两个猪瘟病毒野毒株gp55基因抗原编码序列的分析及比较

利用反转录－ＰＣＲ方法扩增了吉林省猪瘟病毒（ＨＣＶ）两个野毒株ｇｐ５５基因的主要保护性抗原编码区,并将其克隆到ＰＧＥＭ＿Ｔ载体中,然后用Ｓａｎｇｅｒ双地测定了其核苷酸序列,并推导了其氨基酸序徇。将测定的这两个ＨＣＶ野毒株的部分序更与国内外已知的ＨＣＶ序列进行比较,结果表明：这两个野毒株的核苷酸序理的同生为９４．９％,氨基酸序列同源性为９７．４％,与１９８５－１９９２年意大利中部分离４个野毒的同源性     

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。     

猪瘟病毒糖蛋白E0基因的克隆及其表达研究*

用ＲＴ－ＰＣＲＡ法扩增分别获得了中国猪瘟病毒强毒石门株和免化弱毒株糖蛋白Ｅ０基因ｃＤＮＡ,并克隆到ｐＧＥＭ Ｔ载体中测定其核着酸序列和推导出其对应氨基酸序列;结果表明这两个毒株间的Ｅ０基因核着酸序列同源性为９５．３％,氨基酸序列同源性为９４％,有１４个残基的差异;与几个代表毒株ＡＬＤ株、ＧＰＥ株、Ｂｒｅｓｃｉａ株、Ａｌｆｏｒｔ株和国外测得的免化弱毒Ｃ株相应序列进行比较,所测石门病毒核苷酸序列与上述各株对应序列的同源性分别为９８．０％、９７．１％、９２．７％、８６．８％和９５．４％;氨基酸序列同源性分别为９     

猪瘟病毒石门株与兔化弱毒株E0糖蛋白基因的克隆及序列分析

参考已发表的猪瘟病毒序列,设计并合成了一对引物,应用ＲＴＰＣＲ 技术,扩增了猪瘟兔化弱毒（Ｈｏｇ ｃｈｏｌｅｒａ ｖｉｒｕｓ ｌａｐｉｎｉｚｅｄ Ｃｈｉｎｅｓｅ ｓｔｒａｉｎ , ＨＣＬＶ） 和石门强毒株的Ｅ０ 糖蛋白基因,并将其克隆到ｐＧＥＭＴ 载体中,测定了其核苷酸序列,并推导了其氨基酸序列。结果表明我国这两株强弱不同毒株Ｅ０ 糖蛋白核苷酸序列同源性和推导的氨基酸序列同源性分别为９５－０ ％ 和９４－３ ％ ,有１３ 个氨基酸的差异,ＨＣＬＶ 比石门株多了一个潜在的Ｎ糖基化位点。将我国这两株病毒与国外已报导的ＨＣＶ 毒株Ｅ０ 基因序列进行了比较,发现石门株与日本的两株毒株ＡＬＤ 和ＧＰＥ－ 同源性较高,核苷酸序列同源性分别为９７－４ ％ 和９６－５ ％ ,氨基酸同源性分别为９７－４ ％ 和９６－０ ％ ,而与欧洲Ｂｒｅｓｃｉａ 株和Ａｌｆｏｒｔ 株同源性较低,核苷酸同源性分别为９２－２ ％ 和８６－５ ％ ,氨基酸同源性为９５－２ ％ 和９２－５ ％ , ＨＣＬＶ 与ＡＬＤ、ＧＰＥ－ 、Ｂｒｅｓｃｉａ、Ａｌｆｏｒｔ 株核苷酸同源性分别为９５－６ ％ 、９４－９ ％ 、９１－３ ％ 、８５－５ ％ …     

猪瘟病毒糖蛋白E0基因的克隆及及表达研究

用ＲＴ－ＰＣＲ方法扩增分别获得了中国猪瘟病毒强毒石门株和兔化弱毒株糖蛋白Ｅ０基因ｃＤＮＡ,并克隆到ｐＧＥＭ Ｔ载体中测定其核苷酸序列和推导出其对应氨基酸序列,结果表明这两个毒 间的Ｅ０基因核苷酸序列同源性为９５．３％,氨基酸序列同源性为９４％,有１４个殖基的差异;与几个代表毒株ＡＬＤ株、ＧＰＥ株、Ｂｒｅｓｃｉａ株、Ａｌｆｏｒｔ株和国外测得的兔化弱毒Ｃ株相应序列进行比较,所测石门病毒核苷酸序列与上述     

