Production of infectious hepatitis C virus of various genotypes in cell cultures

A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.     

Extremely divergent song types in the genus Aerotegmina Hemp (Orthoptera: Tettigoniidae: Hexacentrinae) and the description of a new species from the Eastern Arc Mountains of Tanzania (East Africa)

The genus Aerotegmina comprises a group of African canopy-dwelling, predatory bushcrickets (katydids) that is known for its inflated tegmina and the unusually loud and low-frequency calling song of its type species. Here, we describe the songs of another two species of the genus which are much larger than the type species and have an even lower peak frequency. In addition, small and large species differ in song structure. While the type species and closely related forms have a broad banded, multi-peak song with strong components in the audio and ultrasonic range, the species treated here have an extremely narrow banded, resonant song with a carrier frequency of 2 kHz. With Q-values above 100 these sounds belong to the purest songs ever recorded in Orthoptera. Besides describing songs, stridulatory organs and a new species we discuss the possible functions of the song type. A key to the species of Aerotegmina is provided.     

Cyclic di‐GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di‐GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c‐di‐GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c‐di‐GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c‐di‐GMP being a key second messenger that silences energy‐costing systems during early colonization phase and biofilm formation, while low c‐di‐GMP levels unleash T6SS and T4SS to advance plant colonization.     

Phenotypic divergence during speciation is inversely associated with differences in seasonal migration

Differences in seasonal migration might promote reproductive isolation and differentiation by causing populations in migratory divides to arrive on the breeding grounds at different times and/or produce hybrids that take inferior migratory routes. We examined this question by quantifying divergence in song, colour, and morphology between sister pairs of North American migratory birds. We predicted that apparent rates of phenotypic differentiation would differ between pairs that do and do not form migratory divides. Consistent with this prediction, results from mixed effects models and Ornstein–Uhlenbeck models of evolution showed different rates of divergence between these groups; surprisingly, differentiation was greater among non-divide pairs. We interpret this finding as a result of variable rates of population blending and fusion between partially diverged forms. Ancient pairs of populations that subsequently fused are now observed as a single form, whereas those that did not fuse are observable as pairs and included in our study. We propose that fusion of two populations is more likely to occur when they have similar migratory routes and little other phenotypic differentiation that would cause reproductive isolation. By contrast, pairs with migratory divides are more likely to remain reproductively isolated, even when differing little in other phenotypic traits. These findings suggest that migratory differences may be one among several isolating barriers that prevent divergent populations from fusing and thereby increase the likelihood that they will continue differentiating as distinct species.     

Infection of Human Retinal Pigment Epithelium with Chlamydia trachomatis

Purpose

Little is known about the susceptibility of posterior segment tissues, particularly the human retinal pigment epithelium (hRPE), to Chlamydia trachomatis. The purpose of the study was to investigate the possibility of infecting the hRPE with Chlamydia trachomatis, and to examine the infectivity of different Chlamydia trachomatis clinical isolates for hRPE cells and the hRPE cell response to the infection.

Methods

Cultured hRPE and McCoy cells were inoculated with eight Chlamydia trachomatis (serovar E) clinical isolates at multiplicity of infection (MOI) of 2.0 or 0.3. To detect Chlamydia trachomatis, samples were stained immunohistochemically with anti-major outer membrane protein antibodies at 24h, 48h, and 72h postinoculation (PI). The changes in the expression of signaling molecules and proteins of cytoskeleton and extracellular matrix in hRPE cells were examined immunohistochemically.

Results

All eight clinical isolates demonstrated ability to infect hRPE cells. At equal MOI of 0.3, the infectivity of Chlamydia trachomatis clinical isolates for RPE culture was found to be at least as high as that for McCoy cell culture. At 24h PI, the percentage of inclusion-containing cells varied from 1.5 ± 0.52 to 14.6 ± 3.3% in hRPE cell culture infected at MOI of 2.0 against 0.37 ± 0.34 to 8.9 ± 0.2% in McCoy cell culture infected at MOI of 0.3. Collagen type I, collagen type IV, basic fibroblast growth factor, transforming growth factor-beta and interleukin–8 expression at 48h PI were maximally increased, by 2.1-, 1.3-, 1.5-, 1.5- and 1.6-fold, respectively, in the Chlamydia trachomatis-infected compared with control hRPE cell culture specimens (P < 0.05).

Conclusions

This study, for the first time, proved the possibility of infecting hRPE cultured cells with Chlamydia trachomatis, which leads to proproliferative and proinflammatory changes in the expression of signaling molecules and extracellular matrix components.     

Peripheral Blood T Cell Dynamics Predict Relapse in Multiple Sclerosis Patients on Fingolimod

Background

Fingolimod efficiently reduces multiple sclerosis (MS) relapse by inhibiting lymphocyte egress from lymph nodes through down-modulation of sphingosine 1-phosphate (S1P) receptors. We aimed to clarify the alterations in peripheral blood T cell subsets associated with MS relapse on fingolimod.

Methods/Principal Findings

Blood samples successively collected from 23 relapsing-remitting MS patients before and during fingolimod therapy (0.5 mg/day) for 12 months and 18 healthy controls (HCs) were analysed for T cell subsets by flow cytometry. In MS patients, the percentages of central memory T (CCR7⁺CD45RO⁺) cells (TCM) and naïve T (CCR7⁺CD45RO^-) cells decreased significantly, while those of effector memory T (CCR7^-CD45RA^-) and suppressor precursor T (CD28^-) cells increased in both CD4⁺T and CD8⁺T cells from 2 weeks to 12 months during fingolimod therapy. The percentages of regulatory T (CD4⁺CD25^highCD127^low) cells in CD4⁺T cells and CCR7^-CD45RA⁺T cells in CD8⁺T cells also increased significantly. Eight relapsed patients demonstrated greater percentages of CD4⁺TCM than 15 non-relapsed patients at 3 and 6 months (p=0.0051 and p=0.0088, respectively). The IL17-, IL9-, and IL4-producing CD4⁺T cell percentages were significantly higher at pre-treatment in MS patients compared with HCs (p<0.01 for all), while the IL17-producing CD4⁺T cell percentages tended to show a transient increase at 2 weeks of fingolimod therapy (p^corr=0.0834).

Conclusions

The CD4⁺TCM percentages at 2 weeks to 12 months during fingolimod therapy are related to relapse.     

Time Clustered Sampling Can Inflate the Inferred Substitution Rate in Foot-And-Mouth Disease Virus Analyses

With the emergence of analytical software for the inference of viral evolution, a number of studies have focused on estimating important parameters such as the substitution rate and the time to the most recent common ancestor (t _MRCA) for rapidly evolving viruses. Coupled with an increasing abundance of sequence data sampled under widely different schemes, an effort to keep results consistent and comparable is needed. This study emphasizes commonly disregarded problems in the inference of evolutionary rates in viral sequence data when sampling is unevenly distributed on a temporal scale through a study of the foot-and-mouth (FMD) disease virus serotypes SAT 1 and SAT 2. Our study shows that clustered temporal sampling in phylogenetic analyses of FMD viruses will strongly bias the inferences of substitution rates and t _MRCA because the inferred rates in such data sets reflect a rate closer to the mutation rate rather than the substitution rate. Estimating evolutionary parameters from viral sequences should be performed with due consideration of the differences in short-term and longer-term evolutionary processes occurring within sets of temporally sampled viruses, and studies should carefully consider how samples are combined.