狂犬病Flury病毒致弱分子机制及新型疫苗的研究
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摘要
狂犬病(rabies)是一种在全世界范围内流行的,由狂犬病病毒(rabies virus, RV)引起的以中枢神经系统感染(CNS)为特征的人畜共患病。全世界每年死于狂犬病的人数高达55,000,主要发生在发展中国家。目前,狂犬病无法治疗,一旦发病致死率几乎为100%。疫苗免疫是控制狂犬病唯一有效的方法。为研制更加安全、有效的狂犬病疫苗,迫切需要加强对RV病原致病分子机制与免疫机制的基础研究。
I. RV Flury传代致弱的分子机制的研究
1940年,Koprowski等人从病死女孩脑内分离到狂犬病毒Flury株,经1日龄雏鸡脑内、鸡胚卵黄囊传后,再经鸡胚传代178代以上,无论神经内、外接种都对动物丧失致病力,但新生乳鼠和猴在脑内接种时仍有感染性。在鸡胚传代68代以前的称为“LEP”(鸡胚低代株);高于136代的称为“HEP”(鸡胚高代株)。LEP脑内感染成年小鼠致死,而HEP脑内感染成年小鼠不致死。造成LEP和HEP致病力生物学表型差异的分子机制仍未充分阐明。本研究拟就RV Flury传代致弱的分子机制进行系统研究。
对LEP和HEP完整基因组的系统解析表明,LEP和HEP的基因组均含有11,925个核苷酸,相似性达99.3%。LEP和HEP的L基因ORF核苷酸相似性最高为99.7%,N基因(99.6%)、P基因(99.4%)、M基因(99.3%)和G基因(98.9%)依次降低。两株病毒由N、P、M、G和L基因ORF预测的氨基酸序列相似性分别为99.8%、98.3%、99.0%、97.8%和99.6%。比较这两个毒株的5个蛋白的氨基酸序列,共有27个氨基酸发生改变,变化最大的是G蛋白,最保守的是N蛋白。N/P、P/M和M/G间的基因间隔序列完全一致,G/L间的间隔序列仅有一个碱基发生突变。另外,3’、5’端的非编码区及所有基因的转录起始信号和终止信号序列也都一致。
在此基础上,建立了Flury LEP和HEP的反向遗传操作系统。从cDNA克隆拯救获得的rLEP在小鼠神经母细胞(NA)和BHK-21细胞上的多步生长动力学曲线与野生型毒株相似,最高生长滴度分别可达3.0×10~8 FFU/ml和5.0×10~7 FFU/ml,体外嗜神经指数为0.78。rLEP通过脑内(i.c.)、滴鼻(i.n.)及肌肉注射(i.m.)不同途径分别感染成年小鼠均致死,LD50分别1 FFU,474 FFU和3.4×10~5 FFU,与野生型毒株相近。从cDNA克隆拯救获得的rHEP在NA和BHK-21细胞上的多步生长动力学曲线与野生型毒株相似,最高生长滴度均可达2.0×10~7FFU/ml,不具备体外嗜神经性,通过i.c.、i.n.及i.m.不同途径分别感染成年小鼠均不致死,表现出与野生型HEP相似的生物学特性和致病力。
将HEP的G333位的Gln突变为Arg,形成的突变株rHEPG333R体外嗜神经指数由0变为0.8,与LEP相近,表明rHEPG333R已经回复了嗜神经特性。通过i.c.,i.n.和i.m.3种不同途径分别感染小鼠,rHEPG333R均可使小鼠致死,LD50分别为0.3 FFU、887 FFU和1.4×10~6 FFU。结果表明,G333位点的Arg单个氨基酸突变足以使HEP重新获得对成年小鼠的高致病力。
但是,将LEP的G333位点由Arg突变为Gln后,形成的rLEPG333Q突变株尽管体外嗜神经指数下降为0,肌肉接种小鼠全部存活并且无神经症状出现,但通过i.c.或i.n.途径感染小鼠时,rLEPG333Q对小鼠仍显示出高度致死性,LD50分别为36 FFU和2.1×10~5 FFU。结果提示,G333 Arg的替换突变消除了LEP的嗜神经性和外周神经侵入能力,但i.c.或i.n.感染成年小鼠仍保持其高致死力。进一步对rLEPG333Q接种致死小鼠脑内病毒基因组序列进行分析,结果发现,rLEPG333Q在小鼠脑内和NA细胞中复制时,G333位点的Gln人工突变不能保持稳定,发生了嗜神经性的回复突变,因而重新获得对小鼠的致死能力。相反,rHEP无论经i.c或i.n.接种,还是NA细胞连续传代,G333位点的Gln均未发生回复突变。
为了澄清影响G333Q突变在神经组织或细胞复制过程中遗传稳定性的因素,进一步构建了嵌合病毒rHEP-G(L)333Q和rLEP-G(H),前者通过将HEP的G基因替换为LEP的G基因,同时G333位点Arg突变为Gln构建而成的,后者通过将LEP的G基因替换为HEP的G基因构建而成的。rHEP-G(L)333Q体外嗜神经指数也和rHEP一样,仍然为0;经i.c.感染小鼠不致死,病毒在小鼠脑内和NA细胞上复制时G333位点的Gln稳定不变。而嵌合病毒rLEP-G(H),在NA细胞上的生长性能显著地低于rLEP,而与rHEP十分接近;体外嗜神经指数也下降为0。尽管如此,小鼠经i.c.接种105 FFU的rLEP-G(H)后,在感染后12天内全部死亡。提取濒死小鼠脑组织RNA和NA细胞传代的第5代培养物进行RT-PCR和测序分析发现,rLEP-G(H)的G333位点Gln(CAG)已经突变为Arg(CGG)。结果表明,造成rHEP和突变株rLEPG333Q在神经组织或细胞中复制时G333位点Gln的遗传稳定性存在差异是LEP基因组骨架上的某个(或某些)因素而不是G蛋白本身引起的。
