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 |
Production of Equine Infectious Anemia Virus (EIAV) antigen in Pichia pastoris. Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropr...
Amplification of complete gag gene sequences from geographically distinct equine infectious anemia virus isolates. In the current study, primers described previously and modified versions of these primers were evaluated for amplification of full-length gag genes from different equine infectious anemia virus (EIAV) strains from several countries, including the USA, Germany and Japan. Each strain was inoculated into a primary horse leukocyte culture, and the full-length gag gene was amplified by reverse transcription polymerase chain reaction. Each amplified gag gene was cloned into a plasmid vector for sequencing, and the detectable copy numbers of target DNA were determined. Use of a mixture of two forward...
Diagnostic accuracy of a duplex real-time reverse transcription quantitative PCR assay for detection of African horse sickness virus. Blood samples collected from 503 suspect cases of African horse sickness (AHS) and another 503 from uninfected, unvaccinated South African horses, as well as 98 samples from horses from an AHS free country, were tested with an AHS virus (AHSV) specific duplex real-time reverse transcription quantitative PCR (RT-qPCR) assay and virus isolation (VI). The diagnostic sensitivity and specificity of this AHSV RT-qPCR assay and VI were estimated using a 2-test 2-population Bayesian latent class model which made no assumptions about the true infection status of the tested animals and allowed for the p...
Specific detection of all members of the Venezuelan Equine Encephalitis complex: development of a RT-Nested PCR. Venezuelan Equine Encephalitis (VEE) complex belongs to alphavirus genus in the family Togaviridae. Several species of this complex are pathogenic to humans. VEE infections can produce severe or mild disease, and many cases remain undiagnosed. A specific and sensitive reverse transcriptase nested polymerase chain reaction (RT-Nested PCR) method was developed for the detection of all VEE subtypes, including Rio Negro Virus (RNV) (subtype VI), which circulates only in Argentina. Degenerated primers were designed and thermal cycling parameters were standardized. This technique is suitable for rap...
Hendra virus detection using Loop-Mediated Isothermal Amplification. Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amp...
Recombinant envelope protein (rgp90) ELISA for equine infectious anemia virus provides comparable results to the agar gel immunodiffusion. Equine infectious anemia (EIA) is an important viral infection affecting horses worldwide. The course of infection is accompanied generally by three characteristic stages: acute, chronic and inapparent. There is no effective EIA vaccine or treatment, and the control of the disease is based currently on identification of EIAV inapparent carriers by laboratory tests. Recombinant envelope protein (rgp90) was expressed in Escherichia coli and evaluated via enzyme-linked immunosorbent assay (ELISA). There was an excellent agreement (95.42%) between the ELISA results using rgp90 and agar gel immunod...
Development and laboratory evaluation of two lateral flow devices for the detection of vesicular stomatitis virus in clinical samples. Two lateral flow devices (LFD) for the detection of vesicular stomatitis (VS) virus (VSV), types Indiana (VSV-IND) and New Jersey (VSV-NJ) were developed using monoclonal antibodies C1 and F25VSVNJ-45 to the respective VSV serotypes. The performance of the LFDs was evaluated in the laboratory on suspensions of vesicular epithelia and cell culture passage derived supernatants of VSV. The collection of test samples included 105 positive for VSV-IND (92 vesicular epithelial suspensions and 13 cell culture antigens; encompassing 93 samples of subtype 1 [VSV-IND-1], 9 of subtype 2 [VSV-IND-2] and 3...
Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for H3N8 equine influenza virus. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to the detection of equine influenza virus (EIV). Because equine influenza is caused currently by EIV of the H3H8 subtype, the RT-LAMP primer set was designed to target the hemagglutinin gene of this subtype. The detection limit of the RT-LAMP assay was a virus dilution of 10(-5); which was 10(3) times more sensitive than the Espline Influenza A&B-N test and 10 times more sensitive than a reverse transcription polymerase chain reaction (RT-PCR) assay. The specificity of the RT-LAMP assay was examined by usin...
