兔的一种新病毒 IlI.兔出血症病毒形态的超微结构和抗原性的研究

1986年春,武汉市第二制药厂(WSPMM)试验用家兔突然口鼻渗出鲜血,几乎全部猝死。作者观察了罹病死亡的症状,认为此病例与黄印尧等所报道的相同。湖北省中医药研究院(HTCMI)也发生此病的流行。作者对这两种不同来源的病料,进行相同的处理,提取病毒粗纯物,回接同种动物,可引起同样典型的发病症状。进一步将死亡兔的肝、睥等脏器组织匀浆。经低速、高速、超速和蔗糖密度梯度离心后制样,电镜观察可见典型的病毒柱子。以上两种病毒粒子制备的抗血清和南京农业大学的兔出血症病毒抗原在双扩散沉淀中,产生典型的沉淀反应,说明三种病毒抗原的一致性。但是湖北株、南京农业大学病毒株抗原和日本细小病毒的抗血清,在双扩散反应时均无沉淀反应。湖北株病毒的形态大小、超微结构、外壳蛋白的 多肽和血清学特性,均不同于标准兔细小病毒,可能是一新的病毒。     

兔的一种新病毒：Ⅱ.一株兔出血症...

In this paper a strain of Rabbit Hemorrhagic Disease Virus (RHDV) was isolated and purified from the diseased rabbit livers with a method of using chloroform, two-phase of polyethylene-glycol-dextran sulfate sodium and sucrose density gradient centrifugation. Purified virus was nonenveloped, icosahedeal symmetry with a triangulation number of 3, and 33-37 nm in diameter. The capsid was composed of 32 capsomeres with central holes in an outer diameter of about 9nm. Two types of viral particles having different sedimentation coefficient, 130s and 166s could be identified after sucrose density gradient centrifugation. Probably no less than four virion proteins with molecular weight of 66.4, 65.0, 63.5, 41.0 x 10(3) dalton were detected by SDS-polyacrylamide gel electrophoresis. Viral nucleic acid was extracted from purified virus by using SDS-proteinase K-phenol. Tests with diphenylamine, formaldehyde, and staining with acridine orange as well as the curves of thermal denaturation showed that this kind of virus had a single-stranded DNA. The molecular weight of the ssDNA was approx 2.1 x 10(6) dalton as determined by electron microscopy. Data indicate that the RHDV may like the parvovirus of the family Parvoviridae.     

兔的一种新病毒 II．一株兔出血症病毒的某些理化性质的研究

采用氯仿、聚乙二醇。硫酸葡聚糖钠盐二相系统和蔗糖密度梯度离心法,从患病兔肝组织中提取、纯化病毒。该病毒粒子无囊膜,呈20面体对称,直径一般为33—37 nm。其三角剖分数T=3,共有32个子粒,每一子粒为中空的、外径为9 nm左右的圆形轮廓。经蔗糖密度梯度离心后,可获得沉降系数为166s的病毒颗粒,这种颗拉具有很强的感染性。经SDS—PAGE测得病毒粒子有四种多肽,分子量分别为66．4k、65．Ok、63．5k、41．Ok。二苯胺试验,吖啶橙染色、甲醛试验及热变性曲线表明,该病毒是单链DNA病毒。电镜下经甲酰胺法展层的病毒核酸呈单链线状,分子量平均为2．1×106道尔顿。此病毒的上述性质类似于细小病毒。 53s中空的圆形子粒,有很强的血凝性,而对照组铁蛋白和同一条件下所制备正常兔肝 成份无凝血特性,说明53s的9nm粒子应为病毒外壳蛋白子粒。     

