Rapid Detection and Differentiation of Swine-Origin Influenza A Virus (H1N1/2009) from Other Seasonal Influenza A Viruses

Jiangqin Zhao,Zhiping Ye,Indira Hewlett,Viswanath Ragupathy,Wei Tang,Panhe Zhang,Xue Wang,Maryna Eichelberger

doi:10.3390/v4113012

Abstract

We previously developed a rapid and simple gold nanoparticle(NP)-based genomic microarray assay for identification of the avian H5N1 virus and its discrimination from other influenza A virus strains (H1N1, H3N2). In this study, we expanded the platform to detect the 2009 swine-origin influenza A virus (H1N1/2009). Multiple specific capture and intermediate oligonucleotides were designed for the matrix (M), hemagglutinin (HA), and neuraminidase (NA) genes of the H1N1/2009 virus. The H1N1/2009 microarrays were printed in the same format as those of the seasonal influenza H1N1 and H3N2 for the HA, NA, and M genes. Viral RNA was tested using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining. The signal from the 4 capture-target-intermediates of the HA and NA genes was specific for H1N1/2009 virus and showed no cross hybridization with viral RNA from other influenza strains H1N1, H3N2, and H5N1. All of the 3 M gene captures showed strong affinity with H1N1/2009 viral RNA, with 2 out of the 3 M gene captures showing cross hybridization with the H1N1, H3N2, and H5N1 samples tested. The current assay was able to detect H1N1/2009 and distinguish it from other influenza A viruses. This new method may be useful for simultaneous detection and subtyping of influenza A viruses and can be rapidly modified to detect other emerging influenza strains in public health settings.

Highlights

Influenza viruses are classified as A, B, and C based on antigenic differences in their nucleoprotein (NP) and matrix (M) protein
The assay was performed by direct hybridization of PCR products with the capture and the intermediate oligonucleotides to form sandwich complexes, followed by silver staining in a format shown in Figure 1A, with each H1N1/2009 capture oligonucleotide, listed in Table 1, spotted in triplicate
When the PCR products of the HA and NA genes for the seasonal H1N1, H3N2, and H5N1 subtypes were tested in a H1N1/2009 array, no signal was detected

Summary

Introduction

Influenza viruses are classified as A, B, and C based on antigenic differences in their nucleoprotein (NP) and matrix (M) protein. H5 and H7 subtypes are highly pathogenic to domestic fowl due to the presence of a multibasic tryptic cleavage site in the HA [6,7]. It is not transmissible between people, avian H5N1 viruses have infected humans with significant rate of mortality since 1997 [8]. In this study we describe the development of the nanomicroarray assay for rapid detection and subtyping of the H1N1/2009 influenza virus that discriminates between this and other seasonal influenza A viruses

Results and Discussion

Designing Oligonucleotides

Nanomicroarray Assay

Conclusions