【作者】 王喜军；

【导师】 步志高；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2012， 博士

【摘要】 狂犬病（Rabies）是由狂犬病病毒（Rabies virus, RV）引起的一种人兽共患传染病，在全球范围内流行，以急性、渐进性和致死性脑炎为特征。狂犬病是目前病死率最高的传染病之一，一旦出现神经症状病死率几乎高达100%。全球每年死于狂犬病的人数高达55,000，主要发生在非洲和亚洲的发展中国家。近年来，我国狂犬病疫情呈现了快速回升的趋势，每年造成的死亡人数均在2,000以上，已经成为一个严重的公共卫生问题。据统计，99%以上人间狂犬病死亡病例因狂犬病犬咬伤所致。对于人间狂犬病的控制，最有效的措施是通过疫苗接种从源头上控制犬狂犬病。传统的狂犬病弱毒疫苗成本低廉，但存在毒力残留或致病性回复突变的安全隐患；灭活疫苗安全、有效，但成本昂贵。因此，研制安全、有效，成本低廉的动物狂犬病疫苗，仍具现实意义。反向遗传技术的发展与成熟使得不分节段的单股负链RNA病毒作为疫苗载体成为可能。犬瘟热（Canine distemper, CD）是由副粘病毒科的犬瘟热病毒（Canine distemper virus, CDV）引起的犬、貂和狐狸等肉食动物的一种重要传染病，在全球广泛分布，严重影响着养犬业及经济动物养殖业的健康发展。弱毒疫苗接种能有效预防该病，应用广泛。利用CD弱毒疫苗株为载体，构建重组二联活疫苗，可实现一苗两防，有利于提高狂犬病免疫覆盖率，提高免疫质量，节省狂犬病免疫的经济成本。为此，本研究以我国生产中应用的CD弱毒疫苗株CDV/R-20/8为亲本毒株，建立了该毒株的反向遗传操作系统。救获的犬瘟热弱毒疫苗株rCDV保持了野生型CDV/R-20/8的细胞培养生长特性，二者在Vero细胞上的多步生长曲线基本一致，生长滴度在相同的时间内达到峰值，最高生长滴度均为1×10^6.5TCID₅0/mL。为评估CDV作为活病毒载体表达外源蛋白基因的潜力，构建了表达增强型绿色荧光蛋白（EGFP）的重组犬瘟热病毒rCDV-EGFP。重组病毒rCDV-EGFP在Vero细胞上的生长特性与亲本株rCDV相似，最高生长滴度可达1×10^6.5TCID₅0/mL。该重组病毒具有良好的Vero细胞生长适应性及遗传稳定性，在Vero细胞上连续传代10代次后，仍能稳定、高效的表达EGFP。结果表明，CDV疫苗株CDV/R-20/8具有作为活病毒疫苗载体的潜力，为研制防制狂犬病和犬瘟热的重组二联活载体疫苗奠定了基础。在此基础上，进一步构建了表达RV弱毒疫苗株ERA G蛋白的重组犬瘟热弱毒疫苗株rCDV-RVG。该重组CDV保持了亲本毒株rCDV对Vero细胞的良好适应性和稳定性，可达到高水平的生长滴度，最高生长滴度可达1×10^6.25TCID50/mL。rCDV-RVG和rCDV按3×10⁴TCID₅0/30_μL/只和1×10⁵TCID₅0/100_μL/只的剂量，分别经脑内和肌肉注射途径接种小鼠，观察3周，所有接种小鼠未见任何明显的狂犬病症状；rCDV-RVG和rCDV无论肌肉接种小鼠，还是脑内接种小鼠，与相应的PBS接种对照小鼠相比，观察期间的体重变化均无显著性差异。结果表明，RV G蛋白基因的插入、表达并没有增强疫苗载体CDV对小鼠的致病力。同时，rCDV-RVG按1×10⁵TCID₅0/100_μL/只的剂量经肌肉注射途径接种小鼠，可诱导显著的RV中和抗体反应。免疫后3周应用致死剂量的RV街毒GX/09株进行攻击。结果免疫小鼠全部获得保护，而对照小鼠全部发病死亡。结果表明，rCDV-RVG对小鼠具有的良好的生物安全性和的免疫原性。为进一步评估重组病毒对犬的免疫原性，rCDV-RVG和rCDV分别按1×10⁶TCID₅0/1mL/只的剂量，经肌肉注射途径接种3月龄比格犬5只，初次免疫后3周以相同剂量、途径加强免疫一次。免疫后持续观察，定期检测免疫犬血清中CDV和RV中和抗体。结果rCDV-RVG初次免疫后1周和3周，免疫犬血清中的RV中和抗体滴度平均值分别为1.48IU和32.11IU。第二次免疫后2周，RV中和抗体反应显著增强，其平均值可达96.71IU。rCDV-RVG诱导的RV中和抗体反应可持续一年以上，初次免疫后70周，免疫犬血清中的RV中和抗体滴度平均值仍>0.5IU，即保护动物抵抗致死剂量RV街毒攻击的最小滴度。结果提示，重组病毒rCDV-RVG两次免疫接种，可为免疫犬提供1年以上的有效保护。免疫后70周进行第3次免疫，结果诱导更为显著的加强免疫反应，第3次免疫后1周，血清RV中和抗体滴度平均值升至281.4IU。与此同时，rCDV-RVG和rCDV初次免疫后3周，均诱导显著的CDV中和抗体反应，第二次免疫后CDV中和抗体水平迅速上升。一年后进行第3次免疫，二者均可诱导更加显著的加强免疫反应，而且rCDV-RVG诱导的CDV中和抗体滴度峰值与亲本株rCDV的相比无显著性差异。本研究结果表明，重组病毒rCDV-RVG具有良好的生物安全性和免疫原性，是一个有希望的新型狂犬病、犬瘟热重组二联活疫苗候选株。更多还原

【Abstract】 Rabies is a widespread zoonosis caused by the rabies virus （RV） and causes acute, progressive, andfatal encephalitis. The disease is currently one of the deadliest infectious diseases and is almost100%fatal once the neurologic symptoms appear. Worldwide there are approximately55,000human deaths peryear due to rabies, mostly in developing countries of Africa and Asia. Since2003, the annual number ofhuman deaths has been growthing in our country and was above2,000. Now, rabies is a serious publichealth problem in our country. However, the exposure to rabid dogs is still the main cause of over99%of human deaths worldwide. For preventing human rabies, the most