Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.     

乙脑/登革4型嵌合病毒的构建及其小鼠神经毒力测定

目的构建以乙型脑炎病毒(Japanese encephalitis virus,JEV)疫苗株SA14-14-2为基因骨架的乙脑/登革4型嵌合病毒,并分析该嵌合病毒对小鼠的神经毒力。方法通过重叠PCR方法扩增含有登革病毒4型(DENV-4)H241株pr ME基因序列和乙型脑炎病毒疫苗株SA14-14-2的NS1蛋白前177个核苷酸的融合片段,用Nar I和Bgl II双酶切后替换乙型脑炎病毒疫苗株SA14-14-2全长克隆中的相应区域,构建成乙脑/登革4型嵌合全长克隆,通过体外转录和转染BHK21细胞获得嵌合病毒(JEV/DENV-4 chimeric virus,JD4)。通过测定嵌合病毒JD4和2个母本株JEV SA14-14-2株及DENV-4 H241株蚀斑大小、小鼠脑内神经毒力和皮下感染入脑能力、乳鼠脑内神经毒力,比较JD4和母本株之间的差异。通过将JD4在原代地鼠肾(primary hamster kidney,PHK)细胞传代30次,分析传代后嵌合病毒的神经毒力是否减弱及减弱的程度。结果测序结果表明,构建的嵌合病毒JD4基因组序列和预期一致,没有产生新的位点突变。JD4蚀斑较SA14-14-2明显偏小,但和DENV-4 H241株没有明显区别。JD4对3周龄小鼠具有较强的脑内神经毒力,和母本株DENV-4 H241没有差异,对小鼠没有神经侵袭力。乳鼠实验结果表明,嵌合病毒JD4脑内神经毒力虽然略低于母本株DENV-4 H241,但两者之间没有明显差异,都明显强于乙脑疫苗株SA14-14-2。在PHK细胞传代30次后,小鼠神经毒力虽然有所减低,但并不明显。结论成功构建了嵌合病毒JD4,通过测定并比较JD4与母本株的蚀斑特征、小鼠及乳鼠神经毒力等试验,为分析登革疫苗候选株安全性研究奠定了基础。     

中国脊髓灰质炎Ⅱ型疫苗相关分离株病毒性状的观察

对１９９４年中国分离的１３株脊髓灰质炎Ⅱ型疫苗相关株进行了ＰＣＲ－ＲＦＬＰ分析,发现７株为重组病毒,毒力较疫苗株有回复,在Ⅱ型脊髓灰质炎病毒基因序列上,对于神经毒力有重要影响的第４８１位核苷酸发生突变,另一个被视为重要位点的２９０８位核苷酸无一发生变化,反而在２９０９位核苷发生了高频率的点突变,意味着２９０９位点在中国Ⅱ型疫苗相关株的自然变异中可能起着重要作用。     

Yellow fever vaccine: Comparison of the neurovirulence of new 17D-204 Stamaril™ seed lots and RK 168-73 strain

The neurovirulence of two new candidate 17D-204 Stamaril? working seed lots and that of two reference preparations were compared. The Stamaril? working seed lots have been used for more than twenty years for the manufacturing of vaccines of acceptable safety and efficacy. The preparation designated RK 168-73 and provided by the Robert Koch Institute was used as a reference. It was confirmed that RK 168-73 strain was not a good virus control in our study because it has a very low neurovirulence regarding both the clinical and histopathological scores in comparison with Stamaril? strain and is not representative of a vaccine known to be satisfactory in use. The results were reinforced by the phenotypic characterization by plaque assay demonstrating that RK 168-73 was very different from the Stamaril? vaccine, and by sequencing results showing 4 mutations between Stamaril? and RK 168-73 viruses leading to amino acid differences in the NS4B and envelop proteins.     

口服脊髓灰质炎减毒活疫苗猴体神经毒力试验参考品合格标准的统计分析

验证口服脊髓灰质炎减毒活疫苗的猴体神经毒力试验参考品合格标准是否发生改变及其对疫苗猴体神经毒力试验检测结果的影响。按照WHO推荐的猴体神经毒力中枢神经系统病理学检定方法,统计分析了1996~2002年中猴体神经毒力试验三个型参考品各五批以上的实验数据。并对Ⅰ、Ⅱ、Ⅲ型参考品的病变好发部位腰、颈、脑的病变分数进行了比较。结果表明,Ⅰ、Ⅱ型参考品合格标准上、下限及C值有所下降;Ⅲ型参考品合格标准上、下限及C值略有升高。从病理学记分结果显示,三型参考品在腰、颈、脑部的致病力存在着差异;Ⅲ型参考品的致病变力向颈、脑部位扩散的指数明显增大。Ⅰ、Ⅱ型参考品合格标准对疫苗猴体神经毒力试验合格界线趋于严格,而Ⅲ型参考品猴体神经毒力病变指数由腰部向颈、脑部蔓延趋势的增大导致病变扩散指数和强度指数增大,从而使Ⅲ型参考品合格标准对疫苗的猴体神经毒力试验合格界线范围趋大,有可能对Ⅲ型疫苗制品合格率增大。     

转基因小鼠用作脊髓灰质炎活疫苗神经毒力实验动物模型的研究

选用Ⅲ型脊髓灰质炎病毒,３批活疫苗样吕（ＷＨＯ／Ⅲ参考制品,９３／３６３和３Ｊ两批猴体神经毒力实验不合格的疫苗）和１株标准强毒株（Ｌｅｏｎ）,脊髓内注入携带有人细胞脊髓灰质炎病毒受体基因的转基因小鼠（ＰＶＲＴｇ２１）。临床和组织病理学检查表明,Ｌｅｏｎ病毒的毒力极强,２．０ｌｏｇ１０ＴＣＩＤ５０可使１００％小鼠麻痹和死亡,ＷＨＯ／Ⅲ疫苗参考制品毒力最弱,５．５ｌｏｇ１０ＣＩＤ５０才能使８１．７％小     

A single nonsynonymous mutation on ZIKV E protein-coding sequences leads to markedly increased neurovirulence in vivo

Zika virus (ZIKV) can infect a wide range of tissues including the developmental brain of human fetus. Whether specific viral genetic variants are linked to neuropathology is incompletely understood. To address this, we have intracranially serially passaged a clinical ZIKV isolate (SW01) in neonatal mice and discovered variants that exhibit markedly increased virulence and neurotropism. Deep sequencing analysis combining with molecular virology studies revealed that a single 67D (Aspartic acid) to N (Asparagine) substitution on E protein is sufficient to confer the increased virulence and neurotropism in vivo. Notably, virus clones with D67N mutation had higher viral production and caused more severe cytopathic effect (CPE) in human neural astrocytes U251 cells in vitro, indicating its potential neurological toxicity to human brain. These findings revealed that a single mutation D67N on ZIKV envelope may lead to severe neuro lesion that may help to explain the neurovirulence of ZIKV and suggest monitoring the occurrence of this mutation during nature infection may be important.     

乙型脑炎病毒野毒株及其不同株减毒株E蛋白基因序列比较

为了解乙脑减毒活疫苗株SA14-12-1-7的神经毒力减毒机 制,用RT-PCR方法分别扩增不同减毒程度毒株的E基因,克隆、测序,继而对各毒株序列进 行比较.结果表明SA14-12-1-7强毒株与SA14-12-1-7-12-1-7株间只有3个氨基酸发生改变( E-107,E-176,E-439),SA14-12-1-7-12-1-7与SA14-12-1-7-9-7和SA14-12-1-7-5-3 株间有另3个氨基酸发生改变(E-138,E-279,E-315).SA14-12-1-7-9-7株与SA14-12-1-7-5-3株只有一个核苷酸NT -405不同,但未引起氨基酸改变.SA14-12-1-7疫苗株除保留SA14-12-1-7-12-1-7和SA14-12-1-7-9-7所改变的6个氨基酸外,另有2个氨基酸发生了改 变(E-177,E-264),共计在E区共发生8个氨基酸的替代.SA14-12-1-7-12-1-7株的 低神经毒力很不稳定而其余各株的弱毒特征很稳定.因此,E-176(Ile→Val),E-439(Lys→Arg)和 E-107(Leu→Phe)可能与神经外和神经内毒力减弱有关.E-138(Gul→Lys),E-315(Ala→Val)和 E-279(Lys→Met)的突变可能与神经毒力的减弱和稳定性相关.     

乙型脑炎病毒野毒株及其不同株减毒株E蛋白基因序列比较

为了解乙脑减毒活疫苗株SA14 14 2的神经毒力减毒机制 ,用RT PCR方法分别扩增不同减毒程度毒株的E基因 ,克隆、测序 ,继而对各毒株序列进行比较。结果表明SA14 强毒株与SA14 12 1 7株间只有 3个氨基酸发生改变 (E 10 7,E 176 ,E 4 39) ,SA14 12 1 7与SA14 9 7和SA14 5 3株间有另 3个氨基酸发生改变 (E 138,E 2 79,E 315 )。SA14 9 7株与SA14 5 3株只有一个核苷酸NT 4 0 5不同 ,但未引起氨基酸改变。SA14 14 2疫苗株除保留SA14 12 1 7和SA14 9 7所改变的 6个氨基酸外 ,另有 2个氨基酸发生了改变 (E 177,E 2 6 4 ) ,共计在E区共发生 8个氨基酸的替代。SA14 12 1 7株的低神经毒力很不稳定而其余各株的弱毒特征很稳定。因此 ,E 176 (Ile→Val) ,E 4 39(Lys→Arg)和E 10 7(Leu→Phe)可能与神经外和神经内毒力减弱有关。E 138(Gul→Lys) ,E 315 (Ala→Val)和E 2 79(Lys→Met)的突变可能与神经毒力的减弱和稳定性相关     

中国脊髓灰质炎病毒疫苗株基因特征及其神经毒力的初步观察

中国脊髓灰质炎病毒疫苗株普遍存在基因变异的现象,如重组,点突变等。我们选出１０株有代表性的变异株,在具有人脊灰病毒受体基因的转基因小鼠ＰＶＲ－Ｔｇ２１中做毒力分析,发现Ｓａｂｉｎ １基因型与野毒基因型的重组株显示很强的神经毒力,其ＰＤ５０ｉｎＴＣＩＤ５０值为４．５,而Ｓｂｉｎ１标准株的ＰＤ５０值大于８０。另外两株Ｉ型疫苗株只有关键性核苷酸位点发生突变,只在位点５２５－发生突变的一株,其ＰＤ５０值为