水痘病毒野毒株及减毒株在恒河猴中的免疫原性

作者将30只恒河猴分为6组,分别接种1.0×10~4PFU水痘病毒OKA及KMcC野毒株、减毒株及灭活OKA株,3个月后再用同株或异株病毒加强免疫。OKA野毒株在HEL细胞传6代,Flow 5000细胞传9代;减毒株分别在HEL、豚鼠胚胎细胞、WI-38细胞及Flow5000细胞传11、12、2及5代。KMcC野毒株在WI-38细胞传11代;减毒株分别在WI-38细胞传40及50代。接种后所有动物均未见人水痘感染的临床症状。血标本、咽拭子及脑脊液病毒分离     

水痘减毒活疫苗病毒的单核苷酸多态性分析

水痘-带状疱疹病毒（varicella-zoster virus,VZV）是引起水痘和带状疱疹的主要病原体,Oka株水痘减毒活疫苗（V-Oka）能有效预防水痘发生。V-Oka株与Oka亲本株之间存在至少42个单核苷酸多态性（single nucleotide polymorphism,SNP）位点差异,部分位点与V-Oka株的减毒特性有关。目前已知有6个SNP位点为疫苗型位点,可用于区分V-Oka病毒与野生型病毒。近期研究发现某些SNP位点与疫苗相关皮疹有关。此文对水痘减毒活疫苗的重要SNP位点做一综述。     

水痘-带状疱疹病毒Oka株的减毒和实验室标志

作者从1例典型水痘患儿的水疱中分离出水痘-带状疱疹病毒(VZV)Oka株,在人胚肺(HEL)细胞内34℃传11代;在豚鼠胚成纤维(GPEF)细胞内37℃传12代;然后在人二倍体细胞内传代数次制成疫苗。从接种该减毒活疫苗的儿童所产生的水疱中分离了9株病毒。研究这些病毒的生物学和生物物理学特性,并与从水痘或带状疱疹患者中分离的VZV野毒株以及亲本Oka株作了比较。结果发现:(1)Oka疫苗株有较高的温度敏感性,在39℃和34℃所形成的蚀斑数之     

Oka疫苗株再活化可增强对水痘的免疫力

美国食品和药物管理局 Krause等的血清流行病学研究显示 ,水痘疫苗接种后血清抗体滴度降低时 ,Oka疫苗株可再活化 ,增强宿主对水痘病毒的免疫力。对野型病毒的再接触和潜伏疫苗病毒的再活化相结合可保持对野型水痘病毒的终身免疫力。　　Krause等分析了 46 31名儿童的血清抗体滴度和水痘感染率的资料。低滴度儿童抗体滴度每年平均增高 6 6 % ,而高滴度儿童抗体滴度每年降低 2 5 %。此外 ,低滴度儿童临床感染和免疫增强发生率明显较高 ,超过每年野型水痘病毒接触率 (1 3% ) ,符合疫苗病毒株再活化的假说。　　 Krause认为 ,这项研究可减轻…     

麻疹病毒的抗原性漂移

八十年代以来,一些国家分离的麻疹野毒株,其主要抗原与现行麻疹疫苗株Moraten (MOR)、Schwarz以及Edmonston(ED)株在核苷酸水平及氯基酸水平上有差异,发现麻疹野毒株 有抗原性漂移,这将为制定控制麻疹计划提供新动向。     

用人二倍体细胞制备新的流行性腮腺炎减毒活疫苗

以往制备的流行性腮腺炎疫苗主要是将病毒接种于鸡胚腔内,再经鸡胚细胞减毒培养而成。这些制品要求鸡蛋不能带有痕量的病原。本文作者旨在分离出流行性腮腺炎病毒,用人二倍体细胞(HDC)减毒制成HDC麻疹、流行性腮腺炎、风疹(MMR)联合疫苗。从患儿口腔中采集病毒,经绿猴肾细胞(GHKC)传3代适应,作为本试验野毒株。核株直接传入HDC中减毒,连续移种14代后作为弱毒株S-12用于疫苗。选用3株国际参考株来检测新分离病毒的特征和免疫原性及安全性。并作了改变培养温度、噬斑比     

