Detection of bovine viral diarrhea virus by spot hybridization with probes prepared from cloned cDNA sequences

A cytopathic strain of bovine viral diarrhea virus (BVDV) was purified from infected cell culture fluids by isopycnic density-gradient centrifugation. Genomic RNA was extracted and tailed with adenine residues at the 3' end with poly-A polymerase. Double-stranded complementary DNA (cDNA) was synthesized, using the poly-A-tailed RNA as a template and oligo-dT as a primer, and then cloned into the pUC9 plasmid. Virus-specific cDNA sequences, varying in length from 0.5 to 2.5 kilobases (kb), were obtained. One BVDV-specific sequence of cloned cDNA, 1.1 kb in length and with an internal Pst I restriction endonuclease cleavage site, was selected for use as a probe. The cloned cDNA insert was removed from the plasmid either with or without flanking plasmid sequences and labeled with 32P-nucleotides by nick translation for use as hybridization probes for BVDV. The performance of probes of smaller fragments of the insert was compared to that of the intact sequence in hybridization assays. In addition, 2 methods of specimen preparation were compared to establish optimum parameters for hybridization. The hybridization assay was 10-100 times more sensitive than infectivity assays for BVDV in infected cell cultures. Freezing of specimens reduced by 10-fold the sensitivity of hybridization for BVDV target sequences. The probes prepared from the cloned cDNA hybridized with all cytopathic and noncytopathic BVDV strains tested but not with uninfected cell cultures, cellular ribosomal RNA, bovine coronavirus, bluetongue virus, or bovine adenovirus 3. Probes prepared with native plasmid DNA did not hybridize with BVDV or uninfected cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of a genomic probe for malignant catarrhal fever virus

A genomic probe specific for malignant catarrhal fever (MCF) virus was cloned by using purified viral DNA from MCF-virus strain WCll. Restriction endonuclease analysis of the purified viral DNA was used to identify the cloned viral genomic fragment. Dot blot hybridization by use of the genomic probe (pRP-5) indicated that the probe hybridized specifically with WCll-MCF virus, as well as with one other isolate of MCF-associated herpesvirus. Hybridization also was observed to a non-MCF virus strain of bovine herpesvirus.     

Detection of cattle infected with bovine viral diarrhea virus using nucleic acid hybridization.

A ribonucleic acid (RNA) hybridization assay to identify cattle infected by bovine viral diarrhea virus (BVDV) is described. The RNA probe was derived from the coding region at the 3' end of the genome of the NADL strain of BVDV. Total RNA from infected cell cultures or peripheral blood leukocytes from suspect animals was extracted and applied to nylon membranes with a slot blot apparatus. Peripheral blood leukocytes were tested concurrently for BVDV by virus isolation. The results of hybridization and virus isolation were in agreement for 92% of the cases. When compared with virus isolation, hybridization had a sensitivity of detection of 59.5% and a specificity of 95%. Cross-reactivity to RNA extracts of border disease virus-infected cells was noted. No cross-reactivity was detected to other common bovine viruses (bovine herpesvirus-1, bovine respiratory syncytial virus, parainfluenza-3 virus, and bluetongue virus), to viruses classified in related families (equine arteritis virus and Venezuelan equine encephalitis virus), or to viruses having similar genomic organization (dengue virus type 2 and Japanese encephalitis virus).     

Detection of bovine viral diarrhea virus genome in leukocytes from persistently infected cattle by RNA-cDNA hybridization.

A bovine viral diarrhea virus (BVDV) cDNA library was constructed. One cloned complementary DNA sequence was used as a probe to detect BVDV RNA by hybridization in infected cell cultures and in mononuclear leukocytes from persistently infected cattle by dot blot and in situ hybridization. The cDNA probe hybridized with all cytopathic and noncytopathic BVDV isolates tested. The hybridization results were consistent with results obtained using conventional subculturing and immunofluorescent staining methods and by inoculation of seronegative test cattle.     

牛病毒性腹泻病毒基因2型代表株全基因组序列分析

为了解我国牛病毒性腹泻(BVD)的分子流行病学情况,本研究对分离的3株牛病毒性腹泻病毒基因2型(BVDV-2)代表株全基因组进行序列分析,应用RT-PCR法分段扩增3株BVDV-2(XJ-04、SD-09和QH-09)的全基因组序列,共分为18个片段:A～Q以及5'-UTR和3'-UTR的部分序列。除5'-UTR和3'-UTR部分序列外,A～Q基因片段末端相互重叠。经序列拼接获得3株全基因组序列,全长均为12 284 bp。将测序结果与GenBank登录的瘟病毒科代表毒株序列、自我裂解酶(Npro)、结构蛋白(C、Erns、E1、E2)以及标准株BVDV-1 NADL和BVDV-2 890进行核苷酸以及氨基酸序列同源性分析。结果表明:XJ-04、SD-09和QH-09的全基因组序列同源性高于99.6%,3株BVDV-2分离株与890和NewYork93株亲缘关系最近,属于BVDV-2a亚型。在致细胞病变型的XJ-04、SD-09、QH-09株的NS2/3基因上无外源基因的插入。本研究分离的BVDV-2株为国内首次鉴定国内分离株全基因组序列,为我国BVD的分子流行病调查提供依据。     

