A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication.

RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.

Wild bird surveillance around outbreaks of highly pathogenic avian influenza A(H5N8) virus in the Netherlands, 2014, within the context of global flyways.

Highly pathogenic avian influenza (HPAI) A(H5N8) viruses that emerged in poultry in east Asia since 2010 spread to Europe and North America by late 2014. Despite detections in migrating birds, the role of free-living wild birds in the global dispersal of H5N8 virus is unclear. Here, wild bird sampling activities in response to the H5N8 virus outbreaks in poultry in the Netherlands are summarised along with a review on ring recoveries. HPAI H5N8 virus was detected exclusively in two samples from ducks of the Eurasian wigeon species, among 4,018 birds sampled within a three months period from mid-November 2014. The H5N8 viruses isolated from wild birds in the Netherlands were genetically closely related to and had the same gene constellation as H5N8 viruses detected elsewhere in Europe, in Asia and in North America, suggesting a common origin. Ring recoveries of migratory duck species from which H5N8 viruses have been isolated overall provide evidence for indirect migratory connections between East Asia and Western Europe and between East Asia and North America. This study is useful for better understanding the role of wild birds in the global epidemiology of H5N8 viruses. The need for sampling large numbers of wild birds for the detection of H5N8 virus and H5N8-virus-specific antibodies in a variety of species globally is highlighted, with specific emphasis in north-eastern Europe, Russia and northern China.

New rat models of iron sucrose-induced iron overload.

The majority of murine models of iron sucrose-induced iron overload were carried out in adult subjects. This cannot reflect the high risk of iron overload in children who have an increased need for iron. In this study, we developed four experimental iron overload models in young rats using iron sucrose and evaluated different markers of iron overload, tissue oxidative stress and inflammation as its consequences. Iron overload was observed in all iron-treated rats, as evidenced by significant increases in serum iron indices, expression of liver hepcidin gene and total tissue iron content compared with control rats. We also showed that total tissue iron content was mainly associated with the dose of iron whereas serum iron indices depended essentially on the duration of iron administration. However, no differences in tissue inflammatory and antioxidant parameters from controls were observed. Furthermore, only rats exposed to daily iron injection at a dose of 75 mg/kg body weight for one week revealed a significant increase in lipid peroxidation in iron-treated rats compared with their controls. The present results suggest a correlation between iron overload levels and the dose of iron, as well as the duration and frequency of iron injection and confirm that iron sucrose may not play a crucial role in inflammation and oxidative stress. This study provides important information about iron sucrose-induced iron overload in rats and may be useful for iron sucrose therapy for iron deficiency anemia as well as for the prevention and diagnosis of iron sucrose-induced iron overload in pediatric patients.

Repository of Eurasian influenza A virus hemagglutinin and neuraminidase reverse genetics vectors and recombinant viruses.

Reverse genetics can be used to produce recombinant influenza A viruses containing virtually every desired combination of hemagglutinin (HA) and neuraminidase (NA) genes using the virus backbone of choice. Here, a repository of plasmids and recombinant viruses representing all contemporary Eurasian HA and NA subtypes, H1-H16 and N1-N9, was established. HA and NA genes were selected based on sequence analyses of influenza virus genes available from public databases. Prototype Eurasian HA and NA genes were cloned in bidirectional reverse genetics plasmids. Recombinant viruses based on the virus backbone of A/PR/8/34, and containing a variety of HA and NA genes were produced in 293T cells. Virus stocks were produced in MDCK cells and embryonated chicken eggs. These plasmids and viruses may be useful for numerous purposes, including influenza virus research projects, vaccination studies, and to serve as reference reagents in diagnostic settings.