一种新病毒——兔出血症病毒的鉴定初报

从我国新发生的一种家兔急性败血性传染病死兔内脏抽提物中,观察到典型的病毒粒子,回归兔可引起典型发病,再从病死兔内脏回收到同样病毒,证明该病系病毒性传染病,暂定名为“兔病毒性出血症”,病原暂定为“兔出血症病毒”。经初步鉴定,认为本病毒可能是一种首次发现的新病毒,属双股RNA病毒。但从病毒大小和核酸节段看,又不同于呼肠病毒科。最终归属正在进一步研究。     

兔出血症病毒与细小病毒抗原相关性试验

用间接ELISA、ELISA交叉阻断法和交叉血凝抑制试验对兔出血症病毒(RHDV)与6种细小病毒进行抗原相关性试验。用间接ELISA证实,RFIDV与它们有轻度交叉关系,其抗原相关值分别为:小鼠细小病毒(MVM)5.59％;鹅细小病毒(GPV)3.54％;猪细小病毒(PPV)1.76％;水貂肠炎病毒0.7％。细小病毒间的抗原相关值:MEV与PPV为31.6％,MEV与MVM为35.36％;而CPV与MEM、PPV、MVM的相关值均为零,即无相关性。在ELISA交叉阻断法中证实:犬细小病毒(CPV)、猫泛白细胞减少症病毒(FPV)和MEV均不能阻断RHDV与其抗体结合,仅GPV有轻度阻断作用,其最大阻断率为40％。在血凝交叉抑制试验中,未发现RHDV与细小病毒及其相应抗体间存在交叉抑制现象。以上结果表明RHDV与细小病毒在血清学方面有轻度相关性。     

兔出血症病毒四个分离株的病毒多肽和血清学关系研究

使用PEG-DS两相系统和超离心提纯的兔出血症病毒(RHDV)四个分离株病毒,再经Sepharose 4B柱层析进一步提纯后,得到较纯的病毒粒子,回收率可达70％以上。应用常规双向免疫扩散试验,交叉血凝抑制试验和酶联免疫吸附试验(ELISA)对四个不同地区分离株间的血清学关系进行了比较研究。结果表明实验中的四个分离株病毒均属同一血清型。SDS-PAGE结果表明,这四个分离株病毒均含有四条多肽,分子量为28—64KD。各株病毒多肽的分子量和各多肽在病毒粒子总蛋白中所占比例略有差异。因此四个分离株的RHDV在蛋白结构上可能存在地区差异。     

兔细小病毒肺炎的诊断与防治

兔细小病毒肺炎(常称兔瘟)病原体为一种新RNA病毒,是1984年首次在我国发现的一种家兔烈性传染病。本病来势凶猛,传播迅速,流行面广,病程短,发病率(90—100%)和死亡率(90%以上)极高。病兔、死兔、隐性感染兔为主要传染源,成年兔发病急,死亡快,死亡率最高;育成兔死亡率稍低;哺乳期仔兔很少发病死亡。感染兔无性别差异。一年四季均可发病,春冬常见,主要经过消化道及呼吸道传播。发病机制至今尚不清楚。许益民等(1986)认为,病毒感染兔后作用于靶细胞一血管内皮细胞,病毒毒素侵害并损伤血管内皮细胞而启动体内的凝血机制和继发性纤溶而导致弥散…     

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.     

Resistance to RHD virus in wild Australian rabbits: Comparison of susceptible and resistant individuals using a genomewide approach

Deciphering the genes involved in disease resistance is essential if we are to understand host–pathogen coevolutionary processes. The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus). During the first outbreaks of the disease, RHDV caused mortality rates of up to 97%. Recently, however, increased genetic resistance to RHDV has been reported. Here, we have aimed to identify genomic differences between rabbits that survived a natural infection with RHDV and those that died in the field using a genomewide next‐generation sequencing (NGS) approach. We detected 72 SNPs corresponding to 133 genes associated with survival of a RHD infection. Most of the identified genes have known functions in virus infections and replication, immune responses or apoptosis, or have previously been found to be regulated during RHD. Some of the genes identified in experimental studies, however, did not seem to play a role under natural selection regimes, highlighting the importance of field studies to complement the genomic background of wildlife diseases. Our study provides a set of candidate markers as a tool for the future scanning of wild rabbits for their resistance to RHDV. This is important both for wild rabbit populations in southern Europe where RHD is regarded as a serious problem decimating the prey of endangered predator species and for assessing the success of currently planned RHDV variant biocontrol releases in Australia.     

