新疆猪瘟病毒分子流行病的研究

采用RT-PCR方法从新疆临床发病猪场采集的病料中扩增了CSFV流行株E2基因的主要抗原位点编码区，测定了15株CSFV的核苷酸序列。应用DNAstar计算机软件对6个地区6个代表株CSFV的核苷酸序列和推导的氨基酸序列进行了比较分析，结果表明：新疆6株CSFV流行株与猪瘟兔化弱毒株(C株)核苷酸序列的同源性为80．9％～82．8％，氨基酸的同源性为86．2％～88．3％，说明新疆CSFV流行株E2基因发生了部分变异，与疫苗株相比抗原性产生一定的差异。     

H5N1亚型禽流感病毒M基因的克隆与分子进化分析

以一株H5N1亚型禽流感病毒RNA为模板,用RT-PCR方法,扩增M基因全长,将PCR产物克隆于pMD18-T载体,测序结果表明所克隆的982个核苷酸的片段包含了M1和M2基因的完整阅读框架,通过软件推导M1和M2基因分别编码252和97个氨基酸,将M全长序列与Genbank收录的10株H5N1亚型流感病毒M基因序列进行比较,病毒株之间M基因核苷酸序列同源性为92.3%～99.3%,编码的两个蛋白M1和M2氨基酸序列间同源性分别为为96.0%～98.8%和92.9%～99%,分子进化分析揭示了病毒株间的亲源关系.     

PRV广东株gE基因去信号肽片段的克隆与序列测定

根据伪狂犬病病毒（PRV）Rice株gE基因的序列设计并合成了1对引物,以我国PRV地方毒株广东株的基因组DNA为模板,通过PCR方法获得了一大小约1.6kb的DNA片段,并将其克隆到pMD18-T载体上进行测序,序列测定结果显示,该片段长1665bp,编码555个氨基酸,与PRV Rice株gE基因的核苷酸序列同源性为97.7%,氨基酸序列同源性为95.9%。     

鹅细小病毒C日v强毒株VP3基因克隆及遗传进化

根据Genbank中的鹅细小病毒(GPV)B株全基因序列,设计合成一对引物,应用PCR技术扩增了GPV强毒株CHv的VP3基因片段,将扩增后的VP3基因重组到pMD18-T质粒载体上,并对擂人片段进行序列测定,将测序结果及由该结果推导的氨基酸序列与国内外分离的GPV,M DPV,PPV和CPV等不同宿主的细小病毒的VP3进行比对分析.结果表明:中国四川分离的GPV CHv株VP3基因长1 605 bp,编码 534个氨基酸,与国内外10株GPV的VP3基因进行比较,核苷酸同源性为93.4%-99.8%,氨基酸同源性为96.5%99.3%,其变异较小,是GPV保持一个血清型的分子基础.与番鸭细小病毒的核苷酸和氨基酸同源性分别为79.6%和89.9%,而与其他种属的细小病毒同源性均在30%以下,表明它们与GPV CHv株亲缘关系较远.     

鹅细小病毒CHv强毒株VP3基因克隆及遗传进化

根据Genbank中的鹅细小病毒(GPV)B株全基因序列,设计合成一对引物,应用PCR技术扩增了GPV强毒株CHv的VP3基因片段,将扩增后的VP3基因重组到pMD18-T质粒载体上,并对插入片段进行序列测定,将测序结果及由该结果推导的氨基酸序列与国内外分离的GPV、MDPV、PPV和CPV等不同宿主的细小病毒的VP3进行比对分析。结果表明:中国四川分离的GPV CHv株VP3基因长1 605 bp,编码534个氨基酸,与国内外10株GPV的VP3基因进行比较,核苷酸同源性为93.4%~99.8%,氨基酸同源性为96.5%~99.3%,其变异较小,是GPV保持一个血清型的分子基础。与番鸭细小病毒的核苷酸和氨基酸同源性分别为79.6%和89.9%,而与其他种属的细小病毒同源性均在30%以下,表明它们与GPV CHv株亲缘关系较远。     

鸡新城疫强毒分离株SC01的分子特性及与La Sota和F48E8的交叉保护

对NDV四川分离鸡源强毒株SC01的F基因进行克隆测序,克隆的SC01 F基因片段长1600bp,编码的F蛋白裂解位点序列为112R-R-Q-K-R-F117,具有强毒株序列特征,聚类分析属基因Ⅶe型;F基因与国内外NDV代表株的核苷酸同源性为75.4 %～95.4 %,其中与鹅源毒株JS501Go 的同源性高达95.4%,与F48E8同源性为84.3%,与La Sota 的同源性为82%;La Sota弱毒活疫苗及F48E8灭活疫苗免疫小鸡2周后,对SC01的致死性攻击能提供完全保护.     