狂犬病病毒的抗原变异株

抗狂犬病单克隆抗体可用以研究疫苗株与野毒株的抗原变异。业已研制成功单克隆核壳体(Nc)及糖蛋白(G)抗体。本试验所用的狂犬病毒株来自世界各地。31株来自欧洲,4株亚洲,15株美洲,20株非洲大陆及11株实验室减毒株及疫苗株,共81株病毒。除11株实验室试验毒株外,从狂犬病死者中分离     

麻疹病毒缺陷性干扰颗粒的产生及其在减毒活疫苗中的存在

五十年代后期,Enders等首次获得了麻疹病毒减毒株Edmonston,因这株病毒接种反应大而须进一步减毒,在此基础上又获得了3株减毒株(END、SCH和ZAG)。实验发现,病毒在未稀释的细胞培养液中连续传代,产生了同种缺陷性干扰(DI)颗粒,使病毒产量降低,影响了疫苗生产。为了解麻疹减毒活疫苗病毒减毒的机理及改进生产,作者选用3次蚀斑纯化的麻疹野毒株(HU2),在Vero细胞中连续传代3次,获得了无DI的野毒株(PP3)。以此为出发点,再用超声处理的高感染复数(MOI)的细胞培养液经多次连续传代,每代取样检查蚀斑形     

不同毒株对狂犬病疫苗小鼠效力试验的影响

作者按欧洲药典标准效力试验方法,用狂犬病病毒CVS、SAD和LEP株制备的三种疫苗对小鼠进行腹腔免疫后,用三种疫苗病毒株和一种狐狂犬病野毒株(GS7)作交叉攻击试验.结果表明,各免疫组对GS7的攻击保护性最好;其次,对同源毒株的攻击保护性亦好.将上述三种疫苗5倍稀释后分别对小鼠进行腹腔免疫,14天后采血.用小鼠中和试验(MNT)或快速荧光灶抑制试验(RFFIT)测定抗体.发现CVS疫苗免疫动物所获得的抗血清对同源毒株的中和效价(ED_(50))较     

B亚群呼吸道合胞病毒活疫苗在动物中的遗传学稳定性和免疫原性

作者将B亚群呼吸道合胞病毒(RSV)野毒株(wt)经不同细胞传16代,又分别在32～21℃和26～22℃冷传代(cp)12和29代或再用5氟尿嘧啶诱变后空斑纯化,获得包括温度敏感(ts)和非ts(宿主范围)等多种减毒突变株,研究了这些突变株在动物中的复制水平、免疫原性和遗传稳定性.首先观察了wt和cp株于32℃、39℃和42℃时在Vero或HEp-2细胞上的空斑表型和形成效力,wt株皆相等,而cp-23和cp-52的空斑在39℃和40℃时比32℃时缩小50%以上.棉鼠鼻腔接种不同病毒株后在肺和鼻甲中的病毒效价(log_(10)pfu/g),wt分别为5.4～5.8和4.7～5.1,cp-12、cp-23和cp-52的效价分别下降30和100倍、400和500倍、≥20000和1000倍.冷传代次数增加,对病毒的复制限制亦增强.cp-52的减毒表型在用环磷酰胺免疫抑制的棉鼠中长期(14天)     

Comparison between of the attenuated BR-Oka and the wild type strain of Varicella Zoster Virus (VZV) on the DNA level

Oka strain VR-795 (Varicella Zoster Virus, VZV) of American Type Culture Collection (ATCC) has been used for chickenpox vaccine production. In order to use this strain for vaccine production, the strain must be identified and its stability must be confirmed.The identification of the Oka strain has been confirmed using Restriction Fragment Length Polymorphism (RFLP) and DNA sequence analysis of glycoprotein-II (gp-II). The amino acid sequences of Oka deduced from the DNA sequence of gp-II have changed at three amino acids against Ellen and at one amino acid against Webster. To prove the stability of the Oka strain during the passage, RFLP and DNA sequence analyses were also used with 11, 15 and 23 times of virus passage. We found that the Oka strain was stable at passages of up to 23 times, based on the RFLP and DNA sequence analyses. The confirmed Oka strain was renamed as BR-Oka for the purposes of chickenpox vaccine production.     

