PubMed Health⌕ Search

Biomedical subjects

S Zientara

Publications and source records attributed to S Zientara.

At least 19 recordsLinked to original sources
PubMed2006

Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments.

The detection of the bluetongue virus (BTV) by conventional methods is especially difficult and labour-intensive. Molecular diagnosis is also complex because of the high genetic diversity between and within the 24 serotypes of BTV. In the present study, two laboratories joined forces to develop and validate two new RT-qPCRs detecting and amplifying BTV segments 1 and 5. The 2 assays detect strains from all 24 serotypes. They both have a detection limit of 0.01 ECE50 and all 114 samples from BTV-free goats, sheep and cattle were negative. The two assays resulted in similar C(t) values when testing biological samples collected in sheep infected experimentally with a field strain of BTV from the Mediterranean basin. On average, the C(t) values obtained with the 2 methods applied to the 24 serotypes were not significantly different from each other, but some moderate to high differences were seen with a few strains. Therefore these two methods are complementary and could be used in parallel to confirm the diagnosis of a possible new introduction of BTV. An RT-qPCR amplifying a fragment of the beta-actin mRNA was also developed and validated as internal control for the bluetongue specific assays. The three assays described allow a reliable and rapid detection of BTV.

Animals↗
PubMed2006

Bluetongue: characterization of virus types by reverse transcription-polymerase chain reaction.

Bluetongue virus (BTV) is the prototype of the member of the Orbivirus genus within the family Reoviridae. The BTV serogroup contains 24 serotypes. Traditionally, viruses have been isolated in cultured cells, suckling mouse brain or embryonated chicken eggs before their identification and biochemical, antigenic and biological characterization. These procedures are time-consuming and may fail to detect low levels of infectious virus or strains of BTV which fail to replicate in eggs, mice or tissue culture. In the past decade, traditional procedures for virus characterization, such as ELISA and serum neutralisation with serotype-specific antisera, have been supplemented by reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. A number of procedures have been developed to detect the presence of BTV antigens or nucleic acids. RT-PCR technique has appeared to be a powerful tool in the field of BTV diagnosis. Polymerase chain reaction techniques may be used not only to detect the presence of viral nucleic acid but also to 'serogroup' orbiviruses and provide information on the serotype and possible geographical source (topotype or genotype) of BTV isolates within a few days of receipt of a clinical sample such as infected sheep blood. Real-time PCRs have recently been developed.

Animals↗
PubMed2005

Bluetongue virus in the French Island of Reunion.

This paper records the results of a bluetongue virus (BTV) serological survey and reports the first isolation of BTV on the French Island of Reunion. In January 2003, the French Island of Reunion, located off the coast of Madagascar, reported an outbreak of disease in cattle that resembled clinical bluetongue (BT) in sheep. The suspected causal agent was isolated and identified as epizootic haemorrhagic disease of deer virus (EHDV). However, because of the similarity in the clinical signs to those of BT, a retrospective survey against BTV was carried out using sera collected in 2002. Results revealed the presence of antibody in all sera tested indicating that BTV has been resident on the Island since 2002, and probably earlier. Although up to July 2003 no clinical BT had ever been reported in sheep, BTV viral RNA was amplified by RT-PCR from a single sheep blood collected in February that year, which strongly suggested that BTV was currently circulating on the Island. Following a second outbreak of disease in August 2003, this time involving a flock of Merino sheep, infectious BTV was finally isolated, and identified by both traditional and molecular techniques as serotype 3. The nucleotide and amino-acid sequences of the RT-PCR products amplified for BTV segments 7 and 10 from the sheep blood collected in February and August from different areas of the Island, were sufficiently diverse as to suggest that they were of different origins and/or different BTV serotypes.

Animals↗
PubMed2003

Studies on the safety and immunogenicity of the South African bluetongue virus serotype 2 monovalent vaccine: specific detection of the vaccine strain genome by RT-PCR.

In order to study the safety and the immunogenicity of the South African vaccine against the serotype 2 bluetongue virus, two groups of seven sheep were vaccinated with the vaccine used in the French island of Corsica. Vaccinated and non-vaccinated sheep were observed clinically and their rectal temperatures were recorded daily. The serological response in vaccinated animals confirmed the immunogenicity of the vaccine. Post-vaccinal viraemia was investigated and the vaccine genome was detected by reverse transcription polymerase chain reaction (RT-PCR). No viraemia was observed at post-vaccination days 4, 7 and 11 but the vaccine strain of virus was detected by RT-PCR throughout the experiment. The thermostability of the vaccine was also evaluated. The vaccine titre strongly decreased at temperatures higher than 35 degrees C.

Animals↗
PubMed2002

Identification of bluetongue virus serotype 2 (Corsican strain) by reverse-transcriptase PCR reaction analysis of segment 2 of the genome.

In October 2000, bluetongue virus was detected on the French island of Corsica. The disease was also reported in Sardinia, Calabria, Sicily and on the Spanish islands of Majorca and Minorca. This paper describes the use of molecular techniques for a rapid identification and serotype determination of serotype 2 of the virus. The nucleotide sequences of segments 2 and 7 of the genome of the Corsican strain were determined and its phylogenetic relationships are described.

Animals↗
PubMed2002

Detection and isolation of equine herpesviruses 1 and 4 from horses in Normandy: an autopsy study of tissue distribution in relation to vaccination status.

