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Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

Highly pathogenic avian influenza A (HPAI), subtype H5N1, represents a threat to the human population [1]. While respiratory symptoms and fever are typical signs of influenza, H5N1 has a high incidence of neurological sequelae in many animal species and sporadically in humans, but it represents a continuous danger of global pandemic associated with high mortality [2,3]. HPAI viruses have caused millions of deaths in domestic poultry, and hundreds of deaths in humans [4]. Circulating of influenza viruses in wild animals poses the risk to human health [5,6]. Humans can be infected also by animal subtypes, such as avian influenza virus H5N1 and H9N2 and swine influenza virus H1N1 and H3N2 [7–9]. The primary risk factor for human infection appears to be direct or indirect exposure to infected live or dead animals or contaminated environments [10]. H5N1 occurs in two distinct pathotypes in bird population, seasonal low pathogenic avian influenza (LPAI), and highly pathogenic avian influenza (HPAI) [11–14]. LPAI may become HPAI to poultry through mutations after introduction from wild birds to poultry, but only two subtypes (H5 and H7) can become HPAI [15–17]. These viruses may result in 100% mortality within a susceptible poultry species [16]. The mechanism of mutation of LPAI to HPAI is based on passage in susceptible animals, typically poultry during several months. New generated HPAI virus has broken out in flocks of poultry as so as in wild birds, and caused devastation with huge economic and ecologic impact [3,18]. HPAI poses risk not only for birds, there were also reported sporadic human infections with low morbidity but high mortality, nearly 60% [19–21].

HPAI is not capable of droplet infection from human to human. For this reason, spread of H5N1 virus in the human population is limited [22]. There is a concern that H5N1 may obtain this ability and become pose a potential pandemic hazard to public health worldwide [23–27]. Influenza viruses can develop resistance to pharmacological mechanism of neuraminidase inhibitors (NAIs) that is based on the loss of binding affinity of these drugs. Antiviral resistance in influenza may not develop entirely during treatment but also sometimes transmit widely to replace susceptible strains in the absence of drug pressure [28]. Recently, different authors showed different methods for determination of mutations in H5N1. Sparse learning method was developed to identify antigenicity-associated sites in highly pathogenic H5N1 influenza virus HA based on immunologic data sets [29]. The analysis of complete genome sequences, genetic evolution and phylogenetic analyses showed that the sequence analysis of H5N1 influenza virus displayed the drug-resistant mutations in the matrix protein and NA genes [30]. This fact is important for better understanding the prevalence and adaptation of H5N1 influenza viruses in different countries [31,32]. The continuous mutations in NA gene give rise to a great necessity to monitor its sequence for detection of any possibilities to drug resistance in H5N1 [33–36].The current study was targeted to detect possible mutations in NA gene for both diagnosis and treatment purposes. Neuraminidase inhibitors resistance is based on single-point mutations of neuraminidase gene (H 275 Y) [37,38]. Our choice of three various gene sequences for neuraminidase of H5N1 influenza was based on this assumption. There was chosen sections, in which the sequences differed from each other. This way was considered as a model of point mutation.

Further, we report multi-target detection of point mutation in H5N1 NA gene. As a model real sample RNA oligonucleotide (RNA ODN) labeled with CdS was tested. Isolation and detection was carried out under conditions optimized by labeled DNA oligonucleotide. In addition, we described hybridization assay based on automatic isolation by modified paramagnetic particles (MPs). Isolated target molecule labeled by quantum dots (QDs) was detected by electrochemical analysis.

Podpořeno projekty: IGA IP16/2013

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