Development of an antigen-capture ELISA for the detection of equine influenza virus nucleoprotein. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for the detection of the equine influenza virus (EIV), employing monoclonal and polyclonal antibodies against the A/equine/Xingjiang/2007 (H3N8) nucleoprotein (NP). Immunoglobulin G antibodies were purified and used as capture or detector antibodies. The specificity of the optimized AC-ELISA was evaluated using EIV, equine herpesvirus 1 (EHV-1), equine herpesvirus 4 (EHV-4), equine arteritis virus (EAV) and Japanese encephalitis virus (JEV), resulting in only EIV specimens yielding a strong signal. A minimal concentr...
A competitive ELISA for the detection of group-specific antibody to equine encephalosis virus. A polyclonal antibody-based, group-specific, competitive ELISA (C-ELISA) for the detection of antibodies to equine encephalosis virus (EEV) was developed. The assay measures the competition between a specific guinea pig antiserum and a test serum, for a pre-titrated EEV antigen. The C-ELISA detected antibodies to the seven known EEV serotypes. Reference antisera raised against other arboviruses did not cross react with EEV antigen. Negative sera from horses in the United Kingdom were used to establish the baseline for a negative population. Negative and positive populations of South African ho...
Development of a new primer-probe energy transfer method for the differentiation of neuropathogenic and non-neuropathogenic strains of equine herpesvirus-1. Equine herpesvirus-1 (EHV-1) is a major pathogen of horses with worldwide distribution that can cause various clinical signs ranged from mild respiratory disease to neurological symptoms. Comparison of neuropathogenic and non-neuropathogenic EHV-1 strains revealed that a single non-synonymous nucleotide substitution (A/G2254) in the ORF30 region is associated with the altered functions of the viral DNA polymerase and therefore the neuropathogenicity of EHV-1 virus strains. The aim of the present study was the development of a new differentiation method of this particular single nucleotide poly...
Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis virus. An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and...
Immunochromatographic lateral flow test for detection of antibodies to Equine infectious anemia virus. The purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (ICLF) test for specific detection of Equine infectious anemia virus (EIAV) antibodies in equine sera. Viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. The performance of rp26-ICLF was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (AGID) test used as a standard of comparison according to international guidelines. The values obtained for co...
Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus. Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. ...
Development of a blocking ELISA using a recombinant glycoprotein for the detection of antibodies to vesicular stomatitis New Jersey virus. A recombinant glycoprotein (R-GP) of vesicular stomatitis New Jersey virus (VSV-NJ) was expressed in insect cells by a baculovirus system. Its utility as a diagnostic antigen in a blocking ELISA was investigated as an alternative to the current native GP extracted from VSV-NJ. With the cut-off value of 73% inhibition, the R-GP ELISA exhibited 99.1% specificity for naive sera from cattle and horses. It did not cross-react with VSV-Indiana (VSV-IN) positive sera and differentiated from foot-and-mouth disease and swine vesicular disease. Taken together, this is the first report that the R-GP has ...
A multisystem approach for development and evaluation of inactivated vaccines for Venezuelan equine encephalitis virus (VEEV). A multisystem approach was used to assess the efficiency of several methods for inactivation of Venezuelan equine encephalitis virus (VEEV) vaccine candidates. A combination of diverse assays (plaque, in vitro cytopathology and mouse neurovirulence) was used to verify virus inactivation, along with the use of a specific ELISA to measure retention of VEEV envelope glycoprotein epitopes in the development of several inactivated VEEV candidate vaccines derived from an attenuated strain of VEEV (V3526). Incubation of V3526 aliquots at temperatures in excess of 64 degrees C for periods >30 min i...
PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis. A nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR), for rapid detection of African horse sickness virus (AHSV) double-stranded ribonucleic acid (dsRNA) in cell culture and tissue samples, was developed and evaluated. Using an outer pair of primers (P1 and P2), selected from genome segment three of AHSV serotype 6 (AHSV-6), the RT-PCR-based assay resulted in amplification of a 890 base pair (bp) primary PCR product. RNAs from the nine vaccine strains of AHSV, and a number of AHSV field isolates including the Central African isolates of AHSV-9 and AHSV-6, propagated in cell c...
A highly sensitive method for the detection and genotyping of West Nile virus by real-time PCR. In recent years, West Nile virus has been responsible for outbreaks in regions where it has not previously been found. Five genetic lineages with specific geographic distributions exist. Recent outbreaks of WNV associated with the introduction of lineage 1 strains into the western hemisphere, together with the emergence of lineage 2 WNV in Central Europe, has highlighted the potential for spread of pathogenic WNV strains beyond their expected geographical boundaries. Therefore, genotyping of WNV strains may have important applications in surveillance and epidemiology. We report here the develo...
Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens. Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duple...
Comparison of different molecular methods for assessment of equine arteritis virus (EAV) infection: a novel one-step MGB real-time RT-PCR assay, PCR-ELISA and classical RT-PCR for detection of highly diverse sequences of Slovenian EAV variants. In the present study, a new one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) strategy with minor-groove-binder (MGB) technology for the detection of EAV from 40 semen samples of Slovenian carrier stallions was tested. A novel MGB probe (EAVMGBpr) and a reverse primer (EAV-R) based on the multiple sequence alignment of 49 different EAV strain sequences of the highly conserved ORF7 (nucleocapsid gene) were designed. The performance of the assay was compared with different molecular detection methods. Three different primer pairs targeting the ORF1b and ORF7 were used, ...
Development of a real-time RT-PCR assay for improved detection of Borna disease virus. Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan probes and primers specific for the BDV P and BDV L...
Laboratory diagnosis of equine rabies and its implications for human postexposure prophylaxis. Laboratory diagnosis is essential to confirm suspected cases of equine rabies and to determine the medical care needed for human postexposure antirabies prophylaxis. Equine rabies transmitted by the vampire bat, Desmodus rotundus, has increased gradually in the State of São Paulo. The present study has several objectives, the most important being the evaluation of fluorescent antibody test (FAT) and virus-isolation laboratory tests performed with different equine nervous system tissues (cortical, hippocampus, cerebellar, brainstem and cervical medullar) to determine the tissue for which the t...
RT-PCR for detection of all seven genotypes of Lyssavirus genus. The Lyssavirus genus includes seven species or genotypes named 1-7. Rabies genotypes correlate with geographical distribution and specific hosts. Co-circulation of different lyssaviruses, imported cases, and the presence of unknown viruses, such as Aravan, Khujand, Irkut and West Caucasian Bat Virus, make it necessary to use generic methods able to detect all lyssaviruses. Primer sequences were chosen from conserved regions in all genotypes in order to optimise a generic RT-PCR. Serial dilutions of 12 RNA extracts from all seven Lyssavirus genotypes were examined to compare the sensitivity of ...
Detection of equine herpesvirus type 1 by real time PCR. A real-time PCR assay was developed for detection and quantitation of equid herpesvirus type 1 (EHV-1). The sensitivity of the assay was compared with an established nested-PCR (n-PCR). The real-time PCR detected 1 copy of target DNA, with a sensitivity 1 log higher than gel-based n-PCR. The assay was able to detect specifically EHV-1 DNA in equine tissue samples and there was no cross-amplification of other horse herpesviruses. Real-time PCR was applied to determine EHV-1 load in tissue samples from equine aborted fetuses. The high sensitivity and reproducibility of the EHV-1-specific fluorog...
