Oligonucleotide microchip for subtyping of influenza A virus.
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.


OBJECTIVE
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.
Fesenko
Eugeny E
EE
Laboratory of Biological Microchips, Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences, Vavilov Str. 32, Moscow, Russia. fesenko@biochip.ru
Kireyev
Dmitry E
DE
Gryadunov
Dmitry A
DA
Mikhailovich
Vladimir M
VM
Grebennikova
Tatyana V
TV
L'vov
Dmitry K
DK
Zasedatelev
Alexander S
AS
D000818:Animals; D001717:Birds / Q000821:virology; D004196:Disease Outbreaks; D005782:Gels; D006389:Hemagglutinins, Viral / Q000235:genetics; D006801:Humans; D009980:Influenza A virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000453:epidemiology / Q000821:virology; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology*; D009439:Neuraminidase / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012426:Russia; D012680:Sensitivity and Specificity; D013552:Swine / Q000821:virology; D014764:Viral Proteins / Q000235:genetics
D005782:Gels; D006389:Hemagglutinins, Viral; D014764:Viral Proteins; C458881:hemagglutinin fusogenic peptide, influenza virus; C487630:NA protein, influenza A virus; D009439:Neuraminidase