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The presence of human cytomegalovirus (HCMV) in glioblastoma (GBM) and improved outcomes of GBM patients receiving therapies targeting the virus have implicated HCMV in GBM progression. However, a unifying mechanism that accounts for the contribution of HCMV to the malignant phenotype of GBM remains incompletely defined. Here we have identified SOX2, a marker of glioma stem cells (GSCs), as a key determinant of HCMV gene expression in gliomas. Our studies demonstrated that SOX2 downregulated promyelocytic leukemia (PML) and Sp100 and consequently facilitated viral gene expression by decreasing the amount of PML nuclear bodies in HCMV-infected glioma cells. Conversely, the expression of PML antagonized the effects of SOX2 on HCMV gene expression. Furthermore, this regulation of SOX2 on HCMV infection was demonstrated in a neurosphere assay of GSCs and in a murine xenograft model utilizing xenografts from patient-derived glioma tissue. In both cases, SOX2 overexpression facilitated the growth of neurospheres and xenografts implanted in immunodeficient mice. Lastly, the expression of SOX2 and HCMV immediate early 1 (IE1) protein could be correlated in tissues from glioma patients, and interestingly, elevated levels of SOX2 and IE1 were predictive of a worse clinical outcome. These studies argue that HCMV gene expression in gliomas is regulated by SOX2 through its regulation of PML expression and that targeting molecules in this SOX2-PML pathway could identify therapies for glioma treatment.

Coordinated surveillance, vaccination and public information efforts have brought the Chinese rabies epizootic under control, but significant numbers of fatalities are still reported annually with some cases occurring in previously rabies free regions. Tibet has remained virtually rabies free for 16 years, but since 2015 one human rabies case has been reported each year. To better understand the origins of these cases, we sequenced three human samples and an additional sample isolated from a dog in 2012. Three genomes were sequenced from brain samples: human case 1 (reported in 2015), human case 3 (2017), and the 2012 dog case. For human case 2 (2016), the rabies N gene was sequenced from a limited saliva sample. Phylogenetic analysis shows that Case 1 (CXZ1501H) and the dog case (CXZ1201D) belong to China IV lineage (equivalent to Arctic-like-2 in global rabies), suggesting an association with a wildlife spillover event. However, Case 2 (CXZ1601H) is placed within the dominant lineage China I, and was most similar with recent strains from neighboring Yunnan province, indicating the current epizootic has finally reached Tibet. Most surprisingly however, was the finding that Case 3 (CXZ1704H) is distinct from other Chinese isolates. This isolate is placed in the Indian Subcontinent clade, similar to recent Nepal strains, indicating that cross-border transmission is a new source for rabies infections. Thus, the complex mixture of the rabies epizootic in Tibet represents a major new challenge for Tibet and national rabies control.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression by binding to partially complementary regions within the 3'UTR of their target genes. Computational methods play an important role in target prediction and assume that the miRNA "seed region" (nt 2 to 8) is required for functional targeting, but typically only identify ∼80% of known bindings. Recent studies have highlighted a role for the entire miRNA, suggesting that a more flexible methodology is needed. We present a novel approach for miRNA target prediction based on Deep Learning (DL) which, rather than incorporating any knowledge (such as seed regions), investigates the entire miRNA and 3'TR mRNA nucleotides to learn a uninhibited set of feature descriptors related to the targeting process. We collected more than 150,000 experimentally validated homo sapiens miRNA:gene targets and cross referenced them with different CLIP-Seq, CLASH and iPAR-CLIP datasets to obtain ∼20,000 validated miRNA:gene exact target sites. Using this data, we implemented and trained a deep neural network-composed of autoencoders and a feed-forward network-able to automatically learn features describing miRNA-mRNA interactions and assess functionality. Predictions were then refined using information such as site location or site accessibility energy. In a comparison using independent datasets, our DL approach consistently outperformed existing prediction methods, recognizing the seed region as a common feature in the targeting process, but also identifying the role of pairings outside this region. Thermodynamic analysis also suggests that site accessibility plays a role in targeting but that it cannot be used as a sole indicator for functionality. Data and source code available at: https://bitbucket.org/account/user/bipous/projects/MIRAW.

