[The physician facing legal procedures: points of view of the physician and the lawyer].
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France↗
Biomedical subjects
G Le Gall
Publications and source records attributed to G Le Gall.
[The law passed on 4 March 2002, and its implications for medical practice].
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Confidentiality↗
[A court ruling of December 7, 2004 on the legitimate interests surrounding a professional secret].
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Expert Testimony↗
Immunocapture-RT-PCR assay for detection and molecular epidemiology studies of Rabbit Haemorrhagic Disease and European Brown Hare Syndrome viruses.
Rabbit Haemorrhagic Disease Virus and European Brown Hare Syndrome Virus are two members of the genus Lagovirus in the family Caliciviridae. They are the causative agents of highly contagious and fatal diseases of rabbits and hares respectively. We adjusted one assay for the detection and the genomic characterisation of each virus, based on viral purification by immunocapture and genomic amplification by reverse transcription-polymerase chain reaction (IC-RT-PCR). It is carried out directly with the liver exudate obtained after thawing and suppresses the viral nucleic acid preparation step. This assay combines the advantages of an ELISA test (rapidity) because immunocapture and the RT reaction were carried out in the same microtitre plate, and the advantages of PCR (sensitivity). The procedure described allows the processing of large numbers of samples and is suitable for phylogenetic studies of lagomorphs caliciviruses. In addition, it was compared with sandwich-ELISA used for Rabbit Haemorrhagic Disease or European Brown Hare Syndrome diagnosis. A good correlation was found between ELISA and IC-RT-PCR results for Rabbit Haemorrhagic Disease diagnosis, whereas for European Brown Hare Syndrome diagnosis, the results confirmed the higher sensitivity of the molecular method.
Animals↗
Discrimination between orange juice and pulp wash by (1)H Nuclear Magnetic Resonance spectroscopy: identification of marker compounds.
The potential of NMR spectroscopy and multivariate analysis methods to detect the adulteration of orange juice with pulp wash is demonstrated. Principal component analysis has been applied to (1)H NMR spectra of >300 orange and pulp wash juices, and stepwise linear discriminant analysis was used to classify the samples. A model with six principal components gave a high success rate of classification (94%) for both training and validation sets. An important principal component loading showed that dimethylproline played a key role in the discrimination between the two types of juice, with higher levels in pulp wash. Dimethylproline was not previously known as a marker compound for orange juice adulteration. An ANOVA test revealed at least 21 other NMR signals that differed significantly between the authentic and pulp wash groups. The compounds they represent could be seen as potential marker compounds in addition to dimethylproline. This makes NMR with chemometrics an attractive screening tool with advantages in terms of rapidity, simplicity, and diversity of information provided.
Amino Acids↗
Molecular epidemiology of rabbit haemorrhagic disease virus outbreaks in France during 1988 to 1995.
In order to evaluate genetic variation between rabbit haemorrhagic disease virus (RHDV) isolates and to derive phylogenetic relationships, 56 virus isolates collected from various parts of France over a 7 year period (1988 to 1995) were examined. Analyses were carried out by direct nucleotide sequencing of PCR fragments of three genomic regions encoding the capsid protein (VP60) (regions A and B) and a non-structural protein (region C). Multiple sequence alignments revealed maximum nucleotide divergence of 7.6, 9.4 and 8.7% for regions A, B and C, respectively, indicating a high level of conservation between isolates. Irrespective of the genomic region analysed, phylogenetic analyses carried out using various methods allowed the identification of three genogroups; distribution of isolates within these genogroups appears to be more related to the year of their collection than to their geographical origin. The possible evolution of RHDV is discussed.
Animals↗
[Calicivirus infection of the hare and human calicivirus].
The calicivirus outbreak in hares which occurred in Center West of France in the fall of 1996 illustrates the pathogenic role of caliciviruses in animals and in humans. A comparison of these different viruses, based on the RNA nucleic sequences is presented. The aspect of interspecies transmission is also discussed.
Animals↗
Structural, antigenic and immunogenic relationships between European brown hare syndrome virus and rabbit haemorrhagic disease virus.
