Journal of virological methods.
Publisher:
Elsevier/North-Holland Biomedical Press,
Frequency: Sixteen no. a year, 1999-
Country: Netherlands
Language: English
Start Year:1980 -
ISSN:
0166-0934 (Print)
1879-0984 (Electronic)
0166-0934 (Linking)
1879-0984 (Electronic)
0166-0934 (Linking)
Impact Factor
3.1
2022
| NLM ID: | 8005839 |
| (DNLM): | J41255000(s) |
| (OCoLC): | 06403038 |
| Coden: | JVMEDH |
| LCCN: | sn 80013916 |
| Classification: | W1 JO97T |
Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections. Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining dete...
Phage display of the Equine arteritis virus nsp1 ZF domain and examination of its metal interactions. A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp1 protein was described recently to be required for viral transcription. The nsp1 ZF (50 aa) was expressed on the surface of M13KE gIII phage, fused to the N terminus of the phage pIII protein. To evaluate the functionality of the ZF domain, a binding assay was developed, based on the use of immobilized Ni(2+) ions (Ni-NTA). Phages displaying ZF bound significantly better to Ni-NTA than did phages displaying negative-control peptides, which also contained metal-coordinating residues. Also, binding of ZF-displaying phages...
Development of competitive ELISA for serodiagnosis on African horsesickness virus using baculovirus expressed VP7 and monoclonal antibody. VP7, the sero-group common antigen, of African horsesickness virus (AHSV-4) was expressed in insect cells by recombinant baculovirus. To develop a specific diagnostic method, monoclonal antibody (Mab) against VP7 was prepared and investigated as diagnostic reagent with the baculovirus expressed VP7. However, the Mab against VP7 of AHSV cross-reacted with Chuzan virus by the indirect immunofluorescence assay (IFA), confirming the presence of conserved domain of VP7 among Orbiviruses. This study describes two types of ELISA; Mab linked indirect (I-ELISA) and competitive-ELISA (C-ELISA) using bac...
The development of a competitive PCR-ELISA for the detection of equine herpesvirus-1. Equine herpesvirus-1 (EHV-1) infection is of significant animal welfare and economic importance. Yet, no standardised molecular techniques are available for diagnosis or confirmation of viral infection. The purpose of this study was to develop a standardised and quantitative assay system for the reliable detection of EHV-1 infection which was capable of eliminating the likelihood of false negative results. A region within the EHV-1 glycoprotein B gene was amplified by polymerase chain reaction (PCR), cloned and subjected to site-directed mutagenesis to generate a control plasmid, amplifiable b...
Development of a multiplex real-time reverse transcriptase-polymerase chain reaction for equine infectious anemia virus (EIAV). A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) using a fluorogenic real-time PCR detection method is described for the quantitation of equine infectious anemia virus (EIAV) RNA in the plasma of equids. To compensate for variations inherent in sample preparation a multiplex real-time RT-PCR system was developed that permitted the simultaneous calculation of the nucleic acid recovery rate along with the copy number of viral RNA molecules. Detection of EIAV RNA was linear from 10(9) to 10(1) molecules with intra- and inter-assay variability of less than 1% at 10(8), 10(6),...
Detection of equine arteritis virus by real-time TaqMan reverse transcription-PCR assay. A one-tube real-time TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of equine arteritis virus (EAV). The test was validated using the seminal plasma and nasal secretions of infected horses that were proven to contain EAV by traditional virus isolation in rabbit kidney thirteen (RK-13) cells, as well as a variety of cell culture-propagated European and North American strains of EAV. The primers and a fluorogenic TaqMan probe were designed to amplify and detect a highly conserved region of open reading frame 7 (ORF7) of EAV. The real-time Ta...
Serological diagnosis of equine influenza using the hemagglutinin protein produced in a baculovirus expression system. The hemagglutinin (HA) protein of an equine influenza strain, A/equine/La Plata/1/93 (LP/93), was produced using a baculovirus expression system. Silkworm larvae inoculated with recombinant baculovirus expressed high quantities of the HA protein which was then purified to greater than 95% purity by fetuin-affinity chromatography. Purified HA protein was used subsequently in an ELISA for detection of antibodies in horse sera. Two hundred serum samples from vaccinated racehorses were reacted on ELISA plates coated with 40.0 ng/ml of purified HA protein. Subsequent optical density (OD) levels rev...
DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus. In vivo, tissue macrophages have been implicated as an important cell for the replication of equine infectious anemia virus (EIAV). Laboratory investigations of EIAV/macrophage interactions, however, have been hampered by the laborious blood monocyte isolation procedures. In addition, adherent equine macrophage cultures generally have poor long-term viability and are resistant to transfection. This report describes an adherent canine macrophage-like cell line, DH82, that supports the replication of EIAV. This cell line was easily transfectable and supported EIAV Tat transactivation of the LTR....
Detection of horses infected naturally with equine infectious anemia virus by nested polymerase chain reaction. A nested polymerase chain reaction (PCR) amplifying a region of the gag gene of equine infectious anemia virus (EIAV) was developed for the rapid and direct detection of proviral DNA from the peripheral blood of naturally infected horses and was compared with the Coggins test. DNA prepared from white blood cells of 122 field horses from 15 stables with reported cases of EIAV and one seronegative stable were analysed. Amplifications of expected size fragments were obtained by nested PCR for 88 horses using two different sets of primers targeting the gag region. The specificity of the amplified ...
Utilisation of bacteriophage display libraries to identify peptide sequences recognised by equine herpesvirus type 1 specific equine sera. Three filamentous phage random peptide display libraries were used in biopanning experiments with purified IgG from the serum of a gnotobiotic foal infected with equine herpesvirus-1 (EHV-1) to enrich for epitopes binding to anti-EHV-1 antibodies. The sequences of the amino acids displayed were aligned with protein sequences of EHV-1, thereby identifying a number of potential antibody binding regions. Presumptive epitopes were identified within the proteins encoded by genes 7 (DNA helicase/primase complex protein), 11 (tegument protein), 16 (glycoprotein C), 41 (integral membrane protein), 70 ...
Detection of antibodies to equine arteritis virus by enzyme linked immunosorbant assays utilizing G(L), M and N proteins expressed from recombinant baculoviruses. Indirect enzyme linked immunosorbant assays (ELISAs) utilizing the three major structural proteins (M, N, and G(L)) of equine arteritis virus (EAV) expressed from recombinant baculoviruses were developed. A large panel of sera collected from uninfected horses, and from animals experimentally and naturally infected with EAV or vaccinated with the modified live virus vaccine against equine viral arteritis, were used to characterize the humoral immune response of horses to the three major EAV structural proteins. The data suggest that the M protein was the major target of the equine antibody resp...
Screening of horse polyclonal antibodies with a random peptide library displayed on phage: identification of ligands used as antigens in an ELISA test to detect the presence of antibodies to equine arteritis virus. A random hexapeptide fusion-phage library was screened to isolate phages that bind to antibodies present in horse sera positive for equine arteritis virus (EAV). Analysis of the peptide sequences displayed by isolated phages identified seven groups. 25% of the isolated phages used as antigens in an ELISA test were specifically recognised by a pool of sera which was positive for EAV in virus neutralisation test (VN). Five of these, when used as antigen in ELISA, detected greater than 50% of sera (n = 30) containing antibodies to EAV as detected by VN. When these five phages were pooled together...
Equine monocyte-derived macrophage cultures and their applications for infectivity and neutralization studies of equine infectious anemia virus. Equine infectious anemia virus (EIAV) has been shown to infect cells of monocyte/macrophage lineage. These primary cells are intrinsically difficult to obtain, to purify and to culture in vitro for extended periods of time. As a result, most in vitro studies concerning this lentivirus make use of primary equine fibroblasts or transformed canine or feline cell lines. We describe methods that yield reproducibly pure cultures of equine blood monocytes from peripheral blood mononuclear cells. The in vitro differentiation of these cells into mature equine macrophage was verified using various cytoc...
Expression of the nonstructural protein NS1 of equine influenza A virus: detection of anti-NS1 antibody in post infection equine sera. The nucleotide sequence of the nonstructural protein NS1 of the influenza virus A/equine 2/Suffolk/89 was determined and found to be 97% identical to that of A/equine 2/Miami/63. A similar level of identity was shown for the deduced NS1 amino acid sequence. The NS1 gene was expressed, in its entirety and in part, as fusion proteins with glutathione S-transferase using the pGEX-3X expression vector. Antibodies to NS1 protein were detected in serum samples from ponies experimentally infected with influenza virus, but not in animals vaccinated with whole inactivated virus or in unprimed control a...
