Oligonucleotide microchip for subtyping of influenza A virus

Abstract

Background: Influenza A viruses are classified into subtypes depending on the antigenic properties of their two outer glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Sixteen subtypes of HA and nine of NA are known. Lately, the circulation of some subtypes (H7N7, H5N1) has been closely watched because of the epidemiological threat they present.

Objectives: This study assesses the potential of using gel-based microchip technology for fast and sensitive molecular subtyping of the influenza A virus.

Methods: The method employs a microchip of 3D gel-based elements containing immobilized probes. Segments of the HA and NA genes are amplified using multiplex RT-PCR and then hybridized with the microchip.

Results: The developed microchip was validated using a panel of 21 known reference strains of influenza virus. Selected strains represented different HA and NA subtypes derived from avian, swine and human hosts. The whole procedure takes 10 hours and enables one to identify 15 subtypes of HA and two subtypes of NA. Forty-one clinical samples isolated during the poultry fall in Novosibirsk (Russia, 2005) were successfully identified using the proposed technique. The sensitivity and specificity of the method were 76% and 100%, respectively, compared with the 'gold standard' techniques (virus isolation with following characterization by immunoassay).

Conclusions: We conclude that the method of subtyping using gel-based microchips is a promising approach for fast detection and identification of influenza A, which may greatly improve its monitoring.

Figure 1

Scheme of the biochip for molecular typing of influenza A virus. Rows b through g contain oligonucleotides for typing of 15 subtypes of HA, while rows h and i identify subtypes 1 and 2 of NA. Immobilized oligonucleotides are designated as follows: letters H or N indicates HA or NA, correspondingly; next, there is a number corresponding to a subtype; the subscript number is an arbitrary designation of the diagnostic oligonucleotides when there is more than one for the identification of a single subtype. Corner gel elements M (a1, j1, and j7) contain immobilized IMD504 fluorescent dye and serve as marker for computer processing of hybridization pattern. Elements f4 through f6 represent empty gel pads and serve as controls of background fluorescence of the gel elements.

Figure 2

Nucleotide sequence alignment of HA and NA gene fragments of different influenza A subtypes (H1Nx, H5Nx, HxN1) with designed oligonucleotides indicated. Genomic regions covered by variant oligos are shown in gray. Nucleotide designation reads as follows: dot (.), same as reference sequence; R, A or G. Nucleotide positions are numbered according to reference sequences: (*) for H1, H5 subtypes and (**) for N1 subtype. The designations of oligonucleotide probes are at the left from them and correspond to their location in Figure 1.

Figure 3

Hybridization patterns obtained using reference samples of influenza virus A/USSR/90/77(H1N1) (A), A/Duck/Czechoslovakia/56(H4N6) (B) and A/Swine/Hong Kong/9/98(H9N2) (C). Positions of the gel elements with immobilized oligonucleotides are as shown in Figure 1.

Figure 4

Example of hybridization pattern of the field specimen identified as H5N1. RNA was obtained from a suspension of internal organs of a domestic duck which died during an outbreak of influenza in the Novosibirsk region in 2005.

Figure 5

Relative values of fluorescent intensities (arbitrary unit) from the each gel pad averaged among five assays for reference H3N2 subtype. Each column represents the mean value of fluorescent intensity obtained for the defined gel pad. Error bars show the standard deviations of measured fluorescence intensities from mean values.