Susceptibility of protein kinase (ORF47)-deficient varicella-zoster virus strains to anti-herpesvirus nucleosides

To clarify whether varicella-zoster virus (VZV) protein kinase (PK; ORF47) takes part in phosphorylation of anti-herpesvirus nucleosides, thymidine kinase (TK) deficient, and PK/TK double deficient recombinant VZV strains were isolated and their susceptibility, and that of wild type and PK-deficient strains to various nucleoside analogs was evaluated. The PK-deficient VZV strains showed a sensitivity equal to that of the wild type strain against all compounds tested, including ganciclovir. This indicates that PK is not involved in phosphorylation of the tested nucleosides in VZV-infected cells.     

Novel agents for the therapy of varicella-zoster virus infections

Varicella-zoster virus (VZV), a member of the herpesvirus family, is responsible for both primary (varicella or chickenpox) as well as recurrent (zoster or shingles) infections. Acyclovir has been the mainstay for treating VZV infections in both immunocompetent and immunocompromised patients. Recently, newer anti-VZV drugs, i.e., valaciclovir (the oral prodrug form of acyclovir) and famciclovir (the oral prodrug form of penciclovir) have been developed and have enlarged the therapeutic options to treat VZV infections. Both acyclovir and penciclovir are dependent on the virus-encoded thymidine kinase (TK) for their intracellular activation. Although emergence of drug-resistant strains does not occur in immunocompetent patients, several reports have documented the isolation of drug-resistant VZV strains following long-term acyclovir therapy in immunocompromised patients. Mutations at the level of the TK are responsible for development of resistance to drugs that depend on the viral TK for their phosphorylation (i.e., acyclovir and penciclovir). Foscarnet, a direct inhibitor of the viral DNA polymerase, which does not require activation by the viral TK, is the drug of choice for the treatment of TK-deficient VZV mutants emerging under acyclovir therapy. Recently, emergence of foscarnet-resistant strains has also been reported. Both TK-deficient strains and foscarnet-resistant mutants are sensitive to the acyclic nucleoside phosphonate cidofovir, CDV, HPMPC, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine. This agent does not depend on the virus-encoded TK, but on cellular enzymes for its conversion to the diphosphoryl derivative, which then inhibits the viral DNA polymerase.     

Novel agents for the therapy of varicella-zoster virus infections

Varicella-zoster virus (VZV), a member of the herpesvirus family, is responsible for both primary (varicella or chickenpox) as well as recurrent (zoster or shingles) infections. Acyclovir has been the mainstay for treating VZV infections in both immunocompetent and immunocompromised patients. Recently, newer anti-VZV drugs, i.e., valaciclovir (the oral prodrug form of acyclovir) and famciclovir (the oral prodrug form of penciclovir) have been developed and have enlarged the therapeutic options to treat VZV infections. Both acyclovir and penciclovir are dependent on the virus-encoded thymidine kinase (TK) for their intracellular activation. Although emergence of drug-resistant strains does not occur in immunocompetent patients, several reports have documented the isolation of drug-resistant VZV strains following long-term acyclovir therapy in immunocompromised patients. Mutations at the level of the TK are responsible for development of resistance to drugs that depend on the viral TK for their phosphorylation (i.e., acyclovir and penciclovir). Foscarnet, a direct inhibitor of the viral DNA polymerase, which does not require activation by the viral TK, is the drug of choice for the treatment of TK-deficient VZV mutants emerging under acyclovir therapy. Recently, emergence of foscarnet-resistant strains has also been reported. Both TK-deficient strains and foscarnet-resistant mutants are sensitive to the acyclic nucleoside phosphonate cidofovir, CDV, HPMPC, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine. This agent does not depend on the virus-encoded TK, but on cellular enzymes for its conversion to the diphosphoryl derivative, which then inhibits the viral DNA polymerase.     

轮状病毒基因重配株Ls(G3型)在Vero细胞上的适应性培养及免疫原性分析

目的:研究轮状病毒基因重配株Ls(G3型)在Vero细胞上的适应性及其免疫原性。方法:将轮状病毒基因重配株Ls(G3型)在Vero细胞上连续传代20代,进行适应培养,通过RNA-PAGE、PCR、核酸电泳、病毒滴度及小鼠免疫试验鉴定其遗传稳定性和免疫原性。结果:筛选出1株Vero细胞适应株(Ls)。该毒株在Vero细胞上稳定传代20代,具有遗传稳定性,且自第5代后病毒滴度均稳定在7.0 lgTCID50.mL-1以上。动物免疫结果表明,Vero细胞适应株Ls诱导小鼠产生高滴度的血清抗体。结论:Ls株可在Vero细胞上稳定增殖,具有遗传稳定性和良好的免疫原性。     