First case of rabbit haemorrhagic disease in Canada: contaminated flying insect, vs. long-term infection hypothesis

Following the announcement of the first case of rabbit haemorrhagic disease (RHD) in a pet rabbit, housed indoors in Canada for more than 1 year, I submitted an evidence‐based explanation to ProMed explaining how RHD might have caused the death of ‘one’ of the three pet rabbits. I suggested with supporting evidence, that it may have been persistently infected with rabbit haemorrhagic disease virus (RHDV) which may have reactivated to cause the fatal disease. However, in this issue, Peacock et al. have proposed an alternative ‘hypothesis’ for the appearance of RHD in the pet rabbit. They hypothesise that a non‐identified insect or fomite might have become contaminated by a Chinese strain of RHDV somewhere in the US. This insect/fomite then flew or was windborne, from the US to Canada where it entered the house containing three pet rabbits and infected one of them. RHD is non‐endemic and is rarely reported in the US, where it has only been observed in domestic European rabbits, held in rabbitries. My proposal was based on the details provided by ProMed, the veterinary report from Canada, where RHDV has never previously been identified and the epidemiological, ecological and evolutionary history of RHDV which includes serological and phylogenetic evidence that ancestral RHDV lineages circulated before 1984. The flying insect hypothesis of Peacock et al. is based on circumstantial evidence and, I believe, has a lower probability of being correct than my evidence‐based long‐term infection proposal.     

Histo-blood group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-dependent manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50-90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level.     

Detection and preliminary characterization of a new rabbit calicivirus related to rabbit hemorrhagic disease virus but nonpathogenic.

A new rabbit calicivirus related to the rabbit hemorrhagic disease virus (RHDV) was identified. The new virus contains significant differences from the previously characterized RHDV isolates in terms of pathogenicity, viral titer, tropism, and primary sequence of the structural protein. Cross-protection experiments, antigenic data, and sequence comparisons demonstrate that the new virus is more closely related to RHDV than to the European brown hare syndrome virus, another member of the caliciviruses of the lagomorph group. The existence of a nonpathogenic calicivirus, which we propose to name rabbit calicivirus (RCV), provides an explanation for the early discrepancies found in the course of serological surveys of the rabbit population in European countries.     

Adaptive changes in the genomes of wild rabbits after 16 years of viral epidemics

Since its introduction to control overabundant invasive European rabbits (Oryctolagus cuniculus), the highly virulent rabbit haemorrhagic disease virus (RHDV) has caused regular annual disease outbreaks in Australian rabbit populations. Although initially reducing rabbit abundance by 60%, continent‐wide, experimental evidence has since indicated increased genetic resistance in wild rabbits that have experienced RHDV‐driven selection. To identify genetic adaptations, which explain the increased resistance to this biocontrol virus, we investigated genome‐wide SNP (single nucleotide polymorphism) allele frequency changes in a South Australian rabbit population that was sampled in 1996 (pre‐RHD genomes) and after 16 years of RHDV outbreaks. We identified several SNPs with changed allele frequencies within or close to genes potentially important for increased RHD resistance. The identified genes are known to be involved in virus infections and immune reactions or had previously been identified as being differentially expressed in healthy versus acutely RHDV‐infected rabbits. Furthermore, we show in a simulation study that the allele/genotype frequency changes cannot be explained by drift alone and that several candidate genes had also been identified as being associated with surviving RHD in a different Australian rabbit population. Our unique data set allowed us to identify candidate genes for RHDV resistance that have evolved under natural conditions, and over a time span that would not have been feasible in an experimental setting. Moreover, it provides a rare example of host genetic adaptations to virus‐driven selection in response to a suddenly emerging infectious disease.     