鸡新城疫病毒ND-xx08株F基因的克隆及分析

新城疫病毒（NDV）ND-xx08毒株经10 d龄SPF鸡胚增殖后,提取其基因组RNA并反转录成cDNA,用NDV F基因特异性引物,经PCR扩增后获得与F基因预期大小一致的DNA片段。将NDV F基因片段克隆到pMD18-T载体上,并进行EcoR I和Hind III双酶切鉴定和测序鉴定。结果显示,ND-xx08毒株F基因片段的长度为1 662 bp,共编码554个氨基酸,F蛋白的裂解位点为112R-R-Q-K-R-F117,是典型强毒株氨基酸序列结构。将NDV ND-xx08株F基因的47 bp到420 bp序列与新城疫病毒基因型I至基因型Ⅸ毒株的相同序列绘制病毒基因进化树,显示ND-xx08分离株属于基因Ⅶe型。将NDV ND-xx08株F全基因与国内外发表的23株NDV F基因核苷酸序列和氨基酸序列的同源性比较分析,结果表明,其核苷酸序列的同源性在82.7%~97.8%之间,氨基酸同源性在87.5%~97.7%之间。     

鸭瘟病毒强、弱毒株TK基因的克隆与序列测定

根据GenBank上已发表的鸭瘟病毒TK基因核苷酸序列,设计并合成了1对引物,分别扩增鸭瘟病毒标准强毒株(DPV-F34)和鸭瘟鸡胚化弱毒疫苗株(C-KCE)的TK基因,将它们分别克隆入pBS-T载体,转化TOP10大肠杆菌,对阳性的重组质粒进行序列测定.结果表明:DPV-F34与DPVC-KCE的TK基因长度均为1077bp,编码358个氨基酸,二者的核苷酸与氨基酸组成具有很高的同源性,分别为99.4%和98.9%.     

青海牦牛与云南牦牛乳球蛋白基因5′调控区核苷酸序列同源性比较

利用一对PCR引物,寡聚核苷酸序列的上游引物为5-′gCg,AAT,TCA,TCC,CAC,gTg,CCT,gC-3;′下游引物为5-′gAg,AAT,TCC,Tgg,ggA,ggg,ACC,TT-3′。经68℃退火及30轮循环的PCR程序后,分别克隆来自我国青海和云南地区牦牛的乳球蛋白基因的5′调控区1 447 bp的DNA片段。将该片段从琼脂糖凝胶中回收,克隆在pMD18-T Vector后进行序列测定。序列分析结果表明,青海牦牛该序列的核苷酸序列与文献报道的奶牛该基因的同源性为97.65%,云南牦牛该序列的核苷酸序列与奶牛该基因的同源性为96.8%,青海牦牛与云南牦牛该基因片段的核苷酸序列同源性为99.4%。     

牛结核分枝杆菌PstS3基因克隆与序列分析

以牛结核分枝杆菌（Mycobacterium bovis）基因组DNA为模板,克隆PstS3基因,并进行序列测定,然后利用生物信息学软件对其序列进行分析.结果显示,PstS3基因全长1 113 bp,编码370个氨基酸,与GenBank所发表的人结核分枝杆菌PstS3基因的核苷酸同源性为99.82%,氨基酸同源性为99.4%,有1处位点发生有义突变.     

