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International audience; Equid gammaherpesviruses-2 and -5 are involved in respiratory problems, with potential clinical man-ifestations such as nasal discharge, pharyngitis and swollen lymph nodes. These viruses are sometimesassociated with a poor-performance syndrome, which may result in a significant and negative economicimpact for the horse industry. The aim of the present study was to develop and validate quantitative PCRmethods for the detection and quantitation of EHV-2 and EHV-5 in equine respiratory fluids. Two dis-tinct tests were characterised: (a) for the qPCR alone and (b) for the whole method (extraction and qPCR)according to the standard model AFNOR XP U47-600-2 (viz., specificity, quantifiable sensibility, linearity,accuracy, range of application, trueness, precision, repeatability and precision of reproducibility). EHV-2and EHV-5 detection were performed on nasal swabs collected from 172 horses, all of which exhibitedclinical signs of respiratory disease. The data revealed a high rate of EHV-2/EHV-5 co-detection that wascorrelated significantly with age. Viral load of EHV-2 was significantly higher in young horses whereasviral load of EHV-5 was not significantly different with age.

The nucleocapsid (N) gene of human coronavirus strain OC43 (HCoV-OC43) was amplified by reverse transcriptase-polymerase chain reaction, and cloned in pENTR™/D-TOPO® plasmid. This plasmid containing the N gene was recombined with in a BaculoDirect™ baculovirus DNA designed in order to express N protein in fusion with a C-terminal polyhistidine tag containing V5 epitope. Sf21 cells were transfected with recombinant baculovirus DNA. Recombinant N protein was extracted from infected cells, analysed by SDS-PAGE and Western blot, and purified by Ni2+ affinity procedure. Sera from 100 healthcare workers and five 2–3-year-old children were tested in a Western blot assay using the purified recombinant N protein. All of the sera from adults and two of the sera from children have a positive result.

Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1-4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.