禽痘疫苗病毒中网状内皮组织增生病病毒5'LTR整合位点序列分析

参照国外发表的禽网状内皮组织增生病病毒（REV）5’长末端重复序列（LTR）在禽痘病毒（FPV）疫苗株基因组上的整合位点及相关序列,合成一对来自FPV的引物,从国内5个不同厂家生产的禽痘疫苗中经PCR均扩增到REV-5’LTR。通过序列比较发现,我国5个FPV疫苗毒株中REV-5’LTR整合位点与美国和澳大利亚的天然重组禽痘疫苗完全一致。其中,有3个的REV-5’LTR插入序列也与美国的Vac-3-Am株和澳大利亚的Vac-M3-Au株有100％的同源性。另2个中国疫苗毒株中的REV．LTR插入序列与美国疫苗毒株Vac-1-A。中的REV-LTR插入序列有99．6％的同源性。但是,这5个中国禽痘疫苗毒株中整合的REVLTR与中国近年分离到的REV野毒株HA9901的5’LTR的同源性只有75．4％-92．4％。     

整合进禽反转录病毒基因组片段的鸡马立克氏病病毒重组野毒株的发现

用对禽网状内皮组织增生病病毒(REV)的单抗从广东和广西分离到的二株马立克氏病病毒(MDV)野毒株做间接荧光抗体试验, 用MDV感染细胞的基因组DNA做斑点分子杂交及PCR显示, 这二株MDV基因组中已整合进REV的LTR序列. 根据MDV基因组上易插入REV的LTR的高频位点的序列合成7条引物, 根据REV的LTR合成4条引物, 由此交叉组成28对引物, 分别从这二个MDV野毒株扩增和克隆已整合进MDV的REV-LTR序列及其相连的MDV序列. 测序证明, 二株中的REV-LTR插入序列及在MDV基因组中短独特序列(US)上的插入位点完全相同. 表明这二个毒株很可能是一次重组事件在鸡群中形成的流行毒株.     

含有禽网状内皮组织增生病病毒基因组片段的天然重组禽痘病毒的研究

将国内5个不同生产厂家来源的禽痘弱毒疫苗株和1株禽痘野毒株在鸡胚成纤维细胞(CEF)上连续传5代,用禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)特异性的单克隆抗体进行间接免疫荧光试验(Immunonuoreseellee assay,IFA),均检测不到传染性REV。但以6株禽痘病毒(FPV)感染的第2代和第5代细胞基因组提取物为模板,通过PCR均能扩增出REV的长末端重复序列(LTR)和囊膜蛋白(env)基因片段。用特异性核酸探针作分子斑点杂交(Dot blot),结果显示所扩增的PCR条带为特异的REV—LTR和REV—env)基因片段。实验结果表明,国内的一些痘病毒疫苗和野毒株基因组中,已稳定地整合进了REV的基因组成分。     

网状内皮组织增生症病毒中国分离株HA9901全基因组核苷酸序列的测定与分析

以网状内皮组织增生症病毒(REV)中国分离株HA9901感染的鸡胚成纤维细胞(CEF)基因组DNA作为其前病毒基因组模板, 根据已发表序列设计合成6对引物, 经PCR扩增出6段连续的、相互部分重叠的DNA片段和闭合环形前病毒两末端LTR的连接区段, 并分别连入T载体进行克隆、测序. 用DNAstar软件对测序结果进行剪辑和拼接, 完成了REV第一个中国分离株HA9901前病毒全基因组核苷酸序列. 在从截然不同的地区、不同年份、不同禽类分离到的毒株中, 将该序列与另两个毒株已完成的全基因组序列的比较表明, REV的整个基因组相对保守, 各毒株间对应基因的同源性都在92%以上. 其中, 从我国鸡体分离到的野毒株HA9901与美国鸡源分离株FA在整个基因组上的同源性均显著高于美国的鸭源SNV株.     