负链RNA病毒的RNA聚合酶的低保真性是病毒突变最主要的原因。为此,将rLEPG333Q的L基因替换为HEP的L基因,构建了嵌合病毒rLEPG333Q-L(H)。成年小鼠脑内接种105 FFU的rLEPG333Q-L(H)全部存活且没有出现神经症状,rLEPG333Q-L(H)的G333位点Gln(CAA)在鼠脑和NA细胞传代时稳定不变。结果表明,L蛋白与G333位点的Gln突变的遗传稳定性密切相关。L基因与G基因的协同变异、进化导致Flury在鸡胚传代过程获得致弱。
同时,我们还研究了GR333Q突变对Flury诱导细胞凋亡能力的影响。结果发现,rLEP和rHEPG333R感染的NA细胞中,NA细胞发生凋亡的比例分别为2.9±0.7%和2.8±0.6%,与空白对照组的细胞凋亡水平相似(2.6±0.6 %)。相比之下,rHEP、rLEPG333Q、rHEP-G(L)333Q和LEPG333Q-L(H)诱导的凋亡水平则高于对照组,分别为对照组的2.1、2.4、2.1和2.1倍。进一步利用western blot分析比较在病毒感染的细胞中不同蛋白的表达水平。结果发现,rLEP与rLEPG333Q、rHEP-G(L)333Q或rLEPG333Q-L(H)相比,G蛋白表达水平无明显差异;rHEP与rHEPG333R的G蛋白表达量也十分相近。结果显示,Flury病毒诱导NA细胞凋亡能力与G蛋白的表达量并无一定相关性。这一结果与前人报道G蛋白的表达量与凋亡诱导水平呈正相关的结果有所不同,提示狂犬病病毒诱导神经细胞凋亡的分子机制仍然有待进一步研究。
嵌合病毒rHEP-G(L)333Q和rLEPG333Q-L(H)高度致弱,失去嗜神经特性,并且遗传稳定。对小鼠的免疫和攻毒试验结果显示:rHEP-G(L)333Q和rLEPG333Q-L(H)免疫小鼠无论诱导的RV中和抗体水平,还是对街毒攻击的保护效力,均显著高于LEP和HEP,具有开发成为分子修饰弱毒疫苗的潜力。
II.狂犬病灭活疫苗的研制
RV灭活疫苗在生物安全性上具有突出优势,但是制造成本高,价格相对昂贵,免疫期相对较短,在发展中国家特别是农村地区难以推广应用。提高灭活疫苗抗原的单位生产效率,将有助于降低生产成本,同时提高免疫效力,延长疫苗接种间隔期。
本研究构建了表达双重G蛋白基因的重组RV Flury LEP病毒株rLEP-G。生物学特性分析显示,rLEP-G在NA和BHK-21细胞的生长滴度分别可达7.0×10~8 FFU/ml和1.0×10~8 FFU/ml,体外嗜神经指数为0.85,与LEP相近。rLEP-G通过i.c.接种感染小鼠的LD50为1 FFU,与LEP的致病性无显著差异。Western blot结果显示,rLEP-G感染BHK-21细胞后G蛋白的表达量显著提高3倍。分别以rLEP-G和LEP为种毒,按相同细胞培养量制备成灭活疫苗,对进行小鼠和比格犬的免疫效力试验。结果表明,无论小鼠还是比格犬,rLEP-G灭活疫苗诱导RV中和抗体的能力均显著高于LEP灭活疫苗,表现出良好的免疫原性,适用于狂犬病灭活疫苗的开发。
Rabies is an enzootic viral disease widespread throughout the world and causes severe destruction to the central nervous system. The annual number of human deaths worldwide caused by rabies is estimated to be 55,000, mostly in rural areas of Africa and Asia. To date, no effective medical therapy has been established for overt rabies. Once neurologic symptoms of the disease develop, rabies is fatal to both animals and humans. Vaccination is the only effective way to control rabies. Therefore, we should take a deep insight into the pathogenic and attenuation mechanism of rabies virus (RV) and development of new types of rabies vaccines.
