【作者】 刘萍；

【导师】 姚火春； 孙志；

【作者基本信息】 南京农业大学 ， 兽医博士（专业学位）， 2020， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)出现于20世纪90年代初,是目前危害全球养猪业最重要的传染病之一,该疾病的主要临床表现包括乳猪、育肥猪的呼吸系统疾病和母猪的生殖障碍。猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)是引起该疾病的主要病原。疫苗免疫是当前防治该疾病最有效的手段,目前减毒活疫苗和灭活疫苗均在广泛的使用。减毒活疫苗能诱导体液免疫反应,并且减轻同源强毒引起的临床发病。但PRRSV变异率高,减毒活疫苗对异源毒株感染也不能提供完全有效的保护,并且存在散毒和毒力返强的风险,这些都给PRRS的防控提出了巨大的挑战。本研究初期旨在分离一株PRRSV流行毒株,采用体外细胞连续传代的方法降低病毒在动物体上的致病力,并且保留其免疫原性,最终获得一株安全且有效的疫苗候选毒株。随后,通过一系列动物试验验证传代后毒株的安全性和有效性。在筛选毒株过程中,我们经过病毒分离和细胞传代致弱获得了两株不同代次的传代毒株PRRSV/0007-01-P85和P115,同时为了增加疫苗研发的成功率,加入一株来源于高致病性毒株JX143毒株的传代致弱毒株JXM105(转让自上海兽医研究所)作为减毒活疫苗候选株进行后续研究,最终研究表明:两株在仔猪上有良好安全性和效力性的候选株PRRSV/0007-01-P85和JXM105均表现出不同程度的致病力返强。随后本研究分析了这两个毒株体内传代后基因序列的变化,鉴定出多个可能与毒力返强相关的位点,并利用反向遗传学方法和动物回归试验对几处氨基酸位点与毒力的相关性进行了验证。初步解析了两株致弱毒株毒力返强的机制,为未来PRRS疫苗开发和人工致弱病毒提供理论基础和参考。现将研究内容简述如下:1 PRRSV分离、传代致弱及其体外鉴定从2012年的临床样品中分离到一株PRRSV,命名为PRRSV/0007-01。该毒株在nsp2的第533-561位存在29个氨基酸的缺失以及在第469-489位含有21个连续氨基酸的缺失。遗传进化分析的结果显示,该毒株在进化树上位于HP-PRRSV和经典PRRSV毒株之间。将该毒株在MA104细胞上连续传代1 15次,对传代病毒生长特性和基因组序列分析的研究结果显示:传代病毒与亲本病毒有着相似的生长特征,但传代病毒有较高的滴度;病毒基因组上的突变也随着代次的增加而增多,80代后的病毒的突变数量显著增多。对P85和P115病毒进行全长基因组序列分析的结果表明,与亲本病毒序列相比,P85病毒共出现50处核苷酸突变,P115病毒共有60处核苷酸突变,其中氨基酸突变均为21处。根据病毒生长特性和序列分析结果,将P85和P115病毒选为疫苗候选株,并进行大规模扩增制备病毒库。2 PRRSV传代致弱毒株安全性和免疫效力性评价PRRS减毒活疫苗候选毒株都必须在靶动物猪上验证其安全性和效力性,并且通过动物回传试验验证其有无致病力返强。为了验证致弱毒株的安全性和有效性,本研究首先对亲本毒株PRRSV/0007-01-P3进行动物回归试验,并建立了 JX143毒株的攻毒模型。将传代毒株 PRRSV/0007-01-P85、PRRSV/0007-01-P115 和 JXM105 接种 4周龄仔猪并连续观察21天,通过临床症状和肺病理变化等初步评价候选毒株的安全性。免疫后第28天,部分免疫猪接受HP-PRRSV毒株JX143攻击,后通过各项指标评价候选疫苗株提供的保护效力。随后,将已验证初步安全且有效的候选毒株PRRSV/0007-01-P85和JXM105在仔猪体内回传5代,观察二者是否存在毒力返强的现象。最后,将候选毒株及其回传5代的猪血清接种妊娠约90天的怀孕母猪,通过观察母猪产仔情况评价致弱毒株的安全性。以上试验结果表明,PRRSV/0007-01-P3分离毒株在猪体上的发病率和致死率分别为100%和40%,而JX143毒株发病率和致死率分别为100%和90%。在细胞上连续传代获得的三株疫苗候选毒株对仔猪均无致病性。面对高致病性毒株JX143的攻击,PRRSV/0007-01-P85和JXM105毒株免疫动物无PRRS相关临床症状,表明二者均可以提供100%保护;而PRRSV/0007-01-P115仅能提供部分保护。PRRSV/0007-01-P85在仔猪上回传5代期间对仔猪无致病力,但其回传5代后的病毒严重影响母猪产仔性能;而JX143毒株的细胞传代弱化的疫苗候选株JXM105在仔猪传代4代出现毒力返强。本研究表明,PRRSV/0007-01-P85和JXM105对仔猪无致病力,提供的保护率可以达到100%,但二者表现出不同程度的毒力返强风险。3 PRRSV传代致弱毒株毒力返强机制初步研究病毒基因组上的某些变异以及病毒的准种多样性与病毒的致病力密切相关。本研究运用二代测序的方法,对病毒在体内传代过程中基因组序列和病毒准种多样性演变进行了跟踪和分析。初步分析了不同亲本病毒致弱毒株回归动物体毒力返强的机制。序列比对的结果表明,JXM105毒株上有4处回复性突变为可能与毒力相关的位点,分别位于 nsp1β-52、nsp10-275、nsp10-313、GP5a-2;而 PRRSV/0007-01-P85 在仔猪上回传5代后继续在妊娠母猪上传代1代后,出现7处可能与毒力相关的回复突变分别位于 nsp1α-113、nsp2-722、nsp4-20、nsp9-246、nsp9-302、nsp9-670 和 nsp10-72。这些回复突变随着体内传代次数的增加在病毒准种群体中的占比逐渐增加并且成为病毒准种优势序列。利用反向遗传学方法和动物回归试验证明,将PRRSV/0007-01上的7处潜在毒力相关位点突变为致弱型显著降低突变病毒在3周龄仔猪上的致病力。这些研究表明PRRSV在动物体内适应性进化与准种变化相关,毒力相关位点的回复突变的出现和累积并且成为优势准种是PRRSV致弱毒株毒力返强的重要原因之综上所述,本研究分离了一株PRRSV高致病性的毒株PRRSV/0007-01;将PRRSV/0007-01在MA104细胞上连续传代后获得两株疫苗候选毒株P85和P115,二者在仔猪上均有良好的安全性;PRRSV/0007-01-P85和JXM105对高致病性PRRSV毒株攻击有较好的免疫保护作用而PRRSV/0007-01-P115毒株只能提供部分保护;PRRS V/0007-01-P85和JXM105都存在不同程度的毒力返强,前者回传5代后严重影响妊娠母猪产仔率,后者回传至第4代可致仔猪发病;通过二代测序分析了PRRSV/0007-01-P85和JXM105体内传代后基因序列的变化,鉴定到多个可能与毒力返强相关的位点并运用反向遗传学方法进行了验证,初步解析了两株致弱毒株毒力返强的机制。本论文的研究结果可以为PRRS疫苗研发和人工致弱病毒提供理论基础和参考。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome(PRRS)first emerged in North America and Europe in 1990s has caused significant economic loss to global swine industry during the past thirty years.The hallmark clinical signs of PRRS are respiratory disease of piglets and growing pigs and reproductive failure of sows.Porcine reproductive and respiratory syndrome virus(PRRSV)was determined to be the causative agent of PRRS.Currently,vaccination is the most efficient strategy in the prevention of PRRS.In China,classic PRRSV was first isolated in 1996.Since 2006,a large number of new strains emerged in China,although many inactivated and live-attenuated PRRS vaccines were used to combat PRRS in China.Due to the high mutation rate and strong immunosuppression nature of PRRSV,the inactivated vaccines have failed