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.     

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…     

Molecular cloning and nucleotide sequence of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 paris of partially overlapping primers which span the entire genome of CSFV strain Shimen were designed and synthesized. Each cDNA fragment of strain Shimen was amplified by RT-PCR method from the anticoagulant blood of strain Shimen infected pig. The PCR fragments were cloned into pGEM-T vector respectively and sequenced. The results show that we have obtained the nucleotide sequence of strain Shimen. The viral RNA consists of 12 297 nucleotides including noncoding regions of 373 and 227 bases at the 5′ and 3′ end, respectively, and a single large open reading frame spanning 11 697 nucleotides in the middle, which encodes an amino acid sequence of 3 989 residues with a calculated molecular weight of 437.6×10³. The precisely sequencing of 5′ and 3′ termini is undertaking. Supported by the National Pandeng Project Huang Qianhua: born in 1968. Graduate student     

猪瘟病毒分子生物学诊断的研究进展

猪瘟是由猪瘟病毒引起的猪的急性、热性、高度接触性传染病，严重危害我国养猪业。尽管中国很早就研制成功了猪瘟兔化弱毒疫苗。但近年来猪瘟的流行和发病特点发生了很大的变化，很多地区和猪场不断出现非典型猪瘟、温和型猪瘟，给该病的诊断带来了困难。近年来，猪瘟病毒分子生物学诊断方法的研究进展迅速，为猪瘟的快速、准确诊断起到了重要的作用。从聚合酶链式反应技术、核酸探针技术、DNA芯片技术等方面对猪瘟病毒的分子生物学诊断方法进行了综述。     

猪瘟病毒分子生物学与致病机制研究进展

猪瘟是由猪瘟病毒感染导致的高度接触性传染病,家猪和野猪对该病原易感.该病主要特征是高热、微血管变性而引起实质器官出血、坏死,是世界上危害最严重猪病之一,给养猪业带来重大损失.综述了猪瘟病毒基因组、蛋白质功能以及致病机理的最新研究进展,为相关研究人员参考.     

Construction of cytopathic PK-15 cell model of classical swine fever virus

No cytopathic effect (CPE) can be observed on classical swine fever virus (CSFV) infected cell culture in vitro. This brings an obstacle to the researches on reciprocity between CSFV and host cells. Based on the construction of full-length genomic infectious cDNA clone of Chinese CSFV standard virulent Shimen strain, partial deletion is introduced into genomic cDNA to obtain a 7.5 kb subgenomic cDNA. A new subgenomic CSFV is derived from transfection with the subgenomic cDNA on PK-15 cells pre-infected by CSFV Shimen virus. Typical CPE induced by this subgenomic virus is observed on PK-15 cells. Coexistence of wildtype and subgenomic virus in cytopathic cell culture is demonstrated by RT-PCR detection in cytopathic cells. For conclusion, the construction of cytopathic cell model exploited a new way for researches on the molecular mechanism of CSFV pathogenesis.     

乙型脑炎病毒(JEV)免疫荧光(IFA)检测方法的建立

目的建立乙型脑炎病毒(JEV)免疫荧光(IFA)检测方法,应用于猪及猪源性生物材料JEV的检测。方法滴定病毒TCID50,筛选JEV敏感细胞,依据国标IFA法制备抗原片,并进行特异性、敏感性和稳定性试验。结果选取BHK21细胞作为JEV敏感细胞,病毒感染力滴度(TCID50)为10-8.9.mL-1;与猪瘟病毒(CSFV)、猪细小病毒(PPV)均无交叉反应;稳定性和敏感性试验显示,不同时间IFA检测灵敏度均为1∶2560;可检测到的病毒滴度最低为10-6.5.mL-1。结论建立的IFA法敏感性、特异性强,稳定性好,可用于猪及猪源性生物材料JEV的检测。     

Construction of cDNA library, nucleotide sequence and analysis of entire genome of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 pairs of primers have been designed and synthesized, which cover the entire genome of CSFV strain Shimen. Each cDNA fragment has been amplified by RT-PCR from the anticoagulant blood of strain Shimen infected pig. The PCR products have been cloned respectively and sequenced. Results show that the cDNA library of strain Shimen and its nucleotide sequence have been obtained. The genomic RNA of strain Shimen is 12 298 nucleotides in length, containing a 5′ and a 3′ noncoding region 373 and 231 nt long respectively. The center of genome is a single large open reading frame of 11 697 nt which encodes a polyprotein of 3 898 amino acids. The entire sequence of strain Shimen has also been compared with that of other CSFV strains.     

Characterization of the Antigenicity and Immunogenicity of the Escherichia Coli Produced RNase Domain of the Classical Swine Fever Virus Glycoprotein E~(rns)

Erns is a highly glycosylated envelope protein of classical swine fever virus (CSFV) with RNase activity. Erns can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the Erns was produced in Escherichia coli. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the Erns retained i...     

