Detection of Resistance Mutations to Antivirals Oseltamivir and Zanamivir in Avian Influenza A Viruses Isolated from Wild Birds

The neuraminidase (NA) inhibitors oseltamivir and zanamivir are the first-line of defense against potentially fatal variants of influenza A pandemic strains. However, if resistant virus strains start to arise easily or at a high frequency, a new anti-influenza strategy will be necessary. This study aimed to investigate if and to what extent NA inhibitor–resistant mutants exist in the wild population of influenza A viruses that inhabit wild birds. NA sequences of all NA subtypes available from 5490 avian, 379 swine and 122 environmental isolates were extracted from NCBI databases. In addition, a dataset containing 230 virus isolates from mallard collected at Ottenby Bird Observatory (Öland, Sweden) was analyzed. Isolated NA RNA fragments from Ottenby were transformed to cDNA by RT-PCR, which was followed by sequencing. The analysis of genotypic profiles for NAs from both data sets in regard to antiviral resistance mutations was performed using bioinformatics tools. All 6221 sequences were scanned for oseltamivir- (I117V, E119V, D198N, I222V, H274Y, R292K, N294S and I314V) and zanamivir-related mutations (V116A, R118K, E119G/A/D, Q136K, D151E, R152K, R224K, E276D, R292K and R371K). Of the sequences from the avian NCBI dataset, 132 (2.4%) carried at least one, or in two cases even two and three, NA inhibitor resistance mutations. Swine and environmental isolates from the same data set had 18 (4.75%) and one (0.82%) mutant, respectively, with at least one mutation. The Ottenby sequences carried at least one mutation in 15 cases (6.52%). Therefore, resistant strains were more frequently found in Ottenby samples than in NCBI data sets. However, it is still uncertain if these mutations are the result of natural variations in the viruses or if they are induced by the selective pressure of xenobiotics (e.g., oseltamivir, zanamivir)

Environmental Levels of the Antiviral Oseltamivir Induce Development of Resistance Mutation H274Y in Influenza A/H1N1 Virus in Mallards

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals

Study of oseltamivir and zanamivir resistance-related mutations in influenza viruses isolated from wild mallards in Sweden

Resistance to neuraminidase inhibitors (NAIs) is a growing problem in battle against influenza A virus. However, little is known about the resistance of viruses isolated from dabbling ducks, the natural reservoir of the influenza virus. To our knowledge, no low-pathogenic avian influenza (LPAI) virus resistant to NAIs has been detected. The aim of this study was to investigate mallard isolates of influenza A virus previously identified to carry oseltamivir carboxylate (OC) or zanamivir (ZA) resistance-related mutations. In this work, 21 viruses belonging to the N1, N3, N6 and N9 subtypes were analyzed using a colorimetric NA inhibition assay. The results of assay showed no NAIs-resistant phenotype for any of the viruses. The R118K mutation was the most recurrent, as it was observed in all subtypes except for N6. IC50 values confirmed the differences in sensitivity to OC or ZA observed in the N1 and N2 groups of NAs. Furthermore, both wild types (WTs) in the N6 and one WT in the N9 subtype were less sensitive to ZA than were genotypically related mutants with R152K and R118K change in the respective subtypes. This may indicate that these and probably even other NAIs resistance-related mutations found in our virus collection were not induced by NAIs residuals in the environment and that the impact of such mutations in an avian influenza could be dependent on subtype, strain and host species

Portrayal of Protagonist Relationships and Identity in Anime "Neon Genesis Evangelion" (1995)