啤酒花潜隐病毒新疆分离物的全基因组序列分析

对新疆啤酒花上获得的HpLV分离物HpLV-XJ进行了全长克隆和基因组序列分析。结果显示:HpLV-XJ的全基因组序列为8612个核苷酸(nt)(不包括poly A),含有6个开放阅读框(ORF),分别编码224 kDa(ORF1)、25kDa(ORF2)、11 kDa(ORF3)、7 kDa(ORF4)、34 kDa(ORF5)、和12 kDa(ORF6)蛋白。序列相似性分析结果表明,HpLV-XJ与HpLV(GenBank:AB032469)序列相似性达98.5%,6个开放阅读框的核苷酸序列相似性分别为98.3%、99.0%、97.6%、96.7%、99.5%和98.4%;由此推导的氨基酸序列相似性分别为98.6%、98.7%、97.2%、95.0%、99.4%和98.1%,各个基因的核苷酸序列和蛋白的氨基酸之间存在着一定的差异。     

副猪嗜血杆菌hhdB-A基因的鉴定和分析

为了明确副猪嗜血杆菌致病菌株的毒力相关因子,从广东省不同地区发病猪场送检病猪中分离到9株细菌(H1～H9),并对其hhdB-A基因进行了鉴定和分析。结果显示,所分离的9株细菌经形态观察、生化特性鉴定和PCR鉴定为副猪嗜血杆菌阳性,并具有卫星现象和不溶血特征。用hhdB-A基因引物从分离菌株中扩增到了特异性片段,序列分析表明,所获得的hhdA基因序列与GenBank中副猪嗜血杆菌SH0165,HPS59的hhdA基因同源性为98%～100%,分离菌株hhdA基因之间序列同源性为99.0%～99.9%;hhdB基因序列与SH0165的hhdB基因同源性为99%,分离菌株hhdB基因之间序列同源性为97.5%～99.8%;推导的氨基酸序列与少数放线杆菌、杜克雷嗜血杆菌的溶血素蛋白具有一定的相似性。RT-PCR扩增到了hhdB-A基因的目的条带,表明该基因在分离菌株中得到了相应的表达。     

番木瓜环斑病毒复制酶基因的克隆和序列分析

用ＲＴ－ＰＣＲ技术从ＰＲＶ－ＡＬ中分离到复制酶（ＲＰ）基因，将基因克隆进载体ｐＵＣ１８，用双脱氧链终止法测定了基因序列，表明其全长为１６０２ｂｐ，与国内外报道的ＨＡ５－１、ＹＫ和Ｓｍ的ＲＰ基因相比，同源性分别达８２．８０％，９５．０７％和９１．８３％．     

猪传染性胃肠炎病毒、猪流行性腹泻病毒和猪轮状病毒三重PCR检测方法的建立

通过设计三对引物分别扩增猪传染性胃肠炎病毒(TGEV)、猪流行性腹泻病毒(PEDV)及猪轮状病毒(RV)的特异性基因,建立了一个能对TGEV、PEDV及RV感染鉴别诊断的三重PCR方法。该方法能同时检测TGEV的M基因252bp片段、PEDV的N基因540bp片段及RV的VP7基因412bp片段,而对CSFV、PCV2、PRRSV、PRV等无扩增,其检测TGEV、PEDV及RV的极限为100pg核酸/μL;用该方法对临床收集的26份粪便样品进行检测,结果表明:建立的多重PCR方法具有特异性强,强灵敏度高等特点,能用于临床诊断及流行病学调查。     

Identification of a nanovirus-like DNA molecule associated with Tobacco curly shoot virus isolates containing satellite DNA

A circular single-stranded DNA molecule, designated DNA1, was identified from Tobacco curly shoot virus (TbCSV) isolates Y35 and Y115 containing satellite DNAβ using abutting primers based on the two reported DNA1 sequences of whitefly-transmitted geminiviruses, while DNA1 molecule was not found in TbCSV isolates Y1 and Y121 without DNAβ. The immunotrapping PCR test showed that DNA1 could be encapsidated in virus particles. Southern blot further confirmed that DNA1 molecules were only associated with TbCSV isolates (Y35 and Y115) containing DNAβ. Sequences of Y35 and Y115 DNA1 comprise 1367 and 1368 nucleotides, respectively, each having a conserved ORF encoding nanovirus-like replication-associated protein (Rep). A low nucleotide sequence identity was found between DNA1 molecules and their cognate DNA-As. Y35 and Y115 DNA1 shared 92% overall nucleotide sequence identity and 96% amino acid sequence identity for Rep, while 69%～79% overall nucleotide sequence identity and 87%～90% amino acid sequence identity were found when compared with two reported DNA1 molecules associated with Ageratum yellow vein virus and Cotton leaf curl Multon virus. Sequence analysis showed that DNA1 was less related to nanovirus DNA.     

