A dengue receptor as possible genetic marker of vector competence in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.     

Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells

Background

Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/Principal Findings

We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4B^C74A mutant.

Conclusions/Significance

Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.     

Paracoccidioides brasiliensis pancreatic destruction in Calomys callosus experimentally infected

Background  

The wild rodent Calomys callosus is notably resistant to Trypanosoma cruzi infection. In order to better characterize this animal model for experimental infections, we inoculated C. callosus intraperitoneally with Paracoccidioides brasiliensis, a thermally dimorphic fungus that causes a chronic disease with severe granuloma formation in the mouse and humans. The dissemination of P. brasiliensis cells through the lungs, liver, pancreas, and spleen was assessed by histological analysis.     

Generation of Phaseolus vulgaris ESTs and investigation of their regulation upon Uromyces appendiculatus infection

Background  

Phaseolus vulgaris (common bean) is the second most important legume crop in the world after soybean. Consequently, yield losses due to fungal infection, like Uromyces appendiculatus (bean rust), have strong consequences. Several resistant genes were identified that confer resistance to bean rust infection. However, the downstream genes and mechanisms involved in bean resistance to infection are poorly characterized.     

BST2/Tetherin Inhibits Dengue Virus Release from Human Hepatoma Cells

Type I interferons (IFN) have been shown to play an important role for inhibiting Dengue virus (DENV) infection. Identifying IFN-induced cellular proteins are essential for understanding its mechanisms against DENV. Here we established stable Huh7-derived cell lines expressing the IFN-induced cell membrane protein BST2 (Huh7-BST2) or its variant bearing a V5 tag at the C-terminal (Huh7-BST5CV5). These cell lines were infected with DENV to determine proteins modulating their anti-DENV response. We found that expression of BST2 did not affect the efficiency of DENV infection and intracellular replication. Rather, it significantly reduced the virion yield of the infected cells, particularly at low MOI infection. In addition, BST2 also decreased the foci formation and the size of infectious foci in cultured Huh7 monolayers with media containing methocellulose. The addition of the V5 tag at C-terminal inhibited the GPI modification of BST2 and blocked its shift from endoplasm to cytoplastic membrane. BST2CV5 did not affect DENV infection and foci formation in Huh7 cells but reduced virion yield by 1 log at low MOI infection. Interestingly, intracellular BST2CV5 expression was reduced by high level of DENV production.

Conclusion

Our results imply that BST2 is a functional mediator of the IFN response against DENV infection. BST2 inhibits the release of DENV virions from Huh7 cells and limits viral cell-to-cell transmission. BST2CV5 variant is unable to inhibit DENV release but impairs viral infection in cells.     

Small RNA profiling of Dengue virus-mosquito interactions implicates the PIWI RNA pathway in anti-viral defense

Background  

Small RNA (sRNA) regulatory pathways (SRRPs) are important to anti-viral defence in mosquitoes. To identify critical features of the virus infection process in Dengue serotype 2 (DENV2)-infected Ae. aegypti, we deep-sequenced small non-coding RNAs. Triplicate biological replicates were used so that rigorous statistical metrics could be applied.     

Bacillus anthracis spore interactions with mammalian cells: Relationship between germination state and the outcome of in vitro

Background  

During inhalational anthrax, internalization of Bacillus anthracis spores by host cells within the lung is believed to be a key step for initiating the transition from the localized to disseminated stages of infection. Despite compelling in vivo evidence that spores remain dormant within the bronchioalveolar spaces of the lungs, and germinate only after uptake into host cells, most in vitro studies of infection have been conducted under conditions that promote rapid germination of spores within the culture medium.     

Synergy between type 1 fimbriae expression and C3 opsonisation increases internalisation of E. coli by human tubular epithelial cells

Background  

Bacterial infection of the urinary tract is a common clinical problem with E. coli being the most common urinary pathogen. Bacterial uptake into epithelial cells is increasingly recognised as an important feature of infection. Bacterial virulence factors, especially fimbrial adhesins, have been conclusively shown to promote host cell invasion. Our recent study reported that C3 opsonisation markedly increases the ability of E. coli strain J96 to internalise into human proximal tubular epithelial cells via CD46, a complement regulatory protein expressed on host cell membrane. In this study, we further assessed whether C3-dependent internalisation by human tubular epithelial cells is a general feature of uropathogenic E. coli and investigated features of the bacterial phenotype that may account for any heterogeneity.     

