马铃薯X病毒湖南分离物的鉴定与分组研究

从湖南石门采集表现重花叶症状的马铃薯叶片中分离纯化到一株线状病毒HN021.经双链RNA(ds-RNA)抽提、寄主反应测定、病毒粒子和内含体的形状观察, 初步确定该病毒为马铃薯X病毒 (Potato virus X).以ds-RNA作为模板,用相应引物对HN021分离物的ORF4-UTR-ORF5片段进行RT-PCR,得到1kb左右的双链cDNA片段.对该片段进行克隆和测序,并将测序所得的核苷酸序列与Genbank登录的11株不同分离物的相应片段的核苷酸序列进行同源性比较和分析.结果表明,HN021与分离自南美洲的三株分离物(COAT,KPA和HB)的同源性为78.4%~79.4%,与其它8株(分离自亚洲、欧洲、大洋州和北美洲)分离物的同源性为96.4%-97.8%.从氨基酸水平比较,HN021与COAT,KPA和HB三者CP和8kDa蛋白氨基酸序列同源性分别为86.5%~89.0%和74.3%~75.7%,相应地与其它8株分离物的同源性分别为97.1%-98.7% 和97.1%-100%.序列分析的结果证实了HN021分离物为马铃薯X病毒,同时表明PVX明显存在两个组(组Ⅰ和组Ⅱ),HN021和其它来自亚洲、欧洲、大洋州、北美洲分离物的组II,3个南美洲分离物属于组I.     

鹅Ⅰ型禽副粘病毒GPMV/QY97-1株HN基因的克隆和序列分析

在华南地区进行鹅病病因的调查过程中,从患病鹅群中分离到一株对鹅和鸡都有致病力的病毒,初步判定该病毒属于Ⅰ型禽副粘病毒,命名为GPMV／QY97—1株。以GPMV／QY97—1毒株的基因组RNA为模板,通过RT—PCR方法,扩增出血凝素-神经氨酸酶(HN)基因3’端和5’端的cDNA片段,分别将其克隆至pGEM—TEasy载体中,对其进行序列测定。序列分析表明,HN基因的cDNA全长为2024nt,编码571个氨基酸。序列中含有13个半胱氨酸残基和6个潜在的糖基化位点,其HN基因及编码蛋白结构与新城疫病毒株相符。将GPMV／QY97-1株HN基因序列和推导的氨基酸序列与新城疫病毒株的HN基因相应序列做比较后发现,它们的核苷酸序列同源性分别在89．6％～83．6％,氨基酸序列同源性在93．3％～87．9％。在同源性比较的基础上,进一步绘制了Ⅰ型禽副粘病毒株HN基因的系统发育树。这对于Ⅰ型禽副粘病毒毒力基因的功能分析和该病的分子流行病学调查有着重要意义。     

山东省玉米矮花叶病毒的生物学特性及基因组全序列测定

对山东省玉米矮花叶病毒原分离物(SD)进行了寄主范围、血清学等普通生物学鉴定,测定了该病毒的基因组核苷酸全序列。该病毒基因组RNA由9596个核苷酸组成(不包括3’—polyA的长度),整个基因组按一个ORF编码一个3063个氨基酸的多聚蛋白。序列比较表明,该病毒分离物(SD)与玉米矮花叶病河南分离物(HN,EMBL登录号：AF94510)核苷酸全序列同源性最高,为98．2％,与已报道的甘蔗花叶病毒(SCMV)7个分离物同源性也高达79．5％-98．2％,但与玉米矮花叶病毒保加利亚分离物(MDMV—B8,AJ001691)和高梁花叶病毒萧山甘蔗分离物(SrMV—XoS,AJ310197)的同源性仅为67．8％和69．3％,与约翰逊草花叶病毒(226920,JGMV)差异最大。系统进化树分析也表明,该病毒与SCMV分离物位于同一进化簇,而与MDMV进化关系很远。     

