ABSTRACT
The production, preliminary characterisation and applications of monoclonal antibodies (mAbs) against two novel swine bocaviruses isolated in cell culture from swine in Northern Ireland are described. Of the 17 stable final clones produced, four were characterised. All were of the IgG2a isotype and showed no cross-reactivity with either bocavirus strain. Partial neutralisation was observed with PBoV4 mAbs and homologous virus. The two mAbs selected for use in antigen-detecting ELISAs were successful in highlighting those fractions containing infectious virus within sucrose gradients. This is the first report of the production of specific reagents that will prove useful in the study of the biology of these viruses and swine bocavirus-associated diseases.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Antibodies, Viral/biosynthesis , Bocavirus/immunology , Bocavirus/isolation & purification , Sus scrofa/virology , Animals , Antigens, Viral/analysis , Bocavirus/pathogenicity , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Immunoglobulin G/biosynthesis , Mice , Mice, Inbred BALB C , Neutralization Tests , Northern Ireland , Parvoviridae Infections/veterinary , Parvoviridae Infections/virology , Swine , Swine Diseases/virologyABSTRACT
In this study the design and development of two real-time PCR assays for the rapid, sensitive and specific detection of infectious laryngotracheitis virus (ILTV) DNA is described. A Primer-Probe Energy Transfer (PriProET) assay and 5' conjugated Minor Groove Binder (MGB) method are compared and contrasted. Both have been designed to target the thymidine kinase gene of the ILTV genome. Both PriProET and MGB assays are capable of detecting 20 copies of a DNA standard per reaction and are linear from 2×10(8) to 2×10(2)copies/µl. Neither PriProET, nor MGB reacted with heterologous herpesviruses, indicating a high specificity of the two methods as novel tools for virus detection and identification. This study demonstrates the suitability of PriProET and 5' conjugated MGB probes as real-time PCR chemistries for the diagnosis of respiratory diseases caused by ILTV.
Subject(s)
DNA Primers/chemistry , DNA Primers/genetics , DNA, Viral/isolation & purification , Herpesviridae Infections/veterinary , Herpesvirus 1, Gallid/isolation & purification , Polymerase Chain Reaction/methods , Poultry Diseases/diagnosis , Animals , Base Sequence , DNA, Viral/genetics , Herpesviridae Infections/diagnosis , Herpesviridae Infections/virology , Herpesvirus 1, Gallid/genetics , Molecular Sequence Data , Poultry , Poultry Diseases/virology , Sensitivity and SpecificityABSTRACT
The design and development of a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR assay are described for rapid, sensitive and specific detection of swine vesicular disease virus (SVDV) RNA. The assay is designed to target the 2C gene of the SVDV genome and is capable of detecting 2×10(2) copies of an RNA standard per reaction. It does not detect any of the other RNA viruses that cause vesicular disease in pigs, or the human enterovirus, Coxsackie B5 virus (CVB5) which is closely related antigenically to SVDV. The linear range of this test was from 2×10(2) to 2×10(8) copies/µl. The assay is rapid and can detect SVDV RNA in just over 3.5 h including the time required for nucleic acid extraction. The development of this assay provides a useful tool for the differential diagnosis of SVD or for the detection of SVDV in research applications. This study demonstrates the suitability of MGB probes as a real-time PCR chemistry for the diagnosis of swine vesicular disease.
Subject(s)
DNA Primers/genetics , Enterovirus B, Human/isolation & purification , Enterovirus Infections/diagnosis , Reverse Transcriptase Polymerase Chain Reaction/methods , Swine Diseases/diagnosis , Swine Diseases/virology , Virology/methods , Animals , Enterovirus B, Human/genetics , Enterovirus Infections/virology , Sensitivity and Specificity , Swine , Time FactorsABSTRACT
Nine viral diseases included in the World Organization for Animal Health list of notifiable diseases (former list A) were chosen for their contagiousness and high capacity of spreading to improve their diagnosis using new and emerging technologies. All the selected diseases--foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease and highly pathogenic avian influenza--are considered as transboundary diseases, which detection causes the prohibition of livestock exportation, and, thus, it leads to high economical losses. The applied diagnostic techniques can fall into two categories: (i) nucleic-acid detection, including padlock probes, real-time PCR with TaqMan, minor groove binding probes and fluorescence energy transfer reaction probes, isothermal amplification like the Cleavase/Invader assay or the loop-mediated amplification technology and the development of rapid kits for 'mobile' PCR and (ii) antigen-antibody detection systems like simplified and more sensitive ELISA tests. Besides, internal controls have been improved for nucleic acid-detecting methods by using an RNA plant virus--Cowpea Mosaic Virus--to ensure the stability of the RNA used as a positive control in diagnostic real-time RT-PCR assays. The development of these diagnosis techniques has required the joint efforts of a European consortium in which nine diagnostic laboratories and an SME who have collaborated since 2004 within the European Union-funded Lab-on-site project. The results obtained are shown in this paper.
