阿卡斑病毒云南分离株全基因组序列特征研究

为阐明分离自云南省的蚊媒阿卡斑病毒(Akabane virus,AKV)DHL10M110株(简称云南株)的分子生物学特征,采用RT-PCR和核苷酸序列测定方法,对云南株进行全基因组序列测定和分析。结果获得了云南株全基因组核苷酸序列,该病毒株的L、M和S基因核苷酸序列全长分别为6 869bp、4 309bp和856bp,其中开放读码框(Open reading frame,ORF)核苷酸序列依次为6 756bp、4 206bp和702bp,并分别编码2252、1402和234个氨基酸。这三个基因片段ORF的系统进化分析表明,云南株L基因与AKV标准株OBE-1(日本)和AKVS-7/SKR/2010株(韩国)处于同一进化分支;在M和S基因进化树中,云南株与日本、韩国和台湾地区的AKV流行株亲缘关系较近,但云南株单独处于一个小的进化分支。同源性分析表明,云南株L、M和S片段ORF与OBE-1株的核苷酸(氨基酸)同源性分别为92.6%(98%)、88.5%(94%)和96.4%(99.1%)。云南株与OBE-1株的L、M和S片段分别有45、84和2个氨基酸位点的差异。此外,无论L、M和S基因核苷酸序列的同源性或系统进化分析均表明,云南株与肯尼亚和澳大利亚AKV分离株进化关系较远,同源性较低。本研究进一步通过病毒全基因组序列分析证实DHL10M110病毒株为AKV,属亚洲进化群,但与日本和韩国株相比仍有一定差异,云南株地域特征明显。这是首次在中国大陆地区测定到该病毒的全基因组序列。     

我国2009年新分离乙脑病毒全基因组序列特征研究

本研究对我国2009年新分离的两株乙脑病毒进行全基因组序列测定和分析,以了解病毒全基因组分子特征。通过RT-PCR和核苷酸序列测定方法获得病毒全基因组序列,采用ClustalX、DNASTAR、MEGA等生物学软件完成核苷酸序列及氨基酸序列分析和系统进化分析等。研究结果显示,新分离两株乙脑病毒YN0911和YN0967株基因组全长均为10 965个核苷酸,编码3 432个氨基酸。这2株乙脑病毒之间核苷酸同源性为98.7%,氨基酸同源性为99.8%。与国际乙脑病毒流行株相比,核苷酸同源性为83.5%～98.9%,氨基酸同源性为94.8%～99.7%。与乙脑病毒疫苗株SA14-14-2相比,在E蛋白有13个氨基酸差异位点,但都位于抗原关键位点之外。这2株病毒在3′UTR区域存在11nt缺失。基于C/PrM区段、E基因、全基因组系统进化分析结果均显示新分离2株乙脑病毒为G I乙脑病毒,并且和越南、四川、贵州、广西以往的分离株遗传进化关系较近。本研究提示我国新分离的2株乙脑病毒均为G I乙脑病毒,决定病毒毒力的关键氨基酸位点未见明显变化。     

汉坦病毒汉滩型特殊新亚型的发现

应用RT－PCR扩增了皖南山区分离株AH09的M和S片段全基因,克隆于T载体,纯化后测定序列。结果AH09株M片段的全基因序列共3625个核苷酸,编码1135个氨基酸;S片段的全基因序列共1724个核苷酸,编码430个氨基酸。M和S片段全基因核苷酸和氨基酸与汉坦病毒各型株的代表株和HTN型毒株的同源性比较表明,AH09株分枝与HTN型接近,与其它各型病毒则相距较远,故确定为HTN型毒株,但AH09株与HTN型毒株的M和S片段全基因序列有差异,其差异分别高达23．6％和20．4％,经种系发生分析,AH09株是迄今为止所发现的HTN型病毒中差异最大的新基因亚型病毒株,AH09株病毒M片段的氨基酸与HTN型相差13．5％至14．8％,而S片段仅相差7％－8．1％,说明AH09毒株的变异主要发生在M片段。而ORF和3‘端的NCR区核苷酸序列分析比较说明,病毒的变异更主要集中在该片段的3‘端的NCR区。     