甜菜坏死黄脉病毒75kDa通读蛋白基因54kDa片段的克隆及其序列分析

以甜菜坏死黄脉病毒(BNYVV)内蒙分离物的总RNA为模板,通过反转录和PcR扩增获得75kDa通读蛋白基因54kDa片段的目的片段。将其克隆到pGEM－7Zf(+)上并转化DH5α得到了含有完整s4kDa片段的重组子pGBW52。采用双脱氧终止法进行序列分析。结果表明内蒙分离物的54kDa片段全长为1509nt,与法国的F13分离物相比缺失了3个核苷酸。其核苷酸序列和由此推导的氨基酸序列的同源性分别为94．97％和96．42％．     

兔出血症病毒中国株衣壳蛋白基因的克隆和序列分析

应用RT—PCR技术,从兔出血症病毒中国分离株WX84中成功扩增出预期大小为1．7kb的特异性条带,将扩增产物提纯后克隆入pGEM^R—T载体,经转化、筛选及酶切鉴定后,获得了该株病毒衣壳蛋白基因的克隆,序列分析表明扩增的中国株BHD衣壳蛋白基因片段长度为1740bp,共编码580个氨基酸。该核酸序列与其它国家报道的多株BHDV序列相互间同源性高达98．2％一99．0％,其推导的氨基酸序列同源性也达98．3％--99．1％,为极度保守片段。     

大豆花叶病毒外壳蛋白基因的克隆和其在大肠杆菌中的表达

通过多聚酶连锁反应(PCR),我们合成了包括病毒外壳蛋白基因在内的病毒基因组3’端区域,并完成了其全部序列分析,比较SMV(北京分离物)和SMv—N株的序列发现;外壳蛋白基因的核苷酸序列同源性达93．D％,其氨氢基酸序列同源性高达98．5％,就基因组3,端非编码序列而言,其同源性达88．8％。Western b1ot分析结果表明所克隆的cDNA片段在大肠杆菌JMl07中能表达正常的病毒外壳蛋白。     

马铃薯卷叶病毒外壳蛋白基因的合成,分子克隆和全序列分析

以分离自马铃薯主栽品种“紫花白”的马铃薯卷叶病毒(PLRV)分离物的RNA为模板,用人工合成的引物,用反转录和随后PCR扩增的方法合成了PLRV外壳蛋白(CP)基因的cDNA,并克隆于pUC19中。进一步用限制酶切和核苷酸序列分析表明,合成的cDNA由627个核苷酸组成(包括起始和终止密码),序列中有Hinc Ⅱ和BamH Ⅰ两个酶切位点,与国外报道一致。和国外的4个PLRV分离物CP基因序列对比结果,具有高度同源性,其同源率达99.0—99.7％。     

一个引起玉米矮花叶病的甘蔗花叶病毒基因组全序列测定及其结构分析

测定了从浙江省呈现矮花叶病症状的玉米上分离得到的一个马铃薯Y病毒属病毒RNA的核苷酸全序列. 该病毒分离物的RNA基因组由9596个核苷酸组成(不包括polyA尾). 单一的ORF由9192个核苷酸组成, 编码一个分子量为346.1 ku的聚合蛋白. 该蛋白结构特征与高粱花叶病毒(SrMV)中国甘蔗分离物和一个玉米矮花叶病毒(MDMV)保加利亚分离物基因组编码的蛋白非常相似. 序列分析表明, 该病毒分离物与甘蔗花叶病毒(SCMV)各分离物(已报道的仅为基因组3′末端序列)同源性最高, 与SrMV和MDMV同源性次之, 而与约翰逊草花叶病毒(JGMV)同源性最低. 根据马铃薯Y病毒属区分不同病毒和株系的分类标准, 报道的玉米病毒分离物应当鉴定为SCMV的一个株系. 然而, 该分离物在HC-Pro, P3和CI蛋白区域和SrMV中国甘蔗分离物具有极高的氨基酸同源性.     