地高辛标记核酸探针检测牛粘膜病病毒

本课题建立了地高辛标记核酸探针检测牛病毒性腹泻－粘膜病病毒（ＢＶＤＶ）的诊断方法。根据ＢＶＤＶ基因序列的结构特点,在编码非结构蛋白ｐ１２５高度保守基因区内,设计合成一对特异性引物。以ＰＣＲ技术从ＢＶＤＶ基因重组质粒中扩增出了ＢＶＤＶ特异的、保守的４００ｂｐ核苷酸片段,经纯化后,地高辛标记,制备牛病毒性腹泻病毒的特异性核酸探针。通过检测ＢＶＤＶ细胞培养物、相关病毒及核酸载体和ＢＶＤＶ细胞培养物的总Ｒ     

Expression of fowl adenovirus type 10 antigens in Escherichia coli

In an attempt to construct a genetic map of the fowl adenovirus (FAV) and to determine which viral proteins are natural immunogens in chickens and hence may be relevant to protective immunity we have constructed an expression library of FAV type 10 DNA. The genomic DNA was partially digested with the restriction endonuclease Sau3A, and this DNA was inserted into the 3' terminal end of the beta-galactosidase gene in a plasmid vector. To date, approximately 600 clones have been identified that express FAV type 10 antigens as determined by immunological screening with rabbit antisera to purified virus, including one that has amino acid homology with the 100 kDa protein of human adenovirus type 5. These antigen positive clones were found to contain DNA from FAV type 10 genome as determined by hybridisation to FAV DNA. These clones will allow the further characterisation of FAV and possibly the identification of potential vaccine molecules.     

牛病毒性腹泻病毒RT-PCR诊断方法的建立

根据已发表的牛病毒性腹泻病毒株的基因序列,分析合成了一对扩增跨幅为190bp左右的引物,对牛病毒性腹泻病基因I型或基因II型的毒株进行RT-PCR扩增,结果是取得了与预期大小一致的RT-PCR产物,而对照样品的扩增全为阴性;该方法最低可检测到0.10ng的牛病毒性腹泻病毒RNA。     

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.     

Recombinant DNA probe for serotype-specific identification of bluetongue virus 17

The double-stranded RNA genome from 117 field isolates of bluetongue virus (BTV) serotypes 10, 11, 13, and 17 was blotted onto nitrocellulose paper and hybridized with a radioactively labeled cloned copy of DNA genome segment 2 of BTV-17. Viral RNA from BTV prototype strains 2, 10, 11, 13, and 17 were used as controls. The probe hybridized only with the viral RNA from prototype BTV-17 virus and field isolates of BTV-17. There was no cross hybridization with field isolates of BTV serotypes 10, 11, and 13. A complementary DNA probe developed from genes coding for BTV serotype specificity was effectively used in a slot-blot hybridization system for efficiently characterizing the viral serotype.     

牛病毒性腹泻病毒BVDV-JL分离株全基因序列测定与分析

为了解中国农业科学院特产研究所分子生物学重点实验室前期分离的牛病毒性腹泻病毒BVDV-JL毒株完整的基因序列信息,本试验对BVDV-JL F6代毒株进行了完整基因序列测定。利用反转录—聚合酶链式反应(RT-PCR)方法分段扩增了BVDV-JL分离毒株的7段cDNA片段,分别克隆于pMD18-T载体并进行测序。BVDV-JL株基因组序列全长为12276 bp,编码3901个氨基酸。基因组两端为非编码区5'UTR和3'UTR。基因比对及进化树分析结果表明BVDV-JL与BVDV CP7同源性最高,核苷酸同源性为93.2%,归类于BVDV-1b2基因亚型。BVDV-JL是第1个在中国报道的BVDV-1b2亚型毒株。了解BVDV-JL完整基因序列有利于中国BVDV流行病学调查。     

Expression of a 50 kDa putative receptor for bovine viral diarrhea virus in bovine fetal tissues.

The expression of a 50 kDa bovine viral diarrhea virus putative receptor in different bovine fetal tissues from 3-month old fetuses was studied. The receptor expression was examined by immunocytochemical staining and by immunoblotting using antiidiotypic probe (anti-D89). Intense specific staining in enterocytes of the small and large intestines, cortical tubular epithelial cells of kidneys, respiratory epithelial cells of the trachea and esophageal mucosal epithelial cells was observed, demonstrating the strong expression of bovine viral diarrhea virus receptor in the tissues. Weak staining was found in cerebellum, thymus, spleen, liver, cerebrum, and lung tissues; however, heart tissues were negative. Immunoblotting results correlated with the immunoperoxidase staining assays. Thus, the expression levels of the receptor are variable in different tissues. This pattern of expression may provide clues to the pathogenic potential of bovine viral diarrhea virus in the bovine fetus.     

