Work package 2: Development of diagnostic tools

All centers involved

The main goals of WP2 are:

Multiplex RT-PCR

A molecular diagnostic approach has been developed to avoid bio-safety constraints (BSL3 facilities) when highly pathogenic respiratory viruses are isolated in cell culture and to allow identification of some bacteria that cannot grow in regular media (e.g. Mycoplasma pneumoniae, Chlamydia pneumoniae,…). This approach aims to implement cutting-edge techniques, like real-time PCR, for the diagnosis of infections with high epidemic potential. Virus-specific PCR are first developed prior to inclusion in multiplex reactions.  We are now using 5 multiplex RT-PCR (Bellau-Pujol S et al., J Virol Methods, 2005) to simultaneously detect 17 RNA respiratory viruses: Influenza A, B and C, hRSV, hMPV, PIV 1-4, four human coronaviruses (OC43, 229E, NL63 and HKU1), human bocavirus, adenovirus, enterovirus and rhinovirus.  The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex semi-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. IP-Cambodia has also set up multiplexes for the detection of Bordetella pertussis, Legionella pneumophila, Chlamydia psittaci and pneumoniae and Mycoplasma pneumoniae.

Virus isolation and microarrays

IP-Shanghai and IPC are focusing on the identification and study of emerging/new viruses from samples of patients with acute respiratory diseases that are negative for H5N1 and SARS-CoV, by inoculating four different cell lines that are subsequently analyzed by immunofluorescence and cytopathic effects (CPE). If immunofluorescence is negative but CPE is observed, supernatant fluids are processed for analysis by microarrays.

DNA microarrays can detect and analyse the pattern of expression of thousands of genes in a single experiment. Wang et al. (PLoS Biol, 2003) have developed DNA microarray diagnostic systems for rapid, sensitive, multiplex discrimination of microbial gene sequences and have reported their application for detecting 22 respiratory pathogens in clinical samples, demonstrating that this approach is a suitable diagnostic tool. IPS is developing a platform for rapid virus identification using cell biology and molecular biology techniques.

Sero-diagnosis

The epizootic of avian influenza H5N1 and the consequent pandemic threat is presently the major concern that has to be confronted by both public and veterinary health authorities in Asia and beyond. This infection poses significant challenges in diagnosis, in understanding its pathogenesis and for therapeutics. Serological diagnosis for H5N1 infection in humans (and other mammalian species) necessitates micro-neutralization tests which require BSL-3 biosafety containment laboratories. Hence, there is an urgent need for development of novel high throughput methodologies that can be used for seroepidemiological studies that can be carried out in BSL-2 containment and for differentiating vaccine- vs infection-induced immunity in poultry.

A new diagnostic tool has been developed at HKU-PRC and IPS, based on influenza pseudotyped lentiviral particles bearing on their surface H5 hemagglutinin and encapsidating a luciferase reporter gene. Each part (virion, surface glycoprotein and reporter gene) can be independently and easily changed leading to a great flexibility of the tool. This assay has been successfully implemented in a high throughput screen and validated against microneutralization for sero-diagnosis in the detection of neutralizing antibodies against H5 influenza (>1500 sera tested). Other pseudotyped particles expressing combinations of the 3 main surface glycoproteins of the H5N1 virus are being developed.