I.Attenuation molecular basis of rabies virus Flury strain
Attenuated Flury RV low-egg-passage (LEP) and high-egg-passage strains (HEP) were established through serial passage in chicken brain, chicken embryos, and culture cells using a Flury RV isolated from a girl who died of rabies. LEP kills adult mice after intracerebral inoculation, while HEP causes only mild symptoms in adult mice. The molecular mechanisms associated with RV virulence are not fully understood. Therefore, the LEP and HEP strains, showing a high homology of the complete nucleotide sequence, appear to be useful for exploring the pathogenic and attenuation mechanism of RV.
Sequencing analyses revealed that the genomes of the LEP and HEP strains share 99.3% nucleotide sequence identity, and a deduced amino acid homology of 99.8%, 98.3%, 99.0%, 97.8%, and 99.6% between the individual N, P, M, G, and L proteins, respectively. The two strains have identical intergenic regions between N/P, P/M and M/G, and one substitution in G/L intergenic region. Sequences of the 3′and 5′terminal non-coding regions, which include the recognition and initiation site of the viral RNA polymerase, were completely conserved in the two viruses. A total of 27 amino acid substitutions were found between these two strains.
To investigate the molecular mechanism for virulence and attenuation of Flury RV, two infectious viruses, rLEP and rHEP, were rescued from the genomes of LEP and HEP respectively. The growth curves of the rLEP and rHEP strains were similar to wildtype LEP and HEP strains respectively, in both neuronal NA and non-neuronal BHK-21 cells. The rLEP and wildtype LEP strains appeared to be strongly neurotrophic having similar index values of 0.84 and 0.78, respectively. As expected, both rHEP and wildtype HEP did not show any neurotrophic characteristics. Virulence of rLEP, rHEP, wild LEP and HEP in adult mice was determined by different inoculation routes, intracerebral (i.c.), intranasally (i.n.), or intramuscularly (i.m.). The rLEP strain killed adult mice by any inoculation route. The MLD50 of rLEP after i.c., i.n. and i.m. inoculation were 1 FFU, 474 FFU and 3.4×105 FFU, respectively, which were comparable to those of wildtype LEP. In contrast, all mice survived from i.c., i.n. or i.m. inoculation, without exhibiting any neurological symptom except some slight body weight loss in the first few days post inoculation. These results suggested that the rescued viruses had similar biological properties and pathogenicities as their corresponding parental wildtype virus in adult mouse.
We generated a HEP mutant virus, rHEPG333R, in which the Gln at G333 was changed to Arg. The in vitro neurotropism index of rHEPG333R was significantly increased from 0 to 0.8, indicating that rHEPG333R had acquired the neurotropism property. Infection of adult mice confirmed that rHEPG333R was lethal. The MLD50 by i.c., i.n. and i.m. inoculation routines were 0.3 FFU, 887 FFU and 1.4×10~6 FFU, respectively. Theses results indicated that the mutation at G333 is sufficient to cause HEP to become highly pathogenic in adult mice.
However, rLEPG333Q, in which the Arg at G333 was changed to Gln, lost its neurotropism property and had a neurotropism index value of 0 in cell culture. Infection by the i.m. route with the maximum dosage of 3×10~6 FFU did not kill adult mice, and all surviving mice did not show any signs of neural disease. However, when inoculated by i.c. and i.n. routes, rLEPG333Q remained highly lethal in adult mice. The MLD50 by i.c. and i.n. routes were 36 FFU and 2.1×10~5 FFU, respectively. These results suggested that substitution of Arg with Gln at G333 only eliminated the peripheral neuroinvasiveness of LEP by i.m. inoculation but not its lethal phenotype in adult mice by i.c. or i.n. inoculation. Further analysis revealed that the Gln (CAA) at G333 of rLEPG333Q completely reverted to Arg (CGA) in all mice after one i.c. inoculation. Passage of the virus in NA cells showed that the Gln (CAA) at G333 in rLEPG333Q was not stably maintained in NA cells in vitro, and partially mutated back to Arg (CGA) within five passages. These results indicated that rLEPG333Q could not stably maintain the GR333Q mutation in mouse neural tissue and NA cells. The virus reverted back to regain its neurotropism and highly pathogenic phenotype. In contrast, rHEP stably maintained Gln (CAG) at G333, in both in vivo infection in mice by i.c. inoculation and in vitro passaging in NA cells for up to five passages.