甘蔗花叶病毒3’末端基因的克隆及外壳蛋白序列分析比较

选取我国SCMV优势株系A株系的分离物SCMV-CA为材料,经过病毒和病毒RNA的提纯,反转录获得病毒cDNA,并克隆到载体pUC19的SmaI位点上,筛选得到多个重组质粒。选取其中一个克隆SCMV-CA54进行测序,得到一个全长为1296 bp的核苷酸序列。这段序列由一个长为1044 bp的开放阅读框架（ORF）和一个长279 bp的3’末端非编码区序列（3'-UTR）及poly(A)尾巴组成。这个ORF包括病毒完整的外壳蛋白（CP）及部分核内含体蛋白b（NIb）基因序列。将所得序列同已知SCMV亚组中各株系分离物的核苷酸和氨基酸进行同源性比较,结果表明该序列与其它株系分离物CP核苷酸序列的同源性介于63.7%～77.6%之间,氨基酸的同源性介于64%～89%之间。根据马铃薯Y病毒属的序列同源性划分标准,SCMV-CA与其它株系或分离物的同源性关系均介于种与株系划分标准之间。这是我国首次报道SCMVCP基因序列。     

水稻矮缩病毒第一号组份基因和编码蛋白的序列分析

水稻矮缩病毒（RiceDwarfVirus,简称RDV）是我国南方水稻病毒病的重要病原,属植物呼肠孤病毒。从中国福建分离物中克隆了基因组第一号片段（S1）的全长cDNA并对其进行全序列分析,结果表明RDV福建分离物S1克隆片段全长4422bp,含有一个长4332bp的开放阅读框架,编码一个由1444个氨基酸组成的多肽（P1）,分子量为164kD．根据基因序列,对推测的P1氨基酸序列分析表明,序列中含有依赖于RNA的RNA聚合酶（RNA-dependentpolymerase-RDRP）保守序列：motifＩ（DXXXXD）、motifⅡ（SGXXXTXXXN）和motifⅢ（GDD）,除此之外,在模式Ⅲ后还存在一个很保守的区域EXXKXY。由此说明RDVS1编码的蛋白P1可能是病毒的一种RDRP。将RDV福建分离物引核苷酸和编码蛋白氨基酸序列与日本流行株系相比,同源性分别为95％和97％。RDV福建分离物S1序列已被DenBank接受,号码为U73201。     

Complete Genome Sequence of a Chinese Isolate of Potato virus X and Analysis of Genetic Diversity

The complete genomic sequence of a Chinese Potato virus X isolate FX21 (PVX‐FX21) was determined from three overlapping cDNA clones. The genome of PVX‐FX21 is 6435 nucleotides in length excluding the poly(A) tail and contains five open reading frames (ORFs). Its entire genomic sequence shares 95.2–96.3% identities with Asian and European isolates, and 77.3–77.8% with American isolates. Phylogenetic analysis of the complete genomic sequence reveals two groups: the Eurasian group and the American group. PVX‐FX21 belongs to the Eurasian group and forms a separate sub‐branch with three Asian isolates. Similar analyses of the coat protein genes of 37 PVX isolates also reveal two major groups. All PVX isolates from Asia are clustered to group I, whereas isolates from Europe and America are clustered to both groups. Nucleotide sequence diversity analyses show that there is no geographical differentiation between PVX isolates and that constraint on the ORF encoding RNA‐dependent RNA polymerase is much higher than those on the other four ORFs.     

Phylogeny, coat protein genetic variability, and transmission via seeds of Hosta Virus X

The complete genome of Hosta Virus X (HVX), which is thought to be a distinct species of Potexvirus, was sequenced. Nucleotide sequences of HVX were compared with those of other members of the genus Potexvirus and phylogenetic tree was constructed. The range of identities of viral replicase open reading frame 1 (ORF1) between HVX and other potexviruses were 43.1%-55.1% and 35.9%-46.6% at the nucleotide and amino acid levels, respectively. Phylogenetic analysis was performed according to the amino acid sequence of the replicase to determine the position of HVX in the genus Potexvirus. Results from the phylogenetic analysis demonstrated that HVX was in the same group as Cassava common mosaic virus (CsCMV), Plantago asiatica mosaic virus (PlAMV), Tulip virus X (TVX), and Hydrangea ring spot virus (HdRSV). In particular, coat protein (CP) sequences among viruses from different Hosta cultivars were revealed to be less variable than those from different isolates of Potato virus X (PVX), a Potexvirus type species. In the present study, HVX was transmissible by seeds of the Hosta "Blue Cadet" cultivar. Moreover, HVX was detected in the embryo but not in the seed coat or endosperm of the seed.     