Resistance testing of clinical varicella-zoster virus strains

Acyclovir resistance of varicella-zoster virus (VZV) has been reported in rare cases of immunocompromised patients. In this study, the natural polymorphism of the thymidine kinase (TK) and DNA polymerase (pol) genes was examined in 51 clinical VZV isolates sensitive to acyclovir (ACV). In addition, 16 VZV strains with clinical resistance to ACV were analyzed. None of the ACV-sensitive strains of the clades 1, 3 and 5 showed gene polymorphism of the TK. By contrast, the DNA pol gene exhibited polymorphism-related substitutions as a function of the VZV clade. The novel substitutions M286I, E824Q, R984H and H1089Y were detected in strains of clades 3 and 5. In the TK gene of 7 VZV strains with clinical ACV resistance, the novel substitutions L73I, A163stop, W225R, T256M, N334stop and the deletion of nucleotides 19-223 were found to be associated most likely with resistance. In one strain showing the substitution W225R, ACV resistance could be confirmed by the viral phenotype. In the DNA pol gene, the novel amino acid substitutions T237K and A955T could be detected, but their significance remains unclear. In conclusion, the characterization of resistance using genetic analysis of the TK and DNA pol genes has to be considered the method of choice for the determination of VZV resistance to antiviral drugs. In a considerable number of patients with clinical ACV-resistant VZV infections, resistance cannot be verified by virological methods.     

The occurrence of rare rpoB mutations in rifampicin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait

Mutations associated with rifampicin (RIF) resistance in two regions of the rpoB gene were studied by line probe (INNO-LiPA Rif. TB) assay (LiPA) and/or DNA sequencing in 32 RIF-resistant and 21 RIF-susceptible Mycobacterium tuberculosis strains isolated from 53 tuberculosis patients in Kuwait. The LiPA identified all susceptible strains as RIF sensitive, and the DNA sequences of the 81bp rifampicin resistance-determining region (RRDR) and the N-terminal region of the rpoB gene from five isolates were identical to the wild-type sequences from M. tuberculosis H(37)Rv. The LiPA identified 24 of 32 (75%) phenotypically documented RIF-resistant M. tuberculosis isolates as RIF resistant, with specific detection of mutation in 19 isolates, whilst 8 strains were identified as RIF susceptible. DNA sequencing of the RRDR confirmed the results for the 19 RIF-resistant isolates and identified precise mutations in five isolates for which specific base changes could not be determined by LiPA, as well as identifying a mutation (insertion 514TTC) in three isolates and no mutation in five of the eight isolates that were detected as RIF susceptible by LiPA. Two of the latter five isolates contained a V146F mutation in the N-terminal region of the rpoB gene and were recovered from patients of Middle Eastern origin. These analyses identified 11 different mutations in two regions of the rpoB gene. The majority of the isolates carrying identical or no mutations in the rpoB gene exhibited unique DNA banding patterns in fingerprinting studies. The occurrence of rare mutations in the rpoB gene, namely insertion 514TTC in 3 (9%) and V146F in 2 (6%) of 32 RIF-resistant M. tuberculosis isolates from Kuwait, is the highest reported so far.     

一株具有抗肿瘤活性的土壤放线菌的筛选与菌种鉴定

目的快速高效的筛选到有抗肿瘤活性的土壤放线菌。方法初筛采用MTT法检测样品作用后A549细胞、HGC-27细胞和正常对照细胞N9的成活率,PI染色流式细胞检测技术复筛可能致细胞凋亡的菌株,以及对活性菌株进行种属鉴定。结果与结论从350株土壤放线菌中初筛得到16株菌种,它们的代谢产物对A549及HGC-27细胞有抑制作用,但对N9细胞生长无影响;复筛得到1株编号为1070的菌株的代谢产物能使A549细胞经过处理后,经流式细胞仪检测到亚二倍体峰,峰高为32.07%。从菌株菌落形态、孢子丝形态、生理生化特性及16SrDNA序列、系统进化分析鉴定该菌种为Streptomyces capillispiralis。