Detection of neuraminidase stalk motifs associated with enhanced N1 subtype influenza A virulence via Raman spectroscopy

Abstract

Oligonucleotides corresponding to neuraminidase (NA) stalk motifs that have been associated with enhanced influenza virulence have been identified using surface-enhanced Raman spectroscopy (SERS).. 5′-thiolated ssDNA oligonucleotides were immobilized onto a hexadecyltrimethylammonium bromide (CTAB) coated Au nanoparticles (AuNP). Three synthetic RNA sequences corresponding to specific amino acid deletions in the influenza NA stalk region were attached to the CTAB-modified AuNPs. Two of these sequences were specific to sequences with amino acid deletions associated with increased virulence, and one was a low virulence sequence with no amino acid deletions. Hybridization of synthetic matched and mismatched DNA-RNA complexes were detected based on the intrinsic SERS spectra. In addition, this platform was used to analyze RNA sequences isolated from laboratory grown influenza viruses having the NA stalk motif associated with enhanced virulence, including A/WSN/33/H1N1, A/Anhui/1/2005/H5N, and A/Vietnam/1203/2004/H5N1 strains. Multivariate feature selection methods were employed to determine the specific wavenumbers in the Raman spectra that contributed the most information for class discrimination. A one-way analysis of variance (ANOVA) test identified 884 and 1196 wavenumbers as being highly significant in the high and low virulence spectra, respectively (p < 0.01). A post-hoc Tukey Honestly Significance Difference (HSD) test identified the wavenumbers that played a major role in differentiating the DNA-RNA hybrid classes. An estimate of the spectral variability, based on the Wilcoxon rank sum test, found the major source of variation to be predominately between the different classes, and not within the classes, thus confirming that the spectra reflected real class differences and not sampling artifacts. The multivariate classification methods partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) were able to distinguish between different NA stalk-motifs linked to NA-enhanced influenza virus virulence (NA-EIV) with >95% sensitivity and specificity in both synthetic RNA sequences as well as the isolated viral RNA. This study demonstrates the feasibility of SERS for direct identification of influenza NA stalk mutations associated with virulence without sample amplification or labeling.

Silver Nanoparticle Based Codelivery of Oseltamivir to Inhibit the Activity of the H1N1 Influenza Virus through ROS-Mediated Signaling Pathways

“…Finally, the influenza protein and new viron are expressed and released. − Hemagglutinin (HA) is a cell-anchoring viral glycoprotein which plays an important role in viral infection by combining sialic acid-containing receptors on host cells and mediating the entry and fusion of the virus . Neuraminidase (NA) and hemagglutinin are the most significant glycoproteins on the surface of the influenza virus. , When mature viruses separate from the host cell surface, neuraminidase plays an important role in assisting the virus to cleave the linkage between sialic acid and hemagglutinin . Vaccination is the common avenue to restraining the spread of influenza infections .…”

“…11,12 When mature viruses separate from the host cell surface, neuraminidase plays an important role in assisting the virus to cleave the linkage between sialic acid and hemagglutinin. 13 Vaccination is the common avenue to restraining the spread of influenza infections. 14 Nevertheless, in the long period between the rapid virus evolution and vaccine development, there is an urgent need for the exploration of new antiviral agents to inhibit the spreading of the influenza virus.…”

Label‐Free Detection of Glycan–Protein Interactions for Array Development by Surface‐Enhanced Raman Spectroscopy (SERS)

“…A SERS substrate for the analysis of HA binding to glycans was fabricated by first treating a glass microscope slide with the bifunctional reagent 6 for capturing AuNPs (Figure ). A monolayer of AuNPs was self‐assembled onto this treated glass slide by using an ionic surfactant‐mediated Langmuir–Blodgett transfer methodology (Figure S5a in the Supporting Information, SEM image of the AuNP monolayer). The reproducibility of the fabricated AuNP substrate was examined using 4‐aminothiophenol (4‐ATP).…”

“…A monolayer of AuNPs was self-assembled onto this treated glass slide by using an ionic surfactant-mediated Langmuir–Blodgett transfer methodology [17] (Figure S5a in the Supporting Information, SEM image of the AuNP monolayer). The reproducibility of the fabricated AuNP substrate was examined using 4-aminothiophenol (4-ATP).…”

Inhibitory activity of selenium nanoparticles functionalized with oseltamivir on H1N1 influenza virus

“…11,12 NA plays an important role in assisting the virus cleave the linkage between sialic acid and HA, when mature viruses separate from the host cell surface. 13 Vaccination is a common method of restraining the spread of influenza infections.…”