基因重组抗原酶联免疫法检测兔出血症病毒抗体及其标准化

以重组兔出血症病毒(RHDV)VP60蛋白为抗原，建立了RHDV抗体间接ELISA检测方法。优化的试验反应务件为：重组VP60的包被质量浓度为1．0mg／L，用10％牛血清封闭，以大肠杆菌提取物稀释被检血清以消除非特异性反应。将所建立的ELISA与现行血凝抑制(HI)试验比较发现，不同免疫状态的兔血清的RHDV ELISA抗体与HI抗体均呈正相关。对11个RHD免疫兔场1130份血清样品的抗体检测表明，各免疫兔群血清RHDV抗体水平不完全一致，D值在1．09～1．76之间，显著高于非免疫兔(0．05)及SPF兔(0．02)，低于高免兔(2．34)。在此基础上，研制了RHDV抗体酶联免疫检测试剂盒，测定了其主要指标，制定了各成分的质量控制标准，为兔群进行免疫学监测及评价疫苗的免疫效果提供了便利。     

No virus replication in domestic cats fed with RHDV-infected rabbit livers

Previous studies have shown that feral cats (Felis catus) from rabbit haemorrhagic disease (RHD) epidemic areas in New Zealand had antibodies against RHD Virus (RHDV) and RHDV RNA was identified by nested RT-PCR from one seropositive feral cat liver. To assess whether RHDV replicates and produces clinical consequences in cats following the consumption of RHDV-infected rabbit, a challenge trial was conducted by feeding cats RHDV-infected rabbit livers. Antibodies against RHDV were detected by immunoassay from sera of cats collected 10 days after the consumption of RHDV-infected livers. Animals fed four times with RHDV-infected livers, had higher antibody titres than animals fed only once. RHDV RNA was detected by nested RT-PCR from mesenteric lymph nodes, tonsil, spleen and liver of cats fed with RHDV-infected livers. RHDV anti-genomic RNA was also detected by nested RT-PCR from mesenteric lymph nodes collected from one animal 2 days after the fourth feed. RHDV was detected by antigen ELISA from cat faeces 1-2 days after the consumption of RHDV-infected livers. Even though a large amount of RHDV has been used, cats did not show any signs of disease. Although abortive RHDV replication could not be ruled out, active RHDV replication was not demonstrated.     

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.     

A serial cross-sectional study of the prevalence of rabbit haemorrhagic disease on three farms in the lower North Island of New Zealand

AIM: To estimate over a 3-year period following the first release of rabbit haemorrhagic disease virus (RHDV) the prevalence of rabbit haemorrhagic disease (RHD) and the abundance of rabbits (Oryctolagus cuniculus) in an area that historically had low rabbit densities. METHODS: Three farms grazing predominantly sheep and beef cattle, located close together and with low initial rabbit densities, were selected for study. RHDV had been deliberately released on all farms in December 1997. Farms were visited 2-3 times per year between June 1998 and April 2001. At each visit, rabbits were shot with the aid of spotlights at night and blood samples were collected for detection of RHDV antibodies. Rabbit carcasses were necropsied and the age of the animals was determined. Rabbit abundance on each property was measured throughout the study using spotlight night counts. Logistic regression was used to identify factors associated with the risk of carcasses being seropositive for RHDV. RESULTS: Rabbit density differed initially between farms (8.2, 9.9, 2.3 rabbits per spotlight km in June 1998), and declined on all three properties over time (1.2, 2.4, 1.1 rabbits per spotlight km in November 2000). Highest antibody titres to RHDV were initially evident on the farm on which rabbits were most abundant. The average prevalence of seropositive rabbits overall was 21% (95% CI=15-28%). Female rabbits tended to be less likely to be seropositive for RHDV than males (OR=0.47; 95% CI=0.21-1.02). The odds of becoming seropositive were reduced for rabbits born in the breeding season of 1999-2000 (OR=0.17; 95% CI=0.05-0.64). CONCLUSIONS: The temporal pattern of outbreaks measured by peaks of seroprevalence differed between closely-spaced farms when they had different rabbit densities, but were similar when rabbit densities were similar. Microclimate and vegetation influencing abundance of insect vectors for RHDV and intrinsic population-related factors like rabbit breeding behaviour are also likely to be involved in local patterns of spread.     

