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Dobrá díla jsou plodem dobrého charakteru a protože je chvályhodnější příčina než následek, chval více dobrý charakter bez vzdělání než vzdělance bez charakteru.

Leonardo da Vinci

mendel

Výzkum

Electrochemical microarrays as a rapid method for an identification of mutation in influenza viruses

Influenza is an infectious disease caused by ssRNA viruses 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 outpatients and inpatients accompanied by substantial economic burden both on health care systems and society [1]. The risk of complications comming 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 influenza A (H1N1) pandemic in 2009, pregnant women were at risk for severe influenza illness [2, 3]. Moreover, the mentioned pandemic disproportionately affected children and resulted in a substantially increased number of hospitalizations and deaths among them [4, 5]. The threat of an influenza pandemic, which could be virulent and 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 Centre, Centres 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 be transmitted to human in several countries [7-9], whereas china had high severity and fatality of human infections with avian influenza A(H7N9) infection [9, 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 neuraminidase inhibitors and the M2 protein blockators belongs to the current antivirals. The neuraminidase inhibitors (NAIs) are the most commonly used class of influenza antiviral drugs for the treatment of infected patients [11]. The M2 protein blockators 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 [12-14]. The most common NAIs drugs are zanamivir, oseltamivir, peramivir, and a long-acting NAIs, laninamivir [15]. 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 [16]. Oseltamivir resistance was reported for the first time in A(H3N2) virus strains during the 2011-2012 influenza season [17]. 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 occurence during treatment with neuraminidase inhibitors [18]. 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 [19]. Recently, a study reported that an I223R/H275Y double mutant of neuraminidase (NA) creates a multidrug resistant form of the pandemic influenza A (H1N1) virus [20].

Based on the above mentioned facts, new molecular techniques are required for rapid detection of the influenza mutation to monitor its transmission in the population. Application of oligonucleotide microarrays in different areas of molecular biology and clinical studies has been rapidly growing during the last decade [21-24]. The microarrays are currently combined 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 [25-29]. 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 [30]. 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 (Fig. 1). The CombiMatrix influenza detection system is an effective methodology for influenza A and B subtype analysis [28, 31]. In this study, the electrochemical method (Combimatrix ElectraSenseTM platform) was 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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