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S Zientara

Publications and source records attributed to S Zientara.

At least 37 records · Page 2Linked to original sources
PubMed1998

Molecular epidemiology of African horse sickness virus based on analyses and comparisons of genome segments 7 and 10.

This paper describes a method to rapidly identify African horse sickness virus (AHSV), using a single tube reverse transcription polymerase chain reaction (PCR). This method was used to amplify cDNA copies of genome segments 7 and 10 from several different AHSV strains, of different serotypes, which were then analysed by sequencing and/or endonuclease digestion. AHSV VP7 (encoded by genome segment 7) is one of the two major capsid proteins of the inner capsid layer, forming the outer surface of the core particle. VP7 is highly conserved and is the major serogroup specific antigen common to all nine AHSV serotypes. Digestion of the 1179 bp cDNA with restriction enzymes, allowed differentiation of several strains of different serotypes and identified six distinct groups containing AHSV-1, 3, 6 and 8; AHSV-2; AHSV-4; AHSV-5; AHSV-7; and AHSV-9. Differences were detected between wild type viruses and vaccine strains that had been attenuated by multiple passage in suckling mouse brain or in tissue cultures. RFLP analysis was also used to study variation the 758 bp cDNA copies of AHSV genome segment 10, which encodes the two small non-structural membrane proteins NS3 and NS3a. In this way it was possible to distinguish each of the strains tested, except AHSV 4 (USDA) and AHSV 9 (USDA). However, these isolates could be distinguished by RFLP analysis of genome segment 7 cDNA. Using sequence analysis of genome segment 10 we were able to classify the virus isolates into three groups: AHSV-1, 2 and 8; AHSV-3 and 7; AHSV 4, 5, 6 and 9. These studies confirmed that the virus which first appeared in central Spain in July 1987, subsequently spread into northern Morocco in October 1989.

African Horse Sickness↗
PubMed1998

Structural studies of orbivirus particles.

We are using crystallographic methods to investigate the structure of AHSV and BTV. Our initial approach was to investigate the structure of the major protein component of the viral core, VP7(T13). This trimeric protein has been studied in several crystal forms from both orbiviruses and reveals a structure made up of conserved domains, capable of conformational changes and possessing a cleavage site. Further crystallographic analyses of native particles have provided a picture of the VP7(T13) and VP3(T2) layers of the BTV core. The VP7(T13) layer consists of 260 trimers arranged rather symmetrically and possessing very similar structures, thereby following the rules of quasi equivalence. The VP3(T2) layer is thin and contains 120 copies of 100 kDa protein arranged as 60 approximate dimers. This type of icosahedral construction has not been observed before and appears to contain a genome which is highly ordered. We anticipate that all of these features will be common to AHSV.

African Horse Sickness Virus↗
PubMed1998

Validation of ELISA for the detection of African horse sickness virus antigens and antibodies.

The mortality rate in susceptible populations of horses during an epizootic of African horse sickness (AHS) may be in excess of 90%. Rapid and reliable assays are therefore essential for the confirmation of clinical diagnoses and to enable control strategies to be implemented without undue delay. One of the major objectives of a recent European Union funded project was the validation of newly developed diagnostic assays which are rapid, sensitive, highly reproducible and inexpensive, for the detection of African horse sickness virus (AHSV) antigens and antibodies. The Laboratorio de Sanidad y Produccion Animal (LSPA) in Algete, Spain was charged with the responsibility of co-ordinating and supplying samples of viruses and antisera to the participating laboratories in Spain, France and the United Kingdom. The panels comprised 76 antigen samples for assay by indirect sandwich ELISAs and 53 serum samples for antibody detection by either indirect or competitive ELISAs. Results generated by ELISA for each laboratory were analysed in LSPA in terms of their relative sensitivities and specificities. There was a good agreement between the ELISAs used for either antigen or antibody detection. The participating groups agreed that any field sample giving a doubtful result would always be retested by ELISA and an alternative assay.

African Horse Sickness↗
PubMed1998

Use of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridisation for the detection and identification of African horse sickness virus nucleic acids.

A coupled reverse transcriptase-polymerase chain reaction assay (RT-PCR) for the detection of African horse sickness virus (AHSV) dsRNA, has been developed using genome segment 7 as the target template for primers. RNA from isolates of all nine AHSV serotypes were readily detected. The potential inhibitory effects of either ethylene diamine tetra acetic acid (EDTA) or heparin on the RT-PCR were eliminated by washing blood samples before lysis of the red blood cells and storage. There was a close agreement in the sensitivity and the specificity of the RT-PCR and an indirect sandwich ELISA. Confirmation of the presence of AHSV using RT-PCR and dot-blot hybridization on blood samples collected from horses experimentally infected with AHSV serotype 4 (AHSV 4) and AHSV serotype 9 (AHSV 9), was achieved within 24 hours, compared to the period of several days required for virus isolation. The RT-PCR and virus isolation methods showed similar levels of sensitivity when used for the detection of AHSV in 3 horses infected with AHSV 4, and in 2 out of 3 horses infected with a less virulent isolate of AHSV 9. Although viraemia was detected in the third horse by virus isolation, from 6 to 14 days after infection, this animal remained consistently negative by RT-PCR. Conversely, AHSV viral RNA was detected by RT-PCR in the blood of 4 donkeys and 4 mules up to 55 days after their experimental infection despite the absence of any detectable infectious virus. RT-PCR is a sensitive and rapid method for detecting AHSV nucleic acids during either the incubation period at the start of an African horse sickness (AHS) epizootic, or for epidemiological investigations in species where clinical signs may be inapparent.