The capsid protein of a French isolate of the European brown hare syndrome virus (EBHSV) was expressed in the baculovirus system. The recombinant EBHSV (rEBHSV) capsid protein was able to self-assemble into virus-like particles (VLPs). The VLPs were indistinguishable from the infectious EBHSV and displayed morphological characteristics similar to those we have described for the VLPs resulting from the expression of the capsid protein of rabbit haemorrhagic disease virus (RHDV), a closely related calicivirus. Cross-protection experiments showed that vaccination with rEBHSV particles did not protect rabbits against an RHDV challenge. A set of monoclonal antibodies (MAbs) was raised against rEBHSV capsid protein and used together with anti-RHDV and anti-EBHSV MAbs produced against native viruses to study the antigenic relationships between the two caliciviruses. All six anti-EBHSV MAbs delineated discontinuous epitopes; two of them reacted with specific surface epitopes and the remaining four reacted with internal epitopes which were also present in rRHDV. All anti-RHDV MAbs were monospecific; three reacted with surface linear epitope(s), two reacted with internal linear epitope(s) and one with a surface conformational epitope. On the basis of all these results, a classification of RHDV and EBHSV as two serotypes of a single serogroup is proposed.
Animals↗
Protection of rabbits against rabbit viral haemorrhagic disease with a vaccinia-RHDV recombinant virus.
In order to protect domestic and wild rabbits against RVHD, we constructed a recombinant vaccinia-RHDV virus, using the Copenhagen strain of the vaccinia virus. This recombinant virus expressed the RHDV capsid protein (VP60). Analysis of the expressed product showed that the recombinant protein, which is 60 kDa in size, was antigenic as revealed by its reactions in immunoprecipitation and indirect immunofluorescence with the antibodies raised against RHDV. The recombinant virus induced high level of RHDV specific antibodies in rabbits following immunization. Inoculations by both the intradermal and oral routes allow protection of animals against a challenge with virulent RHDV.
Administration, Oral↗
European brown hare syndrome virus: molecular cloning and sequencing of the genome.
The genome of the European brown hare syndrome virus (EBHSV), a calicivirus related to rabbit haemorrhagic disease virus (RHDV), was fully sequenced. It was 7442 bases long and contained two ORFs. In RHDV, the 5' large ORF (ORF1) is predicted to encode a polyprotein precursor to the non-structural and capsid proteins. The small ORF (ORF2) encodes a predicted protein of 12 kDa. Alignment of sequences of EBHSV and RHDV showed 71% nucleotide identity; the changes were uniformly scattered over the whole genome. Minor differences could be detected when comparing two EBHSV sequences, indicating that EBHSV could vary to the same extent as RHDV. Four cleavage sites previously identified on the RHDV polyprotein were conserved in EBHSV. These sequencing data clearly show that EBHSV and RHDV share a similar genomic organization and confirm that EBHSV and RHDV are two distinct members within the family Caliciviridae.
Amino Acid Sequence↗
Protection against myxomatosis and rabbit viral hemorrhagic disease with recombinant myxoma viruses expressing rabbit hemorrhagic disease virus capsid protein.
Two myxoma virus-rabbit hemorrhagic disease virus (RHDV) recombinant viruses were constructed with the SG33 strain of myxoma virus to protect rabbits against myxomatosis and rabbit viral hemorrhagic disease. These recombinant viruses expressed the RHDV capsid protein (VP60). The recombinant protein, which is 60 kDa in size, was antigenic, as revealed by its reaction in immunoprecipitation with antibodies raised against RHDV. Both recombinant viruses induced high levels of RHDV- and myxoma virus-specific antibodies in rabbits after immunization. Inoculations by the intradermal route protected animals against virulent RHDV and myxoma virus challenges.
Animals↗
Detection of rabbit haemorrhagic disease virus isolates and sequence comparison of the N-terminus of the capsid protein gene by the polymerase chain reaction.
At present there is no sensitive method for the detection of rabbit haemorrhagic disease virus (RHDV), a calicivirus causing high mortality in rabbit populations. For this purpose a reverse transcriptase polymerase chain reaction (RT-PCR) was established in the N-terminal portion of the RHDV capsid region. The RT-PCR was 10(4)-fold more sensitive than ELISA testing for the detection of the virus and was able to detect as few as 12 copies of template cDNA. By using the RT-PCR test and sequencing, 96.6 to 98.7 per cent homology was demonstrated in the N-terminal portion of the capsid protein of three isolates from geographically and temporally separate outbreaks of viral haemorrhagic disease, indicating that this portion of the RHDV capsid protein is highly conserved.
