重新评价G-480位点变异对Ⅰ型脊髓灰质炎疫苗病毒神经毒力的影响

在对分离于中国贵州省的9株Ⅰ型循环的疫苗衍生脊髓灰质炎病毒（cVDPVs）进行全基因组核苷酸序列分析后,发现已知最重要的决定病毒神经毒力的位点G-480和U-525并没有发生回复野生型突变;另外一些已知的神经毒力决定位点,如A-2438、A-2795、C-6203和G-7441等均已经发生了回复野生型突变。根据核苷酸序列的不同,从9株Ⅰ型cVDPVs毒株中选取5株病毒感染转人脊髓灰质炎病毒受体基因的小鼠进行神经毒力实验,发现它们的神经毒力都有所升高,其中CHN8184株和CHN8229-1．1株的神经毒力已经十分接近P1／Mahoney株,CHN8229-1．1株、CHN8229-2株和CHN8229-3株神经毒力依次递减,但仍处于较高水平,在它们的全基因组中分别只有7个和2个核苷酸的差异,而毒力却相差很多,提示有新的未鉴别的神经毒力决定位点的存在。对这些毒株5′非编码区（5′NCR）的第Ⅴ结构域进行二级结构预测,发现它们的二级结构很稳定。在G-480位点没有发生回复突变的情况下,部分毒株的神经毒力已经非常接近P1／Mahoney的水平,提示先前的研究中关于G-480突变对Ⅰ型脊髓灰质炎病毒神经毒力的作用可能被估计过高,G-480位点不是唯一重要的神经毒力决定位点,可能多个核苷酸联合突变才能达到减毒的效果。要真正全面了解P1／Sabin株的减毒机制,还需要进行更加深入的研究。     

Ⅰ型脊髓灰质炎病毒壳蛋白肽图谱分析

脊髓灰质炎病毒三个血清型的野毒株与疫苗株的全基因序列业已测出,有学者比较发现,疫苗株病毒在减毒过程中有许多基因位点发生了突变。本文用单向肽图谱分析法,对Ⅰ型脊髓灰质炎病毒野毒株(Mahoney株)和减毒株(Sabin Ⅰ株)的外壳蛋白VP1,VP2,VP3分别作了比较分析。结果发现四个有差     

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。     

急性弛缓性麻痹聚集病例病原毒力和分子特征分析

为探索脊灰病毒型内重组、位点突变等分子特征与神经毒力的关系,对2002年6月四川省攀枝花地区发生的急性弛缓性麻痹(acute flaccid paralysis,AFP)聚集病例的分离病毒株进行了转基因小鼠(PVR-Tg21 mice)神经毒力实验和全基因组核苷酸序列测定。结果显示:所测4株分离病毒(均为Ⅱ型脊灰病毒)神经毒力没有明显升高。4株病毒基因全长都是7 439bp,编码区翻译的氨基酸全长2 207aa。4株病毒的VP1区核苷酸序列完全相同。6208和6235c两株病毒是由Ⅱ型和Ⅲ型脊灰病毒在3A区重组而来,6209和6236两株病毒是由Ⅱ型和Ⅰ型脊灰病毒在2C区重组而来。4株病毒在5'非编码区nt481和VP1区的nt2 909两个强减毒位点都发生回复突变,6209和6236两株病毒重组的SabinⅠ的重要减毒位点nt6 203亦发生回复突变。结合神经毒力实验和序列分析结果可以确定脊灰病毒的型别间的重组与神经毒力没有直接关系,同时可以推论SabinⅢ的nt5 832,SabinⅡ的nt1 450和nt2 386这3个位点可能是毒力相关位点。     

脊髓灰质炎病毒Sabin株减毒的遗传学基础的研究进展

用Ｓａｂｉｎ株生产的口服脊髓灰质炎减毒活疫苗（ＯＰＶ）由１、２、３型组成,疫苗株是由强毒株经连续传代突变而来,对每种疫苗株而言只有少数位点与减毒有关,特别是５′端非编码区的第５个茎 环结构。现已证实Ｓａｂｉｎ株病毒在人体中复制时由于病毒的回复突变或第２位点的抑制突变导致毒力回升,可能在疫苗接种者和接触者中引发脊髓灰质炎病例。用聚合酶链式反应和限制性内切酶酶切技术作突变分析（ＭＡＰＲＥＣ）对疫苗中的痕量回复突变进行定量分析已成为可能,正逐步用于从分子水平监测ＯＰＶ生产的连续性,成为评价３型ＯＰＶ质量的一个重要指标。     