我国肾综合征出血热疫苗生产株LR1株的全基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列 ,了解该株分子基础 ,从提取的细胞总RNA逆转录PCR扩增 ,产物纯化后克隆T载体纯化后测序 ,结果证明 ,LR1株全基因组序列由L6 5 33、M36 16、S片段的16 92个核苷酸组成 ,依各自读码框架分别编码 2 15 1、1135、42 9个氨基酸。序列同源比较分析表明 ,LR1毒株与国外HTN型毒株高度同源 ,属同一亚型 ,尤其与HTN代表株 76 - 1183个片段同源率高达 99 3%～ 99 8% ,而与国内的HTN型病毒差异较大 ,同源率仅为 79 4%～ 84 6 %。氨基酸比较也显示了同样的结果。     

中国6株狂犬病病毒街毒株全基因组测序与分析

实验研究中对分离于中国的6株狂犬病病毒街毒株进行了全基因组测序,对基因组的5个结构基因(N、P、M、G和L)的核苷酸和推断的氨基酸序列以及非编码区序列进行了分析与比较,并与来自GenBank的40株毒株从全基因组水平进行了分子进化分析。所测6株中国狂犬病病毒街毒株的全基因组核苷酸序列长度介于11 907 nt(CQ92)和11 924 nt(SH06和gg4)之间,基因组结构相同,用全基因组和不同的结构基因构建的进化树拓扑结构相似,基因组3′和5′末端高度保守而且末端11个核苷酸互补配对,5个结构基因的保守性依次是NLMGP,核苷酸同源性的最小值依次分别是81.9%、81.7%、80.7%、78.3%和76.7%。     

肾综合征出血热疫苗候选毒株A16株的分子特性

为研究肾综合征出血热疫苗候选毒株A16株的分子基础,应用RT－PCR方法扩增并测定了A16株的M和S片段的序列,结果A16株M片段的全基因序列共3615个核苷酸,编码1133个氨基酸。S片段的全基因序列共1770个核苷酸,编码429个氨基酸。A16株M片段核苷酸全基因序列及其的氨基酸与HTN型毒株同源率为75.5%-90.3%和85.9%-97.1%,即A16与HTN同型毒株间的差异高达9.7%-24.3%和2.9%-14.1%,而与SEO型的同源率比较,核苷酸和氨基酸分别仅为70.2%-70.8%和73.4%-76.7%.S片段的序列同源率与M片段的结果相类似,即核苷酸和氨基酸的同源率与HTN型毒株分别为76.0%－905和92.1%-97.7%,与SEO型为67.0%-67.7%和81.7%-82.6%。结果显示,A16株虽然属于HTN型,但该毒株为HTN型病毒的新亚型病毒株。     

中国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30基质蛋白基因和融合蛋白基因的分子特征分析

对我国西藏小反刍兽疫病毒野生株China/Tib/Gej/07-30进行基质蛋白(M)和融合蛋白(F)基因序列测定,并进行分子生物学特征分析。首先应用逆转录聚合酶链式反应扩增出M和F基因片段,对聚合酶链式反应产物进行直接测序,然后对测定的核苷酸和推测的氨基酸序列进行比较分析。China/Tib/Gej/07-30的M基因由1483个核苷酸组成,编码335个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为92.4%～97.7%和97.0%～98.2%。F基因由2411个核苷酸组成,编码546个氨基酸,与其他分离株核苷酸和氨基酸序列同源性分别为85.5%～96.1%和94.3%～98.2%。China/Tib/Gej/07-30的F蛋白含有信号肽序列和跨膜结构域,序列高度变异。F蛋白第104～108位和第109～133位氨基酸位点分别是高度保守的裂解位点和融合肽结构域。F蛋白还含有序列高度保守的三个七肽重复区。China/Tib/Gej/07-30的M基因3′端的非编码区(UTR)长度为443个核苷酸,GC含量高达68.4%,与其他PPRV毒株的同源性为82.4%～93.5%。China/Tib/Gej/07-30的F基因5′UTR区长度为634个核苷酸,GC含量高达70.0%,与其他PPRV毒株序列相似性为76.2%～91.7%。     