A serial cross-sectional study of the prevalence of rabbit haemorrhagic disease on three farms in the lower North Island of New Zealand

AIM: To estimate over a 3-year period following the first release of rabbit haemorrhagic disease virus (RHDV) the prevalence of rabbit haemorrhagic disease (RHD) and the abundance of rabbits (Oryctolagus cuniculus) in an area that historically had low rabbit densities.

METHODS: Three farms grazing predominantly sheep and beef cattle, located close together and with low initial rabbit densities, were selected for study. RHDV had been deliberately released on all farms in December 1997. Farms were visited 2–3 times per year between June 1998 and April 2001. At each visit, rabbits were shot with the aid of spotlights at night and blood samples were collected for detection of RHDV antibodies. Rabbit carcasses were necropsied and the age of the animals was determined. Rabbit abundance on each property was measured throughout the study using spotlight night counts. Logistic regression was used to identify factors associated with the risk of carcasses being seropositive for RHDV.

RESULTS: Rabbit density differed initially between farms (8.2, 9.9, 2.3 rabbits per spotlight km in June 1998), and declined on all three properties over time (1.2, 2.4, 1.1 rabbits per spotlight km in November 2000). Highest antibody titres to RHDV were initially evident on the farm on which rabbits were most abundant. The average prevalence of seropositive rabbits overall was 21% (95% CI=15–28%). Female rabbits tended to be less likely to be seropositive for RHDV than males (OR=0.47; 95% CI=0.21–1.02). The odds of becoming seropositive were reduced for rabbits born in the breeding season of 1999–2000 (OR=0.17; 95% CI=0.05–0.64).

CONCLUSIONS: The temporal pattern of outbreaks measured by peaks of seroprevalence differed between closely-spaced farms when they had different rabbit densities, but were similar when rabbit densities were similar. Microclimate and vegetation influencing abundance of insect vectors for RHDV and intrinsic population-related factors like rabbit breeding behaviour are also likely to be involved in local patterns of spread.     

Antibody responses to rabbit haemorrhagic disease virus in predators, scavengers, and hares in New Zealand during epidemics in sympatric rabbit populations

AIM: To test for antibodies to rabbit haemorrhagic disease (RHD) virus (RHDV) in sera from mammals and birds associated with rabbit populations infected with RHDV. METHODS: Sera from feral and domestic cats, feral ferrets, stoats, hedgehogs, hares, harrier hawks, and black-backed gulls were taken (apart from some of the hares) from areas in New Zealand where RHD was active among rabbit populations. The presence of antibodies to RHD was investigated using a competition enzyme-linked immunosorbent assay (ELISA). RESULTS: Some individual animals of all species were seropositive. Thirty eight of 71 feral cats, but only 1/80 domestic cats were seropositive at a 1:40 dilution. The latter had not been exposed to RHDV. Also reactive in the ELISA were 2/8 stoats; 11/115 ferrets, with significantly more females having antibodies than males; 4/73 hedgehogs; 2/18 hawks, and 1/30 gulls. Three of 66 hares, comprising 3/14 from one population, were seropositive. CONCLUSIONS: Apart from the hares, all these species are known to prey upon rabbits or scavenge their carcasses, a possible means of exposure to RHDV. The possibility that the positive test reactions were due to cross-reactions with other caliciviruses cannot be ruled out, especially for the hares. Nor could the study differentiate whether the positive results were due to an antigenic reaction to ingestion of RHDV, as suggested by overseas work, or to infection of new species by RHDV. These possibilities are being investigated further.     