乙型脑炎病毒减毒中间株SA14—12—1—7基因组全序列的测定

本研究通过对乙型脑炎活疫苗减毒过程中间株SA14 12 1 7株进行全序列测定和分析 ,进一步了解乙脑活疫苗减毒及其稳定性的分子机制。根据已发表的SA14 14 2株及SA14 株的序列 ,设计 6对重叠引物 ,涵括整个乙脑病毒的基因组 ,通过RT PCR扩增出SA14 12 1 7株的各cDNA片段 ,分别克隆到pGEM T载体 ,转化至TG1受体菌中 ,挑取阳性克隆进行鉴定后测序。结果表明SA14 12 1 7株基因组全序列长 10 976个核苷酸 ,从 96到 10 394为一个长开放读码框 ,编码 3432个氨基酸。与野毒株SA14 和疫苗株SA14 14 2的核苷酸序列和氨基酸序列相比 ,同源性均在 99%以上 ,突变位点分散于各个区域 ,E区有 5个位点与疫苗株一致而与野毒株不同 ,3个位点与野毒株一致而与疫苗株不同 ,推测与其容易产生回复突变、恢复毒力有关。此外 ,NS3、NS5和 3′NTR的几个位点可能与病毒毒力稳定性相关。综上所述 ,乙脑病毒减毒中间株的基因组全序列基本类似于已发表的序列 ,若干突变位点影响病毒的弱毒性及毒力的稳定性。全序列的测定对于研究疫苗株的减毒机理具有重要意义     

2010年山东省环境污水中脊髓灰质炎病毒的监测及其基因特征分析

本研究在山东省开展了脊髓灰质炎病毒(Poliovirus,PV)的外环境监测,从济南、临沂两地采集污水标本,浓缩处理后进行病毒分离,对分离到的PV采用中和试验进行血清定型,并对其VP1及3D区进行序列测定,分析其基因突变和重组情况。2010年,共采集污水标本32份,PV阳性10份,阳性率31.3%;分离到18株PV(PV1型3株,PV2型9株,PV3型6株),均为疫苗相关株,VP1完整编码区核苷酸变异数在0~4个之间,在3株PV2型病毒和4株PV3型病毒的基因组中发现重组;对VP1区影响神经毒力的减毒位点分析发现,PV1型病毒中有1株在nt 2 749发生突变(A→G),PV2型病毒中有1株在nt2 908发生A→G突变,3株在nt2 909发生U→C突变,6株PV3型病毒全部在nt2 493发生C→U突变。环境污水中可以分离到PV,其基因重组率和主要减毒位点的回复突变率较高,未发现脊灰野毒株和疫苗衍生株脊灰病毒(Vaccine-derived poliovirus,VDPV)。     

云南省疫苗衍生脊髓灰质炎病毒全基因组序列遗传特征分析

为了解云南省疫苗衍生脊髓灰质炎病毒(Vaccine-derived Poliovirus,VDPV)的基因组特征,对2010年及2012年监测到的4株VDPV进行全基因组序列测定。结果显示,2株Ⅱ型VDPV的基因组全长均为7439nt,与Sabin Ⅱ疫苗株全基因组核苷酸和氨基酸的序列同源性分别为95.4%和97.7%;2株I型VDPV基因组全长均为7441nt,与Sabin I疫苗株全基因组核苷酸和氨基酸序列的同源性分别为93.9%和97.9%。减毒位点分析发现II型和I型VDPV毒株分别有两个(nt 481和nt 2909)和三个减毒位点(nt 480、nt 2795和nt 6203)发生了回复突变。VP1序列分析显示II型和I型VDPV毒株与相应Sabin株的变异分别为1%和2.3%,重组分析显示II型和I型VDPV的基因组结构分别为S2/S3和S1/S2/S1/S3,后者的重组次数高达3次,显示了重组的普遍性和复杂性,也表明了病毒在人体内复制和传播的持久性与重组的多样性成正相关。因此,从分子水平分析VDPV的特性,可掌握病毒的变异动态,为制定科学可行的VDPV控制策略提供理论依据。     

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     

乙型脑炎病毒E264位点回复突变对疫苗安全性的影响

目的通过反向遗传学技术,构建乙型脑炎病毒减毒疫苗株SA14-14-2囊膜蛋白(envelope protein,E蛋白)第264位氨基酸的回复突变株,并分析该位点突变对疫苗株小鼠毒力的影响。方法通过融合PCR扩增含基因组第1 769核苷酸位点回复突变的基因片段(T→G),替换SA14-14-2相应区域,使得编码E264位点的氨基酸位点由SA14-14-2的组氨酸(H)回复突变成强毒株SA14的谷氨酰胺(Q),将该回复突变株命名为r JEV264(H→Q),通过测定蚀斑大小和一步生长曲线,分析r JEV264的增殖特征;通过测定该突变株的昆明种小鼠脑内神经毒力、皮下感染入脑能力、腹腔感染入脑能力,分析E264位点回复突变后对小鼠毒力的影响。结果成功构建了回复突变株r JEV264,该突变株蚀斑大小与SA14-14-2相似,在PHK细胞上的增殖特征与SA14-14-2没有明显差异。r JEV264对小鼠有可检测的脑内神经毒力,毒力为5.51 lg PFU/LD50,但无论是皮下注射还是腹腔注射,都没有使小鼠发病。结论在分子水平证明了E264位点是影响乙脑减毒活疫苗安全性的关键位点之一,E264位点的回复突变增强了SA14-14-2的小鼠脑内神经毒力,但没有增强小鼠神经侵袭力。     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418