To investigate if a specific property of the G gene itself or if the genome background of LEP is responsible for the reversion of the Gln mutation at G333 to Arg, we constructed two chimeric viruses. The rHEP-G(L)333Q virus was generated by replacing the ORF of the G gene of HEP with that of LEP in which the amino acid at G333 was mutated from Arg to Gln. The neurotropism index of rHEP-G(L)333Q was 0 and was the same as that of rHEP. All mice inoculated with 105 FFU of rHEP-G(L)333Q survived the infection and did not show any signs of neural disease. Another chimeric virus, rLEP-G(H), was generated by replacing the G gene ORF of LEP with that of HEP. The titers of rLEP-G(H) in both NA and BHK-21 cells were lower than that of rLEP and similar to those of rHEP. The in vitro neurotropism index of rLEP-G(H) was 0. All mice inoculated with 105 FFU of rLEP-G(H) died within 12 days post-infection. Genome analysis revealed that G333 of rLEP-G(H) had changed to Arg (CGG) from Gln (CAG) in both in vivo infection in mice by i.c. inoculation and in vitro passaging in NA cells for up to five passages. In comparison, the Gln (CAA) at G333 of rHEP-G(L)333Q was stably maintained when propagated in mice brain or NA cells. These results strongly suggest that mutation from Gln to Arg at G333 does not happen randomly. Certain viral element(s) in the LEP genome backbone other than the G gene might be responsible for the instability of Gln mutation at G333.
The low fidelity of the RNA polymerase of negative-strand RNA viruses is the major reason for virus mutation. For this reason, we constructed another chimeric virus, rLEPG333Q-L(H), in which the ORF of L gene of rLEPG333Q was replaced with that of rHEP. Multi-step growth kinetics analysis showed that rLEPG333Q-L(H) replicated at a relatively lower rate, about 10-fold lower than that of the rLEPG333Q. All mice infected by the i.c. route with rLEPG333Q-L(H) survived and showed no signs of neural disease except slight loss of body weight. Genome analysis revealed the Gln (CAA) at G333 of rLEPG333Q-L(H) was stably maintained when propagated in mice brain or NA cells. These results indicated that the L protein is responsible for the stability of the Gln mutation at G333 of Flury RV.
Moreover, we employed the annexin V binding assay to investigate if the attenuation of Flury RV changed its ability to induce apoptosis in neural cells. The ability of the different RVs to induce apoptosis in infected NA cells at 24 hours post infection was compared. The percentage of cells that were positively stained by the FITC-labeled annexin V in rLEP- or rHEPG333R-infected NA cells was about 2.9±0.7% and 2.8±0.6%, respectively. These values were similar to that of uninfected cells (2.6±0.6%). In contrast, rHEP, rLEPG333Q, rHEP-G(L)333Q and rLEPG333Q-L(H) induced significantly higher levels of apoptosis in NA cells, and the percentage of the cells that were positively stained by FITC-labeled annexin V were 2.1, 2.4, 2.1, and 2.1 times higher respectively than that of the uninfected cells. Further, we compared the expression level of viral proteins in infected cells. The rLEP virus expressed similar amounts of G and N proteins to rLEPG333Q, rHEP-G(L)333Q and rLEPG333Q-L(H) in NA cells. Meanwhile, there was no significant difference in the expression level of G or N protein between NA cells infected by rHEP and rHEPG333R. These results indicated that the higher levels of early apoptosis in NA cells induced by the recombinant viruses containing G333Q may not be associated with an increase in the expression of G protein.
Two chimeric strains rHEP-G(L)333Q and rLEPG333Q-L(H) were highly attenuated and genetically stable. Immunization and challenge study results showed that rHEP-G(L)333Q or rLEPG333Q-L(H) induced significantly higher VNA responses and more effective protection than rLEP and rHEP, indicating two viruses would be able to serve as modified live vaccine candidates.
II.Development of improved rabies vaccines
Inactivated rabies vaccines have a distinct advantage in the biological safety, however can not be widely in developing countries especially in rural areas due to its high production cost and relatively short immunization persistent period. Improvement of vaccine antigen production efficiency will help to reduce production costs, while improve the immune efficiency and extend vaccination interval.