兔出血症病毒TP株VP60基因DNA免疫小鼠的实验研究

为评价兔出血症病毒(RHDV)VP60基因在小鼠体内诱导产生体液免疫和细胞免疫情况,本研究构建了RHDV VP60基因的真核表达质粒pcDNA-VP60,并将其免疫小鼠。利用ELISA方法检测特异性抗体和小鼠外周血细胞因子水平,MTT法和流式细胞术(FACS)检测小鼠外周血T淋巴细胞增殖情况和T淋巴细胞亚群的动态变化。抗体检测结果显示,重组质粒免疫组抗体水平在免疫后5周～6周达到峰值,而且与对照组比较差异显著(p<0.05);细胞因子检测结果显示,重组质粒免疫组血清中IFN-γ、IL-2、IL-4因子水平随免疫时间延长而升高,并且能够维持较高水平,与对照组比较差异显著(p<0.05);T淋巴细胞检测结果显示,重组质粒免疫组T淋巴细胞明显增殖,CD4+T淋巴细胞数量免疫后明显增高,与对照组比较差异显著(p<0.05);所有检测指标显示重组质粒免疫组和灭活苗免疫组比较均无显著差异(p>0.05)。以上结果表明pcDNA-VP60重组质粒可以诱导小鼠产生特异性体液免疫和细胞免疫,为研制预防兔病毒性出血症的候选DNA疫苗提供了实验依据。     

Egg yolk IgY against RHDV capsid protein VP60 promotes rabbit defense against RHDV infection

VP60 capsid protein is the major structural and immunogenicity protein of RHDV (Rabbit hemorrhagic disease virus, RHDV), and has been implicated as a main protein antigen in RHDV diagnosis and vaccine design. In this report, egg yolk antibody (IgY) against N-terminal of VP60 was evaluated and developed as a new strategy for RHDV therapy. Briefly, N-terminal of VP60 (～250aa) fragment was cloned and inserted into pET28a expression vector, and then the resultant plasmid, pET28a/VP60-N, was transformed into E. coli BL21(DE3) for recombinant VP60-N protein (rVP60-N) expression. Next, the rVP60-N was purified by Ni⁺-affinity purification chromatography and identified by Western blotting with RHDV antiserum. After immunizing the chickens with rVP60-N, the anti-rVP60-N IgY was isolated, and the activity and specificity of the IgY antibody were analyzed by ELISA and Western blotting. In our results, the rVP60-N could be expressed in E. coli as soluble fraction, and the isolated anti-rVP60-N IgY demonstrated a high specificity and titer (1:22,000) against rVP60-N antigen. For further evaluation of the IgY efficacy in vivo, rabbits were grouped randomly and challenged with RHDV, and the results showed that anti-rVP60-N IgY could significantly protect rabbits from virus infection and promote the host survival after a sustained treatment with anti-rVP60-N IgY for 5 days. Taken together, our study demonstrates evidence that production of IgY against VP60 could be as a novel strategy for the RHDV therapy.     

An outbreak of rabbit hemorrhagic disease (RHD) caused by Lagovirus europaeus GI.2/rabbit hemorrhagic disease virus 2 (RHDV2) in Ehime, Japan

A total of ten 1–2-year-old rabbits died within 2 weeks at a facility in Ehime prefecture in May 2019. Necropsy revealed liver discoloration and fragility, hemorrhage of some organs and blood coagulation failure. On histopathologic examination, necrotizing hepatitis was a common finding, together with fibrin thrombi in the small vessels and hemorrhage in some organs. Rabbit hemorrhagic disease (RHD) virus gene was detected in liver samples, and viral particles of approximately 32 nm in diameter were found in the cytoplasm of degenerated hepatocytes by electron microscopy. Phylogenetic analysis based on the partial VP60 gene sequence classified it as Lagovirus europaeus GI.2/RHDV2. This is the first confirmed outbreak of RHD caused by globally emerging GI.2/RHDV2 in Japan.     

The recombinant rabbit hemorrhagic disease virus VP60 protein obtained from Pichia pastoris induces a strong humoral and cell-mediated immune response following intranasal immunization in mice

Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.     