汉坦病毒K24株M和S片段基因的结构特征

为研究汉坦病毒K24株M和S片段基因的结构特征,从病毒感染细胞提取细胞总RNA,经35个循环的一次性PCR得到了与理论值相符合产物,将RT－PCR产物直接克隆T载体,要K24毒株M片段的全基因序列共3653个核苷酸,四种核苷酸的酶切和PCR鉴定正确的克隆通过柱纯化后进行序列测定,结果比较分别为A30.44%,T30.14%,G20.72%,C18.70%,GC含量为39.42%,AT含量为60.58%,编码1133个氨基酸。S片段的全基因序列共1772个核苷酸,四种核苷酸的比例分别为A31.30%,G26.05%,T22.79%,C19.77%,GC含量为45.81%,AT含量为54.19%,共编码429个氨基酸。K24株M片段核苷酸全基因和 在酸与HTN型毒株同源率分别为70.7%－71.2%和75.9%-76.9%,与SEO型为95.2%-99.2%和95.9%-99.4%。S片段与HTN76－118和SEO SR－11病毒的比较,核苷酸同源率分别为74.0%和95.6%,氨基酸同源率为83.3%和97.9%,与M片段的结果相类似。M片段氨基酸与SEO型10个毒株存在4个氨基酸替代。应用DNASTAR软件的系统发生分析方法分别绘出了基于M片段核苷酸及氨基酸的系统发生树。     

狂犬病病毒aG株全基因组序列测定及遗传稳定性分析

目的探究狂犬病病毒(Rabies virus,RV)aG株全基因组序列特征及遗传稳定性。方法严格按疫苗生产工艺进行传代,提取主种子批、工作种子批及疫苗原液病毒RNA,通过RT-PCR技术扩增全基因组各片段基因,然后分别将其克隆到p GEM-T载体中,并进行序列测定;采用DNAStar软件包对aG株与Gen Bank中4aGV参考株(JN234411)以及18株基因1型RV参考株进行同源性分析。结果 aG株全基因组由11 925个核苷酸组成,共编码3 600个氨基酸。疫苗原液与主种子批全基因组核苷酸和氨基酸同源性均为100%,而工作种子批与主种子批的核苷酸与氨基酸同源性分别为99.97%和99.92%;aG株与4aGV参考株全基因组核苷酸与推导的氨基酸同源性均为99.9%,其与18株基因1型参考株核苷酸与氨基酸序列同源性分别为84.2%~97.6%和93.7%~98.3%;aG株传代病毒与4aGV参考株全基因组氨基酸序列高度保守,且各主要功能区未发生变异。结论狂犬病病毒aG株在实验室长期生产传代过程中,全基因组遗传特性稳定。     

汉坦病毒中国疫苗株Z37M片段的克隆及序列分析

汉坦病毒Z37株是从褐家鼠体内分离到的,用于生产双价肾综合征出血热疫苗的病毒毒株之一,血清分型为SEO型。利用RT-PCR方法扩增Z37株M基因片段cDNA,克隆入质粒载体,进行核苷酸序列测定及分析。Z37株M基因片段由3651个核苷酸组成,只有一个开放读码框架,共编码1133个氨基酸。与HTN型病毒(76-118、A9、HV-114)的核苷酸和氨基酸同源性分别为71.8%～72.1%、76.2%～76.7%,与SEO型(R22、L99、80-39)的核苷酸和氨基酸同源性分别为95.3%～96.1%、95.3%～98.9%。这一结果的获得进一步从分子水平确定了Z37株的型别,并为研制M基因片段重组疫苗打下基础。     

Complete sequence of the genome of the human isolate of Andes virus CHI-7913: comparative sequence and protein structure analysis

We report here the complete genomic sequence of the Chilean human isolate of Andes virus CHI-7913. The S, M, and L genome segment sequences of this isolate are 1,802, 3,641 and 6,466 bases in length, with an overall GC content of 38.7%. These genome segments code for a nucleocapsid protein of 428 amino acids, a glycoprotein precursor protein of 1,138 amino acids and a RNA-dependent RNA polymerase of 2,152 amino acids. In addition, the genome also has other ORFs coding for putative proteins of 34 to 103 amino acids. The encoded proteins have greater than 98% overall similarity with the proteins of Andes virus isolates AH-1 and Chile R123. Among other sequenced Hantavirus, CHI-7913 is more closely related to Sin Nombre virus, with an overall protein similarity of 92%. The characteristics of the encoded proteins of this isolate, such as hydrophobic domains, glycosylation sites, and conserved amino acid motifs shared with other Hantavirus and other members of the Bunyaviridae family, are identified and discussed.     

Evidence that the matrix protein of influenza C virus is coded for by a spliced mRNA.

