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Electrochemistry in microarrays as rapid method for identification of influenza viruses mutation

Influenza is an infectious disease caused by ssRNA viruses with negative polarity of the family Orthomyxoviridae. Influenza is considered to be one of the life threatening infectious diseases. Every year, approximately 10-20% of the world's population is infected with influenza viruses, resulting in a significant number of outpatient and hospital visits and substantial economic burden both on health care systems and society [1]. The risk of complications from influenza, including lower respiratory tract infection, admission to hospital, and death vary depending on factors such as age and the type of comorbidity with bacterial infection that may be present. There is significant morbidity through the all ages of the human population and higher mortality in high risk group (children, adults over 60 years old, patients with chronic illnesses and pregnant women. During the 2009 influenza A (H1N1) pandemic, pregnant women were at risk for severe influenza illness [2, 3]. The 2009 influenza A (H1N1) pandemic disproportionately affected the pediatric population and resulted in a substantially increased number of hospitalizations and deaths among children [4, 5]. The threat of an influenza pandemic virulent, highly transmissible has motivated an escalating research effort to identify the transmissible genotypes of avian influenza viruses that cross over into the human population (avian-human transmission) and sustain human-human transmission. On 20 May 2013, the world’s first human-infected case of H6N1 bird flu was reported in Taiwan. A novel avian-origin influenza A(H6N1) virus was confirmed by the National Influenza Center, Centers for Disease Control, Taiwan, and the patient has already recovered [6]. Infections with H7 subtypes, such as H7N2, H7N3, and H7N7, which are usually related to outbreaks of poultry, have been reported to transmission to human in several countries [7-9]. China had high severity and fatality of human infections with avian influenza A(H7N9) infection [8, 10].

Influenza A viruses cause significant mortality worldwide each year. Antiviral inhibitors have become an important alternate means of containing the spread of influenza. The current antivirals are the neuraminidase inhibitors (zanamivir and oseltamivir) and the M2 protein blockators (amantadine and rimantadine). Membrane (M2) ion channel inhibitors that belong to the adamantanes class of compounds were the first generation of influenza antiviral agents. These inhibitors disrupt the viral uncoating inside the host cell and prevent the budding of progeny viruses [11, 12]. The neuraminidase inhibitors (NAIs) are the most commonly used class of influenza antiviral drugs for the treatment of infected patients [13]. The emergence and reemergence of human influenza resistant to medical treatment will present a challenge to the international public health community in the coming decades. The M2 are only effective against influenza A viruses, and resistance arises rapidly. Many of the H5N1 strains circulating in Southeast Asia, especially in Vietnam and Thailand, are also resistant to M2 inhibitors [14-16]. The most common NAIs in the world are zanamivir, oseltamivir, peramivir, and a long-acting NAIs, laninamivir [17]. However, mutations in the influenza viruses induce resistance to antiviral drug. Resistance was more likely to arise to oseltamivir, due to the structural changes needed for oseltamivir to bind with high affinity [18]. Oseltamivir resistance is reported for the first time in A(H3N2) virus strains during the 2011-2012 influenza season [19]. Detection of one mutation in the virus neuraminidase (NA) gene within 2 days of initiating oseltamivir treatment, in the first reported human infection by avian A(H7N9) influenza virus, raised concern about emergence of resistance during treatment with neuraminidase inhibitors [20].

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A and B strains remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir [21]. Recently, one study was reported that an I223R/H275Y double mutant of neuraminidase (NA) creates a multidrug resistant form of the pandemic influenza A (H1N1) virus [22]. New molecular techniques are required urgently for the rapid detection of the influenza mutation to monitor its transmission in the community and begin clinical investigations of disease severity. Application of oligonucleotide microarrays in different areas of molecular biology and clinical studies has been rapidly growing during the last decade [23-26] .The microarrays are currently used with fluorescent detection. However, the underlying electronics employed for the oligonucleotide synthesis can also be utilized for electrochemical detection (Combimatrix ElectraSenseTM) of target molecules bound to the microarray [27-31]. CombiMatrix core technology is based on a specially modified semiconductor adapted for biological applications, which contains arrays of platinum microelectrodes. Electrochemical detection has been developed and assay performances studied for the CombiMatrix oligonucleotide microarray platform that contains 12,544 individually addressable microelectrodes (features) in a semiconductor matrix [32]. The approach is based on the detection of redox active chemistries (such as horseradish peroxidase (HRP) and the associated substrate TMB) proximal to specific microarray electrodes, as shown in Figure 1. The CombiMatrix influenza detection system is an effective methodology for influenza A and B subtype analysis [30, 33]. In the present study, the electrochemical method (Combimatrix ElectraSenseTM platform) has been used to develop nucleic acid assays for highly accurate genotyping of a variety of genes in influenza A virus and detection of mutations in the sequences of the different genes of influenza virus.

Práce je spojená s projektem CEITEC CZ.1.05/1.1.00/02.0068.

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