Rabbit haemorrhagic disease: advantages of cELISA in assessing immunity in wild rabbits (Oryctolagus cuniculus)

Rabbit haemorrhagic disease (RHD) is an acute fatal disease of domestic and wild European rabbits (Oryctolagus cuniculus) caused by RHD virus (RHDV). Accurate assessment of immunity is of great importance for the conservation and control of wild rabbits. We evaluated a competitive ELISA (cELISA) against isotype ELISAs for assessing the protective immunity against the disease by challenging 50 wild-caught rabbits with a lethal dose of RHDV. Death or survival to the challenge was used as a criterion to determine the performance characteristics of the assay for the assessment of immunity in rabbits. At 1:10 dilution, a serum exhibiting ≥ 25% inhibition (1:10(25)) was regarded as the presence of RHDV-specific antibodies. Eleven of 16 (68.8%) rabbits with antibodies at 1:10(25) (<1:40) died of RHD. When the cut-off was moved from 25% to 50% inhibition (1:10(50)) at 1:10 serum dilution, the assay sensitivity, specificity and accuracy for the protective immunity were improved from 84%, 54.2% and 69.4% to 84%, 100% and 91.8%, respectively. We also demonstrated at the epitope amino acid sequence level why the presence of the RHDV-cross reactive benign rabbit calicivirus, which interfered with isotype ELISAs, had little impact on the specificity of the cELISA for the diagnosis of RHDV infection. The presence of RHDV-specific antibody at 1:10(50) by the cELISA is a reliable indicator for the protective immunity. In contrast to isotype ELISAs, the cELISA is a valuable specific tool for monitoring the herd immunity to RHD for the conservation and management of wild rabbits in the field.     

应用单克隆抗体夹心酶联免疫吸附试验检测兔出血症病毒

应用建立的单克隆抗体夹心酶联兔疫吸附试验（ＭｃＡｂ－ＥＬＩＳＡ）,检测了人工感染兔出血症病毒（ＲＨＤＶ）ＤＪＲＫ细胞毒、肝毒的兔以及自然感染ＲＨＤＶ的兔的组织样品。结果表明,感染死亡兔的肝、脾、肾、骨髓样品病毒抗原的检出率为１００％,淋巴结和肌肉的检出率分别为９７．５％和７９．５％。ＭｃＡｂ－ＥＬＩＳＡ能检出肌肉中血凝试验不能检出的ＲＨＤＶ抗原。此外,还用ＭｃＡｂ－ＥＬＩＳＡ检测了肝毒人工感染兔血中ＲＨＤＶ的动态,并对１０份兔出血症脏器灭活苗的效价作了滴定。     

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.     

RABBIT HEMORRHAGIC DISEASE VIRUSES DETECTED IN PET RABBITS IN A COMMERCIAL LABORATORY IN EUROPE

Rabbit hemorrhagic disease (RHD) is an important cause of disease and mortality in wild and domestic European rabbits (Oryctolagus cuniculus) throughout the world. Testing for 2 distinct RHD virus types (RHDV/RHDVa and RHDV2) was carried out on samples collected from 684 rabbits submitted from veterinary practices and private owners throughout Europe between January 2015 and June 2017. Four (0.6%) were positive for RHDV/RHDVa and 257 (37.4%) were positive for RHDV2. RHDV/RHDVa was detected in individual samples from Germany and the Netherlands, while RHDV2 was found in animals from Germany, Great Britain, Luxembourg, The Netherlands, Spain, Switzerland, Poland, Belgium, Austria, Sweden, and Finland.     

兔IL-6基因真核表达载体的构建及其对pcDNA-VP60DNA疫苗佐剂效应研究

为研究兔白介素6（interleukin6,IL-6）真核表达质粒（pcDNA-IL-6）对核酸疫苗免疫效果的影响,本文构建了真核表达质粒pcDNA-IL-6,并将其与核酸疫苗pcDNA-VP60联合免疫家兔,以pcDNA-VP60和质粒载体pcDNA3．1（＋）作对照,用血凝抑制试验检测试验兔体内特异性抗体水平。结果表明：真核重组质粒pcDNA-IL-6对重组质粒pcDNA-VP60均具有免疫增强作用,从免疫后7d到70d,pcDNA-VP60／pcDNA-IL6联合免疫组抗体水平均高于pcDNA-VP60免疫组,差异具有显著统计学意义。     

