Hepatitis C virus genotype 1b chimeric replicon containing genotype 3 NS5A domain

AbstractInfections with hepatitis C virus (HCV) genotype 3 exhibit differences in clinical phenotype including an increase in response to interferon therapy and development of steatosis. To initiate studies on genotype 3, we created a chimeric genotype 1b replicon containing a genotype 3a NS5A domain. The chimera was capable of efficient colony formation after the selection of a novel dominant adaptive mutation. Thus, domains from highly different strains can interact to form a functional replicase. A new genotype 1a replicon was constructed as well. Genotype specific influence on interferon sensitivity was examined using genotype 1a, 1b and chimeric 1b–3a replicons. The genotype 3a NS5A domain did not increase the sensitivity of the chimeric replicon to IFNα. The results suggest that NS5A is not sufficient to convey the increased IFNα response by genotype 3 or the replicon model is not capable of mimicking the events involved in increased sustained viral response

Subclinical infection of macaques and baboons with a baboon simarterivirus

Simarteriviruses (Arteriviridae: Simarterivirinae) are commonly found at high titers in the blood of African monkeys but do not cause overt disease in these hosts. In contrast, simarteriviruses cause severe disease in Asian macaques upon accidental or experimental transmission. Here, we sought to better understand the host-dependent drivers of simarterivirus pathogenesis by infecting olive baboons (n = 4) and rhesus monkeys (n = 4) with the simarterivirus Southwest baboon virus 1 (SWBV-1). Surprisingly, none of the animals in our study showed signs of disease following SWBV-1 inoculation. Three animals (two rhesus monkeys and one olive baboon) became infected and sustained high levels of SWBV-1 viremia for the duration of the study. The course of SWBV-1 infection was highly predictable: plasma viremia peaked between 1 × 107 and 1 × 108 vRNA copies/mL at 3–10 days post-inoculation, which was followed by a relative nadir and then establishment of a stable set-point between 1 × 106 and 1 × 107 vRNA copies/mL for the remainder of the study (56 days). We characterized cellular and antibody responses to SWBV-1 infection in these animals, demonstrating that macaques and baboons mount similar responses to SWBV-1 infection, yet these responses are ineffective at clearing SWBV-1 infection. SWBV-1 sequencing revealed the accumulation of non-synonymous mutations in a region of the genome that corresponds to an immunodominant epitope in the simarterivirus major envelope glycoprotein GP5, which likely contribute to viral persistence by enabling escape from host antibodies

Plant Immune Memory in Systemic Tissue Does Not Involve Changes in Rapid Calcium Signaling

Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an “oscillatory-like” Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus – neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses

Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C

Persistent infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) account for the majority of cases of hepatic cirrhosis and hepatocellular carcinoma (HCC) worldwide. Small, non-coding RNAs play important roles in virus-host interactions. We used high throughput sequencing to conduct an unbiased profiling of small (14-40 nts) RNAs in liver from Japanese subjects with advanced hepatitis B or C and hepatocellular carcinoma (HCC). Small RNAs derived from tRNAs, specifically 30–35 nucleotide-long 5′ tRNA-halves (5′ tRHs), were abundant in non-malignant liver and significantly increased in humans and chimpanzees with chronic viral hepatitis. 5′ tRH abundance exceeded microRNA abundance in most infected non-cancerous tissues. In contrast, in matched cancer tissue, 5′ tRH abundance was reduced, and relative abundance of individual 5′ tRHs was altered. In hepatitis B-associated HCC, 5′ tRH abundance correlated with expression of the tRNA-cleaving ribonuclease, angiogenin. These results demonstrate that tRHs are the most abundant small RNAs in chronically infected liver and that their abundance is altered in liver cancer

Ancient Lowland Maya neighborhoods: Average Nearest Neighbor analysis and kernel density models, environments, and urban scale

Many humans live in large, complex political centers, composed of multi-scalar communities including neighborhoods and districts. Both today and in the past, neighborhoods form a fundamental part of cities and are defined by their spatial, architectural, and material elements. Neighborhoods existed in ancient centers of various scales, and multiple methods have been employed to identify ancient neighborhoods in archaeological contexts. However, the use of different methods for neighborhood identification within the same spatiotemporal setting results in challenges for comparisons within and between ancient societies. Here, we focus on using a single method—combining Average Nearest Neighbor (ANN) and Kernel Density (KD) analyses of household groups—to identify potential neighborhoods based on clusters of households at 23 ancient centers across the Maya Lowlands. While a one-size-fits all model does not work for neighborhood identification everywhere, the ANN/KD method provides quantifiable data on the clustering of ancient households, which can be linked to environmental zones and urban scale. We found that centers in river valleys exhibited greater household clustering compared to centers in upland and escarpment environments. Settlement patterns on flat plains were more dispersed, with little discrete spatial clustering of households. Furthermore, we categorized the ancient Maya centers into discrete urban scales, finding that larger centers had greater variation in household spacing compared to medium-sized and smaller centers. Many larger political centers possess heterogeneity in household clustering between their civic-ceremonial cores, immediate hinterlands, and far peripheries. Smaller centers exhibit greater household clustering compared to larger ones. This paper quantitatively assesses household clustering among nearly two dozen centers across the Maya Lowlands, linking environment and urban scale to settlement patterns. The findings are applicable to ancient societies and modern cities alike; understanding how humans form multi-scalar social groupings, such as neighborhoods, is fundamental to human experience and social organization

Simian Immunodeficiency Virus Infection of Chimpanzees (Pan troglodytes) Shares Features of Both Pathogenic and Non-pathogenic Lentiviral Infections.

The virus-host relationship in simian immunodeficiency virus (SIV) infected chimpanzees is thought to be different from that found in other SIV infected African primates. However, studies of captive SIVcpz infected chimpanzees are limited. Previously, the natural SIVcpz infection of one chimpanzee, and the experimental infection of six chimpanzees was reported, with limited follow-up. Here, we present a long-term study of these seven animals, with a retrospective re-examination of the early stages of infection. The only clinical signs consistent with AIDS or AIDS associated disease was thrombocytopenia in two cases, associated with the development of anti-platelet antibodies. However, compared to uninfected and HIV-1 infected animals, SIVcpz infected animals had significantly lower levels of peripheral blood CD4+ T-cells. Despite this, levels of T-cell activation in chronic infection were not significantly elevated. In addition, while plasma levels of β2 microglobulin, neopterin and soluble TNF-related apoptosis inducing ligand (sTRAIL) were elevated in acute infection, these markers returned to near-normal levels in chronic infection, reminiscent of immune activation patterns in 'natural host' species. Furthermore, plasma soluble CD14 was not elevated in chronic infection. However, examination of the secondary lymphoid environment revealed persistent changes to the lymphoid structure, including follicular hyperplasia in SIVcpz infected animals. In addition, both SIV and HIV-1 infected chimpanzees showed increased levels of deposition of collagen and increased levels of Mx1 expression in the T-cell zones of the lymph node. The outcome of SIVcpz infection of captive chimpanzees therefore shares features of both non-pathogenic and pathogenic lentivirus infections.This work was supported by the Biotechnology and Biological Sciences Research Council and by the Wellcome Trust.This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.ppat.100514

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Analysis of Host Range Phenotypes of Primate Hepadnaviruses by In Vitro Infections of Hepatitis D Virus Pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05–2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy