Analysis of XMRV integration sites from human prostate cancer tissues suggests PCR contamination rather than genuine human infection

XMRV is a gammaretrovirus associated in some studies with human prostate cancer and chronic fatigue syndrome. Central to the hypothesis of XMRV as a human pathogen is the description of integration sites in DNA from prostate tumour tissues. Here we demonstrate that 2 of 14 patient-derived sites are identical to sites cloned in the same laboratory from experimentally infected DU145 cells. Identical integration sites have never previously been described in any retrovirus infection. We propose that the patient-derived sites are the result of PCR contamination. This observation further undermines the notion that XMRV is a genuine human pathogen

Phylogenetic surveillance of viral genetic diversity and the evolving molecular epidemiology of human immunodeficiency virus type 1

With ongoing generation of viral genetic diversity and increasing levels of migration, the global human immunodeficiency virus type 1 (HIV-1) epidemic is becoming increasingly heterogeneous. In this study, we investigate the epidemiological characteristics of 5,675 HIV-1 pol gene sequences sampled from distinct infections in the United Kingdom. These sequences were phylogenetically analyzed in conjunction with 976 complete-genome and 3,201 pol gene reference sequences sampled globally and representing the broad range of HIV-1 genetic diversity, allowing us to estimate the probable geographic origins of the various strains present in the United Kingdom. A statistical analysis of phylogenetic clustering in this data set identified several independent transmission chains within the United Kingdom involving recently introduced strains and indicated that strains more commonly associated with infections acquired heterosexually in East Africa are spreading among men who have sex with men. Coalescent approaches were also used and indicated that the transmission chains that we identify originated in the late 1980s to early 1990s. Similar changes in the epidemiological structuring of HIV epidemics are likely to be taking in place in other industrialized nations with large immigrant populations. The framework implemented here takes advantage of the vast amount of routinely generated HIV-1 sequence data and can provide epidemiological insights not readily obtainable through standard surveillance methods

Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation

Background: Cells transformed by human adenoviruses (Ad) exhibit differential capacities to induce tumours in immunocompetent rodents; for example, Ad12-transformed rodent cells are oncogenic whereas Ad5-transformed cells are not. The E1A gene determines oncogenic phenotype, is a transcriptional regulator and dysregulates host cell gene expression, a key factor in both cellular transformation and oncogenesis. To reveal differences in gene expression between cells transformed with oncogenic and non-oncogenic adenoviruses we have performed comparative analysis of transcript profiles with the aim of identifying candidate genes involved in the process of neoplastic transformation. Results: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells. Gene information was available for 193 transcripts and using gene ontology (GO) classifications and literature searches it was possible to assign known or suggested functions to 166 of these identified genes. A subset of differentially-expressed genes from the microarray was further examined by real-time PCR and Western blotting using BRK cells immortalised by Ad12 E1A or Ad5 E1A in addition to Ad12 E1- or Ad5 E1-transformed BRK cells. Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells. Conclusion: These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation. Further work will focus on investigating which splice variant of Ad E1A is responsible for the observed dysregulation at the pathway level, and the mechanisms of E1A-mediated transcriptional regulation

Host genetic variants and gene expression patterns associated with Epstein-Barr virus copy number in lymphoblastoid cell lines.

Lymphoblastoid cell lines (LCLs) are commonly used in molecular genetics, supplying DNA for the HapMap and 1000 Genomes Projects, used to test chemotherapeutic agents, and informing the basis of a number of population genetics studies of gene expression. The process of transforming human B cells into LCLs requires the presence of Epstein-Barr virus (EBV), a double-stranded DNA virus which through B-cell immortalisation maintains an episomal virus genome in every cell of an LCL at variable copy numbers. Previous studies have reported that EBV alters host-gene expression and EBV copy number may be under host genetic control. We performed a genome-wide association study of EBV genome copy number in LCLs and found the phenotype to be highly heritable, although no individual SNPs achieved a significant association with EBV copy number. The expression of two host genes (CXCL16 and AGL) was positively correlated and expression of ADARB2 was negatively correlated with EBV copy number in a genotype-independent manner. This study shows an association between EBV copy number and the gene expression profile of LCLs, and suggests that EBV copy number should be considered as a covariate in future studies of host gene expression in LCLs

Identification and characterization of Coronaviridae genomes from Vietnamese bats and rats based on conserved protein domains.

The Coronaviridae family of viruses encompasses a group of pathogens with a zoonotic potential as observed from previous outbreaks of the severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus. Accordingly, it seems important to identify and document the coronaviruses in animal reservoirs, many of which are uncharacterized and potentially missed by more standard diagnostic assays. A combination of sensitive deep sequencing technology and computational algorithms is essential for virus surveillance, especially for characterizing novel- or distantly related virus strains. Here, we explore the use of profile Hidden Markov Model-defined Pfam protein domains (Pfam domains) encoded by new sequences as a Coronaviridae sequence classification tool. The encoded domains are used first in a triage to identify potential Coronaviridae sequences and then processed using a Random Forest method to classify the sequences to the Coronaviridae genus level. The application of this algorithm on Coronaviridae genomes assembled from agnostic deep sequencing data from surveillance of bats and rats in Dong Thap province (Vietnam) identified thirty-four Alphacoronavirus and eleven Betacoronavirus genomes. This collection of bat and rat coronaviruses genomes provided essential information on the local diversity of coronaviruses and substantially expanded the number of coronavirus full genomes available from bat and rats and may facilitate further molecular studies on this group of viruses