传染性法氏囊病病毒野毒株的致病性及其vp2基因比较

对4个IBDV野毒株的致病性和它们的vp2基因高变区序列同时做了比较分析。结果表明,4个IBDV毒株在致病性程度上存在较大差异。其中有一个是真正的超强毒,即GX8／99,其他几个虽然达不到真正超强毒的毒力,但也比经典的标准毒的致死性高得多。在vp2基因高变区,这4个IBDV野毒株与超强毒参考株HK46同源性很高,在DNA水平为96．8％～99．5％,在氨基酸(aa)水平为96．6％～100％o而与疫苗毒D78有很大差异,分别只有91．7％～93．6％和91．8％～93．2％。说明IBDV毒株的vp2基因高变区确实与其致病性有一定关系。特别是SD-1／97、SD-3／98、JS-30／99株之间及其与HK46在DNA和aa水平的同源性高达98．4％和98．6％以上,SD-3／99、JS-30／99株之间及其与HK46的氨基酸同源性为100％。然而,致病性特别高的GX8／99株病毒与其他3个野毒株及HK46在DNA和氨基酸水平的同源性相对较低,在DNA和aa水平的同源性只有96．8％～9712％和96．6％～97．9％。相对于国内的流行毒株和香港超强毒参考株HK46,GX8／99株在致病性和VP2高变区都已发生了一定的变异。     

22株猪瘟病毒E2基因部分编码序列的序列分析

RT－PCR方法获得了13株猪瘟病毒分离株、石门系强毒、中国C株及法国温度敏感株Thiverval株的E2基因部分编码序列的拉增片段,并对其进行了测序,得到了251bp的E2基因部分编码序列。利用DNAStar软件对其中224bp的片段进行了序列分析,并与已发表的Alfort、Brescia等毒株进行比较,结果13株猪瘟分离株所测片段均为猪瘟病毒E2基因的序列,与石门系强毒的序列相比所有毒株的碱基替换随机地分布于整个序列,无碱基缺失和碱基插入。其中变化较大的区域位于序列的3′端。2 2株HCV E2基因部分编码序列的核苷酸及氨基酸同源性范围分别为78.1%-100%、78.4%-100%,其中13株猪瘟病毒流行毒株的核苷酸及氨基酸同源性范围分别为:78.1%-100%、78.4%-100%,4株70－80年代分离的毒株的核苷酸及氨基酸同源性范围分别为：79.0%-88.3%、81.1%-87.8%,9株90年代分离的毒株的核苷酸及氨基酸同源性范围分别为：90.3%-100%、83.8%-100％;说明猪病毒流行株的变异呈现一定的多样性。     

PMWS病猪猪圆环病毒2型全基因组序列分析

根据GenBank中发表的猪圆环病毒2型(PCV2)全基因序列,设计一对特异性引物,用PCR方法直接从5省病料中分别扩增出5个PCV2毒株的全基因组,分别命名为FujianPCV、GuangxiPCV、HainanPCV、HunanPCV和ShandongPCV。将扩增片段克隆于pMD18一T载体,进行序列测定,结果表明,除FujianPCV株核苷酸长度为1768bp,其余四株均为1767bp。应用DNAstar序列分析软件分析表明,本实验的5个PCV2分离株与世界上其它地区的PCV2分离株密切相关,核苷酸序列同源性达93％-98．8％,与PCVl毒株的序列同源性只有68．4％～70％。其中ORFl和ORF2所编码的氨基酸序列与国内外PCV2毒株比较,同源性也很高,分别为98．1％499．4％和88％99．1％。     

犬瘟热病毒TN株血凝基因的克隆与序列分析

用RT PCR方法对分离的TN野毒株H基因进行了扩增 ,并将其克隆到pGEM T载体上 ,进行核苷酸序列测定。结果TN野毒株H基因ORF全长 1 ,81 5bp ,编码 60 4个氨基酸。与GenBank中己报道的 7个CDV毒株相比 ,H基因核苷酸序列的同源性在 92 %～ 99%之间 ,推导的H蛋白氨基酸序列的同源性在 91 %～ 99%之间。TN株H蛋白潜在的N 联糖基化位点为 8个 ,而Onderstepoort弱毒株H蛋白为 4个 ;其中N端第 1 9～ 2 1、 30 9～ 31 1、 5 8     

A second envelope glycoprotein mediates neutralization of a pestivirus, hog cholera virus.

Several monoclonal antibodies (MAbs) raised against hog cholera virus (HCV) reacted with the HCV structural glycoprotein gp44/48 and neutralized the virus. The presence of HCV gp44/48 on the viral surface was directly demonstrated by immunogold electron microscopy. Eight anti-HCV gp44/48 MAbs were tested by immunoperoxidase assay against a panel of pestivirus strains. Each MAb showed a distinct pattern of reactivity with HCV strains. It is suggested that the MAbs are well suited for epidemiological investigations of HCV outbreaks.     

Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera

To investigate whether live attenuated pseudorabies virus (PRV) can be used as a vaccine vector, PRV recombinants that expressed envelope glycoprotein E1 of hog cholera virus (HCV) were generated. Pigs inoculated with these recombinants developed high levels of neutralizing antibodies against PRV and HCV and were protected against both pseudorabies and hog cholera (classical swine fever).     

猪瘟病毒兔化弱毒株cDNA片段的克隆及序列分析

猪瘟是猪最重要的传染病之一,往往给养猪业造成重大经济损失,猪瘟的病原为猪瘟病毒（ＨＣＶ）,属黄病毒科,瘟病毒属成员,其基因组为单股正链ＲＮＡ,长度为１２３ｋｂ,仅含有一个大的开放阅读框架,编码一个含３８９８个氨基酸残基（ＡＡ）的多聚前体蛋白［１,２］。目前已经定位的蛋白有５种,即Ｎｐｒｏ、Ｃ、Ｅ０、Ｅ１和Ｅ２,它们均由ＨＣＶＲＮＡ５′端所编码,除Ｎｐｒｏ外,其它４种均为ＨＣＶ的结构蛋白［３］。Ｎｐｒｏ为具有自我催化功能的蛋白水解酶,也是多聚蛋白Ｎ端的第一个蛋白水解酶,分子量为２３ｋＤ,Ｃ为构成…     

Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer.

Neutralizing monoclonal antibodies directed against hog cholera virus (HCV) precipitated two HCV-encoded glycoproteins, HCV gp55 and HCV gp33. Immunoassay with bacterial fusion proteins and Western immunoblotting with extracts from infected cells revealed that the antibodies recognized only HCV gp55. Coprecipitation of HCV gp33 was shown to be due to intermolecular disulfide bridges. One of the antibodies also reacted with the major glycoprotein of another pestivirus, bovine viral diarrhea virus (BVDV). The analogous BVDV glycoproteins exhibited a distribution of cysteine residues which was almost identical to that of HCV gp55 and gp33. The two BVDV glycoproteins were also linked by disulfide bridges.     

Differences in pathogenicity and antigenicity among hog cholera virus strains.

Using antiserum against a particular strain of bovine viral diarrhea virus, the strains of hog cholera virus were divided into two groups, H and B, on the basis of the difference in the degree of neutralization. Group H consisted of strains reacting poorly in neutralization, and group B Consisted of strains reacting well with bovine viral diarrhea antiserum. Most of the strains of group H induced a typical clinical form of hog cholera in experimentally infected pigs. Inoculation of pigs with a strain of group B, however, resulted in a chronic type of illness. When immunized with bovine viral diarrhea virus, pigs succumbed to challenge with group H virus after showing clinical signs of hog cholera, but survived challenge with group B virus without manifesting any clinical sign.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V. Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.     

Naturally occurring BK virus variants (JL and Dik) with deletions in the putative early enhancer-promoter sequences.

The genomes of two independently isolated BK virus (BKV) variants (JL and Dik) were compared with prototype BKV DNA by restriction endonuclease mapping and sequence analysis. Differences were mainly detected in two regions: the BKV (JL) and BKV (Dik) putative early enhancer-promoter regions and the middle of the T-antigen-coding regions. Base sequence analysis of these two regions showed the following. (i) The putative enhancer-promoter regions of BKV (Dik) and BKV (JL) contained only one 68-base-pair (bp) unit of the 68-bp triplication (the central copy of which is missing 18 bp) present in prototype BKV. (ii) In the same region, BKV (JL) and BKV (Dik) contained unique stretches of DNA 33 and 63 bp long, respectively. In these 63 bp, a sequence which was very similar to the proposed simian virus 40 enhancer core sequence (GGAGTGGAAAG) was present. (iii) The altered restriction endonuclease recognition sites in the sequenced part of the T-antigen-coding region of BKV (JL) and BKV (Dik) were due to base sequence changes, leaving the amino acid sequence unchanged.     

我国猪瘟病毒兔化弱毒株囊膜糖蛋白E0基因的克隆及序列测定

采用异硫氰酸胍一步法从４８０代猪瘟病毒兔化弱毒株（ＨＣＬＶ）脾毒中提取总ＲＮＡ,以该ＲＮＡ为模板,进行反转录,然后采用套式ＰＣＲ扩增出ＨＣＬＶ的囊膜糖蛋白Ｅ０基因,琼脂糖凝胶电泳表明其大小与预计相符。将扩增出的Ｅ０基因克隆到ｐＧＥＭＴ载体中,用自动序列分析仪对其进行序列测定。将测得的序列及推导的氨基酸序列与国外测得的Ｃ株相应序列进行比较,结果发现,它们之间核苷酸序列同源性为９９．０８％,氨基酸序列同源性为９８．４２％。     

Secondary structure of the 5'' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.

The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.