兔出血症病毒复制子的构建及其在RK-13细胞中的复制

为研究兔出血症病毒(RHDV)的复制机制、病毒与宿主之间的相互作用以及致病机制等,创建一个安全、有效的技术平台,在前期构建的RHDV侵染性克隆基础上,将病毒的衣壳蛋白编码区删除,保留了RHDV复制必需的所有蛋白酶基因和两端的非编码区,构建了RHDV复制子。试验结果证明,将该复制子RNA导入RK13细胞中后,能够进行高水平的复制和表达。     

一株兔出血症病毒2型毒株的分离鉴定

【背景】我国于2020年4月突发兔出血症病毒2型(Rabbit Hemorrhagic Disease Virus 2,RHDV2)疫情,严重威胁兔养殖业和生态平衡,而且目前国内对RHDV2的病原学以及遗传特征等基础研究匮乏。【目的】分离鉴定RHDV2毒株,对分离株进行全基因测序与遗传进化分析。【方法】对成都某兔场疑似RHDV2感染致死的家兔进行病理剖检,通过RT-qPCR检测和动物回归试验,分离鉴定得到RHDV2毒株,进一步进行全基因测序及遗传进化分析。【结果】病死兔剖检表现为各实质器官出血肿大,以心、肺、肝脏尤为明显,经RT-qPCR确诊为RHDV2,而且不存在其他病原混合感染,试验感染家兔可致相似病变。将分离株命名为SCCN03,其基因序列全长为7464bp,与参考毒株(GenBank登录号为MN901451.1)一致性为99.21%。对比参考株氨基酸序列,分离株的非结构蛋白和结构蛋白氨基酸序列发生了多处错义突变,其中非结构蛋白p16和结构蛋白的几处错义突变可能与毒株变异有关。进化树显示毒株SCCN03属于GI.2基因型。【结论】分离鉴定出一株RHDV2毒株,获得其基因序列,丰富了RHDV2的全基因数据资料,为后续RHDV毒力相关研究和相关疫苗研发奠定了基础。     

European brown hare syndrome virus: relationship to rabbit hemorrhagic disease virus and other caliciviruses.

Monoclonal antibodies directed against the capsid protein of rabbit hemorrhagic disease virus (RHDV) were used to identify field cases of European brown hare syndrome (EBHS) and to distinguish between RHDV and the virus responsible for EBHS. Western blot (immunoblot) analysis of liver extract of an EBHS virus (EBHSV)-infected hare revealed a single major capsid protein species of approximately 60 kDa that shared epitopes with the capsid protein of RHDV. RNA isolated from the liver of an EBHSV-infected hare contained two viral RNA species of 7.5 and 2.2 kb that comigrated with the genomic and subgenomic RNAs of RHDV and were recognized by labeled RHDV cDNA in Northern (RNA) hybridizations. The nucleotide sequence of the 3' 2.8 kb of the EBHSV genome was determined from four overlapping cDNA clones. Sequence analysis revealed an open reading frame that contains part of the putative RNA polymerase gene and the complete capsid protein gene. This particular genome organization is shared by RHDV but not by other known caliciviruses. The deduced amino acid sequence of the capsid protein of EBHSV was compared with the capsid protein sequences of RDDV and other caliciviruses. The amino acid sequence comparisons revealed that EBHSV is closely related to RHDV and distantly related to other caliciviruses. On the basis of their genome organization, it is suggested that caliciviruses be divided into three groups.     

兔出血症病毒遗传变异分析及RT—PCR检测方法的建立

目的获得兔出血症病毒（浙江分离株）近20年间遗传变异概况,建立兔出血症病毒的RT—PCR检测方法。方法对1989～2006年间的7株RHDV浙江分离株的衣壳蛋白基因（VP60）进行了克隆与测序,并与国内、外RHDV的基因组序列进行了遗传比对和分析;根据兔出血症病毒株的VP60设计引物,对20只人工感染兔的实质脏器、全血和350只实验兔全血样本进行了检测。结果7株RHDV的VP60基因均由1740个核苷酸组成,编码580个氨基酸。它们与参考序列之间的氨基酸同源性为94.0%～99.8%。系统发生树分析结果显示,RHDV可划分为2个大的基因群,浙江（中国）的RHDV分离株主要集中于C亚群。RT—PCR检测方法表明20只实验兔全部扩出预期条带,而350只实验兔全血检测中出现13只RHDV可疑样本。经血凝抑制试验检测,13例PCR阳性样品中有10个HAI阳性,3个阴性。检测敏感度达100%,特异性为99.12%,经统计检验,kappa=0.865,表明PCR检出RHDV的结果与HAI高度一致。结论RHDV基因群之间的遗传距离有逐渐加大的趋势。我们建立的RT—PCR法可用于RHDV浙江分离株的检测,用RT—PCR检测全血中RHDV方法的建立为活体检测RHDV打下了基础。     

Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV.     