Dynamic variation of CD5 surface expression levels within individual chronic lymphocytic leukemia clones.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonally derived mature CD5high B cells; however, the cellular origin of CLL is still unknown. Patients with CLL also harbor variable numbers of CD5low B cells, but the clonal relationship of these cells to the bulk disease is unknown and can have important implications for monitoring, treating, and understanding the biology of CLL. Here, we use B-cell receptors (BCRs) as molecular barcodes to first show by single-cell BCR sequencing that the great majority of CD5low B cells in the blood of CLL patients are clonally related to CD5high CLL B cells. We investigate whether CD5 state switching was likely to occur continuously as a common event or as a rare event in CLL by tracking somatic BCR mutations in bulk CLL B cells and using them to reconstruct the phylogenetic relationships and evolutionary history of the CLL in four patients. Using statistical methods, we show that there is no parsimonious route from a single or low number of CD5low switch events to the CD5high population, but rather, large-scale and/or dynamic switching between these CD5 states is the most likely explanation. The overlapping BCR repertoires between CD5high and CD5low cells from CLL patient peripheral blood reveal that CLL exists in a continuum of CD5 expression. The major proportion of CD5low B cells in patients are leukemic, thus identifying CD5low B cells as an important component of CLL, with implications for CLL pathogenesis, clinical monitoring, and the development of anti-CD5-directed therapies

Deep sequencing of norovirus genomes defines evolutionary patterns in an urban tropical setting.

UNLABELLED: Norovirus is a highly transmissible infectious agent that causes epidemic gastroenteritis in susceptible children and adults. Norovirus infections can be severe and can be initiated from an exceptionally small number of viral particles. Detailed genome sequence data are useful for tracking norovirus transmission and evolution. To address this need, we have developed a whole-genome deep-sequencing method that generates entire genome sequences from small amounts of clinical specimens. This novel approach employs an algorithm for reverse transcription and PCR amplification primer design using all of the publically available norovirus sequence data. Deep sequencing and de novo assembly were used to generate norovirus genomes from a large set of diarrheal patients attending three hospitals in Ho Chi Minh City, Vietnam, over a 2.5-year period. Positive-selection analysis and direct examination of protein changes in the virus over time identified codons in the regions encoding proteins VP1, p48 (NS1-2), and p22 (NS4) under positive selection and expands the known targets of norovirus evolutionary pressure. IMPORTANCE: The high transmissibility and rapid evolutionary rate of norovirus, combined with a short-lived host immune responses, are thought to be the reasons why the virus causes the majority of pediatric viral diarrhea cases. The evolutionary patterns of this RNA virus have been described in detail for only a portion of the virus genome and never for a virus from a detailed urban tropical setting. We provide a detailed sequence description of the noroviruses circulating in three Ho Chi Minh City hospitals over a 2.5-year period. This study identified patterns of virus change in known sites of host immune response and identified three additional regions of the virus genome under selection that were not previously recognized. In addition, the method described here provides a robust full-genome sequencing platform for community-based virus surveillance

Genome-Wide Sequence Analysis of Kaposi Sarcoma-Associated Herpesvirus Shows Diversification Driven by Recombination.

BACKGROUND: Kaposi sarcoma-associated herpesvirus (KSHV) establishes lifelong infection in the human host and has been associated with a variety of malignancies. KSHV displays striking geographic variation in prevalence, which is highest in sub-Saharan Africa. The current KSHV genome sequences available are all tumor cell line-derived or primary tumor-associated viruses, which have provided valuable insights into KSHV genetic diversity. METHODS: Here, we sequenced 45 KSHV genomes from a Ugandan population cohort in which KSHV is endemic; these are the only genome sequences obtained from nondiseased individuals and of KSHV DNA isolated from saliva. RESULTS: Population structure analysis, along with the 25 published genome sequences from other parts of the world, showed whole-genome variation, separating sequences and variation within the central genome contributing to clustering of genomes by geography. We reveal new evidence for the presence of intragenic recombination and multiple recombination events contributing to the divergence of genomes into at least 5 distinct types. DISCUSSION: This study shows that large-scale genome-wide sequencing from clinical and epidemiological samples is necessary to capture the full extent of genetic diversity of KSHV, including recombination, and provides evidence to suggest a revision of KSHV genotype nomenclature

Characterization of Posa and Posa-like virus genomes in fecal samples from humans, pigs, rats, and bats collected from a single location in Vietnam.

Porcine stool-associated RNA virus (posavirus), and Human stool-associated RNA virus (husavirus) are viruses in the order Picornavirales recently described in porcine and human fecal samples. The tentative group (Posa and Posa-like viruses: PPLVs) also includes fish stool-associated RNA virus (fisavirus) as well as members detected in insects (Drosophila subobscura and Anopheles sinensis) and parasites (Ascaris suum). As part of an agnostic deep sequencing survey of animal and human viruses in Vietnam, we detected three husaviruses in human fecal samples, two of which share 97-98% amino acid identity to Dutch husavirus strains and one highly divergent husavirus with only 25% amino acid identity to known husaviruses. In addition, the current study found forty-seven complete posavirus genomes from pigs, ten novel rat stool-associated RNA virus genomes (tentatively named rasavirus), and sixteen novel bat stool-associated RNA virus genomes (tentatively named basavirus). The five expected Picornavirales protein domains (helicase, 3C-protease, RNA-dependent RNA polymerase, and two Picornavirus capsid domain) were found to be encoded by all PPLV genomes. In addition, a nucleotide composition analysis revealed that the PPLVs shared compositional properties with arthropod viruses and predicted non-mammalian hosts for all PPLV lineages. The study adds seventy-six genomes to the twenty-nine PPLV genomes currently available and greatly extends our sequence knowledge of this group of viruses within the Picornavirales order