不同致病型马立克氏病病毒1．8kb基因家族序列比较

为了分析鸡马立克氏病病毒(Marek′sdiseasevirus,MDV)的致病性与其1.8kb基因家族的关系,本研究比较了四个不同致病型12个毒株的该基因家族的同源性关系,即弱毒疫苗株、强毒株、超强毒株、特超强毒株和中国野毒株。结果表明:不同毒株间1.8kb基因家族的上游调控序列的同源性在1.8kb基因家族转录方向上大于92.5%,序列间有缺失突变,其中大多数突变发生在弱毒株上。CVI988在TATAbox和上游SP1位点间丢失小片段“5′CTCGG3′”。1.8kb基因家族中存在132bp串连重复序列,CVI988包含37个132bp串连重复序列拷贝,强毒株、超强毒株、特超强毒株通常只包含2个串连重复序列。但是已知的中国疫苗毒株814株也只有2个重复序列,表明重复序列的拷贝数不是引起病毒毒力变化的主要原因。各毒株的1.8kb基因家族同源性在97%以上。其中未剪切的1.69kbcDNA中有两个阅读框,分别编码63和64个氨基酸组成的多肽。不同毒株的这二个多肽都很保守,虽然也有一些氨基酸变异,但这些变异与病毒的致病型或地域分布没有明显的关系。     

不同致病型马立克氏病病毒1.8kb基因家族序列比较

为了分析鸡马立克氏病病毒(Marek's disease virus, MDV)的致病性与其1.8kb基因家族的关系,本研究比较了四个不同致病型12个毒株的该基因家族的同源性关系,即弱毒疫苗株、强毒株、超强毒株、特超强毒株和中国野毒株.结果表明不同毒株间1.8kb基因家族的上游调控序列的同源性在1.8kb基因家族转录方向上大于92.5%,序列间有缺失突变,其中大多数突变发生在弱毒株上.CVI988在TATA box 和上游SP1位点间丢失小片段"5′CTCGG 3′".1.8kb基因家族中存在132bp串连重复序列,CVI988包含3-7个132bp串连重复序列拷贝,强毒株、超强毒株、特超强毒株通常只包含2个串连重复序列.但是已知的中国疫苗毒株814株也只有2个重复序列,表明重复序列的拷贝数不是引起病毒毒力变化的主要原因.各毒株的1.8kb基因家族同源性在97%以上.其中未剪切的1.69kb cDNA中有两个阅读框,分别编码63和64个氨基酸组成的多肽.不同毒株的这二个多肽都很保守,虽然也有一些氨基酸变异,但这些变异与病毒的致病型或地域分布没有明显的关系.     

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     

12株猪瘟病毒E2基因主要抗原区域的序列差异分析

用RTPCR扩增了12个不同时期分离的HCV毒株E2基因主要抗原区域的cDNA片段并对其进行了序列测定。应用DNAstar序列分析软件对所测的12个HCV毒株与国内外已知的6个毒株Alfort株、Ald株、Brescia株、Gpe株、C株、CW株及早期已测定的HCLV株、HCVSM株和北京顺义株(BJSY2/96)3个毒株的相应片段进行了同源性比较分析。E2基因主要区域长度均为224 bp,包括从HCV 2485到2708位的E2基因B、C区域。所测的疫苗株HCLV与国外测得的疫苗株C株核苷酸及氨基酸同源性分别为991%和100%,表明目前应用的疫苗株是稳定的;用目前我国流行的部分野毒株对HCLV株免疫猪的攻击试验表明,HCLV对野毒株均具有很好的免疫力,这与序列分析结果相吻合。根据系统树分析,可将HCV分为两大群,5株90年代的野毒株及1株80年代的野毒株(其中北京3株、河南2株、广东1株)均与国内外C株、标准株属同一群(即第一群),其核苷酸及氨基酸的同源性分别为857%～100%和838%～100%;与Alfort株同属第二群的有6个野毒株(广西北海、辽宁、河北黄骅、吉林、深圳光明、四川成都),其中80年代与90年代的野毒株各有三株,核苷酸及氨基酸的同源性分别为843%～100%和851%～100%;21株HCV的核苷酸及氨基酸的同源性分别为781%～100%和784%～100%。两群之间的特征性差异表现在713和729位氨基酸位点的不同,经分析发现猪瘟野毒株具有复杂性与多样性。     