African Horse Sickness↗
PubMed1998

Serological and virological responses in mules and donkeys following inoculation with African horse sickness virus serotype 4.

Two groups, comprising 4 donkeys and 4 mules (group 1) and 4 donkeys and 3 mules (group 2), were used to determine the duration of viraemia and to monitor the development of antibodies following inoculation with African horse sickness virus (AHSV). One group of animals was given a single dose of attenuated AHSV serotype 4 (AHSV 4) vaccine. The second group was inoculated with a virulent field strain of AHSV 4. Both groups were subsequently challenged with the virulent field strain of AHSV 4, 51 and 58 days, respectively, after their primary inoculation. Blood and serum samples, collected on alternate days after the primary inoculations and also after subsequent challenge, were assayed for virus and antibodies. Seven of the 8 AHSV vaccinated (group 1) and 7 of the 7 AHSV inoculated (group 2) animals showed humoral antibody responses after primary inoculation. Although no infectious virus could be isolated from either group for the duration of the study, reverse transcription-PCR data obtained for the second group did show the presence of AHSV viral RNA from as early as day 5 in mules and day 9 in donkeys after the primary inoculation. Viral RNA was detected consistently up to day 47 in some animals and intermittently thereafter. There was no evidence of a second viraemia in any of the animals after challenge. The detection of specific antibodies, against AHSV 4 NS3 protein, in all animals confirmed that both donkeys and mules were infected and that the virus had replicated.

African Horse Sickness↗
PubMed1998

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 and used as antigen in ELISA, the detection was improved. The sensitivity and specificity of the ELISA were 99 and 71%, respectively, compared with the EAV neutralisation test (n = 200). This study has shown the potential that phage display libraries have for identifying peptide sequences which could be used as antigen in diagnostic ELISAs.

Amino Acid Sequence↗
PubMed1997

Nucleotide sequence comparison of the segments S10 of the nine African horsesickness virus serotypes.

Segments 10 (S10) of the double-stranded RNA (ds RNA) genomes from African horsesickness virus (AHSV) serotypes 2, 4, 5, 6 and 7 were cloned and sequenced. Direct sequencing of previously reverse transcribed amplified (RT)-PCR segments S10 was also performed. Nucleotide sequences of two strains (the virulent Moroccan strain and a vaccine strain) of the same serotype (4) were determined. Sequences of the viral serotypes were analysed and compared to each other. Two in-phase ATG codons were conserved in the S10 of AHSV-2, AHSV-4, AHSV-5, AHSV-6 and AHSV-7 and were considered candidate translation initiation codons of NS3 and NS3A respectively. A close relationship between serotypes 4, 5 and 9 and between serotypes 3 and 7 were described. Closer relationships of serotype 2 to the 1 and 8 group on one hand and of serotype 6 to the 4, 5 and 9 group on the other hand were observed.

African Horse Sickness Virus↗
PubMed1995

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.

African Horse Sickness↗
PubMed1995

Differentiation of African horse sickness viruses by polymerase chain reaction and segments 10 restriction patterns.

This paper describes a single tube reverse transcription-polymerase chain reaction (RT-PCR) method for detection of African horse sickness virus (AHSV). The genomic segments 10 of viruses of the 9 AHSV serotypes were amplified. The 758bp products were digested to completion by restriction enzymes. The restriction fragment length polymorphisms of segments 7 and 10 cDNA allowed the differentiation of the nine serotypes.

Animals↗
PubMed1995

Detection of African horse sickness viruses by dot-blot hybridization using a digoxigenin-labelled probe.

In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other equine virus or orbivirus isolates. The sensitivity and specificity of this probe, together with the simplicity and rapidity of this technique, suggest that a non-radioactive dot blot assay may be useful as a method for the routine and rapid diagnosis of viral infections.

Base Sequence↗
PubMed1994

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 is discussed. Application of the RT-PCR method should improve detection and shorten the time required to confirm a clinical diagnosis of AHSV infection.

African Horse Sickness↗
PubMed1994

[Equine infectious arteritis: molecular biology, epidemiology and preventative measures].

After a brief historical account of the outbreak of infectious arteritis of horses which occurred in 1984 in Kentucky (United States of America), the author reports on the present state of knowledge concerning the organisation of the genome of the virus. Clinical signs of the disease are described, as well as modes and routes of transmission. Finally, currently-available vaccination procedures are discussed and their value is assessed.