In contrast to influenza A and B viruses, which encode their matrix (M) proteins via an unspliced mRNA, the influenza C virus M protein appears to be coded for by a spliced mRNA from RNA segment 6. Although an open reading frame in RNA segment 6 of influenza C/JJ/50 virus could potentially code for a protein of 374 amino acids, a splicing event results in an mRNA coding for a 242-amino-acid M protein. The message for this protein represents the major M gene-specific mRNA species in C virus-infected cells. Despite the difference in coding strategies, there are sequence homologies among the M proteins of influenza A, B, and C viruses which confirm the evolutionary relationship of the three influenza virus types.     

Open reading frame in the minus strand of two plus type RNA viruses

Inspection of the nucleotide sequences of the RNAs complementary to the coat protein mRNAs from two plant viruses with a tripartite genome: alfalfa mosaic virus and brome mosaic virus, showed the presence of open reading frames for 138 and 118 amino acids, respectively. A third virus (cowpea chlorotic mottle virus) from the same family (1) does not show this phenomenon. This suggests that if a protein is coded for by the open reading frames it may be not essential for virus multiplication. Alternatively the open reading frames have no coding function but result from structural requirements of the RNAs.     

Analysis of the Sendai virus M gene and protein.

The nucleotide sequence of the Sendai virus M (matrix or membrane) gene region was determined from cloned genomic DNA, and the limits of the M mRNA were determined by S1 nuclease mapping. The M mRNA is 1,173 nucleotides long and contains a single long open reading frame coding for a protein of 348 amino acids. The amino acid sequences of the N- and C-terminal peptides of the M protein were obtained by mass spectrometric analysis and correspond to those predicted from the open reading frame, with the N terminus modified in vivo by cleavage of the initiating methionine and acetylation of the following amino acid. The amphiphilic nature of the M protein structure is discussed.     

A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

The envelope of hepatitis B virus contains three related glycoproteins (termed L, M and S) produced by alternative translation initiation in a single coding region. The smallest of these, the S protein, is a 24 kDa glycoprotein with multiple transmembrane domains. The M and L proteins contain the entire S domain at their C-termini, but harbor at their N-terminal additional (preS) domains of 55 or 174 amino acids, respectively. Most of these preS residues are displayed on the surface of mature virions and hence would be expected to be translocated into the endoplasmic reticulum (ER) lumen during biosynthesis. Using a coupled, in vitro translation/translocation system we now demonstrate that, contrary to expectation, virtually all preS residues of the L protein are cytoplasmically disposed in the initial translocation product. This includes some preS sequences which in the M protein are indeed translocated into the ER lumen. Since preS sequences are found on the external surface of the virion envelope, our results indicate that during or following budding a dramatic reorganization of either the envelope proteins or the lipid bilayer (or both components) must occur to allow surface display of these sequences. These findings imply that some membrane budding events can have remarkable and previously unsuspected topological consequences.     

Creation of a recombinant Rift Valley fever virus with a two-segmented genome

Rift Valley fever virus (RVFV; family Bunyaviridae) is a clinically important, mosquito-borne pathogen of both livestock and humans, which is found mainly in sub-Saharan Africa and the Arabian Peninsula. RVFV has a trisegmented single-stranded RNA (ssRNA) genome. The L and M segments are negative sense and encode the L protein (viral polymerase) on the L segment and the virion glycoproteins Gn and Gc as well as two other proteins, NSm and 78K, on the M segment. The S segment uses an ambisense coding strategy to express the nucleocapsid protein, N, and the nonstructural protein, NSs. Both the NSs and NSm proteins are dispensable for virus growth in tissue culture. Using reverse genetics, we generated a recombinant virus, designated r2segMP12, containing a two-segmented genome in which the NSs coding sequence was replaced with that for the Gn and Gc precursor. Thus, r2segMP12 lacks an M segment, and although it was attenuated in comparison to the three-segmented parental virus in both mammalian and insect cell cultures, it was genetically stable over multiple passages. We further show that the virus can stably maintain an M-like RNA segment encoding the enhanced green fluorescent protein gene. The implications of these findings for RVFV genome packaging and the potential to develop multivalent live-attenuated vaccines are discussed.     