A family history of DUX4: phylogenetic analysis of DUXA, B, C and Duxbl reveals the ancestral DUX gene

Background  

DUX4 is causally involved in the molecular pathogenesis of the neuromuscular disorder facioscapulohumeral muscular dystrophy (FSHD). It has previously been proposed to have arisen by retrotransposition of DUXC, one of four known intron-containing DUX genes. Here, we investigate the evolutionary history of this multi-member double-homeobox gene family in eutherian mammals.     

Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia

Background  

Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia.     

Activation of mRNA translation by phage protein and low temperature: the case of Lactococcus lactis abortive infection system AbiD1

Background  

Abortive infection (Abi) mechanisms comprise numerous strategies developed by bacteria to avoid being killed by bacteriophage (phage). Escherichia coli Abis are considered as mediators of programmed cell death, which is induced by infecting phage. Abis were also proposed to be stress response elements, but no environmental activation signals have yet been identified. Abis are widespread in Lactococcus lactis, but regulation of their expression remains an open question. We previously showed that development of AbiD1 abortive infection against phage bIL66 depends on orf1, which is expressed in mid-infection. However, molecular basis for this activation remains unclear.     

Consequences of concurrent Ascaridia galli and Escherichia coli infections in chickens

   

Three experiments were carried out to examine the consequences of concurrent infections with Ascaridia galli and Escherichia coli in chickens raised for table egg production. Characteristic pathological lesions including airsacculitis, peritonitis and/or polyserositis were seen in all groups infected with E. coli. Furthermore, a trend for increased mortality rates was observed in groups infected with both organisms which, however, could not be confirmed statistically. The mean worm burden was significantly lower in combined infection groups compared to groups infected only with A. galli. It was also shown that combined infections of E. coli and A. galli had an added significant negative impact on weight gain.     

PCR-based method for targeting 16S-23S rRNA intergenic spacer regions among Vibrio species

Background

Mosquito-borne dengue virus (DENV, genus Flavivirus) has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi) induced by exogenous small interfering RNAs (siRNAs) has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi.

Results

Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI) 0.1 and MOI 10; each strain achieved titers > 4.0 log₁₀pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2), resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

Conclusions

These findings indicate that DENV can replicate in Drosophila S2 cells and that the RNAi pathway plays a role in modulating DENV replication in these cells. S2 cells offer a useful cell culture model for evaluation of the interaction between DENV and the RNAi response.     

The outcome of Cryptococcus neoformans intracellular pathogenesis in human monocytes

Background  

Cryptococcus neoformans is an encapsulated yeast that is a facultative intracellular pathogen. The interaction between macrophages and C. neoformans is critical for extrapulmonary dissemination of this pathogenic yeast. C. neoformans can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. However, most studies of intracellular pathogenesis have been made with mouse cells and their relevance to human infection is uncertain. In this study we extended studies of C. neoformans-macrophage cellular interaction/s to human peripheral blood monocytes.     

Association of HLA Class-I and Inhibitory KIR Genotypes in Gabonese Patients Infected by Chikungunya or Dengue Type-2 Viruses

Background

Natural killer (NK) cells provide defense in the early stages of the immune response against viral infections. Killer cell immunoglobulin-like receptors (KIR) expressed on the surface of NK cells play an important role in regulating NK cell response through recognition of human leukocyte antigen (HLA) class I molecules on target cells. Previous studies have shown that specific KIR/ligand combinations are associated with the outcome of several viral infectious diseases.