兔出血病病毒RT-PCR检测方法的建立及初步应用

为快速检测兔出血病病毒,参照RHDV-TP株VP60基因序列(GenBank Ac-ession AF453761),在其5′端设计一对引物,扩增片段为386 bp,建立了检测兔出血病病毒的RT-PCR检测方法。经过对反应参数的优化,确定了反应的最佳条件。该方法在含多种已知病原的病料中,可特异性检出RHDV,检测病料的最高稀释度为10-5倍,且有很好的稳定性。临床样品检测结果显示,该方法的敏感性要高于血凝抑制试验,从而建立了RHDV特异、敏感的RT-PCR检测技术,可应用于RHDV临床诊断及流行病学调查。     

云南省楚雄州部分地区猪瘟血清学调查

为了解楚雄州部分地区的猪瘟免疫情况,利用酶联免疫法(ELISA)对楚雄市、南华县和禄丰县随机采取的393份血清进行猪瘟抗体检测,并对各县(市)的调查数据加以比较,了解猪瘟在楚雄州部分地区的免疫情况。结果显示,楚雄州部分地区均有较高的猪瘟抗体阳性率,各县(市)的猪瘟抗体阳性率都在80%以上,有的县(市)猪瘟抗体甚至达到了100%。说明楚雄州部分地区的猪瘟免疫效果较好,猪瘟免疫成功。     

Characterisation of monoclonal antibodies to epizootic hemorrhagic disease virus of deer (EHDV) and bluetongue virus by immunisation of mice with EHDV recombinant VP7 antigen.

Immunisation of mice with recombinant VP7 antigen of epizootic hemorrhagic disease virus of deer (EHDV) induced serum antibody responses to EHDV. However, from the 19 monoclonal antibodies (Mab) produced from these mice, 15 were specific for EHDV and four for bluetongue virus (BTV). No Mabs were identified with the specificity for an epitope of VP7 shared by both EHDV and BTV in spite of the fact that they share a large portion of homology in VP7 amino acids composition. These Mabs were divided into five groups based on their specificity and interaction with each other. Group II Mabs, consisting of 13 Mabs, recognises a potential serogroup specific, linear epitope of EHDV VP7 antigen. One of the Mabs to BTV (Group V) was identified as BTV VP7 specific with the possibility of being the serogroup specific and recognizes a potential conformational epitope. Two Mabs from these VP7 specific groups were further analysed and found to be useful in a competitive enzyme-linked immunosorbent assay (C - ELISA) for detection of specific antibodies against EHDV and BTV in bovine sera.     

猪瘟活疫苗(脾淋源)污染外源性兔出血症病毒的检测与启示

在进行2批猪瘟活疫苗（脾淋源）效力检验时,出现效力检验家兔突然死亡现象,为了查明家兔死亡原因,采用无菌检验、支原体检验、血凝试验、兔体中和试验、酶联免疫吸附试验（ELISA）对疫苗或注苗后死亡家兔肝脏、脾脏混合病料进行了检测。结果显示,疫苗的无菌检验、支原体检验结果均为阴性;疫苗及注射疫苗死亡家兔肝脏、脾脏混合病料具有较低的血凝价,血凝试验结果均为可疑;在兔体中和试验中,中和组家兔2／2健康存活,未中和的疫苗对照组家兔2／2死亡;疫苗及注射疫苗死亡后家兔肝脏、脾脏混合病料的ELISA检测结果均为阳性。检测结果证实疫苗中含有兔出血症病毒（Rabbit Haemorrhagic Disease Virus,RHDV）。猪瘟活疫苗（脾淋源）污染RHDV的现象启示：应该加强猪瘟活疫苗（脾淋源）抗原制备过程的控制;同时有必要对猪瘟活疫苗（脾淋源）质量标准进行修订使之进一步补充完善。