to elicit enough protective immunity even against homologous virus.By the contrast,the live-attenuation vaccines are able to combat clinical disease of PRRS,although they are unable to provide enough cross-protection against heterologous strains.The safety concern is another drawback of live-attenuated vaccines,such as the spread of vaccine strains and the incidence of reversion to virulence.Thus,it is still huge challenge for the prevention and control of PRRS in China.The initial aim of this study is to isolate an epidemic strain of PRRSV and obtain an attenuated vaccine candidate by using traditional in vitro cell passage methods to reduce the virulence of virus and retain its good immunogenicity.Subsequently,animal experiments were carried out to verify the safety and efficacy of vaccine candidates.Two attenuated candidates PRRSV/0007-01-P85 and JXM105 did not cause any disease to piglets and provide 100%protection against HP-PRRSV JX143 strain.However,they both showed virulence reversion at different degree during in-vivo passage in piglets.Subsequently,we analyzed PRRSV quasispecies during in-vivo passage and identified several mutations that may be responsible for their virulence reversion.The correlation of these mutations with PRRSV pathogenicity was further assessed by reverse genetics and animal study.Finally,we discussed the mechanism of virulence reversion,which may provide a theoretical foundation for artificial attenuated PRRSV.1 PRRSV isolation,attenuation by passage and characterization in vitroOne PRRSV strain PRRSV/0007-01 was original isolated from a lung sample collected from a pig with PRRS-like symptom in 2012.This virus strain contains a HP-PRRSV characteristic 29+1 amino acids deletion and 21 amino acids deletion in the nsp2 coding region.In line with deletions in nsp2,the phylogenetic analysis showed that this strain located between HP-PRRSV strains and classical PRRSV strains.To attenuate this virus,we passaged PRRSV/0007-01 115 times in MA104 cells.Viral genomic sequences and viral titers were determined every 10 passages.These results suggested that the fitness of PRRSV/0007-01 in MA104 cells increased after serial passage.Of note,the number of mutations accumulated dramatically after passage 80.Based on our results on virus growth in vitro and genomic sequence analysis of passaged viruses,P85 and P115 were chosen as vaccine candidates.Both candidates showed similar growth property to parental strain in cells while passaged viruses showed higher viral peak titer than parental strain.Their full-length genomic sequences were determined using next-generation sequencing method.In comparison with their parental virus,P85 virus contains 50 nucleotide substitutions,while P115 virus contains 60 nucleotide substitutions.Among these mutations,21 mutations lead to amino acid substitutions in both viruses.In conclusion,we isolated PRRSV/0007-01,passaged it 115 times in MA014 cells.The P85 and P115 viruses were selected for further characterization in animal experiments.2 Evaluation of the safety and efficacy of attenuated PRRSV strainsThe live-attenuated PRRS vaccine candidates are required to be tested for their safety and efficacy in pigs and the risk of virulence reversion by back passages in piglets.To evaluate the safety and protective efficacy of live-attenuated vaccine candidates PRRSV/0007-01-P85 and PRRSV/0007-01-P115,the pathogenesis of parental strain was evaluated,and a challenge model was established by using JX143 strain.Three candidates included JXM105 strain were inoculated into four-week old piglets,respectively.The safety of these candidates was determined based on the clinical symptoms and lung lesion of infected animals during 21 days post infection.Some immunized pigs were challenged