Complete Genome Sequence,Phylogenetic Relationships and Molecular Diagnosis of an Indian Isolate of Potato Virus X

The complete genome of a Potato virus X (PVX) isolate from India (ptDel‐9), which occurred symptomlessly in potato but induced ringspots on Nicotiana tabacum cv. Xanthi and necrotic mosaic on Nicotiana benthamiana, was sequenced. The genome was 6435 nucleotides long ( JF430080 ) and contained five open reading frames. The isolate was closely related to those reported from the Eurasian region (95.1–97.1% sequence similarity) and distantly related to those reported from South America (77.2–77.9%). The CP gene was expressed in Escherichia coli as a 76‐kDa fusion protein with maltose‐binding protein and used to generate polyclonal antibodies, which successfully detected PVX in field samples of potato by ELISA. In 20% of field samples, for which ELISA failed, the virus was successfully detected by RT‐PCR. This is the first report of molecular characterization of PVX occurring in India.     

Complete sequencing of potato virus X new strain genome and construction of viral vector for production of target proteins in plants

The complete nucleotide sequence of the genome of a new potato virus X (PVX) strain Tula isolated by us has been determined. Based on comparison of the PVX Tula nucleotide sequence with the sequences of 12 other PVX strains, this strain was assigned to the European cluster of PVX strains. Phylogenetic analysis revealed the same phylogeny for both full genome sequences and nucleotide sequences of polymerase and coat protein genes, suggesting that the PVX evolution did not involve recombination between different strains. The full-size cDNA copy of the PVX Tula genome was cloned and the accumulation of the viral coat protein in infected Nicotiana benthamiana was shown to be about twofold higher than for the PVX strain UK3. Based on the PVX Tula genome, a new vector which contained the target gene instead of the removed triple transport gene block and the coat protein gene has been constructed for expression of target proteins in plants. The productivity of the new vector was about 1.5-2-fold higher than the productivity of the vector of the same structure based on the standard PVX strain genome. The new viral vector can be used for superproduction of recombinant proteins in plants.     

Nucleotide and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates. Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences

Highly virulent Newcastle disease virus (NDV) isolates are List A pathogens for commercial poultry, and reports of their isolation among member nations must be made to the Office of International Epizootes (OIE). The virus is classified as a member of the order Mononegavirales in the family Paramyxoviridae of the subfamily Paramyxovirinae. Two interactive surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, play essential roles in NDV attachment and fusion of cells during infection. Antibodies to the F or HN proteins are capable of virus neutralization; however, no full-length sequences are available for these genes from recently obtained virulent isolates. Therefore, nucleotide and predicted amino acid sequences of the F and HN protein genes from 16 NDV isolates representing highly virulent viruses from worldwide sources were obtained for comparison to older virulent isolates and vaccine strains. The F protein amino acid sequence was relatively conserved among isolates maintaining potential glycosylation sites and C residues for disulfide bonds. A dibasic amino acid motif was present at the cleavage site among more virulent isolates, while the low virulence viruses did not have this sequence. However, a Eurasian collared dove virus had a K114Q substitution at the F cleavage site unique among NDV isolates. The HN protein among NDV isolates maintained predicted catalytic and active site residues necessary for neuraminidase activity and hemagglutination. Length of the HN for the Eurasian collared dove isolate and a previously reported heat resistant virulent isolate were longer relative to other more recent virulent isolates. Phylogenetically NDV isolates separated into four groups with more recent virulent isolates forming a diverse branch, while all the avian paramyxoviruses formed their own clade distinct from other members of the Paramyxoviridae.     

