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1.
目的 腮腺炎疫苗株S79工作种子噬斑纯化及全基因组测序,针对国内已经分离出的毒株,从分子生物学角度探讨疫苗保护力相关问题.方法 噬斑纯化S79疫苗株,利用RT-PCR分段扩增S79株两种亚株的全基因并测序,比较S79株与其来源株Jeryl Lynn(JL)核酸序列差异;从GenBank中获得WHO各基因型参考株和中国腮腺炎流行株疏水蛋白(SH)序列,分析病毒株和疫苗株遗传距离,并构建基因亲缘性关系树.结果 S79疫苗株由两种亚株构成,比例为2∶5(major∶minor),获得全基因序列并递交GenBank;与相应的JL株相比,核苷酸序列同源性分别为99.7%和100%.亚株序列中存在散在区段异源重组.S79疫苗株为A基因型,中国的流行株为F基因型,遗传距离为11.2%~20.0%.结论 S79疫苗株由两种亚株构成,两亚株的全基因序列与JL株基本一致,但亚株的比例与国际上应用的其他JL疫苗株存在较大差异.疫苗株与中国流行株不属于同一基因型,遗传距离较远.S79疫苗免疫保护效果的差异可能主要与疫苗株和流行株不属于同一基因型有关.  相似文献   

2.
目的 阐明吉林省流行麻疹野病毒的基因型别和基因特征,为制定麻疹防控策略措施提供科学依据.方法 用逆转录-聚合酶链反应.限制性片段长度多态性分析(RT-PCR-RFLP)方法对2001-2006年分离的38株麻疹病毒进行基因分型;选择麻疹病毒代表株RT-PCR扩增N基因C-末端450个核苷酸,对比野毒和疫苗株核苷酸和氨基酸同源性,并构建N基因亲缘关系树.结果 经RT-PCR-RFLP基因定型,38株麻疹病毒分离珠均为H1基因型;对其中的29株进一步进行N基因C-末端450个核苷酸的序列测定和分析证实均为H1基因型的H1a亚型.吉林分离株与我国麻疹疫苗S191株450个核苷酸同源性为88.0%~89.4%,所提示氨基酸的同源性为91.8%~92.7%;吉林省内分离株核苷酸平均变异小于1.4%.结论 H1a基因亚型为吉林省近年来流行的麻疹野病毒绝对优势基因亚型.不同年份存在相同麻疹病毒的持续循环传播,同一年份也存有H1a亚型内不同病毒的共循环;与其他省之间存在相同麻疹病毒引起的传播链.  相似文献   

3.
风疹病毒松叶株E1基因遗传变异研究   总被引:1,自引:0,他引:1  
目的 研究风疹病毒(rubella vires,RV)疫苗松叶株(Matsuba)El基因遗传与变异特点,在基因水平评估Matsuba株生产的疫苗对流行株的保护效果. 方法 将风疹病毒松叶株毒种RV14在原代兔肾细胞连续传至第23代,取RV14、RV16、RV17、RV18、RV23代进行研究分析.利用RT-PCR方法扩增松叶株不同代次的E1蛋白基因,将E1基因与pGEM-T载体连接,获得风疹病毒E1基因的克隆并测序,对各代次病毒的E1基因与风疹病毒疫苗Matanba株(GenBank登录号:D50673)及其他参考株的E1基因序列进行比对分析. 结果 风疹病毒Matsuba株传至23代时仍有很高的同源性,RV14、RV16、RV18代次病毒与D50673的核苷酸和氨基酸同源性为100.O%,RVl7、RV23代次病毒与D50673的核苷酸序列同源性分别为99.9%、99.7%,氨基酸序列同源性分别为99.8%、99.2%.各代次病毒与糖基化、抗原性相关的氨基酸位点未发生变异,高度保守;风疹病毒Matsuba株与其他参考株E1基因序列比较结果表明:Matsuba株为1a基因型,中国风疹病毒流行基因型为1E、1F、2A和2B,以1E基因型为主;Matsuba株与各基因型参考株同源性很高,核苷酸和氨基酸同源性分别为92.1%~99.6%和98.1%~99.8%. 结论 风疹病毒Matsuba疫苗株的遗传学特性稳定,从分子水平证明Matsuba疫苗株及其生产的疫苗具有安全性.尽管中国流行不同基因型风疹病毒,由于Matsuba株与各基因型参考株同源性很高,且风疹病毒毒株间抗原表位高度保守,因此Matsuba疫苗株所生产的疫苗可以有效保护不同基因型别的风疹病毒的感染.  相似文献   

