Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses

This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.     

Comparison of the complete genomic sequence of the border disease virus, BD31, to other pestiviruses

The genus Pestivirus is composed of hog cholera virus (HCV) [also known as classical swine fever virus (CSFV)], bovine viral diarrhea virus (BVDV), and border disease virus (BDV). Complete sequences have been published for HCV (or CSFV) and the two genotypes of BVDV (BVDV1 and BVDV2). In this study the complete sequence of the border disease virus (BDV), BD31, was determined. BD31 was isolated from a lamb with hairy shaker syndrome and is the BDV type virus offered by ATCC (ATCC VR-996). The genome was 12268 nucleotides long and had a single large open reading frame (ORF) beginning at nucleotide 357 and ending at nucleotide 12045. The sequence identity of the predicted amino acid sequence of BD31 and other published pestivirus sequences varied from 71% to 78%. Phylogenetic analysis of available complete genomic sequences segregated pestiviruses into two branches. One branch contained BD31 and HCV (or CSFV) isolates while the other branch contained BVDV1 and BVDV2 isolates. Pestiviruses from the same branch were similar in the length of the 5′ and 3′ untranslated regions (UTR). When complete genomic sequences were compared among BD31, HCV (or CSFV), BVDV1 and BVDV2, the highest sequence identity was observed in the 5′ UTR. Within the ORF, the highest sequence identity was observed in the genomic region coding for the nonstructural viral polypeptide p80.     

A short target real-time RT-PCR assay for detection of pestiviruses infecting cattle

A rapid single step real-time duplex TaqMan RT-PCR was developed for detection of bovine viral diarrhoea virus (BVDV)-1, BVDV-2 and border disease virus (BDV). Based on alignment of available and newly generated partial 5′-UTR nucleotide sequences, one forward and two reverse primers were designed, which amplify a 104 bp PCR product. Two TaqMan probes labelled with different fluorochromes were designed to detect BVDV-1/BVDV-2 and BDVs, respectively. The assay was able to detect a selection of strains and isolates that represent the genetic diversity of these three viruses, with an analytical sensitivity that corresponded to 3.6, 48 and 4.8 TCID₅₀ of BVDV-1, BVDV-2 and BDV, respectively. With an overall cycling time of around 70 min, the assay allows rapid diagnosis and efficient use of modern thermocycling machines. Although developed principally for the diagnosis of BVD, the assay should be equally useful for diagnosis of BD in sheep.     

Construction of an infectious chimeric classical swine fever virus containing the 5'UTR of bovine viral diarrhea virus, and its application as a universal internal positive control in real-time RT-PCR

RT-PCR is used widely as a diagnostic method to detect and differentiate pestiviruses. The construction of two chimeric classical swine fever virus (CSFV) recombinants based on a marker virus constructed previously [J. Virol. 72 (1998) 5318-5322] is described. These viruses, termed vA187CAT_5UTRBVD and vA187CAT_IRESBVD, contain the entire 5' untranslated region (5'UTR) or the internal ribosome entry site (IRES) of bovine viral diarrhea virus (BVDV), respectively. Both chimeric viruses proved to be infectious in cell culture. Hence, the 5'UTR as well as the IRES element only of BVDV can substitute for the corresponding genome region of CSFV. Next, two sets of primers and corresponding dual-labeled TaqMan probes were designed; one detecting specifically a conserved but CSFV-specific area within the 5'UTR of wild-type CSFV, the other one targeting the CAT gene inserted in vA187CAT_5UTRBVD. The two primer/probe sets were combined in a closed-tube multiplex one-step RT-PCR. To monitor the entire extraction and detection process limited amounts of vA187CAT_5UTRBVD were added directly to clinical samples before RNA extraction. The multiplex RT-PCR proved to be as sensitive as the single primer/probe set method, but allowed the validation of each sample tested individually, based on the detection of the CAT marker gene. vA187CAT_5UTRBVD was also used successfully for foot-and-mouth disease virus (FMDV) TaqMan RT-PCR. Therefore, it is considered a universal internal positive control for RT-PCR assays to exclude loss of RNA during extraction, or failure of amplification due to inhibitory substances present in the sample.     