Recovery of Swedish Equine arteritis viruses from semen by cell culture isolation and RNA transfection. Recovery of infectious Equine arteritis virus (EAV) from the semen of persistently infected Swedish stallions was attempted by classical cell culture isolation and by transfection of extracted total RNA. Whereas virus from semen samples stored for several months at -20 degrees C or from extended semen could only be recovered by transfection of extracted RNA, isolation in cell culture was achieved readily with fresh, unextended semen stored at -70 degrees C or directly used after sampling. In parallel, the viruses were examined in the variable region of the large glycoprotein GP5 by nested RT-P...
Simultaneous identification of orthopoxviruses and alphaviruses by oligonucleotide macroarray with special emphasis on detection of variola and Venezuelan equine encephalitis viruses. The development of a method in macroarray format for the identification of alphaviruses and orthopoxviruses in samples of concern in biodefense is reported. Capture oligonucleotides designed to bind generic members of the orthopox- or alphavirus families and a collection of additional oligonucleotides to bind specifically nucleic acids from five individual alphaviruses, including Venezuelan equine encephalitis, or DNA from each of four orthopoxviruses, including variola virus (VAR) were deposited onto nylon membranes. Hybridization of digoxigenin labeled PCR products to the macroarray produced...
Detection of equine herpesvirus type 1 using a real-time polymerase chain reaction. Equid herpesvirus 1 (EHV1) is a major disease of equids worldwide causing considerable losses to the horse industry. A variety of techniques, including PCR have been used to diagnose EHV1. Some of these PCRs were used in combination with other techniques such as restriction enzyme analysis (REA) or hybridisation, making them cumbersome for routine diagnostic testing and increasing the chances of cross-contamination. Furthermore, they involve the use of suspected carcinogens such as ethidium bromide and ultraviolet light. In this paper, we describe a real-time PCR, which uses minor groove-bindi...
Development of monoclonal antibody-linked ELISA for sero-diagnosis of vesicular stomatitis virus (VSV-IN) using baculovirus expressed glycoprotein. The gene encoding the envelope glycoprotein (GP) of vesicular stomatitis virus serotype, Indiana (VSV-IN), was expressed under the polyhedron promoter of baculovirus. The recombinant GP was applied as a diagnostic antigen for the detection of cattle and horse antibodies to VSV. In addition, the neutralizing monoclonal antibody (Mab) to GP of VSV-IN was used as trapping antibody in a Mab-linked indirect ELISA (MLI-ELISA) or detecting antibody in a Mab-linked competitive ELISA (MLC-ELISA). The diagnostic efficiencies of MLI-ELISA and MLC-ELISA were evaluated with currently available C-ELISA from...
Preparation of recombinant African horse sickness virus VP7 antigen via a simple method and validation of a VP7-based indirect ELISA for the detection of group-specific IgG antibodies in horse sera. This paper describes the production and purification of a group-specific recombinant protein VP7 of African horse sickness virus serotype 3 (AHSV-3) and validation of an I-ELISA for the detection of IgG-antibodies to VP7 in horse sera. Baculovirus-expressed VP7 crystals were purified from infected insect cells. Analytical accuracy of the I-ELISA was examined using sera (n = 38) from an experimentally infected horse, from foals born to vaccinated mares, from guinea-pigs immunized with nine serotypes of AHSV, and from sera of animals infected with other orbiviruses. Compared to traditional serol...
African horsesickness virus serotyping and identification of multiple co-infecting serotypes with a single genome segment 2 RT-PCR amplification and reverse line blot hybridization. Since protection against African horsesickness (AHS) is serotype-specific, rapid serotyping of AHSV is crucial to identify the correct vaccine serotype for efficient control of the spread of AHS outbreaks, especially when they occur in non-endemic regions. This paper describes the first one-day serotyping procedure that requires only a single RT-PCR and hybridization and which can identify multiple serotypes in mixed infections in one assay. The same region of genome segment 2 of all nine AHSV serotypes is amplified in a single RT-PCR. A universal primer set, designed to amplify the 5'-termina...