Šī darba nosaukums ir “Protagonistu attiecību un identitātes atspoguļojums animē “Jauna gadsimta evaņģēlijs” (1995)”. Animē ir apspriestas tādas indivīdam svarīgas tēmas un jautājumi, kā sevis pieņemšana un nenoliegšana, nebēgšana no realitātes, spēja iekļauties sabiedrībā, nezaudējot savas indivīda vajadzības. Šajā darbā uzsvars ir likts uz tēlu emocijām, psiholoģisko problēmu rašanās iemesliem un sekām, kā arī individualitātes izpausmēm un to nozīmes. Darba ietveros tika tulkoti izvēlēti teksti no datu avota (animes), pētīti psiholoģiskie tipi un induviduālpsiholoģiskie kompleksi. Analizējot tēlu emocijas, runu, ķermeņa valodu, attiecību izmaiņas un to ietekmējošos cēloņus, tika secināts, ka psiholoģiskie tipi ietekmē indivīda pasaules uztveri, bet apmierinātība ar esošo dzīves situāciju un sevis pieņemšana nav atkarīga no tipa. Induviduālpsiholoģiskie kompleksu cēloņi ir atrodami indivīda bērnībā, komunikācijā ar vecākiem, un šī ietekme saglabājās indivīdam pieaugot. Analizējot indivīda kontaktu ar apkārtējiem, var spriest par indivīda piederēšanu pie cilvēkiem ar hikikomori sindromu. Darbs ir sadalīts četrās daļās, un katrā daļā ir 3 nodaļas. Pirmajā, otrajā un trešajā daļā ir izmantotas apakšnodaļas. Pirmajā daļā tiek apskatīti psiholoģiskie tipi, induviduālpsiholoģija un hikikomori teorija. Darba otrajā nodaļā tiek apskatītas trīs protagonistu attiecības ar vecākiem un citiem tēliem. Trešajā daļā tiek apskatītas protagonistu seksuālās problēmas un kā tās izpaužas. Savukārt ceturtajā daļā tiek apskatītas tēlu attiecības ar savu induviduālitāti.The topic of this thesis is “PORTRAYAL OF PROTAGONIST RELATIONSHIPS AND IDENTITY IN ANIME “NEON GENESIS EVANGELION”(1995)”.This anime discusses topics and issues important to the individual such as self-acceptance and non-denial, not escaping from reality, the ability to integrate into society without losing one's individual needs. In this thesis, the emphasis is on the emotions of the characters, the causes and consequences of psychological problems, as well as expression of individuality and its meaning. Within the scope of this thesis, texts selected from data source (anime) were translated, psychological types and individual psychological complexes were studied. Analysing character emotions, speech, body language, changes in relationships and causes affecting them, it was concluded that psychological types influence individuals perception of the world, but satisfaction with the current life situation and self-acceptance does not depend on psychological type. The causes of individual psychological complexes can be found in an individual's childhood, in communication with parents and these influences persist as the individual grows up. Analyzing an individual's contact with others, it is possible to judge whether an individual belongs to people with hikikomori syndrome. This thesis is divided in 4 parts and each part has 3 chapters. First, second and third parts have subdivisions. The first part looks into the psychological types, individual psychology and theory about hikikomori. The second part of the thesis looks into the relationships of the three protagonists with their parents and other characters. The third part looks into the sexual problems of protagonists and how these problems manifest. And the fourth part looks into characters' relationships with their individuality

Performance of virus isolates in CM assay.

1)<p>Code for virus subtypes; *- mutations induced by OC (adopted from literature); without *- mutations induced by ZA (adopted from literature).</p>2)<p>Mutation unrelated to inhibitor resistance.</p>3)<p>F - framework residue; C - catalytic residue; WT- wild type.</p

Sequence alignment of N2 reference, and three N1 sensitive mutants.

<p>The sequence interval between the 121^th and 149^th amino acid (marked with - - - ; N2 reference numbering) is excluded from alignment for practical reasons. The relevant mutation sites are indicated in red. (r) - reference sequence; Number within the parenthesis indicate code for virus isolates according <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089306#pone-0089306-t002" target="_blank">Table 2</a>. (3) - H1N1 R118K mutant; (4) - H1N1 R118K/D151N mutant; (14) - H6N1 R118K/D151N/D198N mutant. Cl.Co- ClustalW consensus sequence, “*” is used to indicate identical residues, “:” is used to indicate conserved substitution, “.” is used to indicate semi-conserved substitution, “ ” (empty space) is used to indicate dissimilar residues.</p

Distribution of mutations with regards to NA subtype.

<p>Mainly adopted from Orozovic et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089306#pone.0089306-Orozovic1" target="_blank">[26]</a>.</p>1)<p>Sum of all R118K mutations.</p>2)<p>Mutation unrelated to inhibitor resistance.</p>3)<p>Isolates that were not analyzed.</p>4)<p>Previously published values.</p

IC₅₀ values of viral isolates analyzed by CM.

<p>Each bar shows mean ± SE IC₅₀ of three replicates. The x-axis shows the viral isolates. NxI and NxII represent wild types (WTs) and 1–14 represent mutants. For codes of enumeration, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089306#pone-0089306-t002" target="_blank">Table 2</a>. z – inhibition by ZA; o – inhibition by OC. On the y-axis, the IC₅₀ values are expressed as concentration of inhibitor in nM. All means were tested by one-way ANOVA and post-hoc Tukey tests. A) N1 subtype; B) N3 subtype; C) N6 subtype and D) N9 subtype.</p

Wild-type and H274Y-containing avian influenza A viruses of N1 subtype in the NCBI database.

<p>* indicates that the four H274Y-positive H5N1 viruses probably represent three distinctly different viruses, see <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024742#s3" target="_blank">Discussion</a></i>.</p

Experimental set-up.

<p>Set-up for 80 ng/L and 1 µg/L experiments shown with blue blocks, 80 µg/L with red dotted lines.</p