In this study, we generated a recombinant RV strain rLEP-G carrying double glycoprotein genes in the genome background of LEP strain. Biological analysis showed that introduction of an identical G gene did not affect virus replication in vitro and pathogenicity in adult mice, however, leaded to higher production of G protein in infected cells. The inactivated vaccine prepared from rLEP-G strain grown in BHK-21 cells produced higher VNA titer than that of wtLEP by intramuscular inoculation both in mice and dogs. The strong increase in immunogenicity in both mice and dogs makes rLEP-G strain a superb candidate for an inactivated RV vaccine.
I. RV Flury传代致弱的分子机制的研究
1940年,Koprowski等人从病死女孩脑内分离到狂犬病毒Flury株,经1日龄雏鸡脑内、鸡胚卵黄囊传后,再经鸡胚传代178代以上,无论神经内、外接种都对动物丧失致病力,但新生乳鼠和猴在脑内接种时仍有感染性。在鸡胚传代68代以前的称为“LEP”(鸡胚低代株);高于136代的称为“HEP”(鸡胚高代株)。LEP脑内感染成年小鼠致死,而HEP脑内感染成年小鼠不致死。造成LEP和HEP致病力生物学表型差异的分子机制仍未充分阐明。本研究拟就RV Flury传代致弱的分子机制进行系统研究。
对LEP和HEP完整基因组的系统解析表明,LEP和HEP的基因组均含有11,925个核苷酸,相似性达99.3%。LEP和HEP的L基因ORF核苷酸相似性最高为99.7%,N基因(99.6%)、P基因(99.4%)、M基因(99.3%)和G基因(98.9%)依次降低。两株病毒由N、P、M、G和L基因ORF预测的氨基酸序列相似性分别为99.8%、98.3%、99.0%、97.8%和99.6%。比较这两个毒株的5个蛋白的氨基酸序列,共有27个氨基酸发生改变,变化最大的是G蛋白,最保守的是N蛋白。N/P、P/M和M/G间的基因间隔序列完全一致,G/L间的间隔序列仅有一个碱基发生突变。另外,3’、5’端的非编码区及所有基因的转录起始信号和终止信号序列也都一致。
在此基础上,建立了Flury LEP和HEP的反向遗传操作系统。从cDNA克隆拯救获得的rLEP在小鼠神经母细胞(NA)和BHK-21细胞上的多步生长动力学曲线与野生型毒株相似,最高生长滴度分别可达3.0×10~8 FFU/ml和5.0×10~7 FFU/ml,体外嗜神经指数为0.78。rLEP通过脑内(i.c.)、滴鼻(i.n.)及肌肉注射(i.m.)不同途径分别感染成年小鼠均致死,LD50分别1 FFU,474 FFU和3.4×10~5 FFU,与野生型毒株相近。从cDNA克隆拯救获得的rHEP在NA和BHK-21细胞上的多步生长动力学曲线与野生型毒株相似,最高生长滴度均可达2.0×10~7FFU/ml,不具备体外嗜神经性,通过i.c.、i.n.及i.m.不同途径分别感染成年小鼠均不致死,表现出与野生型HEP相似的生物学特性和致病力。
将HEP的G333位的Gln突变为Arg,形成的突变株rHEPG333R体外嗜神经指数由0变为0.8,与LEP相近,表明rHEPG333R已经回复了嗜神经特性。通过i.c.,i.n.和i.m.3种不同途径分别感染小鼠,rHEPG333R均可使小鼠致死,LD50分别为0.3 FFU、887 FFU和1.4×10~6 FFU。结果表明,G333位点的Arg单个氨基酸突变足以使HEP重新获得对成年小鼠的高致病力。
但是,将LEP的G333位点由Arg突变为Gln后,形成的rLEPG333Q突变株尽管体外嗜神经指数下降为0,肌肉接种小鼠全部存活并且无神经症状出现,但通过i.c.或i.n.途径感染小鼠时,rLEPG333Q对小鼠仍显示出高度致死性,LD50分别为36 FFU和2.1×10~5 FFU。结果提示,G333 Arg的替换突变消除了LEP的嗜神经性和外周神经侵入能力,但i.c.或i.n.感染成年小鼠仍保持其高致死力。进一步对rLEPG333Q接种致死小鼠脑内病毒基因组序列进行分析,结果发现,rLEPG333Q在小鼠脑内和NA细胞中复制时,G333位点的Gln人工突变不能保持稳定,发生了嗜神经性的回复突变,因而重新获得对小鼠的致死能力。相反,rHEP无论经i.c或i.n.接种,还是NA细胞连续传代,G333位点的Gln均未发生回复突变。