Congenital human cytomegalovirus (HCMV) infection is the leading cause of neurological disabilities in children worldwide, but the mechanisms underlying these disorders are far from well-defined. HCMV infection has been shown to dysregulate the Notch signaling pathway in human neural progenitor cells (NPCs). As an important downstream effector of Notch signaling, the transcriptional regulator Hairy and Enhancer of Split 1 (Hes1) is essential for governing NPC fate and fetal brain development. In the present study, we report that HCMV infection downregulates Hes1 protein levels in infected NPCs. The HCMV 72-kDa immediate-early 1 protein (IE1) is involved in Hes1 degradation by assembling a ubiquitination complex and promoting Hes1 ubiquitination as a potential E3 ubiquitin ligase, followed by proteasomal degradation of Hes1. Sp100A, an important component of PML nuclear bodies, is identified to be another target of IE1-mediated ubiquitination. A C-terminal acidic region in IE1, spanning amino acids 451 to 475, is required for IE1/Hes1 physical interaction and IE1-mediated Hes1 ubiquitination, but is dispensable for IE1/Sp100A interaction and ubiquitination. Our study suggests a novel mechanism linking downregulation of Hes1 protein to neurodevelopmental disorders caused by HCMV infection. Our findings also complement the current knowledge of herpesviruses by identifying IE1 as the first potential HCMV-encoded E3 ubiquitin ligase.

In China, rabies remains an ongoing threat to public health. Although control efforts have been effective in reducing the number of annual cases, the virus continues to spread into new areas. Tibet, Qinghai, Gansu and Ningxia in western China have, until recently, reported only a handful of events. However, since 2011, there have been increasing numbers of cases recorded in these areas. In this study, we report the collection and analysis of samples collected from these regions. We find that cases originate from two different sources. Strains collected from Gansu and Ningxia are closely related to the primary lineage associated with the current epizootic, whereas those from Tibet and Qinghai are related to the Arctic-like-2 lineage that is most commonly associated with wildlife cases in China. Thus, it appears that while the epizootic is beginning to encroach into Gansu and Ningxia, Tibet and Qinghai a significant number of rabies cases originate from wildlife.

Identifying patients most responsive to specific chemotherapy agents in neoadjuvant settings can help to maximize the benefits of treatment and minimize unnecessary side effects. Metagene approaches that predict response based on gene expression signatures derived from an associative analysis of clinical data can identify chance associations caused by the heterogeneity of a tumor, leading to reproducibility issues in independent validations. In this study, to incorporate information from drug mechanisms of action, we explore the potential of microRNA regulation networks as a new feature space for identifying predictive markers. We introduce a measure we term the CoMi (Context-specific-miRNA-regulation) pattern to represent a descriptive feature of the miRNA regulation network in the transcriptome. We examine whether the modifications to the CoMi pattern on specific biological processes are a useful representation of drug action by predicting the response to neoadjuvant Paclitaxel treatment in breast cancer and show that the drug counteracts the CoMi network dysregulation induced by tumorigenesis. We then generate a quantitative testbed to investigate the ability of the CoMi pattern to distinguish FDA approved breast cancer drugs from other FDA approved drugs not related to breast cancer. We also compare the ability of the CoMi and metagene methods to predict response to neoadjuvant Paclitaxel treatment in clinical cohorts. We find the CoMi method outperforms the metagene method, achieving area under curve (AUC) values of 0.78 and 0.66 respectively. Furthermore, several of the predicted CoMi features highlight the network-based mechanism of drug resistance. Thus, our study suggests that explicitly modeling the drug action using network biology provides a promising approach for predictive marker discovery.

China has seen a massive resurgence of rabies cases in the last 15 years with more than 25,000 human fatalities. Initial cases were reported in the southwest but are now reported in almost every province. There have been several phylogenetic investigations into the origin and spread of the virus within China but few reports investigating the impact of the epidemic on neighboring countries. We therefore collected nucleoprotein sequences from China and South East Asia and investigated their phylogenetic and phylogeographic relationship. Our results indicate that within South East Asia, isolates mainly cluster according to their geographic origin. We found evidence of sporadic exchange of strains between neighboring countries, but it appears that the major strain responsible for the current Chinese epidemic has not been exported. This suggests that national geographical boundaries and border controls are effective at halting the spread of rabies from China into adjacent regions. We further investigated the geographic structure of Chinese sequences and found that the current epidemic is dominated by variant strains that were likely present at low levels in previous domestic epidemics. We also identified epidemiological linkages between high incidence provinces consistent with observations based on surveillance data from human rabies cases.