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。     

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.     

Comparative site-directed mutagenesis in the catalytic amino acid triad in calicivirus proteases

Calicivirus proteases cleave the viral precursor polyprotein encoded by open reading frame 1 (ORF1) into multiple intermediate and mature proteins. These proteases have conserved histidine (His), glutamic acid (Glu) or aspartic acid (Asp), and cysteine (Cys) residues that are thought to act as a catalytic triad (i.e. general base, acid and nucleophile, respectively). However, is the triad critical for processing the polyprotein? In the present study, we examined these amino acids in viruses representing the four major genera of Caliciviridae: Norwalk virus (NoV), Rabbit hemorrhagic disease virus (RHDV), Sapporo virus (SaV) and Feline calicivirus (FCV). Using single amino‐acid substitutions, we found that an acidic amino acid (Glu or Asp), as well as the His and Cys in the putative catalytic triad, cannot be replaced by Ala for normal processing activity of the ORF1 polyprotein in vitro. Similarly, normal activity is not retained if the nucleophile Cys is replaced with Ser. These results showed the calicivirus protease is a Cys protease and the catalytic triad formation is important for protease activity. Our study is the first to directly compare the proteases of the four representative calicivirus genera. Interestingly, we found that RHDV and SaV proteases critically need the acidic residues during catalysis, whereas proteolytic cleavage occurs normally at several cleavage sites in the ORF1 polyprotein without a functional acid residue in the NoV and FCV proteases. Thus, the substrate recognition mechanism may be different between the SaV and RHDV proteases and the NoV and FCV proteases.     

兔出血症病毒2型理化特性研究

[目的]本研究旨在研究该病毒的理化特性,评价不同处理条件对RHDV2的杀灭效果.[方法]本研究拟对临床疑似RHDV2感染致死的家兔进行RT-PCR鉴定病原,并利用不同pH值、不同温度、常用兽用消毒剂、不同浓度甲醛处理RHDV2,通过PMA-RT-qPCR对病毒理化特性进行研究.[结果]经RT-PCR检测与测序分析,确诊...     

兔出血症病毒中国株衣壳蛋白基因的克隆和序列分析

应用RT—PCR技术,从兔出血症病毒中国分离株WX84中成功扩增出预期大小为1．7kb的特异性条带,将扩增产物提纯后克隆入pGEM^R—T载体,经转化、筛选及酶切鉴定后,获得了该株病毒衣壳蛋白基因的克隆,序列分析表明扩增的中国株BHD衣壳蛋白基因片段长度为1740bp,共编码580个氨基酸。该核酸序列与其它国家报道的多株BHDV序列相互间同源性高达98．2％一99．0％,其推导的氨基酸序列同源性也达98．3％--99．1％,为极度保守片段。     

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp,nonstructural proteins p16 and p23, playing a role in virus replication

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.     

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。     

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.     

Gene transfer using recombinant rabbit hemorrhagic disease virus capsids with genetically modified DNA encapsidation capacity by addition of packaging sequences from the L1 or L2 protein of human papillomavirus type 16

The aim of this study was to produce gene transfer vectors consisting of plasmid DNA packaged into virus-like particles (VLPs) with different cell tropisms. For this purpose, we have fused the N-terminally truncated VP60 capsid protein of the rabbit hemorrhagic disease virus (RHDV) with sequences which are expected to be sufficient to confer DNA packaging and gene transfer properties to the chimeric VLPs. Each of the two putative DNA-binding sequences of major L1 and minor L2 capsid proteins of human papillomavirus type 16 (HPV-16) were fused at the N terminus of the truncated VP60 protein. The two recombinant chimeric proteins expressed in insect cells self-assembled into VLPs similar in size and appearance to authentic RHDV virions. The chimeric proteins had acquired the ability to bind DNA. The two chimeric VLPs were therefore able to package plasmid DNA. However, only the chimeric VLPs containing the DNA packaging signal of the L1 protein were able efficiently to transfer genes into Cos-7 cells at a rate similar to that observed with papillomavirus L1 VLPs. It was possible to transfect only a very limited number of RK13 rabbit cells with the chimeric RHDV capsids containing the L2-binding sequence. The chimeric RHDV capsids containing the L1-binding sequence transfer genes into rabbit and hare cells at a higher rate than do HPV-16 L1 VLPs. However, no gene transfer was observed in human cell lines. The findings of this study demonstrate that the insertion of a DNA packaging sequence into a VLP which is not able to encapsidate DNA transforms this capsid into an artificial virus that could be used as a gene transfer vector. This possibility opens the way to designing new vectors with different cell tropisms by inserting such DNA packaging sequences into the major capsid proteins of other viruses.     