为了澄清影响G333Q突变在神经组织或细胞复制过程中遗传稳定性的因素,进一步构建了嵌合病毒rHEP-G(L)333Q和rLEP-G(H),前者通过将HEP的G基因替换为LEP的G基因,同时G333位点Arg突变为Gln构建而成的,后者通过将LEP的G基因替换为HEP的G基因构建而成的。rHEP-G(L)333Q体外嗜神经指数也和rHEP一样,仍然为0;经i.c.感染小鼠不致死,病毒在小鼠脑内和NA细胞上复制时G333位点的Gln稳定不变。而嵌合病毒rLEP-G(H),在NA细胞上的生长性能显著地低于rLEP,而与rHEP十分接近;体外嗜神经指数也下降为0。尽管如此,小鼠经i.c.接种105 FFU的rLEP-G(H)后,在感染后12天内全部死亡。提取濒死小鼠脑组织RNA和NA细胞传代的第5代培养物进行RT-PCR和测序分析发现,rLEP-G(H)的G333位点Gln(CAG)已经突变为Arg(CGG)。结果表明,造成rHEP和突变株rLEPG333Q在神经组织或细胞中复制时G333位点Gln的遗传稳定性存在差异是LEP基因组骨架上的某个(或某些)因素而不是G蛋白本身引起的。
负链RNA病毒的RNA聚合酶的低保真性是病毒突变最主要的原因。为此,将rLEPG333Q的L基因替换为HEP的L基因,构建了嵌合病毒rLEPG333Q-L(H)。成年小鼠脑内接种105 FFU的rLEPG333Q-L(H)全部存活且没有出现神经症状,rLEPG333Q-L(H)的G333位点Gln(CAA)在鼠脑和NA细胞传代时稳定不变。结果表明,L蛋白与G333位点的Gln突变的遗传稳定性密切相关。L基因与G基因的协同变异、进化导致Flury在鸡胚传代过程获得致弱。
同时,我们还研究了GR333Q突变对Flury诱导细胞凋亡能力的影响。结果发现,rLEP和rHEPG333R感染的NA细胞中,NA细胞发生凋亡的比例分别为2.9±0.7%和2.8±0.6%,与空白对照组的细胞凋亡水平相似(2.6±0.6 %)。相比之下,rHEP、rLEPG333Q、rHEP-G(L)333Q和LEPG333Q-L(H)诱导的凋亡水平则高于对照组,分别为对照组的2.1、2.4、2.1和2.1倍。进一步利用western blot分析比较在病毒感染的细胞中不同蛋白的表达水平。结果发现,rLEP与rLEPG333Q、rHEP-G(L)333Q或rLEPG333Q-L(H)相比,G蛋白表达水平无明显差异;rHEP与rHEPG333R的G蛋白表达量也十分相近。结果显示,Flury病毒诱导NA细胞凋亡能力与G蛋白的表达量并无一定相关性。这一结果与前人报道G蛋白的表达量与凋亡诱导水平呈正相关的结果有所不同,提示狂犬病病毒诱导神经细胞凋亡的分子机制仍然有待进一步研究。
嵌合病毒rHEP-G(L)333Q和rLEPG333Q-L(H)高度致弱,失去嗜神经特性,并且遗传稳定。对小鼠的免疫和攻毒试验结果显示:rHEP-G(L)333Q和rLEPG333Q-L(H)免疫小鼠无论诱导的RV中和抗体水平,还是对街毒攻击的保护效力,均显著高于LEP和HEP,具有开发成为分子修饰弱毒疫苗的潜力。
II.狂犬病灭活疫苗的研制
RV灭活疫苗在生物安全性上具有突出优势,但是制造成本高,价格相对昂贵,免疫期相对较短,在发展中国家特别是农村地区难以推广应用。提高灭活疫苗抗原的单位生产效率,将有助于降低生产成本,同时提高免疫效力,延长疫苗接种间隔期。
本研究构建了表达双重G蛋白基因的重组RV Flury LEP病毒株rLEP-G。生物学特性分析显示,rLEP-G在NA和BHK-21细胞的生长滴度分别可达7.0×10~8 FFU/ml和1.0×10~8 FFU/ml,体外嗜神经指数为0.85,与LEP相近。rLEP-G通过i.c.接种感染小鼠的LD50为1 FFU,与LEP的致病性无显著差异。Western blot结果显示,rLEP-G感染BHK-21细胞后G蛋白的表达量显著提高3倍。分别以rLEP-G和LEP为种毒,按相同细胞培养量制备成灭活疫苗,对进行小鼠和比格犬的免疫效力试验。结果表明,无论小鼠还是比格犬,rLEP-G灭活疫苗诱导RV中和抗体的能力均显著高于LEP灭活疫苗,表现出良好的免疫原性,适用于狂犬病灭活疫苗的开发。
Rabies is an enzootic viral disease widespread throughout the world and causes severe destruction to the central nervous system. The annual number of human deaths worldwide caused by rabies is estimated to be 55,000, mostly in rural areas of Africa and Asia. To date, no effective medical therapy has been established for overt rabies. Once neurologic symptoms of the disease develop, rabies is fatal to both animals and humans. Vaccination is the only effective way to control rabies. Therefore, we should take a deep insight into the pathogenic and attenuation mechanism of rabies virus (RV) and development of new types of rabies vaccines.