新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

新城疫病毒HN基因的遗传变异与HI相关性的研究

选取国内1999~2004年分离的新城疫野毒10株,经CEF蚀斑纯化和SPF鸡胚增殖,对其血凝素-神经氨酸酶(HN)基因分别进行克隆和测序,结合在GenBank中发表的LaSota、F48E9和Clone30等参考序列,对其氨基酸序列进行遗传变异分析,绘制系统发育树。利用SPF鸡在隔离器中分别制备上述NDV毒株的单因子阳性血清,进行血凝抑制(HI)交叉试验,计算NDV不同株之间的HI相关系数(r)。利用统计学软件SPSS8.0对NDV不同株之间的HN氨基酸同源率和HI相关系数(r)进行相关比较。结果表明:NDV野毒间氨基酸高度同源,同源性为96.5%~99.8%,而与LaSota、F48E9和Clone30同源率仅为87.4%~89.9%;所有野毒均缺乏1个潜在的糖基化位点;HN基因的氨基酸同源性与HI相关系数显著相关(P<0.01,r=0.55)。     

Sequence analysis of the Czech Potato mop-top virus (PMTV) isolate Korneta-Nemilkov

The nucleotide sequence was determined for Czech potato mop-top virus (PMTV) isolate Korneta-Nemilkov, found in the potato field situated in South Bohemia. The nucleotide and amino acid sequences were compared with other PMTV isolates available in databases. The sequence identity was always >99% when Czech isolate RNA 2 and RNA 3 sequences were compared with each of the 3 Danish isolates and with Sw isolate, and slightly lower when compared to Scottish isolates. Similarity of deduced proteins was 100% for 5 out of 6 proteins used in comparison of Czech isolate with Danish isolate 54-15. The only difference between 2 isolates was found in coat protein (CP) gene. Interestingly, the CP of the Czech isolate seems to be 100% identical to the one of Sw, while many changes were found in the region encoding TGBp2, TGBp3 and cysteine-rich protein (CRP) for these 2 isolates. The lowest similarity scores were found when comparing the Czech isolate CRP with CRP of Scottish isolates.     

Sequence Analysis of Segment 8 of Five Chinese Isolates of Rice Gall Dwarf Virus and Expression of a Main Outer Capsid Protein in Escherichia coli

The rice gall dwarf disease, caused by the Rice gall dwarf virus (RGDV) is a serious disease occurring in rice in many regions of Guangdong province. As a basis to control the disease we have studied the genomic diversity of a variety of isolates from different locations. Genome segment 8(S8), encoding a main outer capsid protein (Pns8) of RGDV five isolates (BL, CH, DQ, GZ, XY) from Guangdong province was cloned and sequenced. The results revealed that all the S8 segments of the five isolates consisted of 1 578 nucleotides and had a single open reading frame (ORF) extending for 1 301 nucleotides from nucleotide 21 which encoded a polypeptide of 426 amino acids with an estimated molecular weight of 47.4 kDa. The S8 full-length sequence and the ORF sequence shared 97.3%-98.8% and 97.3%-99.1% nucleotide sequence identities within the five Chinese isolates, and shared 94.8%-95.6% and 95.0%-96.0% identities with those of the Thailand isolate respectively. The deduced amino acid sequence of Pns8 in GZ isolate was identical to that in the Thailand isolate, while the amino acid sequence variability of Pns8 within five Chinese isolates ranged from 0.5% to 2.1%. These results indicate that the S8 segment of RGDV is highly conserved in different isolates from different locations. The S8 cDNA from the XY isolate was cloned into the plasmid vector pET-28b(+) and a fused expression protein with an apparent molecular mass of 51kDa was specifically detected in an analysis of Escherichia coli Rossetta(DE3)Ⅱcells. To our knowledge, this is the first report on analysis of the RGDV segment 8 sequence and genetic comparison of different RGDV isolates and their protein expression.