兔出血症病毒（RHDV）WHNRH株的分离鉴定及基因组全序列的测定与分析

从发病长毛兔中分离鉴定了兔病毒性出血症病毒WHNRH株。参考GenBank中已登录的RHDV毒株序列对RHDV WHNRH分离株进行了全基因组序列测定与分析。设计5对扩增区段相互重叠的RHDV特异性引物,扩增除5′和3′末端以外的序列,采用设计锚引物的5′RACE方法以及针对RHDV 3′末端的polyA结构设计引物获得了RHDV WHNRH株的5′和3′末端序列。胶回收各PCR产物,连接pMD 18-T克隆载体,测得RHDV WHNRH分离株的基因组全长为7437nt(不包括polyA),与GenBank公布的全部共6株RHDV全基因序列进行同源性比较分析,同源性在89.0%~97.1%之间,ORF1同源性为89.0%~97.1%,编码氨基酸序列的同源性为95.2%~98.7%;ORF2的核酸苷序列同源性为92.1%~97.7%,编码氨基酸序列的同源性为94.1%~96.6%。     

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.     

Melatonin modulates mitophagy,innate immunity and circadian clocks in a model of viral‐induced fulminant hepatic failure

The haemorrhagic disease virus (RHDV) is a non‐cultivable virus that promotes in rabbits an acute disease which accomplishes many characteristics of an animal model of fulminant hepatic failure (FHF). Beneficial effects of melatonin have been reported in RHDV‐infected rabbits. This study investigated whether protection against viral‐derived liver injury by melatonin is associated with modulation of mitophagy, innate immunity and clock signalling. Rabbits were experimentally infected with 2 × 10⁴ haemagglutination units of a RHDV isolate and killed at 18, 24 and 30 hours after infection (hpi). Melatonin (20 mg/kg body weight ip) was administered at 0, 12 and 24 hpi. RHDV infection induced mitophagy, with the presence of a high number of mitophagosomes in hepatocytes and increased expression of mitophagy genes. Greater expression of main innate immune intermediaries and inflammasome components was also found in livers with RHDV‐induced FHF. Both mitophagy and innate immunity activation was significantly hindered by melatonin. FHF induction also elicited an early dysregulation in clock signalling, and melatonin was able to prevent such circadian disruption. Our study discloses novel molecular routes contributing to RHDV‐induced damage progression and supports the potential of melatonin as a promising therapeutic option in human FHF.     

Molecular epidemiology of Rabbit Haemorrhagic Disease Virus in Australia: when one became many

Rabbit Haemorrhagic Disease Virus (RHDV) was introduced into Australia in 1995 as a biological control agent against the wild European rabbit (Oryctolagus cuniculus). We evaluated its evolution over a 16‐year period (1995–2011) by examining 50 isolates collected throughout Australia, as well as the original inoculum strains. Phylogenetic analysis of capsid protein VP60 sequences of the Australian isolates, compared with those sampled globally, revealed that they form a monophyletic group with the inoculum strains (CAPM V‐351 and RHDV351INOC). Strikingly, despite more than 3000 rereleases of RHDV351INOC since 1995, only a single viral lineage has sustained its transmission in the long‐term, indicative of a major competitive advantage. In addition, we find evidence for widespread viral gene flow, in which multiple lineages entered individual geographic locations, resulting in a marked turnover of viral lineages with time, as well as a continual increase in viral genetic diversity. The rate of RHDV evolution recorded in Australia ?4.0 (3.3–4.7) × 10^?3 nucleotide substitutions per site per year – was higher than previously observed in RHDV, and evidence for adaptive evolution was obtained at two VP60 residues. Finally, more intensive study of a single rabbit population (Turretfield) in South Australia provided no evidence for viral persistence between outbreaks, with genetic diversity instead generated by continual strain importation.