Development of a real time RT-PCR to detect and type ovine pestiviruses

A real time one-step RT-PCR was designed to detect and type border disease virus (BDV), bovine viral diarrhea virus (BVDV) type 1 and BVDV type 2 in ovine samples. The real time RT-PCR was shown to behave in a linear manner and had limits of detection of 100-1000 copies of viral RNA as judged by in vitro transcribed RNA. The real time RT-PCR was validated on 50 clinical samples from UK flocks and was more sensitive than a virus isolation and a classical nested RT-PCR (nRT-PCR). The results of real time RT-PCR virus typing agreed completely with sequencing. The majority of ovine isolates were BDV; a small proportion were BVDV type 1. BVDV type 2 was not detected in any sample. This test appears reliable and can be used for the typing of ovine pestiviruses in the UK.     

Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829-837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303 epitope in CSFV virulent strain Brescia (BICv) to obtain the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). In vitro growth of mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) was similar to parental BICv, while mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and reduced plaque size. In vivo, T1v, T2v or T3v induced lethal disease, T4v induced mild and transient disease and T5v induced mild clinical signs. Protection against BICv challenge was observed at 3 and 21 days post-T5v infection. These results indicate that E2 residues TAVSPTTLR play a significant role in CSFV virulence.     

Classical swine fever virus: Antigenic architecture of the E2 glycoprotein elucidated by epitope mapping using synthetic peptides

Detailed epitope characterization of the major envelope glycoprotein E2 of the classical swine fever virus (CSFV) is important for our understanding of interactions between the virus and the host immune system, as well as for the development of CSFV-specific diagnostic assays and epitope- or peptide-based marker vaccines. As was shown previously by competitive binding assay, monoclonal antibodies obtained by our group recognize eight epitopes on the E2 protein. Here, we report mapping of five linear, nonoverlapping B-cell epitopes that use a set of synthetic peptides, which encompass the full sequence of the CSFV E2 protein (Shimen strain). Two of the epitopes are located in the antigenic domain A of the E2, while another three belong to a highly structured region of this glycoprotein. The alignment of the identified gene sequences was performed for 12 CSFV strains, three strains of bovine viral diarrhea virus (BVDV), and two strains of the border disease virus (BDV). The data obtained could be used to improve CSFV diagnostic assays, as well as to investigate the effects of aminoacid substitutions in E2 on its antigenic properties.     

Genomic characterization of pestiviruses isolated from lambs and kids in southern Italy

A nested polymerase chain reaction was used to identify 13 pestivirus strains isolated from small ruminants in several mixed (sheep and goats) flocks of Southern Italy, and for classification as bovine viral diarrhoea virus (BVDV) type 1, BVDV type 2, and Border disease virus (BDV) genotypes. Of the nine ovine isolates, two were characterized as BVDV type 1, and seven as BVDV type 2. The four pestiviruses isolated from kids belong to BVDV type 1. None of the pestivirus strains tested could be classified as 'true' BDV (genotype 3). Although BVDV type 2 has been described in Europe rarely, the characterization of BD/90-1M strain as BVDV type 2, isolated in Italy in 1990, demonstrates that this genotype has been circulating in Italy since the 1990s.     

Intracellular virus-induced polypeptides of pestivirus border disease virus

Intracellular virus specific polypeptides of pestivirus, border disease virus (BDV) in bovine turbinate cells were analysed by radio-immunoprecipitation with specific antisera. Eleven viral polypeptides with molecular weights of 220, 165, 118, 84, 66, 58, 55, 53, 45, 37 and 31 kDa, respectively, were detected in infected cells. Of these, the 165, 118, 84, 66, 58, 55, 53, 45 and 31 kDa proteins were found to be glycosylated. Comparative studies indicated that the polypeptides induced by BDV share many antigenic epitopes with those of the polypeptides induced by bovine viral diarrhea virus (BVDV), a serologically related virus of the same genus, pestivirus. The polypeptide profile of BDV appeared to be more similar to that of the noncytopathic BVDV strain NY1 compared to that of cytopathic BVDV strains NADL and Singer. Peptide mapping analysis of homologous polypeptides from BVDV and BDV confirmed their structural relatedness.     