I.Attenuation molecular basis of rabies virus Flury strain
Attenuated Flury RV low-egg-passage (LEP) and high-egg-passage strains (HEP) were established through serial passage in chicken brain, chicken embryos, and culture cells using a Flury RV isolated from a girl who died of rabies. LEP kills adult mice after intracerebral inoculation, while HEP causes only mild symptoms in adult mice. The molecular mechanisms associated with RV virulence are not fully understood. Therefore, the LEP and HEP strains, showing a high homology of the complete nucleotide sequence, appear to be useful for exploring the pathogenic and attenuation mechanism of RV.
Sequencing analyses revealed that the genomes of the LEP and HEP strains share 99.3% nucleotide sequence identity, and a deduced amino acid homology of 99.8%, 98.3%, 99.0%, 97.8%, and 99.6% between the individual N, P, M, G, and L proteins, respectively. The two strains have identical intergenic regions between N/P, P/M and M/G, and one substitution in G/L intergenic region. Sequences of the 3′and 5′terminal non-coding regions, which include the recognition and initiation site of the viral RNA polymerase, were completely conserved in the two viruses. A total of 27 amino acid substitutions were found between these two strains.
To investigate the molecular mechanism for virulence and attenuation of Flury RV, two infectious viruses, rLEP and rHEP, were rescued from the genomes of LEP and HEP respectively. The growth curves of the rLEP and rHEP strains were similar to wildtype LEP and HEP strains respectively, in both neuronal NA and non-neuronal BHK-21 cells. The rLEP and wildtype LEP strains appeared to be strongly neurotrophic having similar index values of 0.84 and 0.78, respectively. As expected, both rHEP and wildtype HEP did not show any neurotrophic characteristics. Virulence of rLEP, rHEP, wild LEP and HEP in adult mice was determined by different inoculation routes, intracerebral (i.c.), intranasally (i.n.), or intramuscularly (i.m.). The rLEP strain killed adult mice by any inoculation route. The MLD50 of rLEP after i.c., i.n. and i.m. inoculation were 1 FFU, 474 FFU and 3.4×105 FFU, respectively, which were comparable to those of wildtype LEP. In contrast, all mice survived from i.c., i.n. or i.m. inoculation, without exhibiting any neurological symptom except some slight body weight loss in the first few days post inoculation. These results suggested that the rescued viruses had similar biological properties and pathogenicities as their corresponding parental wildtype virus in adult mouse.
We generated a HEP mutant virus, rHEPG333R, in which the Gln at G333 was changed to Arg. The in vitro neurotropism index of rHEPG333R was significantly increased from 0 to 0.8, indicating that rHEPG333R had acquired the neurotropism property. Infection of adult mice confirmed that rHEPG333R was lethal. The MLD50 by i.c., i.n. and i.m. inoculation routines were 0.3 FFU, 887 FFU and 1.4×10~6 FFU, respectively. Theses results indicated that the mutation at G333 is sufficient to cause HEP to become highly pathogenic in adult mice.
However, rLEPG333Q, in which the Arg at G333 was changed to Gln, lost its neurotropism property and had a neurotropism index value of 0 in cell culture. Infection by the i.m. route with the maximum dosage of 3×10~6 FFU did not kill adult mice, and all surviving mice did not show any signs of neural disease. However, when inoculated by i.c. and i.n. routes, rLEPG333Q remained highly lethal in adult mice. The MLD50 by i.c. and i.n. routes were 36 FFU and 2.1×10~5 FFU, respectively. These results suggested that substitution of Arg with Gln at G333 only eliminated the peripheral neuroinvasiveness of LEP by i.m. inoculation but not its lethal phenotype in adult mice by i.c. or i.n. inoculation. Further analysis revealed that the Gln (CAA) at G333 of rLEPG333Q completely reverted to Arg (CGA) in all mice after one i.c. inoculation. Passage of the virus in NA cells showed that the Gln (CAA) at G333 in rLEPG333Q was not stably maintained in NA cells in vitro, and partially mutated back to Arg (CGA) within five passages. These results indicated that rLEPG333Q could not stably maintain the GR333Q mutation in mouse neural tissue and NA cells. The virus reverted back to regain its neurotropism and highly pathogenic phenotype. In contrast, rHEP stably maintained Gln (CAG) at G333, in both in vivo infection in mice by i.c. inoculation and in vitro passaging in NA cells for up to five passages.