Candidate Multi-Peptide-Vaccine Against Classical Swine Fever Virus Induces Strong Antibody Response with Predefined Specificity

IntroductionClassical swine fever virus(CSFV) is a pestiviruswhich causes significantmortality and morbidity inpigs.An epidemic of CSFV in a high pig densityarea can result in devastating financial losses,because few infected pigs can be effectively cured.In many countries in Europe and Asia,classicalswine fever (CSF) is controlled by vaccinationwith commercially available vaccine strains,suchas the Chinese vaccine strain(C-strain,i.e.,hogcholera lapinized virus) orsome modified-live vir…     

Expression of human cytomegalovirus DNA polymerase in insect cells using baculovirus expression system: Purification and biochemical characterizations

Human Cytomegalovirus (HCMV) DNA polymerase gene was overexpressed in insect cells using the baculovirus transfer system. A6. 2 kb HCMV Rsr II-EcoRI DNA fragment with intact HCMV pol gene coding sequence was engineered into NheI site of vector pBlueBac under the control of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Recombinant AcNPV carried HCMV pol gene was generated by cotransfection of Spodoptera frugiperta cell (SF21) with AcNPV DNA and baculovirus transfer vector with HCMV pol gene. Infection of SF21 cell with recombinant virus lead to the expression of 140 kD peptide of HCMV specific DNA polymerase at the level approximately 2 mg per 10⁸ cells. The polypeptide was purified from the infected SF21 cells by a series of column chromatography to homogeneity. The purified enzyme had a molecular weight of 140 kD and reacted with antiserum specific for HCMV DNA polymerase. It exhibited both 3′–5′ and 5′–3′ exonuclease activities. This enzyme is also sensitive to phosphono acetate. Ye Linbai: born in Feb. 1948. Professor. Current research interest is in Vitology and Molecular Biology Supported by Public Health Service Grants CA21773, CA15036 and AI12717 from the National Institutes of Health     

Detection of DNA methylation changes during seed germination in rapeseed （Brassica napus）

DNA methylation is a common yet important modi- fication of DNA in eukaryotic organisms. DNA methy- lation, especially methylation of cytosine (m5C), have both epigenetic and mutagenic effects on various cellu- lar activities such as differential gene exp…     

广西猪瘟病毒的调查研究

１９８６～１９８９年，从广西５个地、市的１０个病猪场猪体中分离到１０个病毒。用猪瘟石门系标准强毒作对照，经病原性、抗原性及血清学试验，培养特性，血细胞吸附及病毒的形态结构观察，证明１０个毒株为猪瘟病毒。它们有共同的抗原性。它们之间的区别只是毒力的强弱不同。从临床症状、病理变化和病程方面看，９个为亚急性猪瘟毒株；一个为慢性或温和性猪瘟毒株。     

Characterization of the Antigenicity and Immunogenicity of the Escherichia Coli Produced RNase Domain of the Classical Swine Fever Virus Glycoprotein E

E^rns is a highly glycosylated envelope protein of classical swine fever virus (CSFV) with RNase activity. E^rns can induce neutralizing antibodies and provide immune protection against CSFV infection. In this study, the RNase domain of the E^rns was produced in Escherichia coli. Its reactivity with CSFV-positive sera and its ability to induce antibodies and to provide protective immunity were then investigated. The serological tests showed that the prokaryotically expressed RNase domain of the E^rns retained its antigenicity and induced high titers of humoral responses. However, only partial protection and a limited amount of neutralizing antibodies were demonstrated by an in vitro neutralization test and an immunization/challenge test. The results suggest that other essential factors rather than simply enhancing the immunogenicity of E^rns should be taken into consideration when E^rns is enrolled as one of the components of a candidate vaccine.     

Generation of VP5 deficient mutant of infectious bursal disease virus strain HZ2

Infectious bursal disease virus (IBDV) is one of the most significant viral pathogens in chickens[1]. It causes high mortality in young chickens and establishes an immunosuppression state by destroying the precursorsInfectious bursal disease virus (IBDV) …     

Molecular cloning and nucleotide sequence of the attenuated hog cholera virus Lapinized Chinese strain

The genomic sequence of the attenuated hog cholera virus Lapinized Chinese strain (HCLV) was determined from overlapping cDNA clones. The viral RNA of HCLV stain comprised 12 310 nucleotide (nt) including 374 nt and 239 nt at the 5′ and 3′-noncoding region, respectively. The complete genome sequence contained one large open reading frame which encoded an amino acid sequence of 3 898 residues with a calculated molecular weight of 437×10³. Although there were mostly only small differences between the sequence of the HCLV strain and the published sequences of strains ALD, GPE⁻, Alfort and Brescia, there was one notable insertion of 12 nucleotides, TTTTCTTTTTTC in the 3′ non-coding region of HCLV strain. Supported by the National Pandeng Project, Genbank accession number AF091507 Wang Jiafu: born in 1972, Ph. D.     

Sequence analysis of mRNA polyadenylation signals of rice genes

Most eukaryotic mRNAs receive a poly (A) tail at their 3′-ends through a process involving the cleavage of pre-mRNA and the concomitant polymerization of adenosine residues to the cleaved RNA end[1,2]. As un- translated regions (UTRs) may contain importa…     

Complete nucleotide sequence and genomic organization of Periplaneta fuliginosa densonucleosis virus

We have cloned the replicative form of thePeriplaneta fuliginosa densonucleosis virus (PfDNV) genome and determined its complete sequence. The sequence has 5 454 nucleotides (nt), the genome consists of an internal unique sequence flanked by inverted terminal repeats (201 nt). The first 122 nt at the 5′ end and the terminal 122 nt at the 3′ end of both plus and minus strands can fold into a typical hairpin structure. The genome contains seven major open reading frames (ORFs). The plus strand has 4 ORFs occupying the 5′ half of the plus strand, whereas the others span the 5′ half of the minus strand. Two potential promoters were found at map units (m.u.) 3 and 97. Computer analysis of sequence homologies with other parvoviruses suggests that the plus strand ofPf DNV encodes very likely the nonstructural proteins and the minus strand probably encodes the structural proteins.