4.
目的 构建含有adr、adw、ayw3个血清型的HBV全基因组质粒,并进行测序及序列分析。方法 从HBsAg携带者的血清中扩增出S区克隆,筛选出adr、adw、ayw血清型的血清,然后扩增HBV的全基因组,对扩增产物进行克隆测序。利用DNAStar的MegAlign,Phylogenetic tree对HBV全基因序列以及分段序列进行比较和进化树分析。结果 构建了含有adw、adr、ayw 3种血清型的HBV全基因组质粒,代码分别为:H1、H2、H3,对3株完成序列测定,按照S区核苷酸顺序划分基因型分别为B、C、D,全序列分型显示H1、H2为B、C基因型,与按S区的分型一致,但H3为C基因型,与按S区的分型不同,进一步的序列分析表明H3为一株异常的基因型,按照S区和preS2区分型为D基因型,但是全基因组/C/P/X区的进化树分析,H3株均位于C基因型,提示该异常的基因型可能是由于D/C基因型间基因重组引起,对重组位点进行了分析:3181nt-10nt、799-834nt为可能的重组位点所在区。结论 H3病毒株的发现进一步证明了HBV基因型间的基因重组,但澄清该问题需要在HBV感染的高流行区进一步的积累HBV异常基因型的全序列,以及发现新的基因型资料。  相似文献   

5.
目的 了解近三年北京市朝阳区流行的麻疹野病毒分子流行特征,为本地区消除麻疹提供科学依据.方法 采集北京市朝阳区2010-2012年麻疹疑似病例的咽拭子和(或)尿液标本接种vero/SLAM细胞,对分离的麻疹病毒用逆转录及聚合酶链反应扩增麻疹病毒核蛋白(N)基因羧基末端676个核苷酸片段,对扩增产物进行核苷酸序列测定和分析,并以羧基末端450个核苷酸片段构建基因亲缘关系树,进行遗传距离及核苷酸、氨基酸同源性分析.结果 分离出18株麻疹病毒,均为H1基因型,H1a亚型.这18株野毒株之间核苷酸同源性为98.2%~100.0%(核苷酸差异为0~8bp),氨基酸变异范围为0~2.7%(氨基酸差异为0~4AA).与H1a亚型参考毒株Chin9322相比,核苷酸变异范围为0.7%~1.6%(核苷酸差异为3 ~7 bp).18株野毒株共有7种基因序列,其中2010年5种、2011年1种、2012年3种基因序列.V2基因序列出现在2010和2012年的病例中,V7序列出现在2010和2011年的病例中.其余5种序列只在单一年份的病例中检出.结论 北京近年来流行的本土麻疹病毒为H1a亚型,当年相同基因序列的一组病例提示有麻疹传播链的存在.北京市朝阳区存在基因序列完全相同的麻疹野毒株在2010、2012年循环出现.  相似文献   