Isolation and identification of a novel coronavirus from wild bird

A novel coronavirus strain was isolated from laryngotracheal swab of wild partridge and designated as partridge/GD/S14/2003 (S14). Its whole genomic sequence was obtained (GenBank Accession number: AY646283) through RT-PCR amplification, cloning, nucleotide sequencing, and analysis by the DNASTAR program. To investigate the origin of the virus, we further analyzed the nucleotide sequences of the main structural proteins, and compared those with other available virus isolates. Our results showed that the highest nucleotide homologies between the S1 gene of S14 strain and those of nephrogenic-type strains JX1-99 and TJ2-96 were 94.6% and 93.4%, respectively. In addition, a relatively high genetic identity, 85% and 84.3%, respectively, was detected between S1 gene of S14 and those of strains QXIBV and LX4. The results suggested that the S14 strain may be originated from or related to nephrogenic-type and proventriculus-type infectious bronchitis virus (IBV). The highest nucleotide homology between the S2 gene of S14 strain and those of QXIBV and LX4 was 85% and 84.3%, respectively and all of them belonged to group II coronaviruses. The highest nucleotide homology between the M gene of S14 strain and those of strains SAIB20 and GD6-98 was 90.6% and 90.2%, respectively by which S14 belonged to group III. Although they displayed high level of genetic identity in S1 and S2 gene, there was lower homology of M coding sequences between S14 and BJ, and between S14 and QXIBV strains. Phylogenetic analysis of N gene indicated that group I strains might evolve from RNA recombination between strain H52 and Gray; while group II strains from strain H120 and D1466. S14 strain had the highest N gene homology with strain QXIBV which was 95.7%, thus classified as a group III member. Strains SAIB20 and GD6-98 which were closely related to the M gene of S14 strain belonged to group I and IV, respectively. A possible role of partridge S14 strain may play in the process of coronavirus evolution is discussed.     

Sequencing and rescuing a highly virulent classical swine fever virus： Chinese strain cF114 from a full-length cDNA clone

Classical swine fever is an economically important, highly contagious disease of pigs caused by the classical swine fever virus (CSFV), as referred to as hog cholera virus. CSFV belongs to Pestivirus within the family of Flaviviridae. The virus contains a positivestranded RNA of approximately 12.3 kb in length[1]. The genome is composed of a 5′ non-coding region, a single large open reading frame (ORF) encoding the viral polyprotein with 3898 amino acid residues and a 3′ non-coding reg…     

SpltNPVp49基因的克隆和序列分析

根据新发现的杆状病毒凋亡抑制基因ｐ４９基因的序列设计了一对引物,以ＳｐｌｔＮＰＶ基因组ＤＮＡ为模板,通过ＰＣＲ扩增获得了预期大小的约１．３ｋｂ的ＤＮＡ片段,将此片段克隆到ｐＧＥＭ－Ｔ载体上并直接测序。序列分析表明,该片段为ＳｐｌｔＮＰＶ完整的ｐ４９基因开放读码框。与已知的杆状病毒ｐ４９基因的序列同源性为８７％,与之对应的氨基酸序列的同源性高达９３％。它是首次从ＳｐｌｔＮＰＶ中克隆到的抑制细胞凋亡的     

猪瘟强毒株与兔化弱毒疫苗株的LAMP鉴别检测

为了鉴别猪瘟病毒（CSFV）野毒感染和疫苗接种,系统比较分析CSFV标准强毒株、疫苗株、不同基因亚型的野毒株的全基因组序列差异,设计一套分别针对猪瘟强毒与弱毒NS5B基因的环介导等温扩增（LAMP）引物,建立特异性的猪瘟强毒株与疫苗株的LAMP检测方法.LAMP扩增产物用罗丹明B指示剂或者琼脂糖凝胶电泳进行检测.该方法特异性好,比RT-PCR灵敏度高出1 000倍,且重复性稳定性良好,为快速准确地鉴别CSFV野毒感染和疫苗接种提供了有效的方法.