Development and evaluation of an ELISA using recombinant fusion protein to detect the presence of host antibody to equine arteritis virus. A recombinant glutathione-S-transferase fusion protein expressing amino acids 55-98 of equine arteritis virus (EAV) GL (rGL 55-98) was tested in an ELISA for its ability to detect serum antibodies to EAV. Host antibodies induced following EAV infection bound the recombinant antigen by ELISA. The ELISA specificity and sensitivity were determined with a panel of equine sera including postinfection and postvaccination samples. A good correlation existed between EAV neutralizing antibody titers and ELISA absorbance values (r = 0.827). The sensitivity and specificity of the ELISA were 99.6 and 90.1...
A mouse model for testing the pathogenicity of equine herpes virus-1 strains. A mouse model was developed for testing the pathogenicity of equine herpes virus-1 (EHV-1) strains. The model was validated with EHV-1 strains that are known to be of a low or high pathogenicity in horses. From all parameters tested, the safety index, which was calculated from the body weights of the mice after infection, proved to be the best predictive parameter. When this parameter was used, good and reliable correlations were found with the pathogenicity of the EHV-1 strains in horses. This method enabled the differentiation between the two experimental EHV-1 strains whose genetic backgrou...
Application of the polymerase chain reaction to the detection of African horse sickness viruses. The development of a coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) is described for the detection of African horse sickness virus (AHSV) double-stranded RNA. Genome segments 7 and 10 were chosen as target templates for primers selected for use in the RT-PCR. Using these AHSV-specific primers all 9 serotypes were detectable. The sensitivity and specificity of the RT-PCR results were compared to those obtained by competition ELISA.
Development of PCR assays to detect genetic variation amongst equine herpesvirus-1 isolates as an aid to epidemiological investigation. A search for variable restriction sites has been carried out for equine herpesvirus-1 (EHV-1) in an attempt to develop markers which can be used to group epidemiologically related viruses into groups, and to learn more about the dynamics of EHV-1 disease. Crude viral DNA extracts of EHV-1, prepared by Hirt extraction, were digested with AluI, HaeIII, or RsaI, and Southern blotted following electrophoresis. DNA fingerprints, produced by probing the Southern blots with the EHV-1 EcoR1-I fragment, separated 56 isolates into 16 groups. The variable sites within the EcoR1-I fragment were mapped app...
Rapid, single-step differentiation of equid herpesviruses 1 and 4 from clinical material using the polymerase chain reaction and virus-specific primers. Sets of primers were designed which enabled specific amplification of homologous regions of the glycoprotein C and gene 76 genetic loci of equine herpesviruses 1 and 4 (EHV-1 and EHV-4). The resultant virus-specific polymerase chain reaction (PCR) products arising from each loci could be discriminated easily on the basis of size on an agarose gel, allowing rapid differentiation of the two equine herpesviruses. Specificity of the amplifications were confirmed by Southern hybridization and restriction endonuclease digestion. The PCR test was applied to nasal swab samples from weanling foals and ...
Detection of Borna disease virus RNA in naturally infected animals by a nested polymerase chain reaction. Borna disease virus in naturally infected horses, a donkey and sheep was detected for the first time by amplification of viral RNA using PCR. In contrast to a control group of healthy horses, brain tissue was positive by this assay in all animals with neurological symptoms. The use of a second round of PCR with nested primers following Southern hybridization confirmed the specificity and increased the sensitivity of the test. Comparison with conventional methods recommends this technique for monitoring of BDV infections at a molecular level.
Diagnosis of the African horse sickness virus serotype 4 by a one-tube, one manipulation RT-PCR reaction from infected organs. A single tube reverse transcription-polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV) in splenic tissues from infected horses is described. Double stranded RNA was extracted from infected organs of horses and used to produce complementary DNA (cDNA) with the two primers selected for the PCR. The 1179 bp amplified product (the segment 7 which encodes for VP 7), detected by electrophoresis on agarose gel and ethidium bromide staining, was hydrolysed with eight restriction endonucleases for characterization of the AHSV. The sensitivity of this method i...
