Initial events in infectious salmon anemia virus infection: evidence for the requirement of a low-pH step

We have investigated the initial steps in the interaction between infectious salmon anemia virus (ISAV) and cultured cells from Atlantic salmon (SHK-1 cell line). Using radioactively or fluorescently labelled viral particles we have studied the binding and fusion kinetics and the effect of pH on binding, uptake, and fusion of ISAV to SHK-1 cells and liposomes. As pH in the medium was reduced from 7.5 to 4.5, the association of virus to the cells was nearly doubled. The same effect of pH was observed when fusion between ISAV and liposomes was analyzed. In addition, the binding of ISAV to intact SHK-1 cells and to cell membrane proteins blotted onto filters was neuraminidase sensitive. However, the increased binding induced by low pH was not neuraminidase sensitive, probably reflecting activation of a fusion peptide at low pH. By using confocal fluorescence microscopy, the increased fusion of fluorescently labelled ISAV with the plasma membrane due to low pH could be demonstrated. When vacuolar pH in the cells was raised during inoculation with chloroquine or ammonium chloride, both electron and confocal microscopy showed accumulation of ISAV in endosomes and lysosomes. Production of infectious virus could be increased by lowering the extracellular pH during infection. Furthermore, chloroquine present during virus inoculation also caused a reduction in the synthesis of viral proteins in ISAV-infected cells as well as in the production of infective virus. These results indicate that ISAV binds to sialic acid residues on the cell surface and that the fusion between virus and cell membrane takes place in the acid environment of endosomes. This provides further evidence for a high degree of similarity between ISAV and influenza virus and extends the basis for the classification of this virus as a member of the Orthomyxoviridae family.     

A Novel Functional Human Eukaryotic Translation Initiation Factor 4G

Mammalian eukaryotic translation initiation factor 4F (eIF4F) is a cap-binding protein complex consisting of three subunits: eIF4E, eIF4A, and eIF4G. In yeast and plants, two related eIF4G species are encoded by two different genes. To date, however, only one functional eIF4G polypeptide, referred to here as eIF4GI, has been identified in mammals. Here we describe the discovery and functional characterization of a closely related homolog, referred to as eIF4GII. eIF4GI and eIF4GII share 46% identity at the amino acid level and possess an overall similarity of 56%. The homology is particularly high in certain regions of the central and carboxy portions, while the amino-terminal regions are more divergent. Far-Western analysis and coimmunoprecipitation experiments were used to demonstrate that eIF4GII directly interacts with eIF4E, eIF4A, and eIF3. eIF4GII, like eIF4GI, is also cleaved upon picornavirus infection. eIF4GII restores cap-dependent translation in a reticulocyte lysate which had been pretreated with rhinovirus 2A to cleave endogenous eIF4G. Finally, eIF4GII exists as a complex with eIF4E in HeLa cells, because eIF4GII and eIF4E can be purified together by cap affinity chromatography. Taken together, our findings indicate that eIF4GII is a functional homolog of eIF4GI. These results may have important implications for the understanding of the mechanism of shutoff of host protein synthesis following picornavirus infection.     

A First Insight on the Population Structure of Mycobacterium tuberculosis Complex as Studied by Spoligotyping and MIRU-VNTRs in Santiago,Chile

Tuberculosis (TB) remains a significant public health problem worldwide, but the ecology of the prevalent mycobacterial strains, and their transmission, can vary depending on country and region. Chile is a country with low incidence of TB, that has a geographically isolated location in relation to the rest of South American countries due to the Andes Mountains, but recent migration from neighboring countries has changed this situation. We aimed to assess the genotypic diversity of Mycobacterium tuberculosis complex (MTBC) strains in Santiago, Chile, and compare with reports from other Latin-American countries. We analyzed MTBC isolates from pulmonary tuberculosis cases collected between years 2008 and 2013 in Central Santiago, using two genotyping methods: spoligotyping and 12-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTRs). Data obtained were analyzed and compared to the SITVIT2 database. Mean age of the patients was 47.5 years and 61% were male; 11.6% were migrants. Of 103 strains (1 isolate/patient) included, there were 56 distinct spoligotype patterns. Of these, 16 strains (15.5%) corresponded to orphan strains in the SITVIT2 database, not previously reported. Latin American and Mediterranean (LAM) (34%) and T (33%) lineages were the most prevalent strains, followed by Haarlem lineage (16.5%). Beijing family was scarcely represented with only two cases (1.9%), one of them isolated from a Peruvian migrant. The most frequent clustered spoligotypes were SIT33/LAM3 (10.7%), SIT53/T1 (8.7%), SIT50/H3 (7.8%), and SIT37/T3 (6.8%). We conclude that LAM and T genotypes are the most prevalent genotypes of MTBC in Santiago, Chile, and together correspond to almost two thirds of analyzed strains, which is similar to strain distribution reported from other countries of Latin America. Nevertheless, the high proportion of SIT37/T3, which was rarely found in other Latin American countries, may underline a specific history or demographics of Chile related to probable human migrations and evolutions.     