6.
目的 为获得2015-2016年度中国流行的甲型H1N1亚型流感病毒疫苗候选株,制备流感病毒重配株并对其进行鉴定.方法 采用经典重配的方法,将H1N1亚型流感病毒流行株与H3N2亚型的鸡胚高产重配母本株(X-157株)在鸡胚上进行混合培养.用H3亚型的HA蛋白抗血清和X-157株全病毒抗血清对混合培养病毒进行阴性筛选.阴性筛选后HA滴度较高的病毒用Real-Time PCR法对表面蛋白基因型进行鉴定.对表面蛋白基因型正确的毒株用限制性内切酶酶切鉴定法鉴定其内部基因组成.进一步对HA和NA基因进行Sanger法测序,并用表面基因无氨基酸位点突变的毒株免疫雪貂,进行双向血凝抑制(Two-way hemagglutination inhibition test,HI)试验.结果 Real-Time PCR筛选出5株表面蛋白基因型正确的毒株.经内部基因鉴定其中4株为6+2组成,1株为5+3组成.5株重配株的HA和NA基因均未发生氨基酸位点突变.5株重配株HA滴度均维持在1 024以上.最终选取的12号重配株免疫原性良好,HI滴度达5 120,双向HI试验均通过.重配后疫苗株在鸡胚上的产量是重配前野毒株的64倍.结论 成功制备了2015-2016年度中国流行的甲型H1N1亚型流感病毒疫苗株,为疫苗贮备和疾病防控奠定了基础.  相似文献   

7.
乙型肝炎病毒D基因型系统进化树分析   总被引:1,自引:0,他引:1  
目的:研究HBV D基因型不同毒株全基因进化关系。方法:用聚合酶链式反应(PCR)、克隆及核酸序列测定的方法,测定了1例中国人慢性无症状携带者感染的乙型肝炎病毒D基因型全基因序列。此D基因型毒株全基因序列GenBank Accession为AF280817,将GenBank中已发表的HBV D基因型30株的全序列进行了系统进化树分析。结果:中国株HBV D基因型与源于瑞典(Sweden)的4株HBV D基因型全基因的进化距离最近;地中海地区及欧洲国家系HBV D基因型分布的优势地域,亚洲并非HBV D基因型分布的优势地域。结论:HBV D基因型病毒株传入来源、迁移的方向不同以及病毒株在具有不同遗传和免疫特质的宿主中的长期选择是形成HBV D基因序列病毒进化差异的原因。  相似文献   

8.
目的 建立RT-PCR-RFLP方法 用于天津地区2002-2008年流行的麻疹野病毒基因型研究.方法 采集疑似麻疹患者的尿标本和咽拭子,传代细胞分离病毒.提取病毒液中的RNA,用一步RT-PCR法扩增麻疹病毒核蛋白(nucleoprotein,N)基因C端594个核苷酸片段,扩增产物经Bcn I酶切后琼脂糖凝胶电泳进行限制性片段多态性分析(RFLP),同时与序列分析进行对比验证.根据结果 构建基因系统发生树进行亲缘关系和遗传距离分析.结果 2002-2008年189份标本,分离获得69株麻疹病毒,N基因C端RT-PCR检测全部阳性,RFLP分析H1a是优势流行亚型,占98.55%(68/69),仅1株(1.45%)为H1b基因亚型,分型结果 与序列测定完全一致.系统发生树分析显示H1a亚型毒株分为2个亚枝,变异范围为0.2%~3.8%,存在不同病毒株引起的传播链共循环.结论 基于Bcn I限制性内切酶的RT-PCR-RFLP方法 能够特异性区分A基因型、H1a、H1b基因亚型,具有快速、简便、准确和经济的优点,更适用于国内大规模麻疹病毒的监测.  相似文献   

9.
目的 阐明人呼吸道合胞病毒(human respiratory syncytial virus,HRSV)A和B亚型病毒分离株的核蛋白(nucleoprotein,N)编码基因特征.方法 采用逆转录-聚合酶链反应(revelrse transciptionpolymerase chain reaction,RT-PCR)对北京2004年分离的HRSV代表株(2株A亚型和2株B亚型)进行N基因全长序列扩增、测序,并和GenBank下载的所有HRSV病毒的N基因全长序列进行对比和分析.结果 2株HRSV A亚型分离株与A亚型Long株(标准株)的N蛋白的核苷酸和氨基酸差异分别为36~40个(3.1%~3.4%)和4个(1.0%);2株HRSV B亚型分离株与B亚型CH18537株(标准株)的N蛋白之间的核苷酸和氨基酸差异分别为17(1.4%)和1(0.3%)个.4株HRSV代表株和从GenBank下载的HRSV的N蛋白之间的核苷酸和氨基酸差异分别为3~172个(0.25%~14.63%)和0-18个(0~4.6%).结论 N基因在HRSV基因组中是较为保守的基因,A或B亚型的型内差异相对较小;但A和B亚型之间N基因序列有较大变异,变异平均分配于整个N基因中;本研究为HRSV基因快速诊断试剂的研制提供了基因信息学数据.  相似文献   

