Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

A novel vehicle-mounted sticky trap; an effective sampling tool for savannah tsetse flies Glossina morsitans morsitans Westwood and Glossina morsitans centralis Machado

BackgroundBlack screen fly round (BFR) is a mobile sampling method for Glossina morsitans. This technique relies on the ability of operator(s) to capture flies landing on the screen with hand nets. In this study, we aimed to evaluate a vehicle-mounted sticky panel trap (VST) that is independent of the operator’s ability to capture flies against BFR, for effective and rapid sampling of G. m. morsitans Westwood and G. m. centralis Machado. We also determined the influence of the VST colour (all-blue, all-black or 1:1 blue-black), orientation and presence of odour attractants on tsetse catch.Methodology/Principal findingsUsing randomised block design experiments conducted in Zambia, we compared and modelled the number of tsetse flies caught in the treatment arms using negative binomial regression. There were no significant differences in the catch indices of the three colour designs and for in-line or transversely oriented panels for both subspecies (P > 0.05). When baited with butanone and 1-octen-3-ol, VST caught 1.38 (1.11–1.72; P < 0.01) times more G. m. centralis flies than the un-baited trap. Attractants did not significantly increase the VST catch index for G. m. morsitans (P > 0.05). Overall, the VST caught 2.42 (1.91–3.10; P < 0.001) and 2.60 (1.50–3.21; P < 0.001) times more G. m. centralis and G. m. morsitans respectively, than the BFR. The VST and BFR took 10 and 35 min respectively to cover a 1 km transect.Conclusion/SignificanceThe VST is several times more effective for sampling G. m. morsitans and G. m. centralis than the BFR and we recommend its use as an alternative sampling tool.     

PDCD10 Gene Mutations in Multiple Cerebral Cavernous Malformations

Cerebral cavernous malformations (CCMs) are vascular abnormalities that may cause seizures, intracerebral haemorrhages, and focal neurological deficits. Familial form shows an autosomal dominant pattern of inheritance with incomplete penetrance and variable clinical expression. Three genes have been identified causing familial CCM: KRIT1/CCM1, MGC4607/CCM2, and PDCD10/CCM3. Aim of this study is to report additional PDCD10/CCM3 families poorly described so far which account for 10-15% of hereditary cerebral cavernous malformations. Our group investigated 87 consecutive Italian affected individuals (i.e. positive Magnetic Resonance Imaging) with multiple/familial CCM through direct sequencing and Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis. We identified mutations in over 97.7% of cases, and PDCD10/CCM3 accounts for 13.1%. PDCD10/CCM3 molecular screening revealed four already known mutations and four novel ones. The mutated patients show an earlier onset of clinical manifestations as compared to CCM1/CCM2 mutated patients. The study of further families carrying mutations in PDCD10/CCM3 may help define a possible correlation between genotype and phenotype; an accurate clinical follow up of the subjects would help define more precisely whether mutations in PDCD10/CCM3 lead to a characteristic phenotype.     

Population Genetics of Two Key Mosquito Vectors of Rift Valley Fever Virus Reveals New Insights into the Changing Disease Outbreak Patterns in Kenya

Rift Valley fever (RVF) outbreaks in Kenya have increased in frequency and range to include northeastern Kenya where viruses are increasingly being isolated from known (Aedes mcintoshi) and newly-associated (Ae. ochraceus) vectors. The factors contributing to these changing outbreak patterns are unclear and the population genetic structure of key vectors and/or specific virus-vector associations, in particular, are under-studied. By conducting mitochondrial and nuclear DNA analyses on >220 Kenyan specimens of Ae. mcintoshi and Ae. ochraceus, we uncovered high levels of vector complexity which may partly explain the disease outbreak pattern. Results indicate that Ae. mcintoshi consists of a species complex with one of the member species being unique to the newly-established RVF outbreak-prone northeastern region of Kenya, whereas Ae. ochraceus is a homogeneous population that appears to be undergoing expansion. Characterization of specimens from a RVF-prone site in Senegal, where Ae. ochraceus is a primary vector, revealed direct genetic links between the two Ae. ochraceus populations from both countries. Our data strongly suggest that unlike Ae. mcintoshi, Ae. ochraceus appears to be a relatively recent, single ''introduction'' into Kenya. These results, together with increasing isolations from this vector, indicate that Ae. ochraceus will likely be of greater epidemiological importance in future RVF outbreaks in Kenya. Furthermore, the overall vector complexity calls into question the feasibility of mosquito population control approaches reliant on genetic modification.     