Cloning of the Genomes of Equine Herpesvirus Type 1 (EHV‐1) Strains KyA and RacL11 as Bacterial Artificial Chromosomes (BAC)

The genome of equine herpesvirus type 1 (EHV‐1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Escherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co‐transfection of viral DNA and recombinant plasmid pΔ71‐pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high‐molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full‐length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacL11 and pKyA, respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP‐expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full‐length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2‐negative progeny reconstituted from pRacL11 and pKyA (L11Δgp2 and KyAΔgp2) exhibited different growth properties. Whereas both L11Δgp2 and KyAΔgp2 extracellular titres were reduced by 15‐ to 32‐fold, plaque diameters were only markedly (50%) reduced in the case of KyAΔgp2.     

Rapid fluorescence detection of in situ hybridization with biotinylated bovine herpesvirus-1 DNA probes

The use of biotinylated DNA hybridization probes for clinical detection of bovine herpesvirus-1 was investigated. Biotinylated DNA hybridization probes were prepared from bovine herpesvirus-1 DNA purified from infected cell cultures. The viral DNA was nick translated in the presence of biotin-dUTP with DNA polymerase to incorporate biotin into the newly synthesized strand. The probe was tested for specificity in in situ hybridization assays with bovine herpesvirus-1 DNA. Hybridization was detected using avidin-fluorescein single sandwich systems and an avidin-globulin with anti-globulin-fluorescein double sandwich system. Hybridization was detected by specific fluorescence of infected cells. Fluorescence was present only in bovine herpesvirus-1-infected cell culture and not in noninfected cell culture or cell cultures infected with several other viruses. The assay was performed in 6 hr.     

Cloning of equine herpesvirus type 1 438/77 strain genome as a bacterial artificial chromosome

Equine herpesvirus type 1 (EHV-1) is a major cause of respiratory and reproductive diseases in horses worldwide. The genome of EHV-1 strain 438/77 (isolated from an aborted equine fetus) was cloned as a bacterial artificial chromosome (BAC) in E. coli without any gene deletions. The mini-F plasmid sequence was inserted in the middle of ORF19 and 20 via homologous recombination following co-transfection of viral DNA and plasmid pE19_20/HA into RK13 cells. Circular viral DNA was extracted from RK13 cells infected with purified recombinant virus expressing green fluorescent protein (GFP) and electrophorated into E. coli DH10B cells. The clone harboring the BAC was screened and analyzed by PCR and RFLP. Reconstitution of the recombinant virus was achieved successfully by transfection of the BAC DNA into RK13 cells. The mini-F sequence in the reconstituted virus was subsequently removed by homologous recombination between virus DNA and plasmid pE1920XM, inducing a point mutation in the Xbal site in ORF19. Comparison of RFLP profiles of the rescued, recovered and the wild-type viral genome demonstrated that no unexpected changes occurred during mutagenesis. In vitro replication assays showed that BAC-reconstituted virus mutant growth kinetics and plaque formation morphology/size were indistinguishable to those measured for wild-type virus.     

Use of in situ hybridization for the detection of foot-and-mouth disease virus in cell culture

Biotinylated complementary DNA (cDNA) and RNA probes were prepared from a specific and highly conserved section of the foot-and-mouth disease virus (FMDV) genome coding for the RNA-dependent RNA polymerase. Hybridization was conducted on FMDV-infected, bovine enterovirus (BEV)-infected, and noninfected swine kidney cell cultures. The detection system utilized the enzyme system streptavidin-alkaline phosphatase, the substrate phosphate, and the chromogen nitroblue tetrazolium. Intense cytoplasmic granular staining was present at 2 and 4 hr postinfection (hpi), with less staining observed at 24 hpi. The staining was specific for FMDV, as indicated by a lack of staining of noninfected cells and BEV-infected cells. With the RNA probe, positive cells were detected up to the highest viral dilution assayed, which was approximately 96 TCID50. The cDNA probe was slightly less sensitive, detecting positive cells at 10-fold lower dilutions. This technique could prove useful in the diagnosis of foot-and-mouth disease in animals or in the detection of FMDV in biologics submitted for importation.     

牦牛病毒性腹泻病毒全基因测序及生物信息学分析

从牦牛(Yak)体内分离出牛病毒性腹泻/黏膜病病毒(BVDV/MDV),参考GenBank中已收录的BVDV-Ⅰ型的全基因组序列,设计了19对引物,通过RT-PCR方法,对牛病毒性腹泻病毒牦牛株进行克隆及测序,得到其全基因组序列(GenBank登录号:JQ799141),序列全长12214 nt,其中5'-UTR长288 nt,3'-UTR长232 nt。将牦牛株BVDV全基因序列与GenBank中登录的其他8株BVDV病毒全基因序列进行同源性比对及系统进化分析,结果表明,牦牛株BVDV与BVDV-Ⅰ型毒株出于同一分支,但与其他8株BVDV毒株全基因序列同源性均不高,有可能属于新的独立基因型或基因亚型,仍需进一步研究证实。