10.
中国百日咳疫苗生产株S1和Prn蛋白基因序列分析   总被引:1,自引:0,他引:1  
目的了解我国百日咳疫苗生产株百日咳毒素S1和外膜蛋白(Prm)的基因特征。方法分别克隆疫苗株的S1和Prn基因至PGEM-Teasy载体、进行测序,并与GenBank中收录的百日咳杆菌S1和Prn蛋白基因序列进行比较、分析。结果完成了3株疫苗生产株S1和Prn基因的扩增、克隆和序列测定。序列分析显示3疫苗株间的S1和Prn基因核苷酸序列和氨基酸序列同源性均在99%以上,基因进化树显示与其他菌株的同源性也较高。结论对我国3株疫苗生产株S1和Prn基因序列进行了详细地分析,为我国百日咳流行病学的研究和疫苗的质量控制、研制奠定了基础。  相似文献   

11.
目的检测昆明地区麻疹病毒的优势基因型,为麻疹监测和防治提供依据。方法采集2008年昆明地区临床诊断为麻疹的30例患儿,其中男性18例,女性12例;年龄51天~3岁,0~1岁(≤1岁)的患儿19例,1~2岁(≤2岁)的患儿9例,2~3岁(≤3岁)的患儿2例,平均患病年龄15个月。采集急性传染期静脉血2 mL,应用酶联免疫吸附分析(ELISA)检测麻疹病毒血清IgM抗体;同时收集患儿的尿液标本,运用逆转录-聚合酶链反应(RT-PCR)扩增标本中所分离到的麻疹野病毒株核蛋白(N)基因羧基末端的543个核苷酸,对扩增产物进行核苷酸序列测定和分析,并与麻疹病毒基因库中的参考株进行比较。结果从30份麻疹患儿尿液标本中分离到7份麻疹野病毒阳性;出疹后6 d的尿液标本中仍可检出麻疹野病毒;将其4株麻疹野病毒的核蛋白(N)羧基末端的543个核苷酸与Genebank中的麻疹病毒的23个参考株进行基因亲缘性关系比较,与世界卫生组织(WHO)的H1、H2基因型参考株China 93-7、China 94-1所对应的核苷酸序列的遗传距离分别为0.031~0.047和0.332~0.398;而与H1a的遗传距离最近,为0.010~0.033;4株麻疹野病毒N基因羧基末端543个核苷酸的同源性为96.0%~98.9%;与H1标准株China 93-7和H1a标准株China 93-2的核苷酸同源性分别为95.9%~99.7%和96.9%~99.8%。结论2008年昆明地区所检测到的麻疹野病毒的优势基因型为H1a基因型。  相似文献   

12.
Shi J  Zheng J  Huang H  Hu Y  Bian J  Xu D  Li F 《Virus genes》2011,43(3):319-326
The incidence of measles in China has increased over the last decade. To evaluate the genetic variation of measles strains, 16 measles wild-type virus strains were isolated from 14 vaccinated cases and 2 nonvaccinated cases in Jilin Province during 2005–2006, and their nucleoprotein (N) and hemagglutinin (H) genes were amplified by RT-PCR. The amplified products were sequenced and compared with the Edmonston virus and the existing vaccine strains (Changchun-47 and Shanghai-191). The results showed that the variation rate between the vaccine and wild-type strains was 9.8–12.0% in the N gene and 5.9–6.9% in the H gene, respectively. In addition, cross-neutralization assays revealed that although sera obtained from infants following primary vaccination effectively neutralized vaccine strains, the capacity in neutralizing H1 wild-type measles virus isolates was decreased fourfold. Antigenic ratios testing revealed that the antigenic relatedness between wild-type measles viruses and existing vaccine strains was notably low. These data suggest that the increased incidence of measles in Jilin Province may be attributed to the antigenic drift between wild-type and vaccine strains. Our findings strengthen the recommendation of supplemental immunization with existing vaccines and also strongly suggest a need for developing new vaccines to better control measles virus outbreaks.  相似文献   