INTRODUCTION: The Hepatitis B Virus (HBV) genome contains four ORFs, S (surface), P (polymerase), C (core) and X. S is completely overlapped by P and as a consequence the overlapping region is subject to distinctive evolutionary constraints compared to the remainder of the genome. Specifically, a non-synonymous substitution in one coding frame may produce a synonymous substitution in the alternative frame, suggesting a possible conflict between requirements for diversifying and purifying forces. To examine how these contrasting requirements are balanced within this region, we investigated the relationship amongst positive selection sites, conserved regions, epitopes and elements of protein structure to consider how HBV balances the contrasting evolutionary pressures. METHODOLOGY/RESULTS: 323 HBV genotype D genome sequences were collected and analyzed to identify sites under positive selection and highly conserved regions. Epitopes sequences were retrieved from previously published experimental studies stored in the Immune Epitope Database. Predicted secondary structures were used to investigate the association between structure and conservation. Entropy was used as a measure of conservation and bivariate logistic regression was used to investigate the relationship between positive selection/conserved sites and epitope/secondary structure regions. Our results indicate: (i) conservation in S is primarily dictated by α-helix elements in the protein structure, (ii) variable residues are mainly located in PreS, the major hydrophilic region (MHR) and the C-terminus, (iii) epitopes in S, which are directly targeted by the host immune system, are significantly associated with sites under positive selection. CONCLUSIONS: The highly variable spacer domain in P, which corresponds to PreS in S, appears to act as a harbor for the accumulation of mutations that can provide flexibility for conformational changes and responding to immune pressure.

Rabies remains a serious problem in China with three epidemics since 1949 and the country in the midst of the third epidemic. Significantly, the control of each outbreak has been followed by a rapid reemergence of the disease. In 2005, the government implemented a rabies national surveillance program that included the collection and screening of almost 8,000 samples. In this work, we analyzed a Chinese dataset comprising 320 glycoprotein sequences covering 23 provinces and eight species, spanning the second and third epidemics. Specifically, we investigated whether the three epidemics are associated with a single reemerging lineage or a different lineage was responsible for each epidemic. Consistent with previous results, phylogenetic analysis identified six lineages, China I to VI. Analysis of the geographical composition of these lineages revealed they are consistent with human case data and reflect the gradual emergence of China I in the third epidemic. Initially, China I was restricted to south China and China II was dominant. However, as the epidemic began to spread into new areas, China I began to emerge, whereas China II remained confined to south China. By the latter part of the surveillance period, almost all isolates were China I and contributions from the remaining lineages were minimal. The prevalence of China II in the early stages of the third epidemic and its established presence in wildlife suggests that it too replaced a previously dominant lineage during the second epidemic. This lineage replacement may be a consequence of control programs that were dominated by dog culling efforts as the primary control method in the first two epidemics. This had the effect of reducing dominant strains to levels comparable with other localized background stains. Our results indicate the importance of effective control strategies for long term control of the disease.

Background and objectivesRecent years have seen a rapid increase in the number of rabies cases in China and an expansion in the geographic distribution of the virus. In spite of the seriousness of the outbreak and increasing number of fatalities, little is known about the phylogeography of the disease in China. In this study, we report an analysis of a set of Nucleocapsid sequences consisting of samples collected through the trial Chinese National Surveillance System as well as publicly available sequences. This sequence set represents the most comprehensive dataset from China to date, comprising 210 sequences (including 57 new samples) from 15 provinces and covering all epidemic regions. Using this dataset we investigated genetic diversity, patterns of distribution, and evolutionary history.ResultsOur analysis indicates that the rabies virus in China is primarily defined by two clades that exhibit distinct population subdivision and translocation patterns and that contributed to the epidemic in different ways. The younger clade originated around 1992 and has properties that closely match the observed spread of the recent epidemic. The older clade originated around 1960 and has a dispersion pattern that suggests it represents a strain associated with a previous outbreak that remained at low levels throughout the country and reemerged in the current epidemic.ConclusionsOur findings provide new insight into factors associated with the recent epidemic and are relevant to determining an effective policy for controlling the virus.

Direct cloning of PCR fragments by TA cloning or blunt end ligation are two simple methods which would greatly benefit high-throughput (HTP) cloning constructions if the efficiency can be improved. In this study, we have developed a ribosomal binding site (RBS) switching strategy for direct cloning of PCR fragments. RBS is an A/G rich region upstream of the translational start codon and is essential for gene expression. Change from A/G to T/C in the RBS blocks its activity and thereby abolishes gene expression. Based on this property, we introduced an inactive RBS upstream of a selectable marker gene, and designed a fragment insertion site within this inactive RBS. Forward and reverse insertions of specifically tailed fragments will respectively form an active and inactive RBS, thus all background from vector self-ligation and fragment reverse insertions will be eliminated due to the non-expression of the marker gene. The effectiveness of our strategy for TA cloning and blunt end ligation are confirmed. Application of this strategy to gene over-expression, a bacterial two-hybrid system, a bacterial one-hybrid system, and promoter bank construction are also verified. The advantages of this simple procedure, together with its low cost and high efficiency, makes our strategy extremely useful in HTP cloning constructions.