二株B亚群禽白血病病毒全基因组序列及其在细胞上的复制性比较

本研究比较了从山东地方品系鸡群分离到的二株B亚型禽白血病病毒(ALV)SDAU09E3和SDAU09C2的全基因组序列及它们在细胞培养上的复制动态。这二株ALV-B的同源性为95.4%,与GenBank中3株B亚群参考株之间的同源性也均在91.0%～94.9%间,而与其它亚群参考株的同源性均低于87.9%。与亚群无关的gag、pol基因和LTR的核苷酸序列比较表明,这二株ALV-Bgp85基因的gag和pol基因与所有比较的参考株的同源性均在93%以上。LTR与其他外源性ALV参考株的LTR间的同源性在72.6%～88.3%范围内,但与E亚群内源性ALV的LTR的同源性只有51.5%。然而,这二个ALV-B的LTR的同源性也只有74.8%,远低于其他基因组部分的同源性,特别是它们的LTR的U3区同源性只有68.8%,二者在二个CAAT分布上也显著不同。对这二株ALV-B在DF-1细胞上的复制动态比较表明,它们在细胞培养上清液中的TCID50值非常类似,但SDAU09E3株核衣壳蛋白p27抗原的含量显著高于SDAU09C2株。这表明,同一亚群的不同毒株在复制过程中,所表达的p27抗原量与所形成的具有传染性的病毒量间没有平行关系。这一差异与LTR-U3区的相关性则有待应用感染性克隆技术来做进一步深入研究。     

蛋鸡J亚群白血病病毒的分离鉴定及序列分析

通过接种鸡胚成纤维细胞((CEF)及特异性单抗的间接荧光抗体反应(IFA),从中国商品代蛋鸡群中首次分离到J亚群白血病病毒(ALV-J).对其env基因编码的氨基酸序列及3'-末端(3'-Ter)序列与国内外来源于白羽肉鸡的毒株作了比较分析.结果显示,这两株病毒的gp85基因编码的氨基酸序列与国外5个毒株同源性仅为83.4%～87.3%,与国内来源于白羽肉鸡的8株病毒同源性也仅为86.4%～89.6%.gp37基因编码的氨基酸序列与5个国外毒株同源性为91.8%～97.0%,与8个国内毒株同源性为93.9%～95.9%.另外,国内来源于白羽肉鸡的各毒株的3'-Ter序列在"E"区均有明显缺失,但本次分离的来源于蛋鸡群的毒株在"E"区没有缺失突变.与所列出的13株国内外毒株相比,这两个毒株在3'-Ter的缺失最少,较接近于原型株HPRS-103.显然这两株病毒的来源不同于国内白羽肉鸡.     

Interaction of cucumber mosaic virus and potato virus y with tobacco mosaic virus

A study was performed on the interaction of cucumber mosaic virus (CMV) of potato virus Y (PVY) with tobacco mosaic virus (TMV). Interference was evaluated using tobacco plantsNicotiana tabacum cv. Java responding to CMV and PVY with a systemic infection and to TMV with local necrotic lesions. The decrease in TMV — induced lesion number gave evidence of a decrease in susceptibility caused by the previous infection with CMV or PVY, the decrease of lesion enlargement demonstrated a decreased TMV reproduction in the plants previously infected with CMV or PVY. The interference concerned was incomplete, as evaluated from reproduction of the challenging TMV and from the decrease in susceptibility of the host to TMV brought about by the first infection with CMV or PVY.     