with JX143 on 28 days post immunization.The protection efficacy of candidates was evaluated primarily based on various indicators.The risk of virulence reversion of PRRSV/0007-01-P85 and JXM105 was assessed through passaging vaccine candidates in piglets.PRRSV/0007-01-P85 and serum collected from the piglet infected with PRRSV/0007-01-P85 serial passaged five times in piglet were inoculated to the pregnant sows to further evaluate their impact on the reproductive performance of sows.These results showed that PRRSV/0007-01-P3 resulted in 100%morbidity and 40%mortality,while JX143 showed 100%morbidity and 90%mortality.All candidates showed no pathogenic to pigs after serial passage in cells.PRRSV/0007-01-P85 and JXM105 could provide fully protection against JX143 challenge,while P115 strain provided partial protection.PRRSV/0007-01-P85 did not show virulence reversion within five passages in piglets but its 5th passage serum affected reproductive performance of sows seriously which resulted in no live piglets at the end day of experiment.JXM105 reverted to virulence at passage 4 in piglets.These results suggested that both strains were safe and effective in piglets,but both of them showed virulence reversion potential at different degrees.3 Study on the mechanism of virulence reversion of attenuated PRRSV strainsPoint mutations and viral quasispecies were considered to play critical roles in viral pathogenesis.In this chapter,PRRSV quasispecies diversity during viral passage was tracked and analyzed by next generation sequencing.Several potential virulence-related positions in JXM105 and PRRSV/0007-01-P85 were identified by comparing the mutations and their dynamic changes in consensus sequence.Our results showed that JXM105 had four potential virulence-related positions located at nsp1β-52、nsp10-275、nsp 10-313 and GP5a-2,respectively.PRRSV/0007-01-P85 reverted to virulent type at 7 positions including nsp1α-113、nsp2-722、nsp4-20、nsp9-246、nsp9-302、nsp9-670 and nsp10-72.The mutation frequencies of these positions were increased,and the virulent type of sequence became the dominant in viral quasispecies during passage.Using the reverse genetic system of PRRSV/0007-01,a mutant virus containing the attenuated type of amino acids at these seven residues was constructed.In comparison with parental virus,the mutant virus showed reduced pathogenicity in 3-week old piglets.These results showed that PRRSV evolves rapidly in animals,the accumulation of reverted mutations in virulence-related positions and becoming dominant in quasispecies is one of the critical causes for virulence reversion of PRRSV attenuated strains.Taken together,in this study,a high pathogenic PRRSV/0007-01 was isolated.Two attenuated viruses PRRSV/0007-01-P85 and P115 were obtained by passaging PRRSV/0007-01 strain on MA104 cells for 85 and 115 passages respectively.Both of them were safe in piglets.PRRSV/0007-01-P85 and JXM105 provided fully protection against JX143 strain,while PRRSV/0007-01-P115 only provided partial protection.PRRSV/0007-01-P85 did not show virulence reversion during five back passages in piglets,however its 5th back passage serum impacted sow reproductive performance seriously,while JXM105 strain reverted to virulence at passage 4 in piglets.Both attenuated strains showed the risk of virulence reversion.The evolution of PRRSV quasispecies diversity during in-vivo passage was analyzed by next generation sequencing and several potential virulence-related positions in JXM105 and PRRSV/0007-01-P85 were identified,and PRRSV/0007-01-P85 virulence mutants were further verified using reverse genetic system subsequently.The results of this study may provide a theoretical foundation for PRRS vaccine development and artificial attenuated PRRSV.更多还原