Connective-Tissue Growth Factor (CTGF/CCN2) Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro

Connective-tissue growth factor (CTGF) is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61), CTGF and nephroblastoma overexpressed (NOV). CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling. Additionally, CTGF-induced differentiation of glioblastoma stem cells into a less-tumorigenic state could increase the chances of successful intervention, since differentiated cells are more vulnerable to cancer treatments.     

Phylogeny of Acronychia (Rutaceae) and First Insights into Its Historical Biogeography and the Evolution of Fruit Characters

Background

The genus Acronychia (Citrus family, Rutaceae) contains 49 species of trees and shrubs that are found mainly in rain forest. The genus has a large distributional range from mainland southern Asia to Australia and New Caledonia, but most species are endemic to either New Guinea or Australia. This study aimed to provide the first detailed molecular phylogeny of Acronychia and use it to test the taxonomic value of fruit morphological characters, and infer the historical biogeography of the genus.

Methodology

Phylogenetic analyses (Bayesian Inference, Maximum Likelihood) were undertaken on nucleotide sequence data from two plastid (psbA-trnH, trnL-trnF) and three nuclear markers (ETS, ITS, NIAi3) from 29 Acronychia species (59% of the genus) and representatives of related genera.

Results and Conclusions

The results indicate that the South-East Asian genus Maclurodendron is nested phylogenetically within Acronychia and must be synonymized to render Acronychia monophyletic. Fruit morphological characters have been used previously to infer relationships within Acronychia and our analyses show that these characters are informative for some subclades but are homoplasious for the group as a whole. Apocarpous fruits are the ancestral state in Acronychia and subapocarpous and fully syncarpous fruits are derived. The unisexual flowers of Maclurodendron are derived from bisexual flowers, which are found in all species of Acronychia as well as its relatives. Acronychia probably first evolved on Australia with range expansion to New Guinea via stepping-stone dispersal or direct land connections within the Sahul Shelf, followed by two independent dispersals to areas west of New Guinea. Most species of Acronychia occur in either Australia or New Guinea, but no species occurs in both regions. This is surprising given the close proximity of the landmasses, but might be explained by ecological factors.     

Mosquito-Disseminated Pyriproxyfen Yields High Breeding-Site Coverage and Boosts Juvenile Mosquito Mortality at the Neighborhood Scale

Background

Mosquito-borne pathogens pose major public health challenges worldwide. With vaccines or effective drugs still unavailable for most such pathogens, disease prevention heavily relies on vector control. To date, however, mosquito control has proven difficult, with low breeding-site coverage during control campaigns identified as a major drawback. A novel tactic exploits the egg-laying behavior of mosquitoes to have them disseminate tiny particles of a potent larvicide, pyriproxyfen (PPF), from resting to breeding sites, thus improving coverage. This approach has yielded promising results at small spatial scales, but its wider applicability remains unclear.

Methodology/Principal Findings

We conducted a four-month trial within a 20-month study to investigate mosquito-driven dissemination of PPF dust-particles from 100 ‘dissemination stations’ (DSs) deployed in a 7-ha sub-area to surveillance dwellings and sentinel breeding sites (SBSs) distributed over an urban neighborhood of about 50 ha. We assessed the impact of the trial by measuring juvenile mosquito mortality and adult mosquito emergence in each SBS-month. Using data from 1,075 dwelling-months, 2,988 SBS-months, and 29,922 individual mosquitoes, we show that mosquito-disseminated PPF yielded high coverage of dwellings (up to 100%) and SBSs (up to 94.3%). Juvenile mosquito mortality in SBSs (about 4% at baseline) increased by over one order of magnitude during PPF dissemination (about 75%). This led to a >10-fold decrease of adult mosquito emergence from SBSs, from approximately 1,000–3,000 adults/month before to about 100 adults/month during PPF dissemination.

Conclusions/Significance

By expanding breeding-site coverage and boosting juvenile mosquito mortality, a strategy based on mosquito-disseminated PPF has potential to substantially enhance mosquito control. Sharp declines in adult mosquito emergence can lower vector/host ratios, reducing the risk of disease outbreaks. This approach is a very promising complement to current and novel mosquito control strategies; it will probably be especially relevant for the control of urban disease vectors, such as Aedes and Culex species, that often cause large epidemics.