Reassembly of brome mosaic virus from dissociated virus

The reconstitution of Brome Mosaic Virus (BMV) has been studied using neutron scattering. Experiments were performed on disassembled virus without subsequent separation of components. Phase diagrams of the disassembly and subsequent reassembly of BMV were established as a function of pH and LiCl molarity by analytical centrifugation and quasi-elastic light scattering. Disassembly occurs at a pH above 6.5 and above 0.8 M LiCl. On reassembly, if the pH is lowered first, capsids are formed without subsequent incorporation of RNA. Neutron scattering was used to investigate the formation of virus particles, when the ionic strength was lowered from 1.4 to 0.1 M LiCl at pH 7.8. The reconstitution was followed continuously. As it was driven by a lowering of the ionic strength the kinetics of the process cannot be studied for short times. However the fact that at any given ionic strength no evolution of the scattering was observed with time implies that the reconstitution is complete within a few minutes. The observations in buffers with various amounts of D₂O lead to the conclusion that the reassembly is achieved by co-condensation of the RNA and of the capsid proteins.     

Zika病毒与Zika病毒病

Zika病毒(Zika virus,ZIKV)是一种新现虫媒病毒,属于黄病毒科黄病毒属。其感染后临床表现为轻症发热,因病重住院者罕见。流行病学数据显示Zika病毒病的流行与自身免疫性神经性疾病格林-巴利综合征及先天性小头畸形有关,引起了专家和公众的关注,但具体机制有待深入研究。     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Feline immunodeficiency virus ORF-Ais required for virus particle formation and virus infectivity

The orf-A (orf-2) gene of feline immunodeficiency virus (FIV) is a small open reading frame predicted to encode a 77-amino-acid protein that contains putative domains similar to those of the ungulate lentiviral Tat protein. Orf-A is reported to be critical for efficient viral replication in vitro and in vivo. A series of FIV-pPPR-derived proviruses with in-frame deletions and point mutations within orf-A were constructed and tested for replication in feline lymphoid cells. Orf-A mutant proviruses were also tested for viral gene and protein expression, viral particle formation, and virion infectivity. Deletions within orf-A severely restricted FIV replication in feline peripheral blood mononuclear cells (PBMC) and interleukin-2-dependent T-cell lines. In addition, substitutions of alanines for leucines in the putative leucine-rich domain, for cysteines in the putative cysteine-rich domain, and for a tryptophan at position 43 in Orf-A restricted the replication of FIV mutants. Deletions and point mutations in orf-A imposed a small effect or no effect on FIV long-terminal-repeat-driven viral gene expression and had no effect on viral protein expression. However, release of cell-free, virion-associated viral RNA in supernatants from cells transfected with orf-A mutant proviruses was severely restricted but was rescued by cotransfection with a wild-type Orf-A expression vector. In addition, virions derived from orf-A mutant proviruses expressed reduced infectivity for feline PBMC. Our findings suggest that Orf-A functions involve multiple steps of the FIV life cycle including both virion formation and infectivity. Furthermore, these observations suggest that Orf-A represents an FIV-encoded analog more similar to the accessory gene vpr, vpu, or nef than to the regulatory gene tat encoded by the primate lentiviruses.     

Comparative study of variola virus and monkeypox virus interferon-gamma-binding

DNA fragments containing genes for coding IFN-gamma-binding proteins (IFNgammaBPs) of variola virus (VARV) and monkeypox virus (MPXV) were obtained from viral genomes using PCR. Isolated genes coding desired proteins were expressed in the insect Sf21 cells using baculovirus expression system. Secreted recombinant IFNgammaBPs were isolated from culture medium of infected Sf21 cells through affinity chromatography procedure. SDS-PAAG and Western blot analysis of culture medium of infected insect cells and preparations of purified recombinant IFNgammaBPs indicated that recombinant viral proteins were dimerized even in the absence of ligand (hIFNgamma) unlike their cell (eucaryotic) analogs. Biological activity of the recombinant IFNgammaBPs were studied in the test of protective effect inhibition of hIFNgamma on L68 cells infected with murine encephalomyocarditis virus. It was shown that recombinant IFNgammaBPs had dose-dependent IFNgamma-inhibiting activity. A possibility of the elaboration of new therapeutics for anti-hIFNgamma therapy on the base of IFNgammaBPs is discussed.