efficient strategy is to eliminate thedisease in dogs through vaccination. The manufacturing costs of routine rabies attenuated vaccines arelow, but their residual virulence or pathogenic mutations can sometimes cause the disease of vaccinatedanimals. Although, inactivated cell culture rabies vaccines are safe and effective, their manufacturingcosts are high. Therefore, effective, safe, and affordable rabies vaccines are still being sought.The development of reverse genetics has provided a powerful tool to create recombinantnonsegmented negative-strand RNA virus-based bivalent vaccine. Canine distemper （CD） is awidespread infectious disease of most carnivores, such as dogs, minks, foxes and so on, caused bycanine distemper virus （CDV）, a morbillivirus of family Paramyxoviridae. Now, CD is a very seriousproblem for dogs industry and economic animals industry worldwide. However, vaccination of CDattenuated vaccines is an efficient method to prevent CDV infection. Therefore, CDV-based liveattenuated vaccine serve as a bivalent vaccine can prevent rabies and CD, which will contribute toimprove the rabies vaccine vaccination rate of dogs and the quality of immunization. It will save theeconomic costs of rabies vaccine immunization.In this study, we established a reverse genetics system of the attenuated CDV vaccine strainCDV/R-20/8. The rescued CDV, rCDV, was almost indistinguishable from the parental strainCDV/R-20/8. There are no difference in growth properties between rCDV and the parental wild virus inVero cells. And, the rCDV and the parental strain grew to similar levels and reached peak titers of106.5TCID50/mL at72h post-infection. To investigate the potential of CDV serve as a live vaccine vectorexpressing the foreign proteins, we generated a recombinant CDV, rCDV-EGFP, expressing theenhanced green fluorescent protein （EGFP） by using reverse genetics. The rCDV-EGFP has the similarbiological characteristics with parental rCDV, including high growth titer (10^6.5TCID50/mL) andexcellent adaptability and genetic stability in Vero cell. After serially passages10times in Vero cells, therecombinant CDV still kept a high level expression of EGFP. These results indicated that theCDV/R-20/8attenuated vaccine strain has the potential to serve as a live vaccine vector.CDV is a promising live viral vector to develop a bivalent vaccine against RV and CDV infection.In the present study, we generated a recombinant CDV, rCDV-RVG, expressing the RV vaccine strainERA G protein by using the reverse genetics. The rCDV-RVG grew to similar levels with rCDV and reached peak titer of10^6.25TCID₅0/mL at72h post-infection. This titer was approximately one-quarterof a log lower than that of rCDV. To assess the pathogenicity and immunogenicity of recombinant CDV,mice were injected intracerebrally （i.c.） with3×104TCID50in a