与5个辅助质粒共转染,外源基因均能正确表达;pCI-LFIT质粒转染已接种病毒的EPC细胞组与对照组相比其中的病毒核酸显著减少。     

香菇多糖和黄芪多糖对鲤免疫细胞的活性和IL-1β体外诱生表达的影响

为了探讨香菇和黄芪多糖对鲤免疫细胞的免疫活性作用,采用Percoll密度离心等技术,对鲤的头肾巨噬细胞和外周血白细胞进行分离纯化,并离体培养,从细胞和分子水平上研究了香菇和黄芪多糖的免疫调节作用.巨噬细胞和外周血白细胞分别体外暴露不同浓度的黄芪多糖和香菇多糖后,采用MTT法测定它们对鲤外周血白细胞增殖的影响;NBT还原法和Griess试剂显色法测定对头肾巨噬细胞的呼吸爆发的影响;实时定量PCR法测定头肾巨噬细胞细胞因子IL-1β诱导表达的影响.结果显示,黄芪和香菇多糖作用头肾巨噬细胞24 h后,香菇多糖浓度为1,10,100μg·mL-1时能显著诱导巨噬细胞的氧爆发活性,黄芪多糖则没有显著的诱导作用;黄芪和香菇多糖作用头肾巨噬细胞96 h后,香菇多糖低浓度时对细胞的氮呼吸爆发活性无显著影响,黄芪多糖浓度10μg·mL-1能显著诱导氮呼吸爆发活性,而在浓度为1000μg·mL-1两者均表现为高浓度抑制作用;两者作用外周血白细胞24 h后,香菇多糖浓度为100,1000 μg·mL-1和黄芪多糖浓度为10,100μg·mL-1时都能显著促进鲤外周血淋巴细胞的增殖;两种多糖作用头肾巨噬细胞24 h后能显著增强IL-1β的体外诱导表达.结果表明黄芪多糖和香菇多糖对离体培养鲤免疫细胞有明显活性作用,对鲤非特异性免疫和特异性免疫具有促进作用.     

传染性造血器官坏死病毒新疆株的分离与鉴定

在新疆维吾尔自治区某养殖场取患病虹鳟Oncorhynchus mykiss组织样本,进行传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)分离鉴定、电镜观察、回归感染及遗传进化分析研究。细胞培养结果显示:患病虹鳟组织样本能够感染鲤Cyprinus carpio上皮细胞(carp epithelial cell,EPC)产生典型细胞病变(cytopathic effect,CPE),收集病毒悬液命名为XJ-13。滴度测定实验表明:该病毒为10~(6.15)TCID_(50)/m L。回归感染实验表明:该分离株在浓度为10~5PFU/尾的剂量使体质量5g的虹鳟死亡率达87.5%。通过透射电镜观察发现患病虹鳟组织悬液感染的EPC细胞内存在大量的子弹状病毒粒子,PCR鉴定结果表明该病毒为IHNV。XJ-13的糖蛋白氨基酸序列的聚类分析结果显示,该病毒株与我国IHNV-Sn1203株具有最高的同源性(99%),与美国参考株WRAC的同源性为94.6%。结果表明:IHNV XJ-13是造成该养殖场虹鳟大量死亡的病原。     

κ-卡拉胶寡糖硫酸基含量与其抗疱疹病毒活性的关系

利用酶解法降解κ-卡拉胶,得到κ-卡拉胶寡糖(KOS)。利用甲酰胺-氯磺酸和二甲基亚砜-甲醇-吡啶方法,分别得到κ-卡拉胶寡糖磺化衍生物(SKO)和κ-卡拉胶寡糖脱硫酸基衍生物(DSK)。利用单纯疱疹病毒HSV-1研究了κ-卡拉胶寡糖硫酸基含量与抗病毒活性之间的关系,同时从对病毒的直接作用、阻止病毒对细胞的吸附和影响病毒复制与释放3个途径初步探讨了κ-卡拉胶寡糖抗疱疹病毒的机理。结果表明,KOS及SKO对Vero细胞毒性极低,对单纯疱疹病毒HSV-1无直接灭活作用,也不影响病毒的复制和释放,但是能够阻止病毒对细胞的吸附。相同浓度下,SKO对病毒吸附的抑制率要明显高于KOS,DSK对病毒则无明显作用。实验说明,κ-卡拉胶寡糖硫酸基含量与其抗疱疹病毒活性二者呈正相关,而且κ-卡拉胶寡糖及其磺化衍生物的抗病毒活性主要是通过阻止病毒对细胞的吸附而实现。     

