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

Methods for in-situ porosity determination of moving porous columns and application to horizontal slug flow pneumatic conveying

Two methods were developed to investigate the porosity of moving slugs in situ during horizontal slug flow pneumatic conveying. The first method consists in applying a permeability model in combination with measurements of pressure loss and fluid velocity along the slugs. A review of existing models describing the resistance of porous structures to fluid flow revealed that the semi-empirical model of Ergun is particularly suitable to investigate the porosity profile along moving slugs. The second method consists in a direct determination method involving a slug-catcher able to catch a moving slug in a fraction of a second and simultaneously separate it into three horizontal layers. Those two methods were applied to analyse the porosity of naturally occurring slugs during pneumatic transport of polypropylene pellets. It was found that in contrast to common belief, slugs are slightly fluidised structures that do not display any porosity gradient over the pipe cross-section height. The slug porosity appeared independent of the gas conveying velocity, all slugs displaying an average porosity around 0.41, which is slightly higher than the bulk porosity of 0.38. Most of the slugs displayed a rear that is denser than the front. However, some slugs had a front that is denser than the rear while other slugs displayed a relatively constant porosity over the entire length. Those unique results refuting the commonly used hypothesis that slugs are compact structures give a new incentive to the area of slug flow pneumatic conveying. While bulk solids mechanics can no longer be applied to explain the stresses induced by moving slugs, the validity of other theories that imply that slugs are fluidised structures should be investigated

Physical mechanisms involved in slug transport and pipe blockage during horizontal pneumatic conveying

Moving slugs of plastic pellets were investigated in-situ during low velocity pneumatic conveying in horizontal pipelines. Slug characteristics including the profile of pressure, pressure gradient, particle velocity, porosity, radial and wall shear stresses, aspect and behaviour were combined to obtain a complete picture of moving slugs. The objective was to gain unique knowledge on the physical mechanisms involved in slug formation, transport, and decay and the occurrence of pipe blockage. Slugs in both stable and unstable states were analysed. A strong correlation between particle velocity and wall stresses was found, which suggests that the stresses responsible for the high pressure loss characterising slug flow may result mostly from the transfer of particle impulses to the pipe wall. Most slugs were found to be denser at the rear where particle velocity was the highest, thus leading to slug shortening over time. This phenomenon was successfully modelled using both Newton's 2nd law and the ideal gas law and prediction of particle velocity showed good agreement with experimental values. In contrast, other slugs were found to extend due to the particles at the front moving faster than the particles at the rear. Pipe blockage was found to result from insufficient permeation of the slug by the conveying gas, indicating that sufficient material permeability is a condition for slug flow to occur