Animals↗
PubMed1993

Comparison of the restriction patterns of equine herpesvirus (EHV-1) strains isolated for eight years in France.

The equine herpes viruses strains (EHV) isolated from organs of aborted foetuses or from nasal swabs have been analysed by comparison of their restriction endonucleases patterns using two enzymes, Bam HI and Pst I. The majority of the clinical samples came from the west part of France ("Normandie") after abortions or respiratory disorders. All the viruses isolated were EHV-1 strains whose patterns show considerable homogeneity although some differences can be described. The genomic DNAs of the same twenty strains have been digested by the Pst I enzyme, which induced a great number of restriction fragments. It allows a more precise epidemiological study between strains isolated in the same studs with different Bam HI patterns or between strains with identical Bam HI profiles but with distinct respiratory or abortigenic pathogenicity. No strain isolated from aborted foetuses or nasal swabs presented the vaccinal pattern.

Abortion, Veterinary↗
PubMed1993

Diagnosis and molecular epidemiology of the African horse sickness virus by the polymerase chain reaction and restriction patterns.

African horse sickness is a viral disease caused by an orbivirus belonging to the Reoviridae family. This paper describes a polymerase chain reaction (PCR) for amplifying segments 7, which encode for VP 7, a protein common to the 9 known serotypes of this virus. A reverse transcription step is necessary before amplification. No amplified product could be observed in cell cultures infected with other equine viruses. The amplified DNAs were digested to completion by 8 different restriction enzymes. The restriction fragment length polymorphisms allowed the differentiation of the group of serotypes AHSV-1, 3, 6, 8 and the viruses AHSV-2, AHSV-4, AHSV-5, AHSV-7 and AHSV-9. Differences could also be described between vaccinal strains of the same serotype produced in cell cultures or in brains of suckling mice.

African Horse Sickness Virus↗
PubMed1993

[Equine rhinopneumonitis: molecular epidemiology and diagnosis by molecular probes prepared from organs].

The authors briefly review the clinical forms of equine rhinopneumonitis and indicate changes in the nomenclature of equine herpesviral infections. The value of restriction profiles for epidemiological studies is described, taking as an example the strains of virus isolated in France. A technique is given for preparing molecular probes, as well as the application of these probes in direct diagnosis from biological specimens.

Animals↗
PubMedDate not supplied

Detection of African horse sickness virus in the blood of experimentally infected horses: comparison of virus isolation and a PCR assay.

A reverse transcription-polymerase chain reaction (RT-PCR) assay followed by dot-blot hybridisation was used to detect African horse sickness virus (AHSV); the primers employed amplified the S7 gene that encodes the VP7 protein. The RT-PCR assay was compared with virus isolation for detecting AHSV in blood samples form horses experimentally infected with AHSV-4 and AHSV-9. The influence of sample storage and transportation and the effects of two anticoagulants (EDTA and heparin) were also studied. RT-PCR results were obtained within 48 hours as opposed to a minimum of 15 days for virus isolation. RT-PCR and virus isolation were equally sensitive for detection of AHSV-4. Viraemia was detected more consistently by RT-PCR than by virus isolation from horses infected with the less virulent AHSV-9 isolate except from one animal in which virus was detected only by virus isolation. The sensitivity of virus isolation was increased by passaging samples five times. This study indicates that RT-PCR is a sensitive and rapid method for use in the face of an outbreak of this serious disease, although it has also some limitations as a diagnostic technique.

African Horse Sickness↗
PubMedDate not supplied

Construction of an internal standard used in RT nested PCR for Borna Disease Virus RNA detection in biological samples.

The highly neurotropic Borna Disease Virus (BDV), which belongs to the Mononegavirales order--Bornaviridae family--is generally detected using the RT-nested-PCR. If false positive results (often caused by laboratory contaminations) can be avoided, some false negative results which are mostly due to inhibitory effects of some reaction components and/or to sample preparation errors, can occur. Thus, in order to control the RT-PCR sample, an RNA internal standard molecule named "mimic" was constructed with the same primer recognition sites as the viral nucleic acids, flanking a heterologous DNA fragment of distinct molecular weight. Because of their different sizes, the mimic and viral PCR products can be easily discriminated by agarose gel electrophoresis. The co-amplification of both BDV and mimic RNA was performed on infected cells and on biological tissues such as the brain and blood, commonly known to contain PCR inhibitor components. After mimic sensitivity studies were achieved (2.5 fg of "p40 RNA mimic" and 0.25 fg of "p24 RNA mimic"), the competitive amplification reaction between both BDV and mimic RNA was performed on these tissues. The results confirmed that nervous tissue has an inhibitory effect on RT-PCR, which supports the necessity of BDV detection by a higher sensitive method such as RT nested PCR. Moreover, these results confirmed the interest of an internal standard for BDV RNA detection in biological samples.

Base Sequence↗