传染性造血器官坏死病病毒参考蛋白的研制

为规避活病毒作参考物质存在的生物安全风险,本研究利用毕赤酵母表达系统表达了传染性造血器官坏死病病毒(IHNV)的糖蛋白来制备参考蛋白,作为IHNV免疫学检测参考物质。本研究选用IHNV糖蛋白基因(IHNV-G)为目的基因,根据Gen Bank中IHNV全基因序列设计特异性引物,以IHNV-uk株病毒核酸为模板,通过RT-PCR获得糖蛋白基因片段,将其克隆至真核表达载体p PICZαA,转入毕赤酵母感受态细胞GS115中,使外源基因与酵母基因融合,通过1%甲醇诱导表达外源蛋白,SDS-PAGE分析显示获得可溶性表达蛋白,分子量大于70 ku。经Western Blot和ELISA分析,该表达产物可以被IHNV多抗特异性识别。0.1531 mg的重组蛋白与0.3125TCID50 IHNV在ELISA实验中反应原性相当,–20°C可稳定保存2个月,说明其在一定程度上可替代病毒作为参考物质。该研究为IHNV ELISA检测试剂盒的开发奠定基础。     

贻贝多糖润肤霜的制备及性能评价

贻贝多糖具有免疫调节以及抗紫外等生理活性。本文研究贻贝多糖的吸湿性、保湿性和抗紫外活性,制备添加贻贝多糖的润肤霜。结果表明:低相对湿度(RH43%)条件下,贻贝多糖样品吸湿率和保湿率优于高湿度(RH81%)条件,高于聚乙二醇,低于甘油。低相对湿度(RH43%)条件下,贻贝多糖吸湿率可达18.19%;高相对湿度(RH81%)条件下,贻贝多糖吸湿率达到11.29%。贻贝多糖具有较好的抗紫外UVA和UVB的能力。制备的贻贝多糖润肤霜感官及理化指标符合QB/T 1857-2004,具有很好的离心稳定性。     

四川彭州地区养殖虹鳟传染性造血器官坏死病病毒的分离、鉴定及病理学观察

近期,四川省成都彭州市某虹鳟养殖场暴发流行疾病,导致养殖虹鳟死亡率高达90%。现场采样观察发现患病鱼主要症状为背部发黑,鳔壁、腹膜严重出血,心包积液,空肠、空胃和显著肠炎。同时对病鱼进行细菌学与组织病理学检测,细菌学检查结果为阴性,病理学观察发现脾脏有典型凝固性坏死,肝脏组织广泛变性、坏死,肠道黏膜下层水肿,肠上皮充血及上皮细胞脱落坏死,脑膜和心外膜水肿。将病鱼的脾组织研磨过滤除菌后,腹腔注射60尾健康虹鳟,注射组均表现为急性死亡(累计死亡率达85%),试验鱼出现与自然发病鱼相同的症状而对照组无异常。取病鱼的脾脏组织研磨过滤后接种胖头鲤细胞(fathead minnow cell,FHM),细胞感染3 d后出现典型的细胞病变效应(CPE)。针对编码IHNV糖蛋白(Glycoprotein,G)基因进行逆转录-聚合酶链反应(RTPCR)检测显示,患病鱼、人工感染病鱼和病变细胞均为IHNV阳性,扩增序列与IHNV糖蛋白基因同源性为98.2%。对该病毒分离株的G基因进行系统发育分析,结果显示,该分离株与亚洲分离株聚为一簇,属于JRt基因型。     

Salmonid fish viruses and cell interactions at early steps of the infective cycle

A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds.     

Antiviral activity of casein and αs2 casein hydrolysates against the infectious haematopoietic necrosis virus,a rhabdovirus from salmonid fish

Salmonid fish viruses, such as infectious haematopoietic necrosis virus (IHNV), are responsible for serious losses in the rainbow trout and salmon‐farming industries, and they have been the subject of intense research in the field of aquaculture. Thus, the aim of this work is to study the antiviral effect of milk‐derived proteins as bovine caseins or casein‐derived peptides at different stages during the course of IHNV infection. The results indicate that the 3‐h fraction of casein and α_S2‐casein hydrolysates reduced the yield of infectious IHNV in a dose‐dependent manner and impaired the production of IHNV‐specific antigens. Hydrolysates of total casein and α_S2‐casein target the initial and later stages of viral infection, as demonstrated by the reduction in the infective titre observed throughout multiple stages and cycles. In vivo, more than 50% protection was observed in the casein‐treated fish, and the kidney sections exhibited none of the histopathological characteristics of IHNV infection. The active fractions from casein were identified, as well as one of the individual IHNV‐inhibiting peptides. Further studies will be required to determine which other peptides possess this activity. These findings provide a basis for future investigations on the efficacy of these compounds in treating other viral diseases in farmed fish and to elucidate the underlying molecular mechanisms of action. However, the present results provide convincing evidence in support of a role for several milk casein fractions as suitable candidates to prevent and treat some fish viral infections.     