13.
Two cases of subacute sclerosing panencephalitis (SSPE), diagnosed in Croatia in 2002, were investigated. The coding regions of the matrix (M), hemagglutinin (H) and nucleoprotein (N) genes of measles virus were sequenced following direct RT-PCR amplification of viral RNA extracted from brain tissue. Phylogenetic analysis of the sequences of H and N genes, showed that both strains belonged to genotype D6. No vaccine strain was detected although both patients had been previously immunized. The comparison of analyzed sequences of two SSPE causative viruses with corresponding sequences of D6 genotype and with each other revealed a number of mutations in N and H gene sequences. In comparison to the Edmonston reference strain, the M gene of the SSPE viruses showed the characteristic biased hypermutation and a premature termination codon in one of the patients.  相似文献   

14.
Transmissible gastroenteritis virus (TGEV), the etiological agent of transmissible gastroenteritis (TGE), is the major cause of viral enteritis and fetal diarrhea in swine neonates, resulting in significant economic losses to the swine industry. The Chinese vaccine strain H165 of TGEV was derived from a virulent field strain H16 by serial passage in vitro. Strain H165 has been proven to be safe in piglets and pregnant sows and displays efficacy against TGEV infection. In this study, we report the complete genome sequences of strains H165 and H16, obtained by sequencing several overlapping fragments amplified from viral RNA and our findings from sequence and phylogenetic analyses. The genomes were 28,569 nucleotides in length, including the poly (A) tail. No deletions or insertions were detected in the H16 genome sequence after continuous passage in vitro; however, we found 27 nucleotide mutations in strain H165 compared with strain H16, resulting in 16 amino acid changes distributed among the genes 1, S, 3, and sM. An A to G nucleotide mutation was found in the intergenic region between the 3a and 3b genes. Furthermore, six unique nucleotides identified in the genome sequence of H165 could be used as makers to differentiate the H165 vaccine strain from wild-type TGEV strains. Our findings from phylogenetic analysis may enhance our understanding of the evolution of TGEV, as well as the other coronaviruses.  相似文献   

15.
Despite the marked reduction in the incidence of measles in Korea by the introduction of measles vaccine, a large measles epidemic occurred during 2000-2001. During the epidemic, more than 55,000 measles cases were reported and at least 7 children were dead. In this study, we analyzed the genetic and antigenic properties of 15 measles viruses that isolated during the epidemic. Sequence analyses of entire hemagglutinin (H) and nucleoprotein (N) genes of the viruses indicated that all Korean isolates had a high degree of homology (>99.8%) when compared with each other. They differed from other wild-type viruses by as much as 6.8% in the H gene and 6.5% in the N gene at the nucleotide level. The deduced amino acid variability was up to 6.4% for the H protein and up to 6.5% for the N protein. Phylogenetic analysis of nucleotide sequences and deduced amino acid sequences of the H and N genes revealed that all Korean viruses were grouped into the genotype H1. This strongly demonstrated that single genotype of measles virus has been circulated in Korea during the 2000-2001 epidemic. Plaque reduction neutralizing antibody titers against vaccine strains, Edmonston and Schwarz, and recently isolated Korean strains were measured using sera from vaccinees and recently infected children. Although sera of recently infected children demonstrated higher neutralizing antibody titers against wild-type strains than against vaccine strains, both sera neutralized both strains and the reciprocal geometric mean titers (GMTs) were not significantly different against both strains.  相似文献   