Rapid detection of equine herpesvirus type-1 antigens in nasal swab specimens using an antigen capture enzyme-linked immunosorbent assay. An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine herpesvirus type-1 (EHV-1) antigens in nasal swab specimens. The test was designed as a solid phase, amplified sandwich assay in which an EHV-1 specific monoclonal antibody was used to capture virus antigen and polyclonal antisera used to detect antigen bound to the test plates. Eight monoclonal antibodies were tested for their ability to capture virus antigen and one was selected for routine use. The sensitivity and specificity of the ELISA was compared with that of virus isolation using swa...
Detection of African horsesickness virus in infected spleens by a sandwich ELISA using two monoclonal antibodies specific for VP7. A sandwich enzyme-linked immunsorbent assay (ELISA) for rapid detection of African horsesickness virus (AHSV) in infected spleens or cell culture supernatant was developed. This method uses two monoclonal antibodies (MAbs) which recognize two non-overlapping epitopes of the major core protein (VP7) to coat the solid phase, and one labeled with biotin as second antibody. This ELISA was evaluated for its ability to detect AHSV in infected spleens resulting in a sensitivity of 97.4% and a specificity of 100% compared with virus isolation in cell culture, and can be used for the detection of the n...
Increased sensitivity of a rotavirus serotyping enzyme-linked immunosorbent assay by the incorporation of CaCl2. The sensitivity of a rotavirus serotyping enzyme-linked immunosorbent assay (ELISA) was improved by the addition of 0.5 mM CaCl2 to the washing buffer and reagent diluent. Twenty-nine of 63 (46%) previously untyped bovine and equine faecal rotavirus samples were serotyped in the modified assay. A differential response to Ca2+ ions was noted for different G-serotypes suggesting that serotyping assays performed without the inclusion of CaCl2 in the assay buffers may produce biased results.
An indirect sandwich ELISA utilising F(ab’)2 fragments for the detection of African horsesickness virus. African horsesickness virus (AHSV), an important disease of equines is caused by an orbivirus. Because of the need to contain the spread of the disease, it is often essential to make a rapid diagnosis. For this purpose, an ELISA capable of detecting viral antigen in animal tissue and in cell culture fluid was developed. Immobilised F(ab')2 fragments prepared by digestion of AHSV-specific IgG with pepsin were used to trap virus from tissue homogenates or cell culture supernatant. After addition of intact IgG as detecting antibody, Staphylococcus aureus protein A labelled with horseradish peroxi...
Detection of influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine influenza (H3N8) viruses. An antigen capture indirect enzyme linked immunosorbent assay (ELISA) was developed to detect influenza nucleoprotein antigen in nasal secretions from horses infected with A/equine/H3N8 viruses. Results from this assay were compared with conventional virus isolation in embryonated hens eggs.
Hybridoma cell lines secreting monoclonal antibodies against equine infectious anemia virus. A monoclonal anti-equine infectious anemia virus (anti-EIAV) antibody (1B15) has been generated by fusion of X63 Ag 8.653 myeloma cells and spleen cells from mice hypersensitized with viral antigen p29. Ouchterlony double-diffusion analysis indicated that antibody 1B15 is of the IgG class. The specificity of the immune reaction for p29 was confirmed by cross-over immunoelectrophoresis and disc-gel electrophoresis. MAb 1B15 was used to devise a solid-phase 'capture' RIA for EIAV-p29 antigen. The antigen, bound by 1B15 adsorbed onto wells of flexible microtitre plates, was detected using a rabbi...
Use of indirect and competitive ELISAs to compare isolates of equine influenza A virus. Antigenic differences within equine-1 and equine-2 isolates of influenza were studied by haemagglutination inhibition tests, indirect ELISA and competition ELISA, using the same antisera. Better differentiation was obtained with the competition ELISA than with the other two tests. All three methods produced similar relationships within the equine-1 isolates but differed in their ability to differentiate the equine-2 isolates where the competition ELISA was superior and produced epidemiologically sensible results. In all three tests, post-infection ferret and horse sera were more useful in disc...