Direct and Fe(II)-Mediated Reduction of Technetium by Fe(III)-Reducing Bacteria

The dissimilatory Fe(III)-reducing bacterium Geobacter sulfurreducens reduced and precipitated Tc(VII) by two mechanisms. Washed cell suspensions coupled the oxidation of hydrogen to enzymatic reduction of Tc(VII) to Tc(IV), leading to the precipitation of TcO₂ at the periphery of the cell. An indirect, Fe(II)-mediated mechanism was also identified. Acetate, although not utilized efficiently as an electron donor for direct cell-mediated reduction of technetium, supported the reduction of Fe(III), and the Fe(II) formed was able to transfer electrons abiotically to Tc(VII). Tc(VII) reduction was comparatively inefficient via this indirect mechanism when soluble Fe(III) citrate was supplied to the cultures but was enhanced in the presence of solid Fe(III) oxide. The rate of Tc(VII) reduction was optimal, however, when Fe(III) oxide reduction was stimulated by the addition of the humic analog and electron shuttle anthaquinone-2,6-disulfonate, leading to the rapid formation of the Fe(II)-bearing mineral magnetite. Under these conditions, Tc(VII) was reduced and precipitated abiotically on the nanocrystals of biogenic magnetite as TcO₂ and was removed from solution to concentrations below the limit of detection by scintillation counting. Cultures of Fe(III)-reducing bacteria enriched from radionuclide-contaminated sediment using Fe(III) oxide as an electron acceptor in the presence of 25 μM Tc(VII) contained a single Geobacter sp. detected by 16S ribosomal DNA analysis and were also able to reduce and precipitate the radionuclide via biogenic magnetite. Fe(III) reduction was stimulated in aquifer material, resulting in the formation of Fe(II)-containing minerals that were able to reduce and precipitate Tc(VII). These results suggest that Fe(III)-reducing bacteria may play an important role in immobilizing technetium in sediments via direct and indirect mechanisms.     

Connectivity and information transfer in flow networks: Two magic numbers in ecology?

An ecosystem can be visualized as a graph of certain preassigned trophic compartments; these nodes are then mutually connected through the internal exchanges of material and energy. The mathematical theory of information can be applied to such a graph in order to define two relevant indices: a measure of connectivity (the entropy H of the connections) and a measure of the degree of the “energetic” specialization (the internal transfer of informationI). The computation of these indices in stationary real cases suggests that the observed complexity of ecosystems is conditioned by two competing effects. The first can be interpreted as a “thermodynamical” principle related to the unavoidable irreversibility taking place inside the system, whereas the second can be taken as a “biological” principle concerned with the selection of some particular interactions: those which maximize the information circulating between the compartments.     

Identification of an S-locus glycoprotein allele introgressed from B. napus ssp. rapifera to B. napus ssp. oleifera.

We have previously described a developmentally regulated mRNA in maize that accumulates in mature embryos and is involved in a variety of stress responses in the plant. The sequence of the encoded 16 kDa protein (MA16) predicts that it is an RNA-binding protein, since it possesses a ribonucleoprotein consensus sequence-type RNA-binding domain (CS-RBD). To assess the predicted RNA binding property of the protein and as a starting point to characterize its function we have used ribohomopolymer-binding assays. Here we show that the MA16-encoded protein binds preferentially to uridine- and guanosine-rich RNAs. In light of these results a likely role for this protein in RNA metabolism during late embryogenesis and in the stress response is discussed.