Amino Acid Sequence↗
An alternative method for direct sequencing of PCR products, for epidemiological studies performed by nucleic sequence comparison. Application to rabbit haemorrhagic disease virus.
A sequencing strategy based on the use of commercially available fluorescent-labeled universal primers for directly sequencing polymerase chain reaction (PCR) amplified material has been developed. The PCR reactions were performed with hybrid primers, specific to the viral sequence and possessing the sequence of the standard sequencing primers at their 5' end (M13 and reverse primers). These amplified fragments were sequenced by a classical dye-primer kit on an automated sequencer. As opposed to the use of fluorescent dideoxynucleotides, this sequencing method yielded accurate, high-grade sequences and had several advantages. First, the intensity of the sequencing peaks was much more homogeneous with the dye primer method. In addition, the problem of altered electrophoretic mobility, which may occur during the sequencing of custom-synthesised fluorescent primers, was avoided. This method was successfully reproduced using several different sets of chimeric primers. We believe that it is suitable for epidemiological studies conducted by nucleic sequence comparison, as in the case of rabbit haemorrhagic disease virus, as well as in other systems.
Animals↗
[Recombinant vaccine against rabbit hemorrhagic disease virus].
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Animals↗
Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection.
VP60, the unique component of rabbit hemorrhagic disease virus capsid, was expressed in the baculovirus system. The recombinant VP60, released in the supernatant of infected insect cells, assembled without the need of any other viral component to form viruslike particles (VLPs), structurally and immunologically indistinguishable from the rabbit hemorrhagic disease virion. Intramuscular vaccination of rabbits with the VLPs conferred complete protection in 15 days; this protection was found to be effective from the fifth day after VLP injection and was accompanied by a strong humoral response.
Amino Acid Sequence↗
In vitro bronchial hyperresponsiveness after lung transplantation in the rat.
In this study, we examined the effect of unilateral lung transplantation on in vitro responses of bronchial smooth muscle to electrical field stimulation (EFS) and exogenous acetylcholine (ACh). In syngeneic Lewis rats, we compared the contractile responses of the left hilar transplanted bronchus with that of the nontransplanted right bronchus and the left and right bronchi from control and sham-operated animals. Atropine-sensitive bronchial contraction to EFS was greater on left transplanted than on right nontransplanted bronchi at all frequencies and voltages used. In control and sham-operated animals, contractile responses to EFS were identical on left and right sides and similar to the responses of the right nontransplanted bronchus of the lung transplant recipient. Concentration-response curves to exogenous ACh were similar on transplanted and all control bronchi. Intramural cholinergic nerve cell bodies reactive for acetylcholinesterase were present in the transplanted bronchus. We conclude that EFS-induced cholinergic contraction of rat bronchial smooth muscle is enhanced after lung transplantation. Enhancement is not a result of medication, inflammation, or rejection, absent from our syngeneic transplant, or of upregulation of muscarinic post-junctional receptors because the response to exogenous ACh was normal. Hence, an increased release of ACh from cholinergic nerve terminals may occur, due either to hyperexcitability of postganglionic parasympathetic nerves or to loss of inhibitory mechanism resulting from interruption of neural connections with the central nervous system.
Acetylcholine↗
Bronchial provocation tests in small animals: a quantified and automated procedure.
Bronchial provocation tests using aerosols in laboratory animals are difficult to standardize and quantify, because the amount of drug actually reaching the airways is unknown. To improve the quantification of aerosolized inhaled stimuli, we designed an apparatus that allows, in anesthetized intubated ventilated animals, control of temperature and hygrometry of inspired air, computerized measurement of pulmonary resistance, and fully automated delivery of a known amount of aerosolized drug directly into the trachea. Calibration of the aerosol delivery involved direct measurement of liquid delivered at the tip of the tracheal cannula. Despite all our efforts at standardization and full automation of all steps, reproducibility of aerosol delivery was poor, with stroke-by-stroke differences of 26 or 42%, according to whether an air-jet or an ultrasonic nebulizer was used. Histamine dose-response curves performed in 15 guinea pigs with this device confirmed marked differences among animals and also disclosed large intraindividual changes in bronchial responsiveness.
Aerosols↗