Molecular variation in the nucleoprotein gene (ORF7) of the porcine reproductive and respiratory syndrome virus (PRRSV).

The nucleoprotein gene (ORF7) of 15 European isolates of porcine reproductive and respiratory syndrome virus (PRRSV) was sequenced and compared with corresponding sequences of other PRRSV isolates (2 European and 13 American) and one isolate each of other arteriviruses (the lactate dehydrogenase elevating virus (LDV), the simian haemorrhagic fever virus (SHFV) and the equine arteritis virus (EAV)). Their phylogenetic relationships were established using neighbour-joining and parsimony methods. Four lineages (PRRSV, LDV, SHFV and EAV) were discriminated. Two genotypes of PRRSV, European and American, could be further identified. The European genotype of PRRSV was highly conserved. Analysis of the nucleotide and amino acid substitutions in PRRSV ORF7 revealed four stable regions, probably conserved because of their requirement for nucleocapsid function and/or structure. No constant mutations accumulation in the ORF7 could be determined precisely when either synonymous or non-synonymous mutations were studied. Passage of the European PRRSV in vivo had little influence on the ORF7 sequence: only a small number of synonymous substitutions in ORF7 was detectable, confirming its low variability.

Simplified procedure for detection of enteric pathogenic viruses in shellfish by RT-PCR.

Epidemiological evidence linking the transmission of enteric viral disease to shellfish has been known for a long time. A variety of methods have been described for the detection of viral contaminants in shellfish using RT-PCR. However, these methods generally include numerous, often fastidious and time consuming steps for virus release from shellfish tissues and viral RNA isolation. A simplified procedure based on the enzymatic liquefaction of shellfish digestive tissues without any mechanical homogenisation step, followed by a simple clarification of the lysate using dichloromethane extraction, was developed. Viral RNA is isolated directly from the shellfish extract by a guanidium thiocyanate-silica extraction method, adapted for the use of a vacuum manifold system. Virus-specific RT-PCR assays were set up for detection of genomic sequences of the predominant viral pathogens, HAV, Astrovirus and Norwalk-like viruses (from genogoups I or II). The specificity of the amplicons is confirmed finally by hybridisation with DIG-labelled specific probes. The overall procedure applied to shellfish samples spiked with HAV particles allowed a detection of 20 pfu of HAV per g of hepatopancreas. In addition, up to 20 samples can be tested within 24 h.

Development of a RT-PCR test coupled with a microplate colorimetric assay for the detection of a swine Arterivirus (PRRSV) in boar semen.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) through boar semen has been demonstrated, stressing the need for a reliable semen PRRSV detection test. A diagnostic assay was developed based on amplification of the PRRSV RNA by reverse transcription and polymerase chain reaction (RT-PCR) followed by detection of the amplification products by hybridization and colorimetric assay in microwell plates. A highly reproducible and efficient method of viral RNA isolation from semen samples was set up. A combined RT-PCR procedure was performed, incorporating the use of uracil-N-glycosylase (UNG) in combination with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. An RNA internal control was added during the RNA isolation procedure to detect false negative results. The colorimetric detection in microwell plates of amplification products from either PRRSV or IC RNA gave specific and objective results and was automated. A cut-off value of 1000 RNA copies or 10 TCID50 of PRRSV per ml of semen samples could be detected with this assay. Semen samples collected from experimentally-infected boars were tested with this assay and showed PRRSV excretion early after infection and for an extended period.

Modelling effect of physical and chemical parameters on heat inactivation kinetics of hepatitis A virus in a fruit model system.

While thermal destruction of pathogenic bacteria has been thoroughly studied in food industry, heat inactivation of viruses in food has been poorly investigated. Experiments were carried out to characterize the effects of controlled physical and chemical characteristics of a food matrix upon heat resistance parameters (D and z values) of hepatitis A virus (HAV), taken as model because of its reported heat resistance. Sucrose content (28-52 degrees Brix), calcium concentration (90-1700 mg kg(-1)) and pH (3.3-4.3) were selected for possible influence on thermal inactivation of HAV in strawberry mashes and thus included in an experimental design according to a Doehlert matrix. Use of this design not only allowed to detect and quantify the direct influence of sucrose concentration upon the D85 degrees C value to be higher than the one of pH, but also to reveal a sucrose concentration/pH specific interaction, while no effect of calcium concentration was evidenced. Although the model cannot be directly used to predict heat resistance in real fruit systems, because of differences observed between predicted and measured D85 degrees C values, it is useful for predicting the trends and relative changes in D values due to sucrose concentration and pH variations. Results suggested possible effects of other constituents of strawberry products on heat resistance of HAV and confirmed the importance of experimental validation of any model-derived process. Nevertheless, such a modelling approach using response surface methodology provides a rapid answer to heat resistance evaluation of a food-borne virus as a function of specific physical and chemical parameters of specific food products.