Involvement of the C-terminal tail of Arthrobacter ureafaciens sialidase isoenzyme M in cleavage of the internal sialic acid of ganglioside GM1

Arthrobacter ureafaciens sialidase comprises four isoenzymes, L, M1, M2 and S, of which L, M1, and M2, but not S, have the unique ability to cleave GM1 ganglioside, but the hydrolysis of GM3 and colominic acid by S occurs at a higher rate than that by L, M1 and M2. Since the N-terminal amino acid sequences of L, M1, M2 and S were shown to be identical on protein sequencing, they were suggested to have arisen from the same protein through truncation at different C-terminal sites. A DNA segment containing an open reading frame was cloned from a genomic library, and the structural gene was found to comprise 2,970 bp encoding a protein of 990 amino acids including a signal peptide at the N-terminus, a conserved FYRIP-region and four Asp boxes. The molecular weights of the isoenzymes determined by MALDI-TOFMS revealed that L, M1, M2 and S should comprise amino acids 39-773, 39-653, 39-655 and 39-528, respectively. In fact, recombinant enzymes M2 and S prepared in Escherichia coli exhibited identical substrate specificities toward gangliosides as those of the purified enzymes. Consequently, the C-terminal tail of isoenzyme M2 might be involved in the hydrolysis of the internal sialic acid of GM1.     

The primary structure of rat ribosomal protein L7. The presence near the amino terminus of L7 of five tandem repeats of a sequence of 12 amino acids

The covalent structure of rat ribosomal protein L7 was determined in part from the sequence of nucleotides in a recombinant cDNA and in part from the sequence of amino acids in portions of the protein. The complementary analyses supplemented and confirmed each other. Ribosomal protein L7 contains 258 amino acids and has a molecular weight of 30,040. The protein has an unusual and striking structural feature near the NH2 terminus: five tandem repeats of a sequence of 12 residues. Rat L7 appears to be related to ribosomal protein L7 from the moderate halophile Vibrio costicola and perhaps to L30 from Bacillus stearothermophilus, to L7 from the moderate halophile NRCC 41227, and to L22 from Nicotinia tobaccum chloroplast. In addition, there is a sequence of 24 amino acids in rat protein L7 that may be related to segments of the same number of residues in Escherichia coli ribosomal proteins S10, S15, L9, and L22.     

Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions.

The complete nucleotide sequences of the vesicular stomatitis virus mRNA's encoding the glycoprotein (G) and the matrix protein (M) have been determined from cDNA clones that contain the complete coding sequences from each mRNA. The G protein mRNA is 1,665 nucleotides long, excluding polyadenylic acid, and encodes a protein of 511 amino acids including a signal peptide of 16 amino acids. G protein contains two large hydrophobic domains, one in the signal peptide and the other in the transmembrane segment near the COOH terminus. Two sites of glycosylation are predicted at amino acid residues 178 and 335. The close correspondence of the positions of these sites with the reported timing of the addition of the two oligosaccharides during synthesis of G suggests that glycosylation occurs as soon as the appropriate asparagine residues traverse the membrane of the rough endoplasmic reticulum. The mRNA encoding the vesicular stomatitis virus M protein is 831 nucleotides long, excluding polyadenylic acid, and encodes a protein of 229 amino acids. The predicted M protein sequence does not contain any long hydrophobic or nonpolar domains that might promote membrane association. The protein is rich in basic amino acids and contains a highly basic amino terminal domain. Details of construction of the nearly full-length cDNA clones are presented.     

The M1- and M2-type isozymes of rat pyruvate kinase are produced from the same gene by alternative RNA splicing

The complete nucleotide sequences of rat M1- and M2-type pyruvate kinase mRNAs were determined by sequencing the cDNAs and by analyses of S1 nuclease mapping and primer extension. The sequences have an identical molecular size of about 2220 nucleotides excluding a poly(A) tail and include 1593-nucleotide coding region. Their nucleotide sequences are identical except for 160-nucleotide sequences within the coding regions. The amino acid sequences of the M1- and M2-type subunits deduced from the cDNA sequences differ by only 45 residues within domain C, which constitutes the main region responsible for intersubunit contact. The sequence of this region of the M2-type shows higher homology than that of the M1-type with the corresponding sequence of the L-type. Since the M2- and L-types are allosteric enzymes, unlike to the M1-type, the residues common to the M2- and L-types, but not the M1-type may be important for mediating the allosteric properties. Genomic clones encoding both M1- and M2-type isozyme mRNAs were isolated. By partial sequence analysis of a clone lambda MPK37 four exons were identified, of which two adjacent exons coded the M1- and M2-specific sequences, respectively. The two remaining exons present downstream coded amino acids common to the two isozymes. Thus, we conclude that the M1- and M2-type isozymes of pyruvate kinase are produced from the same gene probably by alternative RNA splicing.