Cytometric microsphere array for subtyping avian influenza virus

Avian influenza is a highly contagious disease, and different subtypes of avian influenza virus (AIV) have different levels of pathogenicity. A microsphere-based fluorescent assay was initially established for subtyping AIV. DNA fragments were amplified with biotinylated primers. AIV subtype-specific DNA probes with an amino-linker at the 5' end were covalently bound with carboxy-modified encoded beads. The modified beads and the denatured DNA fragments were mixed together for hybridization. Then, quantum dots-streptavidin (QDs-streptavidin) was added to conjugated biotinylated PCR products. The reaction products were screened by flow cytometry. AIV strains (such as H5N1 and H9N2) could be determined and subtyped according to their combination of encoded beads and fluorescent QDs. The method's combined sensitivity of the nucleic acids of H5N1 and H9N2 avian influenza virus at a threshold of 74 pg and 1 pg could be detected. This is a powerful method for detecting many pathogens or many types of a pathogen simultaneously.     

Genetic diversity of pestiviruses: identification of novel groups and implications for classification.

The complete Npro coding sequences were determined for 16 pestiviruses isolated from cattle, pig, and several wild ruminant species including reindeer, bison, deer, and bongo. Phylogenetic analysis enabled the segregation of pestiviruses into the established species bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, border disease virus (BDV), and classical swine fever virus (CSFV). For BVDV-1 five distinct subgroups were identified, while BVDV-2, BDV, and CSFV were each subdivided into two subgroups. The virus isolates from bongo and deer as well as one porcine virus isolate belong to BVDV-1. Interestingly, the isolates from reindeer and bison are distinct from the established pestivirus species. The Npro sequences from these two viruses are more similar to BDV than to the other pestivirus species. Calculation of the pairwise evolutionary distances allowed a clear separation of the categories species, subgroup, and isolate only when the reindeer/bison viruses were considered as members of an additional pestivirus species. Furthermore, the entire E2 coding sequences of a representative set of virus isolates covering all recognized species and subgroups were studied. Segregation of pestiviruses based on the E2 region was identical with that obtained with the N(pro) sequences.     

人乳头瘤病毒基因分型液态芯片的构建

目的:通过构建人乳头瘤病毒基因分型的液态芯片,建立高通量、灵敏和特异的HPV型别检测系统.方法:在LuminexTM平台上,采用国际公认的标准HPV质粒建立13种基因分型HPV芯片,选择引物及探针,将探针有效的偶联到微球上.结果:用LuminexTM平台建立了13型HPV芯片,对单一或两种标准质粒混合的样品均可准确分型.对分型检测的阳性标本进行基因测序,经美国NCBI数据库BLAST比对符合其检测型别.结论:利用LuminexTM平台构建了13种HPV 型别液相芯片的诊断体系,具有高通量、快速准确等特点,适用于大规模临床样本的HPV基因分型的诊断.     

Presumptive diagnostic differentiation of hog cholera virus from bovine viral diarrhea and border disease viruses by using a cDNA nested-amplification approach.

Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV), and border disease virus (BDV) are closely related pestiviruses. BVDV and BDV are found worldwide but seldom cause disease in swine. In contrast, HCV has been successfully eradicated from swine in several nations but poses a potentially devastating threat to them because of its great virulence. Rapid differential diagnosis of HCV from BVDV and BDV infections in swine is vital for detection of the possible reintroduction of HCV into national herds from which it has been eradicated. Nested polymerase chain reactions (PCRs) for each of two pestiviral genomic segments are described. Amplification of the relatively conserved 5' genomic terminus identified 59 of 61 HCV, BVDV, and BDV isolates generically as pestiviruses. Nested amplification of the second region was designed to differentiate HCV from BVDV and BDV by exploiting relatively conserved differences in the nucleotide sequences that encode the major envelope glycoprotein. This second PCR correctly identified 36 of 36 diverse HCV isolates while failing to recognize any of 25 BVDV and BDV isolates. Multiple restriction fragment length analyses confirmed the identities of both external and nested PCR products. The two sets of PCRs may help confirm the generic identity of most pestiviruses and may permit presumptive differential diagnosis of HCV from BVDV and BDV.     