Processing of rabbit hemorrhagic disease virus polyprotein.

Expression of rabbit hemorrhagic disease virus (RHDV) cDNAs in vitro with rabbit reticulocyte lysates and in Escherichia coli have been used to study the proteolytic processing of RHDV polyprotein encoded by ORF1. An epitope tag was used for monitoring the gene products by a specific antibody. We have identified four gene products with molecular masses of 80, 43, 73, and 60 kDa, from the amino to the carboxy terminus of the polyprotein. The amino-terminal sequences of the 43- and 73-kDa products were determined and indicated that RHDV 3C proteinase cleaved Glu-Gly peptide bonds.     

Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.

Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.     

表达兔出血症病毒VP60蛋白的重组兔粘液瘤病毒的构建与初步评价

兔出血症病毒 (Rabbit hemorrhagic disease virus,RHDV) 及兔粘液瘤病毒 (Myxoma virus,MYXV) 分别引起兔出血症 (兔瘟) 和兔粘液瘤病,是两种严重危害家兔养殖业以及导致原产地欧洲野兔-穴兔 (Oryctolagus cuniculus) 种群近濒危的重要病原。VP60为构成RHDV衣壳的主要抗原蛋白。为研制能同时免疫预防该两种疫病的重组二联疫苗,本研究分别以MYXV和其复制非必需基因——胸腺激酶 (Thymidine kinase,TK) 基因为重组载体和同源重组靶基因,构建穿梭载体p7.5-VP60-GFP。将p7.5-VP60-GFP载体转染被MYXV感染的兔肾细胞株RK13,经同源重组后,在荧光显微镜下筛选出表达GFP的重组病毒,并将其命名为rMV-VP60-GFP。通过PCR和Western blotting进行重组病毒vp60基因特异性插入和表达验证结果显示,vp60和gfp基因成功插入MYXV基因组中并且可成功表达,表明成功构建了表达RHDV衣壳蛋白基因vp60的重组MYXV。动物攻毒保护试验表明,制备的重组病毒能保护家兔抵抗MYXV的致死性攻击,这为后续疫苗的研发奠定了基础。     

兔出血症病毒VP60基因在昆虫细胞中形成病毒样颗粒及其特性

目的将兔出血症病毒（RHDV）VP60全长基因在昆虫细胞-杆状病毒系统中表达,验证重组蛋白形成病毒样颗粒（VLPs）的能力及其生物学特性,探讨VLPs作为检测抗原及亚单位疫苗的潜力。方法用Bac-to-Bac系统体外表达RHDVVP60全长基因。以免疫荧光及Western blotting检测蛋白表达情况及确定蛋白最佳表达条件;免疫电镜观察VLPs形态,并对VLPs的血凝性、免疫原性进行检测。结果SDS-PAGE电泳分析表明,表达的重组蛋白分子量大小约为68KDa,在免疫荧光、琼脂扩散、ELISA试验中均与RHD多克隆抗血清特异性反应;接种重组病毒的Sf9细胞裂解液在电镜下可观察到与RHDV形态相似的VLPs;该VLPs可凝集人“O”、“B”型红细胞,凝集可被RHD多克隆抗血清所抑制;含VLPs的Sf9细胞裂解液可不经纯化用作间接ELISA抗原,所建立的ELISA方法与进口商品化试剂盒相比,特异性良好,敏感性、检出率稍低;将含VLPs的细胞裂解液加氟氏佐剂免疫兔,HI效价可达1∶40,可经受致死量病毒攻击。结论RHDV-VLPs的获得及其良好的免疫原性,为RHD血清学检测试剂的标准化、亚单位疫苗研制应用奠定基础,同时在转移载体及RHDV受体方面研究亦有潜在应用价值。