16.
The complete genomic sequences of two Chinese measles vaccine viruses, Shanghai‐191 (S‐191) and Changchun‐47 (C‐47), were determined and compared to the sequences of other measles vaccine strains as well as the prototype measles strain, Edmonston wild‐type (Edwt). Compared to Edwt, S‐191 and C‐47 had 49 and 43 nucleotide changes, respectively. These differences were found at 52 nucleotide positions that were not found in other vaccine strains. Phylogenetic analysis of the all of the available genomic sequences for measles vaccines showed that S‐191 and C‐47 were most closely related to the Leningrad‐4 strain. S‐191 and C‐47 shared conserved vaccine virus‐specific amino acid changes in the phosphoprotein (P), V, C, matrix (M), and hemagglutinin (H) that could represent important targets for future studies aimed at understanding the molecular basis of attenuation. In addition, S‐191 and C‐47 had several unique amino acid changes including 13 positions that differed from Edwt. This is the first comparison of the complete genomic sequences of Chinese measles vaccines to the sequences of other vaccine strains. J. Med. Virol. 81:1477–1483, 2009. © 2009 Wiley‐Liss, Inc.  相似文献   

17.
Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide sequence was previously determined (accession numbers , and ). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed. A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s-1970s). L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.  相似文献   

18.
A strain of transmissible gastroenteritis virus (TGEV), designated H16, was isolated in PK-15 cells and passaged serially to level 165. Vaccines based on passages 155–165 in cell cultures are available commercially as vaccines for the prevention and control of infections with TGEV in China. Nucleoprotein (N) sequences of the virus at passages 155 and 165 were aligned and compared using a computer software program. The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiation of the vaccine strain from the other TGEVs was investigated. The RFLP analysis identified a change in the cleavage sites of AclI at passages 155 and 165. This RFLP pattern of the N gene differentiated the Chinese vaccine strain from its parental strain, the 11 TGEVs studied and the other reported TGEVs in the GenBank. Using phylogenetic analysis, the Chinese TGEVs were divided into three groups (G1, G2, and G3). The G3 Chinese TGEVs possessed several specific nucleotides and amino acids that were not found in the G1 and G2 Chinese TGEVs or the other reference TGEVs. Analysis of the phylogenetic trees revealed that the G3 TGEVs represent a separate group that is distinct from the non-Chinese TGEVs and from Chinese TGEVs isolated previously. These findings suggest that Chinese strains of TGEV are evolving continuously.  相似文献   

19.
In Cameroon, genome characterization of influenza virus has been performed only in the Southern regions meanwhile genetic diversity of this virus varies with respect to locality. The Northern region characterized by a Sudan tropical climate might have distinct genetic characterization. This study aimed to better understand the genetic diversity of influenza A(H3N2) viruses circulating in Northern Cameroon. Sequences of three gene segments (hemagglutinin (HA), neuraminidase (NA) and matrix (M) genes) were obtained from 16 A(H3N2) virus strains collected during the 2014 to 2016 influenza seasons in Garoua. The HA gene segments were analysed with respect to reference strains while the NA and M gene was analysed for reported genetic markers of resistance to antivirals. Analysis of the HA sequences revealed that majority of the virus strains grouped together with the 2016-2017 vaccine strain (3C.2a-A/Hong Kong/4801/2014) while 3/5 (60%) of the 2015 viral strains grouped together with the 2015-2016 vaccine strain 3C.3a-A/Switzerland/9715293/2013. Within clade 3C.2a, Northern Cameroon sequences mostly grouped in sub-clade A3 (10/16). Analysis of the coding regions of the NA and M genes showed that none had genetic markers of resistance to neuraminidase inhibitors but all strains possessed the S31N substitution of resistance to amantadine. Due to some discrepancies observed in this region with respect to the Southern regions of Cameroon, there is necessity of including all regions within a country in the sentinel surveillance of influenza. These data will enable to track changes in influenza viruses in Cameroon.  相似文献   

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