BACKGROUND: The family Tymoviridae comprises three plant virus genera, including Tymovirus, Marafivirus, and Maculavirus, which are found in most parts of the world and cause severe agricultural losses. We describe a putatively novel member of the family Tymoviridae, which is isolated from mosquitoes (Culex spp.), referred to as CuTLV. METHODS AND RESULTS: The CuTLV was isolated by cell culture, which replicates and causes cytopathic effects in Aedes albopictus C6/36 cells, but not in mammalian BHK-21 or Vero cells. The complete 6471 nucleotide sequence of CuTLV was determined. The genome of CuTLV is predicted to contain three open reading frames (ORFs). The largest ORF1 is 5307 nucleotides (nt) in length and encodes a putative polypeptide of 1769 amino acids (aa), which contains the conserved motifs for the methyltransferase (MTR), Tymovirus endopeptidase (PRO), helicase (HEL), and RNA-dependent RNA polymerase (RdRp) of the replication-associated proteins (RPs) of positive-stranded RNA viruses. In contrast, the ORF1 sequence does not contain the so-called "tymobox" or "marafibox", the conserved subgenomic RNA promoter present in all tymoviruses or marafiviruses, respectively. ORF2 and ORF3 putatively encode a 248-aa coat protein (CP) and a proline-rich 149-aa polypeptide. The whole genomic nucleotide identity of CuTLV with other members of family Tymoviridae ranged from 46.2% (ChiYMV) to 52.4% (GFkV). Phylogenetic analysis based on the putative RP and CP genes of CuTLV demonstrated that the virus is most closely related to viruses in the genus Maculavirus. CONCLUSIONS: The CuTLV is a novel virus related to members of the family Tymoviridae, with molecular characters that are distinct from those of tymoviruses, marafiviruses, and other maculaviruses or macula-like viruses. This is the first report of the isolation of a Tymoviridae-like virus from mosquitoes. Further investigations are required to clarify the origin, replication strategy, and the public health or agricultural importance of the CuTLV.

BACKGROUND: Plant infection models provide certain advantages over animal models in the study of pathogenesis. However, current plant models face some limitations, e.g., plant and pathogen cannot co-culture in a contained environment. Development of such a plant model is needed to better illustrate host-pathogen interactions. METHODOLOGY/PRINCIPAL FINDINGS: We describe a novel model plant system for the study of human pathogenic bacterial infection on a large scale. This system was initiated by co-cultivation of axenic duckweed (Lemna minor) plants with pathogenic bacteria in 24-well polystyrene cell culture plate. Pathogenesis of bacteria to duckweed was demonstrated with Pseudomonas aeruginosa and Staphylococcus aureus as two model pathogens. P. aeruginosa PAO1 caused severe detriment to duckweed as judged from inhibition to frond multiplication and chlorophyll formation. Using a GFP-marked PAO1 strain, we demonstrated that bacteria colonized on both fronds and roots and formed biofilms. Virulence of PAO1 to duckweed was attenuated in its quorum sensing (QS) mutants and in recombinant strains overexpressing the QS quenching enzymes. RN4220, a virulent strain of S. aureus, caused severe toxicity to duckweed while an avirulent strain showed little effect. Using this system for antimicrobial chemical selection, green tea polyphenols exhibited inhibitory activity against S. aureus virulence. This system was further confirmed to be effective as a pathogenesis model using a number of pathogenic bacterial species. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that duckweed can be used as a fast, inexpensive and reproducible model plant system for the study of host-pathogen interactions, could serve as an alternative choice for the study of some virulence factors, and could also potentially be used in large-scale screening for the discovery of antimicrobial chemicals.

The high rates of failure in oncology drug clinical trials highlight the problems of using pre-clinical data to predict the clinical effects of drugs. Patient population heterogeneity and unpredictable physiology complicate pre-clinical cancer modeling efforts. We hypothesize that gene networks associated with cancer outcome in heterogeneous patient populations could serve as a reference for identifying drug effects. Here we propose a novel in vivo genetic interaction which we call 'synergistic outcome determination' (SOD), a concept similar to 'Synthetic Lethality'. SOD is defined as the synergy of a gene pair with respect to cancer patients' outcome, whose correlation with outcome is due to cooperative, rather than independent, contributions of genes. The method combines microarray gene expression data with cancer prognostic information to identify synergistic gene-gene interactions that are then used to construct interaction networks based on gene modules (a group of genes which share similar function). In this way, we identified a cluster of important epigenetically regulated gene modules. By projecting drug sensitivity-associated genes on to the cancer-specific inter-module network, we defined a perturbation index for each drug based upon its characteristic perturbation pattern on the inter-module network. Finally, by calculating this index for compounds in the NCI Standard Agent Database, we significantly discriminated successful drugs from a broad set of test compounds, and further revealed the mechanisms of drug combinations. Thus, prognosis-guided synergistic gene-gene interaction networks could serve as an efficient in silico tool for pre-clinical drug prioritization and rational design of combinatorial therapies.