To investigate if a specific property of the G gene itself or if the genome background of LEP is responsible for the reversion of the Gln mutation at G333 to Arg, we constructed two chimeric viruses. The rHEP-G(L)333Q virus was generated by replacing the ORF of the G gene of HEP with that of LEP in which the amino acid at G333 was mutated from Arg to Gln. The neurotropism index of rHEP-G(L)333Q was 0 and was the same as that of rHEP. All mice inoculated with 105 FFU of rHEP-G(L)333Q survived the infection and did not show any signs of neural disease. Another chimeric virus, rLEP-G(H), was generated by replacing the G gene ORF of LEP with that of HEP. The titers of rLEP-G(H) in both NA and BHK-21 cells were lower than that of rLEP and similar to those of rHEP. The in vitro neurotropism index of rLEP-G(H) was 0. All mice inoculated with 105 FFU of rLEP-G(H) died within 12 days post-infection. Genome analysis revealed that G333 of rLEP-G(H) had changed to Arg (CGG) from Gln (CAG) in both in vivo infection in mice by i.c. inoculation and in vitro passaging in NA cells for up to five passages. In comparison, the Gln (CAA) at G333 of rHEP-G(L)333Q was stably maintained when propagated in mice brain or NA cells. These results strongly suggest that mutation from Gln to Arg at G333 does not happen randomly. Certain viral element(s) in the LEP genome backbone other than the G gene might be responsible for the instability of Gln mutation at G333.
The low fidelity of the RNA polymerase of negative-strand RNA viruses is the major reason for virus mutation. For this reason, we constructed another chimeric virus, rLEPG333Q-L(H), in which the ORF of L gene of rLEPG333Q was replaced with that of rHEP. Multi-step growth kinetics analysis showed that rLEPG333Q-L(H) replicated at a relatively lower rate, about 10-fold lower than that of the rLEPG333Q. All mice infected by the i.c. route with rLEPG333Q-L(H) survived and showed no signs of neural disease except slight loss of body weight. Genome analysis revealed the Gln (CAA) at G333 of rLEPG333Q-L(H) was stably maintained when propagated in mice brain or NA cells. These results indicated that the L protein is responsible for the stability of the Gln mutation at G333 of Flury RV.
Moreover, we employed the annexin V binding assay to investigate if the attenuation of Flury RV changed its ability to induce apoptosis in neural cells. The ability of the different RVs to induce apoptosis in infected NA cells at 24 hours post infection was compared. The percentage of cells that were positively stained by the FITC-labeled annexin V in rLEP- or rHEPG333R-infected NA cells was about 2.9±0.7% and 2.8±0.6%, respectively. These values were similar to that of uninfected cells (2.6±0.6%). In contrast, rHEP, rLEPG333Q, rHEP-G(L)333Q and rLEPG333Q-L(H) induced significantly higher levels of apoptosis in NA cells, and the percentage of the cells that were positively stained by FITC-labeled annexin V were 2.1, 2.4, 2.1, and 2.1 times higher respectively than that of the uninfected cells. Further, we compared the expression level of viral proteins in infected cells. The rLEP virus expressed similar amounts of G and N proteins to rLEPG333Q, rHEP-G(L)333Q and rLEPG333Q-L(H) in NA cells. Meanwhile, there was no significant difference in the expression level of G or N protein between NA cells infected by rHEP and rHEPG333R. These results indicated that the higher levels of early apoptosis in NA cells induced by the recombinant viruses containing G333Q may not be associated with an increase in the expression of G protein.
Two chimeric strains rHEP-G(L)333Q and rLEPG333Q-L(H) were highly attenuated and genetically stable. Immunization and challenge study results showed that rHEP-G(L)333Q or rLEPG333Q-L(H) induced significantly higher VNA responses and more effective protection than rLEP and rHEP, indicating two viruses would be able to serve as modified live vaccine candidates.
II.Development of improved rabies vaccines
Inactivated rabies vaccines have a distinct advantage in the biological safety, however can not be widely in developing countries especially in rural areas due to its high production cost and relatively short immunization persistent period. Improvement of vaccine antigen production efficiency will help to reduce production costs, while improve the immune efficiency and extend vaccination interval.
In this study, we generated a recombinant RV strain rLEP-G carrying double glycoprotein genes in the genome background of LEP strain. Biological analysis showed that introduction of an identical G gene did not affect virus replication in vitro and pathogenicity in adult mice, however, leaded to higher production of G protein in infected cells. The inactivated vaccine prepared from rLEP-G strain grown in BHK-21 cells produced higher VNA titer than that of wtLEP by intramuscular inoculation both in mice and dogs. The strong increase in immunogenicity in both mice and dogs makes rLEP-G strain a superb candidate for an inactivated RV vaccine.
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