Equine herpesviruses type 1 and 4 (EHV-1 and EHV-4) are ubiquitous in the equine population. One of their main properties is their ability to establish life-long latent infections in their hosts even in those with natural or vaccine-induced immunity. However, effect of vaccination status on prevalence and tissue tropism was not established. In this study, EHV-1 and EHV-4 were detected by polymerase chain reaction and by classical virus isolation from neural, epithelial and lymphoid tissues collected from unvaccinated (33) or vaccinated (23) horses. The percentage of EHV-1- and EHV-4-positive horses between vaccinates and unvaccinates was similar. Both viruses were detected in all tissues of both groups; in particular, lymph nodes draining the respiratory tract, nasal epithelium and nervous ganglia [i.e. trigeminal ganglia (TG)], which represent the main positive sites for EHV-1 and EHV-4. In vaccinated animals, the nervous ganglia (i.e. TG) were less frequently positive than in unvaccinated animals. Detection of positive TG was strongly correlated to the presence of EHV-1 in nasal epithelium.

Animals↗
PubMed2001

Evidence of Borna disease virus genome detection in French domestic animals and in foxes (Vulpes vulpes).

Borna disease virus (BDV) is an enveloped, non-segmented negative-stranded RNA virus which belongs to the Bornaviridae family. BDV is an aetiological agent of encephalitis in horses, sheep and several other vertebrate species. In order to extend our knowledge about the presence of BDV in France, a study based on BDV RNA detection by RT-nested-PCR was done with 196 animal tissues: 171 brain samples collected from different animal species (75 horses, 59 foxes, 31 cattle, 4 dogs, 1 sheep, 1 roe deer) and 25 horse blood samples. An RNA internal standard molecule was constructed and was co-amplified with the test template. This study reports the first detection of BDV RNA in France in 10 brain samples collected from horses, foxes and cattle, and from 14 horse blood samples. Detection of the BDV genome in the brains of six red foxes is the first evidence of BDV infection in this species.

Animals↗
PubMed2001

[Epizootic equine influenza in Tunisia].

The authors describe an equine influenza epizootic that occurred in Tunisia during February and March 1998 in the regions of Tozeur, Sousse and Tunis. They relate the symptoms, the different stages of diagnosis and the serological results.

Animals↗
PubMed2000

Evidence for evolutionary stasis and genetic drift by genetic analysis of two equine influenza H3 viruses isolated in France.

The amino acid sequences of the HA(1) portion of the haemagglutinin of two equine A(H3N8) influenza viruses isolated in France in 1993 and 1998 were analysed to determine their evolutionary relationship with 51 other HA(1) amino acid sequences available in databanks. Our data show that the French strain isolated in 1993 belongs to a group of phylogenetically related viruses branched on the main trunk, illustrating the main lineage of evolution of the equine-2 H3 sequences before its split into two distinct lineages in the late 1980s. By contrast, the 1998 French isolate appears to belong to the more recent 'Eurasian' lineage. These data suggest that equine-2 strains antigenically related to old prototype viruses may cocirculate with the more recent 'Eurasian' and 'American' lineages. In conclusion, it may be necessary to include both strains representative of recent equine influenza variants and an older prototype strain in the current equine influenza vaccines.

Amino Acid Sequence↗
PubMed2000

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 (glycoprotein G), 71 (envelope glycoprotein gp300), and 74 (glycoprotein E). Two groups of sequences, which aligned with either glycoprotein C (gC) or glycoprotein E (gE), identified type-specific epitopes which could be used to distinguish between sera from horses infected with either EHV-1 or EHV-4 in an ELISA using either the phage displaying the peptide or synthetic peptides as antigen. The gC epitope had been previously identified as an immunogenic region by conventional monoclonal antibody screening whereas the gE antibody binding region had not been previously identified. This demonstrates that screening of phage display peptide libraries with post-infection polyclonal sera is a suitable method for identifying diagnostic antigens for viral infections such as EHV-1.

Amino Acid Sequence↗
PubMed2000

Identification and differentiation of the nine African horse sickness virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2.

This paper describes the first RT-PCR for discrimination of the nine African horse sickness virus (AHSV) serotypes. Nine pairs of primers were designed, each being specific for one AHSV serotype. The RT-PCR was sensitive and specific, providing serotyping within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralization for a coded panel of 56 AHSV reference strains and field isolates. Serotyping was achieved successfully with live and formalin-inactivated AHSVs, with isolates of virus after low and high passage through either tissue culture or suckling mouse brain, with viruses isolated from widely separated geographical areas and with viruses isolated up to 37 years apart. Overall, this RT-PCR provides a rapid and reliable method for the identification and differentiation of the nine AHSV serotypes, which is vital at the start of an outbreak to enable the early selection of a vaccine to control the spread of disease.

African Horse Sickness↗
PubMed2000

Isolation and characterization of an equine foamy virus.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.

Amino Acid Sequence↗
PubMed1999

The highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography.

The concentration of double-stranded RNA within the bluetongue virus core renders the genome segments liquid crystalline. Powder diffraction rings confirm this local ordering with a 30 A separation between strands. Determination of the structure of the bluetongue virus core serotype 10 and comparison with that of serotype 1 reveals most of the genomic double-stranded RNA, packaged as well-ordered layers surrounding putative transcription complexes at the apices of the particle. The outer layer of RNA is sufficiently well ordered by interaction with the capsid that a model can be built and extended to the less-ordered inner layers, providing a structural framework for understanding the mechanism of this complex transcriptional machine. We show that the genome segments maintain local order during transcription.

Animals↗