volume of30μL or intramuscularly （i.m.）with105TCID50in a volume of100μL of the rescued viruses. All of the mice showed no apparent signsof disease after inoculation. There was no significant difference in the body weight changes among thethree i.m. inoculated groups or the three i.c. inoculated groups with rCDV-RVG, rCDV and PBS. Theseresults demonstrated that the insertion and expression of RV G gene did not increase the virulence of theCDV vector in mice. Mice inoculated i.m. with10⁵TCID₅0of the rCDV-RVG developed a strong RVneutralizing antibody response, which completely protected mice from challenge with a lethal dose ofstreet virus GX/09strain at3weeks post-vaccination. However, all control mice died of rabies afterchallenge. These results indicated that the rCDV-RVG is safe and immunogenic in mice.To further assess the immunogenicity of the recombinant virus rCDV-RVG, two groups of five3-month-old Beagle dogs were i.m. inoculated with1mL10⁶TCID₅0of rCDV or rCDV-RVG,respectively. At3weeks after initial vaccination, the dogs received a second vaccine dose, respectively.Dogs inoculated with the rCDV-RVG developed a strong and long-lasting RV neutralizing antibodyresponse. The mean titer of RV neutralizing antibodies was1.48IU at1week after the first dose androse to32.11IU at3weeks after the first dose. After receiving the second dose, the rCDV-RVGvaccinated dogs showed signally boost responses to RV neutralizing antibody. The mean titer of RVneutralizing antibodies for the group was96.71IU at2weeks after the second dose. RV neutralizingantibodies induced by rCDV-RVG can sustain for more than a year. At70weeks post-vaccination, themean titer of RV neutralizing antibodies was>0.5IU; the minimum NA titer required to protect animalsfrom challenge with RV street virus. These result suggested that two times vaccination of rCDV-RVGprovides effective protection for>1year in dogs. After receiving the third dose at70weekspost-vaccination, the rCDV-RVG vaccinated dogs showed substantial re-boost responses to RVneutralizing antibody. The mean titer of RV neutralizing antibodies for the group sharply increased to281.4IU at1week after the third dose. Meanwhile, rCDV and rCDV-RVG induced a remarkable CDVneutralizing antibody response in dogs at3weeks after the first dose. The titers of CDV neutralizingantibody for the two groups sharply increased after the second dose. After receiving the third dose at70weeks post-vaccination, all the rCDV-vaccinated and rCDV-RVG-vaccinated dogs showed substantialre-boost responses to CDV neutralizing antibodies. And, there was no significant difference in the peaksof CDV neutralizing antibody titers between the two groups.These results demonstrate that the rCDV-RVG is safe and immunogenic in dogs and a promising,novel and live bivalent vaccine candidate against CD and rabies.更多还原