Detection of somatic phages, infectious enteroviruses and enterovirus genomes as indicators of human enteric viral pollution in surface water.

In the present study, we aimed to determine whether the concentrations of somatic coliphages, infectious enteroviruses or the detection of enterovirus genomes were associated with the detection of human pathogenic viruses in surface water. Four French rivers were sampled monthly or semimonthly for the quantitative detection of somatic coliphages, infectious enteroviruses and the qualitative RT-PCR detection of enterovirus, hepatitis A virus, Norwalk I viruses, Norwalk II viruses, astrovirus and rotavirus genomes over 12 months. All the 68 water samples tested were positive for the quantitative detection of somatic coliphages (range of concentrations: 4 x 10(2) to 1.6 x10(5) FUl(-1)). Infectious enteroviruses were isolated by a cell culture system in only two (3%) of the 68 concentrated water samples tested, whereas enterovirus genomes were detectable in 60 (88%) of the same samples. A positive RT-PCR detection of the genome of hepatitis A virus, Norwalk-like virus genogroup II, astrovirus, rotavirus and Norwalk-like virus genogroup I was demonstrated, respectively, in 1.5% (1/68), 1.5% (1/68), 3% (2/68), 0% and 0% of the 68 concentrated water samples tested. All of these four water samples were positive for the detection of enterovirus genomes, whereas only one of them was positive for the isolation of enteroviruses on cell culture. Moreover, the genomic detection of human pathogenic viruses appeared not to be statistically associated with the concentration levels of somatic coliphages in the 68 concentrated water samples tested (Wilcoxon rank test; P=0.14). Taken together, our findings indicate that the quantitative detection of somatic coliphages and the isolation of enteroviruses on cell culture are not suitable parameters for the control of the viral contamination in surface water, whereas the detection of enterovirus genomes may be useful for predicting the presence of waterborne viruses.

[Tumoral calcinosis of the foot in patient on hemodialysis]. / Calcinose pseudo-tumorale des deux pieds chez un hémodialysé chronique.

We report a new case of tumoral calcinosis in a renal failure patient. In this patient the pseudotumoral formation was found in an unusual localization on the medial border of the foot. The condition progressed with successive development of bilateral tumors and septic contamination. Pathology confirmed the diagnosis. The patient was successfully treated with tumor resection and renal graft. The origin of these pseudotumors remains a question of debate. Several pathogenc mechanisms have been put forward. Pronosis is always favorable. Several authors have suggested a phosphorus calcium work-up can be useful for classifying these tumors, guiding surgical treatment if required and managing the underlying cause.

Validating the use of green fluorescent-marked Escherichia coli O157:H7 for assessing the organism behaviour in foods.

AIMS: Monitoring bacterial kinetics in food is of great importance in food safety. The targeted micro-organism has to be identified accurately among competitive flora. Using green fluorescent protein (GFP)-transformed strains is a possible answer to such issues. However, quantitative studies require that this transformation does not alter the micro-organism behaviour: parent and transformed organisms were thus compared. METHODS AND RESULTS: Three Escherichia coli O157:H7 strains were transformed using a GFP-plasmid expressing. Parent and transformed strains were compared according to their genetic characteristics and serotypes. Growth ability was also assessed in constant and fluctuating temperature profiles. Cardinal values of pH, water activity and temperature were computed. No differences were observed between parent and transformed strains for all these experiments. The plasmid was satisfactorily maintained within transformed strains throughout the studies. Growth was eventually monitored in beef meat. CONCLUSIONS: Using the GFP marker is of great value, as it allows easier enumeration of E. coli O157:H7 in food in the presence of natural microflora. Using transformed strains is legitimate: their behaviour does not differ from that of their parent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: GFP transformation appears to be a valuable and reliable tool for challenge testing studies and predictive microbiology.

Development of a semiquantitative PCR assay using internal standard and colorimetric detection on microwell plate for pseudorabies virus.

We have developed a semiquantitative PCR assay on microtitre plates for quantitation of pseudorabies virus (PRV). The test is based on co-amplification with an internal control (IC) of the target viral DNA, followed by hybridization of the biotin-amplified products on a capture probe covalently immobilized to a Covalink-NH MicroWells plate and then visualization with colorimetric enzymatic reactions. PCR was performed in the presence of uracil-N-glycolsylase (UNG) with dUTP instead of dTTP to prevent false positive results due to carry-over contamination. Our colorimetric test had a 3.5 log dynamic range with a detection level of 30 DNA copies per PCR reaction. A standard curve for quantitation of pseudorabies virus was established from co-amplification of 10 to 10(5) PRV molecules with 1000 IC molecules. Ratios of viral optical density/IC optical density were plotted against the number of PRV DNA target molecules in the PCR amplification. Integration of 96-well formats and automation using robots at different steps of the test ensured a good repeatability. Calibration of the quantitative test using samples from experimentally-infected pigs is in progress.

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma.

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10(3) copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100 microl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.