Methods

We investigated the impact of inhibitory and activating KIR and their HLA-class I ligand genotype on the susceptibility to Chikungunya virus (CHIKV) and Dengue virus (DENV2) infections. From April to July 2010 in Gabon, a large outbreak of CHIKV and DENV2 concomitantly occurred in two provinces of Gabon (Ogooué-Lolo and Haut-Ogooué). We performed the genotypic analysis of KIR in the combination with their cognate HLA-class I ligands in 73 CHIKV and 55 DENV2 adult cases, compared with 54 healthy individuals.

Results

We found in CHIV-infected patients that KIR2DL1 and KIR2DS5 are significantly increased and decreased respectively, as compared to DENV2⁺ patients and healthy donors. The combination of KIR2DL1 and its cognate HLA-C2 ligand was significantly associated with the susceptibility to CHIKV infection. In contrast, no other inhibitory KIR-HLA pairs showed an association with the two mosquito-borne arboviruses.

Conclusion

These observations are strongly suggestive that the NK cell repertoire shaped by the KIR2DL1:HLA-C2 interaction facilitate specific infection by CHIKV.     

Functionality of Dengue Virus Specific Memory T Cell Responses in Individuals Who Were Hospitalized or Who Had Mild or Subclinical Dengue Infection

Background

Although antibody responses to dengue virus (DENV) in naturally infected individuals have been extensively studied, the functionality of DENV specific memory T cell responses in relation to clinical disease severity is incompletely understood.

Methodology/Principal findings

Using ex vivo IFNγ ELISpot assays, and by determining cytokines produced in ELISpot supernatants, we investigated the functionality of DENV-specific memory T cell responses in a large cohort of individuals from Sri Lanka (n=338), who were naturally infected and were either hospitalized due to dengue or had mild or sub clinical dengue infection. We found that T cells of individuals with both past mild or sub clinical dengue infection and who were hospitalized produced multiple cytokines when stimulated with DENV-NS3 peptides. However, while DENV-NS3 specific T cells of those with mild/sub clinical dengue infection were more likely to produce only granzyme B (p=0.02), those who were hospitalized were more likely to produce both TNFα and IFNγ (p=0.03) or TNFα alone.We have also investigated the usefulness of a novel T cell based assay, which can be used to determine the past infecting DENV serotype. 92.4% of DENV seropositive individuals responded to at least one DENV serotype of this assay and none of the seronegatives responded. Individuals who were seronegative, but had received the Japanese encephalitis vaccine too made no responses, suggesting that the peptides used in this assay did not cross react with the Japanese encephalitis virus.

Conclusions/significance

The types of cytokines produced by DENV-specific memory T cells appear to influence the outcome of clinical disease severity. The novel T cell based assay, is likely to be useful in determining the past infecting DENV serotype in immune-epidemiological studies and also in dengue vaccine trials.     

Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

Background  

The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The inc gene CT223 is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested C. trachomatis serovars.     

Diverse accumulation of several dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress

Background  

Dehydrins represent hydrophilic proteins acting mainly during cell dehydration and stress response. Dehydrins are generally thermostable; however, the so-called dehydrin-like (dehydrin-related) proteins show variable thermolability. Both groups immunoreact with antibodies directed against the K-segment of dehydrins. Plant mitochondrial dehydrin-like proteins are poorly characterized. The purpose of this study was to extend previous reports on plant dehydrins by comparing the level of immunoprecipitated dehydrin-like proteins in cauliflower (Brassica oleracea var. botrytis), Arabidopsis thaliana and yellow lupin (Lupinus luteus) mitochondria under cold and heat stress.     

Regulation of callose synthase activity in situ in alamethicin-permeabilized Arabidopsis and tobacco suspension cells

Background  

The cell wall component callose is mainly synthesized at certain developmental stages and after wounding or pathogen attack. Callose synthases are membrane-bound enzymes that have been relatively well characterized in vitro using isolated membrane fractions or purified enzyme. However, little is known about their functional properties in situ, under conditions when the cell wall is intact. To allow in situ investigations of the regulation of callose synthesis, cell suspensions of Arabidopsis thaliana (Col-0), and tobacco (BY-2), were permeabilized with the channel-forming peptide alamethicin.