Rapid detection of a plant virus by solution hybridization using oligonucleotide probes

A novel solution hybridization method for the diagnosis of a plant virus was evaluated. Synthetic oligonucleotide probes were used for the detection of potato virus X (PVX) in crude leaf sap extracts by hybridization in solution. Three 40-nucleotide-long oligonucleotide probes complementary to RNA sequences of potato virus X near the 3' end were synthesized. Two probes were 32P-labelled and one biotinylated. The three probes were allowed to form hybrids with the target viral nucleic acid in solution, and the formed hybrids were isolated with the aid of the biotinylated capture probe using avidin polystyrene beads after the reaction. Alternatively, hybrids were captured from the poly(A) tail of the viral RNA on oligo(dT) cellulose. The maximum signal was obtained after 4 h hybridization. About 70% of the maximum signal was obtained after 2 h hybridization. Sensitivity with the two 32P-labelled oligonucleotide probes was 1-5 x 10(7) molecules of PVX RNA. This corresponds to 0.6-3 ng of the virus. Crude leaf sap did not interfere with the detection of the virus. These results suggest that this solution hybridization method permits rapid detection of a plant virus in crude plant sap without sample pretreatment and may thus open new avenues for the development of a nucleic-acid-based ELISA-like diagnostic test for the detection of plant viruses.     

Application of recombinant GP III a combined Luminex beads for the detection of HPA-la antibody北大核心CSCD

Objective To generate recombinant GP III a as an alternative source for HPA-la antigen and combine it with Luminex xMAP beads for the detection of HPA-la-specific alloantibody. Methods The full coding region of ITGB3gene was amplified and ligated with pcDNA3. 1. The recombinant plasmid was transfected into CHO cells, andthose with stable expression were screened with G418. Expressed protein was identified and coupled with Luminex xMAP beads, which were then reacted with sera samples. Subsequently, phycoerythrin-labeled anti-species IgGantibody was added to the reaction wells and the median fluorescence was determined on a Luminex-100 analyzer.Results DNA sequencing confirmed that the cloned ITGB3 gene was HPA-laa. The recombinant GP III a was coupledwith Luminex xMAP beads. The sensitivity of Luminex beads assay to detect HPA-la antibody was dilution 1/32 (3. 125 U/mL). The Luminex beads assay could specifically identify the HPA-la antibody from the test sera, and theresults were consistent with that of monoclonal antibody-specific immobilization of platelet antigens (MAIPA)technology. Cross-reactivity was not observed with the samples containing HLA, ABO and other HPA antibodies (HPA-3a and HPA-5b). The results illustrated that to detect HPA antibody with Luminex xMAP beads technology isfeasible. Conclusion Recombinant GP III a was successfully obtained and used to establish a Luminextechnology-based method for the detection of HPA antibodies.     

A method for typing polymorphism at the HLA-A locus using PCR amplification and immobilized oligonucleotide probes

Abstract: We have developed a simple and rapid method for DNA typing of the HLA-A locus using PCR amplification and hybridization of the PCR product, labeled with biotinylated primers, to an array of immobilized oligonucleotide probes in a single hybridization reaction (reverse dot or line blot). A single primer set (RAP1007 and DB337) is used to specifically amplify a 990-bp fragment containing the HLA-A locus exons 1, 2, and 3 from genomic DNA. This primer set is locus-specific and amplifies all HLA-A alleles. A set of 51 sequence-specific oligonucleotide (SSO) probes, 25 for exon 2 and 26 for exon 3, was immobilized to a nylon membrane by UV-crosslinking oligonucleotide probes containing a poly-thymidine "tail" added with terminal transferase. In the line blot format, all 50 SSO probes plus a control probe are immobilized on a single nylon membrane strip. The probe array was used for typing in a hybridization reaction with DNA amplified from a variety of samples. These probes can identify 37 homozygous HLA-A alleles. In the analysis of heterozygous samples, 604 heterozygous types out of 633 (95.4%) possible heterozygous probe patterns can be detected as a unique probe reactivity pattern. A simple computer program has been developed to assign the alleles and genotypes based on the probe hybridization pattern.