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.     

Heterogeneous binding of high mobility group chromosomal proteins to nuclei

A dramatic difference is observed in the intracellular distribution of the high mobility group (HMG) proteins when chicken embryo fibroblasts are fractionated into nucleus and cytoplasm by either mass enucleation of cytochalasin-B-treated cells or by differential centrifugation of mechanically disrupted cells. Nuclei (karyoplasts) obtained by cytochalasin B treatment of cells contain more than 90 percent of the HMG 1, while enucleated cytoplasts contain the remainder. A similar distribution between karyoplasts and cytoplasts is observed for the H1 histones and the nucleosomal core histones as anticipated. The presence of these proteins, in low amounts, in the cytoplast preparation can be accounted for by the small percentage of unenucleated cells present. In contrast, the nuclei isolated from mechanically disrupted cells contain only 30-40 percent of the total HMGs 1 and 2, the remainder being recovered in the cytosol fraction. No histone is observed in the cytosol fraction. Unike the higher molecular weight HMGs, most of the HMGs 14 and 17 sediment with the nuclei after cell lysis by mechanical disruption. The distribution of HMGs is unaffected by incubating cells with cytochalasin B and mechanically fractionating rather than enucleating them. Therefore, the dramatic difference in HMG 1 distribution observed using the two fractionation techniques cannot be explained by a cytochalasin-B-induced redistribution. On reextraction and sedimentation of isolated nuclei obtained by mechanical cell disruption, only 8 percent of the HMG 1 is released to the supernate. Thus, the majority of the HMG 1 originally isolated with these nuclei, representing 35 percent of the total HMG 1, is stably bound, as is all the HMGs 14 and 17. The remaining 65 percent of the HMGs 1 and 2 is unstably bound and leaks to the cytosol fraction under the conditions of mechanical disruption. It is suggested that the unstably bound HMGs form a protein pool capable of equilibrating between cytoplasm and stably bound HMGs.     

Efficiency clustering for low-density microarrays and its application to QPCR

Background  

Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency.     

AIR: A batch-oriented web program package for construction of supermatrices ready for phylogenomic analyses

Background  

Large multigene sequence alignments have over recent years been increasingly employed for phylogenomic reconstruction of the eukaryote tree of life. Such supermatrices of sequence data are preferred over single gene alignments as they contain vastly more information about ancient sequence characteristics, and are thus more suitable for resolving deeply diverging relationships. However, as alignments are expanded, increasingly numbers of sites with misleading phylogenetic information are also added. Therefore, a major goal in phylogenomic analyses is to maximize the ratio of information to noise; this can be achieved by the reduction of fast evolving sites.     

Ovarian activity and plasma sex steroid levels of Distichodus antonii in relation to environmental conditions in the upper basin of the Congo River

Distichodus antonii is an endemic fish species of the Congo River basin in which the stocks of wild populations are threatened by overfishing pressure. Knowledge of its reproductive biology would be useful in consideration of conservation and management options for the species. Therefore, this study investigated changes in ovarian activity and levels of steroid profiles in wild populations in relation to variation in temperature and rainfall. Adult females (n = 101, body weight of 3 183 ± 14.75 g, SE) were captured monthly over one year (2013–2014). Apart from evaluation of oocyte diameters and gonad developmental stages, gonado-, hepato-, lipososomatic indices (GSI, HSI, LSI) and plasma levels of sex steroids (testosterone-T, estradiol-17β-E2) were determined. The results suggested a synchronous development of oocytes with two annual reproductive seasons over the one-year study. Plasma T and E2 levels peaked during spawning periods likely reflecting active oogenesis. The highest values of morphosomatic indices were observed during the longest rainfall period in September, and were associated with high steroidogenic activity evidenced by increased E2 production. In addition, more vitellogenic oocytes (September and October) were observed during the latter season than during the short rainy season (in May).     

Comparative analysis of the zeta-crystallin/quinone reductase gene in guinea pig and mouse

zeta-Crystallin is a novel nicotinamide adenine dinucleotide phosphate:quinone reductase, present at enzymatic levels in various tissues of different species, which is highly expressed in the lens of some hystricomorph rodents and camelids. We report here the complementary DNA (cDNA) cloning of zeta-crystallin from liver libraries in guinea pig (Cavia porcellus), where zeta-crystallin is highly expressed in the lens, and in the laboratory mouse (Mus musculus), where expression in the lens occurs only at enzymatic levels. A 5' untranslated sequence different from the one previously reported for the guinea pig lens cDNA was found in these clones. We also report the isolation of genomic clones including the complete guinea pig zeta-crystallin gene and the 5' region of this gene in mouse. These results show the presence of two promoters in the guinea pig zeta-crystallin gene, one responsible for expression at enzymatic levels and the other responsible for the high expression in the lens. The guinea pig lens promoter is not present in the mouse gene. This is the first example in which the recruitment of an enzyme as a lens crystallin can be explained by the acquisition of an alternative lens- specific promoter.      

Invasion status of Florida bass Micropterus floridanus (Lesueur, 1822) in South Africa

Largemouth bass Micropterus salmoides are a popular North American angling species that was introduced into South Africa in 1928. To enhance the largemouth bass fisheries, Florida bass Micropterus floridanus were introduced into KwaZulu Natal, South Africa, in 1980. Knowledge on the status of M. floridanus in South Africa is required, because it lives longer and reaches larger sizes than M. salmoides, which may result in heightened impacts on native biota. Because M. floridanus are morphologically similar, but genetically distinct from M. salmoides, the distribution of this species was assessed by genetically screening 185 Micropterus sp. individuals sampled from 20 localities across South Africa using the mitochondrial ND2 gene. Individuals with mitochondrial DNA matching M. salmoides were recovered from 16 localities, whereas M. floridanus mitochondrial DNA was recovered from 13 localities. At nine localities (45%), the mitochondrial DNA of both species was detected. These results demonstrate M. floridanus dispersal to multiple sites across South Africa.     

Evidence for independent recruitment of zeta-crystallin/quinone reductase (CRYZ) as a crystallin in camelids and hystricomorph rodents

Zeta-crystallin/quinone reductase (CRYZ) is an NADPH oxidoreductase expressed at very high levels in the lenses of two groups of mammals: camelids and some hystricomorph rodents. It is also expressed at very low levels in all other species tested. Comparative analysis of the mechanisms mediating the high expression of this enzyme/crystallin in the lens of the Ilama (Lama guanacoe) and the guinea pig (Cavia porcellus) provided evidence for independent recruitment of this enzyme as a lens crystallin in both species and allowed us to elucidate for the first time the mechanism of lens recruitment of an enzyme- crystallin. The data presented here show that in both species such recruitment most likely occurred through the generation of new lens promoters from nonfunctional intron sequences by the accumulation of point mutations and/or small deletions and insertions. These results further support the idea that recruitment of CRYZ resulted from an adaptive process in which the high expression of CRYZ in the lens provides some selective advantage rather than from a purely neutral evolutionary process.