Automated Detection and Subtyping of Avian Influenza Viruses

Many wild and domestic animals harbour influenza viruses that regularly give rise to new variants that can have a severe impact on animal and public health. Avian influenza viruses (AIV) are currently classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. Rapid identification of subtypes is critical for outbreak response and disease management. A fully automated microfluidic device that integrates/automates the nucleic acid extraction, RT-PCR and probe-based subtyping process for differentiation of all 16 AIV HA and 9 NA subtypes was developed. Bioinformatic analysis of 10,225 AIV matrix, 16,744 HA and 9,709 NA sequences led to new RT-PCRs for amplification of the HA and NA coding region and subtype specific capture probes for differentiating the different HA and NA subtypes. The RT-PCR and capture probes successfully amplified and differentiated a panel of over 40 strains representing all 16 HA subtypes and 9 NA subtypes. The fully-automated subtyping tests can be completed within one working day as compared to sequencing and hemagglutinin inhibition and neuraminidase inhibition tests that may take up to several days. The new tests will reduce the time, labour and cost associated with influenza subtyping, facilitate disease diagnosis and surveillance, and shorten the time needed to acquire important information in outbreak situations.