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.     

First evidence of infectious hematopoietic necrosis virus (IHNV) in the Netherlands

In spring 2008, infectious hematopoietic necrosis virus (IHNV) was detected for the first time in the Netherlands. The virus was isolated from rainbow trout, Oncorhynchus mykiss (Walbaum), from a put‐and‐take fishery with angling ponds. IHNV is the causative agent of a serious fish disease, infectious hematopoietic necrosis (IHN). From 2008 to 2011, we diagnosed eight IHNV infections in rainbow trout originating from six put‐and‐take fisheries (symptomatic and asymptomatic fish), and four IHNV infections from three rainbow trout farms (of which two were co‐infected by infectious pancreatic necrosis virus, IPNV), at water temperatures between 5 and 15 °C. At least one farm delivered trout to four of these eight IHNV‐positive farms. Mortalities related to IHNV were mostly <40%, but increased to nearly 100% in case of IHNV and IPNV co‐infection. Subsequent phylogenetic analysis revealed that these 12 isolates clustered into two different monophyletic groups within the European IHNV genogroup E. One of these two groups indicates a virus‐introduction event by a German trout import, whereas the second group indicates that IHNV was already (several years) in the Netherlands before its discovery in 2008.     

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.     

Comparative stability of two salmonid viruses and poliovirus in fresh, estuarine and marine waters

Abstract. The inactivation rates in the aquatic environment of two fish pathogens, infectious pancieatic necrosis virus (IPNV) and infectious haematopoietic necrosis virus (IHNV), were compared with that of poliovirus type 1 as a representative of the human enterovirus group. The survival studies were performed using untreated fresh, estuarine and sea water samples held at 15 and 20°C. The results indicated that the salmonid viruses survived longer than poliovirus in the saline waters, whereas in fresh water poliovirus was the most stable of the three viruses. IPN virus proved to be most stable in estuarine water at 15°C, whereas the survival of IHN virus was favoured in fresh water. We also observed that at 20°C the inactivation rate for each virus was independent of salt concentration in estuarine and sea water. Although temperature exhibited a marked effect on virus stability in fresh water, the salmonid viruses presented similar survival patterns at both temperatures in sea water. In general the period of greatest viral inactivation correlated with higher bacterial numbers in the waters.     

QTL for IHNV resistance and growth identified in a rainbow (Oncorhynchus mykiss) × Yellowstone cutthroat (Oncorhynchus clarki bouvieri) trout cross

Infectious hematopoietic necrosis virus (IHNV) is a major constraint to rainbow trout culture. Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) have greater resistance to this virus than do rainbow trout (O. mykiss), but the genetic mechanism of this resistance is not understood. We conducted a genome scan using a backcross of cutthroat trout into a rainbow trout background to estimate the number and locations of quantitative trait loci (QTL) associated with IHNV resistance and growth in trout. IHNV resistance was considered in terms of both survival (binary trait) and days to death (quantitative trait). The genetic map was scanned using interval mapping via two different approaches: one model considered survival alone and a second two-part model combined both survival and days to death. Three QTL were significantly (P ≤ 0.05) associated with virus resistance genome-wide, explaining 32.5% of the phenotypic variation. Cutthroat alleles at two of these QTL resulted in increased resistance to the pathogen, as expected. No growth QTL were detected in this cross. We suggest that these traits are genetically independent.     

Appearance of Infectious Hematopoietic Necrosis Virus in Rainbow Trout,Oncorhynchus mykiss,During Seawater Adaptation

About 7% mortality occurred in rainbow trout, Oncorhynchus mykiss, during seawater adaptation at a marine farm in the South Sea of Korea during the winter of 2014. Most diseased fish showed petechial hemorrhaging of gills and internal fat with enlarged spleen. Although no parasites or bacteria were isolated from the diseased fish, all tissue filtrates produced cytopathic effects (CPEs) in fathead minnow and Chinook salmon embryo‐214 cells. The cell culture supernatant showing CPE contained specific 1527‐bp fragment for the infectious hematopoietic necrosis virus (IHNV) glycoprotein gene by polymerase chain reaction. Their nucleotide sequences shared 98.1–98.2% identities with IHNV RtUi02 isolated from rainbow trout in Korea. This isolate (RtGoH14) was closely related to Korean IHNV isolates of genogroup JRt rather than to those of North American and European genogroups. These results suggest that this IHNV isolate might have been introduced to rainbow trout farm (land‐based culture system) in Korea. This is the first report of IHNV infection in rainbow trout during seawater adaptation in Korea.