Direct Sequence Detection of Human H5 Influenza Viral RNA

Rapid subtype identification of the H5N1 influenza virus is vital for mediating the proper public health response. This article presents an isothermal application of sequence-specific molecular beacons (dual labeled DNA probes) designed to identify the H5 subtype, cleavage motif and receptor specificity when hybridized directly with viral RNA (vRNA). These probes identify the target vRNA sequence without thermal cycling while maintaining exceptional sensitivity and specificity. The hemagglutinin (HA) segment from a highly pathogenic H5N1 strain (A/Hanoi/30408/2005(H5N1)) isolated from humans was used as a template for in vitro transcription of sense strand vRNA. The hybridization behavior of vRNA and a conserved subtype probe, H5G1112, was characterized experimentally by varying conditions of time, temperature and Mg2+ to optimize detection by fluorescence emission. A comparison of the hybridization rates of probe to DNA and RNA targets appear to indicate that conformational switching of influenza RNA structure is a rate limiting step and secondary structure of vRNA dominates the binding kinetics. The sensitivity and specificity of probe recognition to other H5 strains was calculated from sequence matches to the NCBI influenza database. The hybridization specificity of the subtype probes, H5G1112 and H5G1192, were experimentally verified with point mutations within the probe loop at 5 locations corresponding to the other human H5 strains. The abundance frequencies of the HA cleavage motif and sialic acid recognition sequences are detailed and experimentally tested for H5 in humans and all-host viral species. These new probes are designed to form the basis of a portable, point-of-care diagnostic device for influenza subtyping to provide benefit to clinicians, epidemiologists and public health personnel.